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    Drug-perturbation-based stratification of blood cancer. Dietrich Sascha,Oleś Małgorzata,Lu Junyan,Sellner Leopold,Anders Simon,Velten Britta,Wu Bian,Hüllein Jennifer,da Silva Liberio Michelle,Walther Tatjana,Wagner Lena,Rabe Sophie,Ghidelli-Disse Sonja,Bantscheff Marcus,Oleś Andrzej K,Słabicki Mikołaj,Mock Andreas,Oakes Christopher C,Wang Shihui,Oppermann Sina,Lukas Marina,Kim Vladislav,Sill Martin,Benner Axel,Jauch Anna,Sutton Lesley Ann,Young Emma,Rosenquist Richard,Liu Xiyang,Jethwa Alexander,Lee Kwang Seok,Lewis Joe,Putzker Kerstin,Lutz Christoph,Rossi Davide,Mokhir Andriy,Oellerich Thomas,Zirlik Katja,Herling Marco,Nguyen-Khac Florence,Plass Christoph,Andersson Emma,Mustjoki Satu,von Kalle Christof,Ho Anthony D,Hensel Manfred,Dürig Jan,Ringshausen Ingo,Zapatka Marc,Huber Wolfgang,Zenz Thorsten The Journal of clinical investigation As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care. 10.1172/JCI93801
    <i>SF3B1</i>-mutated chronic lymphocytic leukemia shows evidence of NOTCH1 pathway activation including CD20 downregulation Pozzo Federico,Bittolo Tamara,Tissino Erika,Vit Filippo,Vendramini Elena,Laurenti Luca,D'Arena Giovanni,Olivieri Jacopo,Pozzato Gabriele,Zaja Francesco,Chiarenza Annalisa,Di Raimondo Francesco,Zucchetto Antonella,Bomben Riccardo,Rossi Francesca Maria,Del Poeta Giovanni,Dal Bo Michele,Gattei Valter Haematologica Chronic lymphocytic leukemia (CLL) is characterized by a low CD20 expression, in part explained by an epigenetic-driven downregulation triggered by mutations of the NOTCH1 gene. In the present study, by taking advantage of a wide and well-characterized CLL cohort (n=537), we demonstrate that CD20 expression is downregulated in SF3B1-mutated CLL in an extent similar to NOTCH1-mutated CLL. In fact, SF3B1-mutated CLL cells show common features with NOTCH1-mutated CLL cells, including a gene expression profile enriched of NOTCH1-related gene sets and elevated expression of the active intracytoplasmic NOTCH1. Activation of the NOTCH1 signaling and down-regulation of surface CD20 in SF3B1-mutated CLL cells correlate with over-expression of an alternatively spliced form of DVL2, a component of the Wnt pathway and negative regulator of the NOTCH1 pathway. These findings are confirmed by separately analyzing the CD20-dim and CD20-bright cell fractions from SF3B1-mutated cases as well as by DVL2 knock-out experiments in CLL-like cell models. Altogether, the clinical and biological features that characterize NOTCH1-mutated CLL may also be recapitulated in SF3B1-mutated CLL, contributing to explain the poor prognosis of this CLL subset and providing the rationale for expanding novel agents-based therapies to SF3B1-mutated CLL. 10.3324/haematol.2020.261891
    Effects of CD20 antibodies and kinase inhibitors on B-cell receptor signalling and survival of chronic lymphocytic leukaemia cells. Cavallini Chiara,Galasso Marilisa,Pozza Elisa Dalla,Chignola Roberto,Lovato Ornella,Dando Ilaria,Romanelli Maria G,Krampera Mauro,Pizzolo Giovanni,Donadelli Massimo,Scupoli Maria T British journal of haematology Recently, clinical trial results have established inhibitors of B-cell receptor (BCR)-associated kinase (BAKi), with or without CD20 moniclonal antibodies (mAbs), as the preferred first-line treatment for most chronic lymphocytic leukaemia (CLL) patients. Using phosphospecific flow cytometry, we showed that in leukaemic cells from CLL patients the CD20 therapeutic antibodies - rituximab, ofatumumab, and obinutuzumab - inhibited BCR signalling pathways targeting preferentially pBTK - but not BTK - and pAKT. On the contrary, ibrutinib and idelalisib reduced pBTK to a higher extent than pBTK . The strong reduction of pAKT induced by idelalisib was enhanced by its combination with rituximab or ofatumumab. Moreover, CD20 mAbs and BAKi induced the death of leukaemia cells that was significantly potentiated by their combination. Analysis of the enhancement of cell death in these combinations revealed an approximately additive enhancement induced by rituximab or obinutuzumab combined with ibrutinib or idelalisib. Taken together, our data identified negative regulatory effects of CD20 mAbs and their combinations with BAKi on BCR signalling and cell survival in CLL. In conclusion, this study advances our understanding of mechanisms of action of CD20 mAbs as single agents or in combination with BAKi and could inform on the potential of combined therapies in ongoing and future clinical trials in patients with CLL. 10.1111/bjh.17139
    Targeting CD20 takes the backseat in CLL. Byrd John C Blood 10.1182/blood-2019-01-892695
    Autologous T-cell activation fosters ABT-199 resistance in chronic lymphocytic leukemia: rationale for a combined therapy with SYK inhibitors and anti-CD20 monoclonal antibodies. Elías Esteban Enrique,Almejún María Belén,Colado Ana,Cordini Gregorio,Vergara-Rubio Maricef,Podaza Enrique,Risnik Denise,Cabrejo María,Fernández-Grecco Horacio,Bezares Raimundo Fernando,Custidiano María Del Rosario,Sánchez-Ávalos Julio César,Vicente Ángeles,Garate Gonzalo Martín,Borge Mercedes,Giordano Mirta,Gamberale Romina Haematologica 10.3324/haematol.2018.188680
    HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies. Bobrowicz Malgorzata,Dwojak Michal,Pyrzynska Beata,Stachura Joanna,Muchowicz Angelika,Berthel Elise,Dalla-Venezia Nicole,Kozikowski Mieszko,Siernicka Marta,Miazek Nina,Zapala Piotr,Domagala Antoni,Bojarczuk Kamil,Malenda Agata,Barankiewicz Joanna,Graczyk-Jarzynka Agnieszka,Zagozdzon Agnieszka,Gabrysiak Magdalena,Diaz Jean-Jacques,Karp Marta,Lech-Maranda Ewa,Firczuk Malgorzata,Giannopoulos Krzysztof,Efremov Dimitar G,Laurenti Luca,Baatout Dunja,Frenzel Lukas,Malinowska Agata,Slabicki Mikolaj,Zenz Thorsten,Zerrouqi Abdessamad,Golab Jakub,Winiarska Magdalena Blood Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene () has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors. 10.1182/blood-2016-08-736066
    Obinutuzumab: what is there to learn from clinical trials? Cartron Guillaume,Watier Hervé Blood Obinutuzumab (OBZ) is a recombinant type II anti-CD20 and immunoglobulin G1 Fc-optimized monoclonal antibody (mAb), recently approved in chronic lymphocytic leukemia (CLL; B-cell CLL) and follicular lymphoma (FL). Rituximab (RTX) is frequently considered as its "ancestor" and OBZ clinical development was justified by the importance of FcγRIIIA-mediated mechanisms in RTX clinical activity. However, RTX differs from OBZ in 2 critical independent properties: being a type I anti-CD20 mAb and not being Fc-optimized. Moreover, the use of a different dosing regimen for RTX and OBZ further complicates any interpretation of clinical results. The results obtained for OBZ in CLL provide new arguments for FcγRIIIA-mediated mechanisms when the target antigen is expressed at a low density. Results of OBZ in FL confirm the interest for FcγRIIIA-mediated mechanisms, with some limitations, some of them being possibly due to lack of OBZ-induced complement activation. The situation in diffuse large B-cell lymphoma is deceiving, as the possible gains of activity of OBZ appear to be annihilated by the lack of complement activation. Although RTX was by chance an anti-CD20 mAb with equilibrated pharmacodynamic properties, the reinforcement of some of these properties, which has been done at the expense of complement activation, has conferred an advantage in some B-cell disorders while restricting OBZ indications. The OBZ story nicely demonstrates that the future of naked mAbs is to design agents with optimized and tailored properties, and that this must be done step by step, with a full clinical validation. 10.1182/blood-2017-03-771832
    Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B cells opsonized with anti-CD20 antibodies. Valgardsdottir Rut,Cattaneo Irene,Klein Christian,Introna Martino,Figliuzzi Marina,Golay Josée Blood Polymorphonuclear neutrophils (PMNs) have previously been reported to mediate phagocytosis of anti-CD20-opsonized B cells from patients with chronic lymphocytic leukemia (CLL). However, recent data have suggested that PMNs, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and to discriminate between trogocytosis and phagocytosis. In live-cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMNs of anti-CD20-opsonized CLL B cells, but could detect only the repeated close contact between effectors and targets, which suggested trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we observed that a mean of 50% and 75% of PMNs had taken a fraction of the dye from CLL B cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis occurs, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B cells, which was evident with rituximab but not obinutuzumab. We conclude that PMNs mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B cells, and we discuss the implications of this finding in patients with CLL treated with rituximab or obinutuzumab in vivo. 10.1182/blood-2016-08-735605
    miR-125b and miR-532-3p predict the efficiency of rituximab-mediated lymphodepletion in chronic lymphocytic leukemia patients. A French Innovative Leukemia Organization study. Gagez Anne-Laure,Duroux-Richard Isabelle,Leprêtre Stéphane,Orsini-Piocelle Frédérique,Letestu Rémi,De Guibert Sophie,Tuaillon Edouard,Leblond Véronique,Khalifa Olfa,Gouilleux-Gruart Valérie,Banos Anne,Tournilhac Olivier,Dupuis Jehan,Jorgensen Christian,Cartron Guillaume,Apparailly Florence Haematologica The underlying mechanisms of rituximab action remain incompletely understood in chronic lymphocytic leukemia. Recent data suggest that circulating micro-ribonucleic acids correlate with chronic lymphocytic leukemia progression and response to rituximab. Our study aimed at identifying circulating micro-ribonucleic acids that predict response to rituximab monotherapy in chronic lymphocytic leukemia patients. Using a hierarchical clustering of micro-ribonucleic acid expression profiles discriminating 10 untreated patients with low or high lymphocyte counts, we found 26 micro-ribonucleic acids significantly deregulated. Using individual real-time reverse transcription polymerase chain reaction, the expression levels of micro-ribonucleic acids representative of these two clusters were further validated in a larger cohort (n=61). MiR-125b and miR-532-3p were inversely correlated with rituximab-induced lymphodepletion (=0.020 and =0.001, respectively) and with the CD20 expression on CD19 cells (=0.0007 and <0.0001, respectively). analyses of genes putatively targeted by both micro-ribonucleic acids revealed a central role of the interleukin-10 pathway and CD20 (MS4A1) family members. Interestingly, both micro-ribonucleic acids were negatively correlated with expression, while they were positively correlated with and Our results identify novel circulating predictive biomarkers for rituximab-mediated lymphodepletion efficacy in chronic lymphocytic leukemia, and suggest a novel molecular mechanism responsible for the rituximab mode of action that bridges miR-125b and miR-532-3p and CD20 family members. (). 10.3324/haematol.2016.153189
    Ibrutinib inhibits CD20 upregulation on CLL B cells mediated by the CXCR4/SDF-1 axis. Pavlasova Gabriela,Borsky Marek,Seda Vaclav,Cerna Katerina,Osickova Jitka,Doubek Michael,Mayer Jiri,Calogero Raffaele,Trbusek Martin,Pospisilova Sarka,Davids Matthew S,Kipps Thomas J,Brown Jennifer R,Mraz Marek Blood Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies. 10.1182/blood-2016-04-709519
    NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation. Pozzo F,Bittolo T,Arruga F,Bulian P,Macor P,Tissino E,Gizdic B,Rossi F M,Bomben R,Zucchetto A,Benedetti D,Degan M,D'Arena G,Chiarenza A,Zaja F,Pozzato G,Rossi D,Gaidano G,Del Poeta G,Deaglio S,Gattei V,Dal Bo M Leukemia In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression. 10.1038/leu.2015.182
    Blood Cancers Diminish Response to COVID-19 Vaccine. Cancer discovery Recent research suggests that patients with chronic lymphocytic leukemia and multiple myeloma may have a diminished immune response to COVID-19 vaccination. In a pair of studies, these patients produced lower levels of SARS-CoV2 antibodies than healthy people-and responses were particularly low in certain patients, such as those actively receiving treatment. 10.1158/2159-8290.CD-ND2021-0108
    Clinical characteristics and mortality of patients with hematologic malignancies and COVID-19: a systematic review. Kim J S,Lee K H,Kim G E,Kim S,Yang J W,Li H,Hong S H,Ghayda R A,Kronbichler A,Koyanagi A,Jacob L,Shin J I,Smith L European review for medical and pharmacological sciences OBJECTIVE:Hematologic cancer patients with Coronavirus Disease 2019 (COVID-19) tend to have a more serious disease course than observed in the general population. Herein, we comprehensively reviewed existing literature and analyzed clinical characteristics and mortality of patients with hematologic malignancies and COVID-19. MATERIALS AND METHODS:Through searching PubMed until June 03, 2020, we identified 16 relevant case studies (33 cases) from a total of 45 studies that have reported on patients with COVID-19 and hematologic malignancies. We investigated the clinical and laboratory characteristics including type of hematologic malignancies, initial symptoms, laboratory findings, and clinical outcomes. Then, we compared those characteristics and outcomes of patients with hematologic malignancies and COVID-19 to the general population infected with COVID-19. RESULTS:The median age was 66-year-old. Chronic lymphocytic leukemia was the most common type of hematologic malignancy (39.4%). Fever was the most common symptom (75.9%). Most patients had normal leukocyte counts (55.6%), lymphocytosis (45.4%), and normal platelet counts (68.8%). In comparison to patients with COVID-19 without underlying hematologic malignancies, dyspnea was more prevalent (45.0 vs. 24.9%, p=0.025). Leukocytosis (38.9 vs. 9.8%, p=0.001), lymphocytosis (45.4 vs. 8.2%, p=0.001), and thrombocytopenia (31.3 vs. 11.4%, p=0.036) were significantly more prevalent and lymphopenia (18.2 vs. 57.4%, p=0.012) less prevalent in patients with hematologic malignancies. There were no clinical and laboratory characteristics predicting mortality in patients with hematologic malignancies. Mortality was much higher in patients with hematologic malignancies compared to those without this condition (40.0 vs. 3.6%, p<0.001). CONCLUSIONS:Co-occurrence of hematologic malignancies and COVID-19 is rare. However, due to the high mortality rate from COVID-19 in this vulnerable population, further investigation on tailored treatment and management is required. 10.26355/eurrev_202011_23852
    Evaluation of vecabrutinib as a model for noncovalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia. Jebaraj Billy Michael Chelliah,Müller Annika,Dheenadayalan Rashmi Priyadharshini,Endres Sascha,Roessner Philipp M,Seyfried Felix,Walliser Claudia,Wist Martin,Qi Jialei,Tausch Eugen,Mertens Daniel,Fox Judith A,Debatin Klaus-Michael,Meyer Lüder Hinrich,Taverna Pietro,Seiffert Martina,Gierschik Peter,Stilgenbauer Stephan Blood Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eμ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eμ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib. 10.1182/blood.2021011516
    Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. Hallek Michael,Al-Sawaf Othman American journal of hematology DISEASE OVERVIEW:Chronic lymphocytic leukemia (CLL) is one of the most frequent types of leukemia. It typically occurs in elderly patients and has a highly variable clinical course. Leukemic transformation is initiated by specific genomic alterations that interfere with the regulation of proliferation and of apoptosis in clonal B-cells. DIAGNOSIS:The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes, which identify a clonal B-cell population carrying the CD5 antigen as well as typical B-cell markers. PROGNOSIS AND STAGING:The clinical staging systems provide prognostic information by using the results of physical examination and blood counts. Various biological and genetic markers provide additional prognostic information. Deletions of the short arm of chromosome 17 (del[17p]) and/or mutations of the TP53 gene predict resistance to chemoimmunotherapy and a shorter time to progression with most targeted therapies. The CLL international prognostic index integrates genetic, biological, and clinical variables to identify distinct risk groups of patients with CLL. THERAPY:Only patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. When treatment is indicated, several therapeutic options exist: a combination of the B-cell lymphoma 2 (BCL2) inhibitor venetoclax with obinutuzumab, monotherapy with inhibitors of Bruton tyrosine kinase (BTK) such as ibrutinib and acalabrutinib, or chemoimmunotherapy. At relapse, the initial treatment may be repeated, if the treatment-free interval exceeds 3 years. If the disease relapses earlier, therapy should be changed using an alternative regimen. Patients with a del(17p) or TP53 mutation are usually resistant to chemotherapy and should, therefore, be treated with targeted agents. FUTURE CHALLENGES:Combinations of targeted agents are now being investigated to create efficient, potentially curative therapies of CLL with fixed duration. One of the most relevant questions currently addressed in clinical trials is the comparison of monotherapies with BTK inhibitors with fixed duration combination therapies. Moreover, the optimal sequencing of targeted therapies remains to be determined. Alternative therapies are needed for patients with BTK and BCL2 inhibitor double-refractory disease. 10.1002/ajh.26367
    Impact of venetoclax monotherapy on the quality of life of patients with relapsed or refractory chronic lymphocytic leukemia: results from the phase 3b VENICE II trial. Cochrane Tara,Enrico Alicia,Gomez-Almaguer David,Hadjiev Evgueniy,Lech-Maranda Ewa,Masszi Tamas,Nikitin Eugene,Robak Tadeusz,Weinkove Robert,Wu Shang-Ju,Sail Kavita R,Pesko John,Pai Madhavi,Komlosi Viktor,Anderson Mary Ann Leukemia & lymphoma Venetoclax, a potent B-cell lymphoma-2 (BCL-2) inhibitor, has demonstrated clinical efficacy in chronic lymphocytic leukemia (CLL). VENICE II is an open-label, single-arm, phase 3b study (NCT02980731) evaluating the impact of venetoclax monotherapy (400 mg once daily) for ≤2 years on health-related quality of life (HRQoL) of patients with relapsed/refractory CLL. The primary endpoint was mean change in the global health status (GHS)/quality of life (QoL) subscale of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) from baseline to Week 48. Overall, 210 patients received ≥1 dose of venetoclax; median treatment duration was 67.4 weeks. The primary endpoint was met with mean improvement of +9.3 points ( = 156, 95% confidence interval 6.1-12.5; =.004) in GHS/QoL. At Week 48, clinically meaningful improvements were observed for role functioning, fatigue, and insomnia domains of EORTC QLQ-C30, suggesting venetoclax monotherapy has a positive impact on HRQoL. No new safety signals were reported. 10.1080/10428194.2021.1986217
    Low prevalence and independent prognostic role of del(11q) in Chinese patients with chronic lymphocytic leukemia. Zou Yi-Xin,Tang Han-Ning,Zhang Jing,Tang Xiao-Lu,Qin Shu-Chao,Xia Yi,Zhu Hua-Yuan,Qiao Chun,Wang Li,Fan Lei,Xu Wei,Li Jian-Yong,Miao Yi Translational oncology The 11q deletion (del(11q)) is a conventional cytogenetic aberration observed in chronic lymphocytic leukemia (CLL) patients. However, the prevalence and the prognostic value of del(11q) are still controversial. In this research, we retrospectively explored the prevalence, association, and prognostic significance of del(11q) in 352 untreated and 99 relapsed/refractory Chinese CLL patients. Totally 11.4% of untreated and 19.2% of relapsed/refractory patients harbored del(11q). Del(11q) was more common in patients with β2-microglobulin > 3.5 mg/L, positive CD38, positive zeta-chain associated protein kinase 70, unmutated immunoglobulin heavy variable-region gene and ataxia telangiectasia mutated mutation. Kaplan-Meier method and univariate Cox regression indicated that del(11q) was an independent prognostic factor for overall survival (OS). Based on the results of univariate Cox regression analysis, two nomograms that included del(11q) were established to predict survival. Desirable area under curve of receiver operating characteristic curves was obtained in the training and validation cohorts. In addition, the calibration curves for the probability of survival showed good agreement between the prediction by nomogram and actual observation. In summary, the prevalence of del(11q) is relatively low in our cohort and del(11q) is an unfavorable prognostic factor for untreated CLL patients. Besides, these two nomograms could be used to accurately predict the prognosis of untreated CLL patients. 10.1016/j.tranon.2021.101176
    Novel phospho-specific monoclonal antibodies reveal differential regulation of tyrosine phosphorylation within the immunoreceptor tyrosine-based activation motif of the Fc receptor γ subunit leading to fine tuning of Syk activation. Yamashita Yumi,Yamashita Toshiyuki Biochemical and biophysical research communications The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is Y47. Consistent with this, an in vitro kinase assay using these phospho-specific antibodies demonstrated that the Src family kinase Lyn, which is primarily responsible for ITAM phosphorylation, phosphorylates Y58 more efficiently than Y47. These results indicate that Y58 is more susceptible to dephosphorylation and phosphorylation than is Y47. Because a phosphate group on Y58 is more important for Syk binding than is a phosphate group on Y47, the preferential phosphorylation and dephosphorylation of Y58 may contribute to the fine tuning of Syk activity by promoting rapid recruitment and reducing excessive activation. 10.1016/j.bbrc.2021.02.042
    Phosphoinositide 3-Kinase Signaling in the Tumor Microenvironment: What Do We Need to Consider When Treating Chronic Lymphocytic Leukemia With PI3K Inhibitors? Aydin Ebru,Faehling Sebastian,Saleh Mariam,Llaó Cid Laura,Seiffert Martina,Roessner Philipp M Frontiers in immunology Phosphoinositide 3-kinases (PI3Ks) and their downstream proteins constitute a signaling pathway that is involved in both normal cell growth and malignant transformation of cells. Under physiological conditions, PI3K signaling regulates various cellular functions such as apoptosis, survival, proliferation, and growth, depending on the extracellular signals. A deterioration of these extracellular signals caused by mutational damage in oncogenes or growth factor receptors may result in hyperactivation of this signaling cascade, which is recognized as a hallmark of cancer. Although higher activation of PI3K pathway is common in many types of cancer, it has been therapeutically targeted for the first time in chronic lymphocytic leukemia (CLL), demonstrating its significance in B-cell receptor (BCR) signaling and malignant B-cell expansion. The biological activity of the PI3K pathway is not only limited to cancer cells but is also crucial for many components of the tumor microenvironment, as PI3K signaling regulates cytokine responses, and ensures the development and function of immune cells. Therefore, the success or failure of the PI3K inhibition is strongly related to microenvironmental stimuli. In this review, we outline the impacts of PI3K inhibition on the tumor microenvironment with a specific focus on CLL. Acknowledging the effects of PI3K inhibitor-based therapies on the tumor microenvironment in CLL can serve as a rationale for improved drug development, explain treatment-associated adverse events, and suggest novel combinatory treatment strategies in CLL. 10.3389/fimmu.2020.595818
    Final Results of a Randomized, Phase III Study of Rituximab With or Without Idelalisib Followed by Open-Label Idelalisib in Patients With Relapsed Chronic Lymphocytic Leukemia. Sharman Jeff P,Coutre Steven E,Furman Richard R,Cheson Bruce D,Pagel John M,Hillmen Peter,Barrientos Jacqueline C,Zelenetz Andrew D,Kipps Thomas J,Flinn Ian W,Ghia Paolo,Eradat Herbert,Ervin Thomas,Lamanna Nicole,Coiffier Bertrand,Pettitt Andrew R,Ma Shuo,Tausch Eugen,Cramer Paula,Huang Julie,Mitra Siddhartha,Hallek Michael,O'Brien Susan M,Stilgenbauer Stephan Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:A randomized, double-blind, phase III study of idelalisib (IDELA) plus rituximab versus placebo plus rituximab in patients with relapsed chronic lymphocytic leukemia (CLL) was terminated early because of superior efficacy of the IDELA-plus-rituximab (IDELA/R) arm. Patients in either arm could then enroll in an extension study to receive IDELA monotherapy. Here, we report the long-term efficacy and safety data for IDELA-treated patients across the primary and extension studies. PATIENTS AND METHODS:Patients were randomly assigned to receive rituximab in combination with either IDELA 150 mg twice daily (IDELA/R; n = 110) or placebo (placebo/R; n = 110). Key end points were progression-free survival (PFS), overall response rate (ORR), overall survival (OS), and safety. RESULTS:The long-term efficacy and safety of treatment with IDELA was assessed in 110 patients who received at least one dose of IDELA in the primary study, 75 of whom enrolled in the extension study. The IDELA/R-to-IDELA group had a median PFS of 20.3 months (95% CI, 17.3 to 26.3 months) after a median follow-up time of 18 months (range, 0.3 to 67.6 months). The ORR was 85.5% (94 of 110 patients; n = 1 complete response). The median OS was 40.6 months (95% CI, 28.5 to 57.3 months) and 34.6 months (95% CI, 16.0 months to not reached) for patients randomly assigned to the IDELA/R and placebo/R groups, respectively. Prolonged exposure to IDELA increased the incidence of all-grade, grade 2, and grade 3 or greater diarrhea (46.4%, 17.3%, and 16.4%, respectively), all-grade and grade 3 or greater colitis (10.9% and 8.2%, respectively) and all-grade and grade 3 or greater pneumonitis (10.0% and 6.4%, respectively) but did not increase the incidence of elevated hepatic aminotransferases. CONCLUSION:IDELA improved PFS and OS compared with rituximab alone in patients with relapsed CLL. Long-term IDELA was effective and had an expected safety profile. No new IDELA-related adverse events were identified with longer exposure. 10.1200/JCO.18.01460
    Classic and Novel Signaling Pathways Involved in Cancer: Targeting the NF-κB and Syk Signaling Pathways. Tang Cong,Zhu Guodong Current stem cell research & therapy The nuclear factor kappa B (NF-κB) consists of a family of transcription factors involved in the regulation of a wide variety of biological responses. Growing evidence support that NF-κB plays a major role in oncogenesis as well as its well-known function in the regulation of immune responses and inflammation. Therefore, we made a review of the diverse molecular mechanisms by which the NF-κB pathway is constitutively activated in different types of human cancers and the potential role of various oncogenic genes regulated by this transcription factor in cancer development and progression. We also discussed various pharmacological approaches employed to target the deregulated NF-κB signaling pathway and their possible therapeutic potential in cancer therapy. Moreover, Syk (Spleen tyrosine kinase), non-receptor tyrosine kinase which mediates signal transduction downstream of a variety of transmembrane receptors including classical immune-receptors like the B-cell receptor (BCR), which can also activate the inflammasome and NF-κB-mediated transcription of chemokines and cytokines in the presence of pathogens would be discussed as well. The highlight of this review article is to summarize the classic and novel signaling pathways involved in NF-κB and Syk signaling and then raise some possibilities for cancer therapy. 10.2174/1574888X13666180723104340
    Prognostic Factors for Complete Response to Ibrutinib in Patients With Chronic Lymphocytic Leukemia: A Pooled Analysis of 2 Clinical Trials. O'Brien Susan M,Jaglowski Samantha,Byrd John C,Bannerji Rajat,Blum Kristie A,Fox Christopher P,Furman Richard R,Hillmen Peter,Kipps Thomas J,Montillo Marco,Sharman Jeff,Suzuki Sam,James Danelle F,Chu Alvina D,Coutre Steven E JAMA oncology Importance:Ibrutinib, a first-in-class Bruton tyrosine kinase inhibitor taken once daily, is approved in the United States for patients with chronic lymphocytic leukemia/small lymphocytic lymphoma and allows for treatment without chemotherapy. Extended treatment with ibrutinib has demonstrated increased complete response (CR) rates over time. Objective:To analyze baseline factors that predict CR in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma treated with ibrutinib. Design, Setting, and Participants:Univariate and multivariate analyses of pooled data from 2 clinical trials were used to assess the prognostic value of baseline factors associated with CR in 327 patients from the PCYC-1102 and PCYC-1112 studies treated with single-agent ibrutinib. Participants were followed up in academic and community medical centers in the United States, the United Kingdom, Australia, France, Italy, Ireland, Poland, Spain, and Austria. Main Outcomes and Measures:Odds ratio (OR) of CR rate. Results:The 327 patients included in this analysis had a median age of 67 years (range, 30-86 years) and 227 (69.4%) were male. At baseline, 185 patients (56.6%) had bulky disease (lymph node ≥5 cm), 184 (56.3%) had advanced-stage disease, and 182 (55.7%) had an Eastern Cooperative Oncology Group performance status of 1 or higher. Thirty-one patients (9.5%) were in the first-line setting; 38 (11.6%) had undergone 1 previous therapy, 81 (24.8%) had undergone 2, and 177 (54.1%) had undergone 3 or more; patients with relapsed/refractory disease had undergone a median of 3 (range, 0-12) previous therapies. Median time on study was 26.4 months (range, 0.3-55.6 months). Thirty-two of the 327 patients (9.8%) treated with ibrutinib had a CR (PCYC-1102: relapsed/refractory, 12 of 101 [11.9%]; treatment-naive, 8 of 31 [25.8%]; and PCYC-1112: 12 of 195 [6.2%]). The median time to CR for these patients was 14.7 months (range, 4.6-47.1 months). Univariate analysis of baseline factors showed that bulky disease, clinical stage, number of previous therapies, and β2-microglobulin concentration had a significant effect on the odds of CR. The final multivariate model showed that patients with no previous therapy vs patients with at least 1 previous therapy (OR, 2.65; 95% CI, 1.01-6.95; P = .047) and patients without bulky disease (lymph node <5 cm) vs those with bulky disease (lymph node ≥5 cm [OR, 4.97; 95% CI, 1.91-12.91; P = .001]) had an increased likelihood of CR. Conclusions and Relevance:Patients receiving ibrutinib as a first-line therapy for chronic lymphocytic leukemia and those without bulky disease had a better likelihood of CR to treatment. The CR rate with continued longer-term ibrutinib treatment was higher than in previous reports. Trial Registration:clinicaltrials.gov Identifiers: NCT01105247 and NCT01578707. 10.1001/jamaoncol.2017.5604
    Idelalisib in Combination With Rituximab or Bendamustine or Both in Patients With Relapsed/Refractory Chronic Lymphocytic Leukemia. Coutre Steven E,Flinn Ian W,de Vos Sven,Barrientos Jacqueline C,Schreeder Marshall T,Wagner-Johnson Nina D,Sharman Jeff P,Boyd Thomas E,Fowler Nathan,Dreiling Lyndah,Kim Yeonhee,Mitra Siddhartha,Rai Kanti,Leonard John P,Furman Richard R HemaSphere Phosphatidylinositol 3-kinase-delta (PI3Kδ) signaling is critical for proliferation, survival, homing, and tissue retention of malignant B cells. Idelalisib, a selective oral inhibitor of PI3Kδ, has shown considerable single-agent activity in patients with heavily pretreated chronic lymphocytic leukemia (CLL). This study evaluated the safety and clinical activity of idelalisib in combination with bendamustine (IB) or rituximab (IR) or both (IBR) in patients with relapsed or refractory (R/R) CLL. Idelalisib was given continuously at 100 or 150 mg twice daily in combination with rituximab (375 mg/m weekly × 8 doses), bendamustine (70 or 90 mg/m, days 1 and 2 every 4 weeks × 6 cycles) or BR (rituximab, 375 mg/m every 4 weeks and bendamustine, 70 mg/m, days 1 and 2 every 4 weeks × 6 cycles). The primary endpoint was safety; secondary endpoints included overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS). Fifty-two patients (median age 64 years) with a median of 3 prior therapies were enrolled. ORR was 84.6% (89.5% IR group, 77.8% IB group, and 86.7% IBR group). The overall median PFS was 25.6 months, and median DOR was 26.6 months. The most common grade ≥3 adverse events (≥10% of patients) were pneumonia (19.2%), diarrhea (13.5%), and febrile neutropenia (17.3%). Idelalisib-based combination therapy with bendamustine and/or rituximab was highly active, resulting in durable tumor control in patients with heavily pretreated R/R CLL. However, its tolerability profile suggests that these regimens should be used cautiously in this patient population. ClinicalTrials.gov ID: NCT01088048. 10.1097/HS9.0000000000000039
    Idelalisib may have the potential to increase radiotherapy side effects. Gryc Thomas,Putz Florian,Goerig Nicole,Ziegler Sonia,Fietkau Rainer,Distel Luitpold V,Schuster Barbara Radiation oncology (London, England) INTRODUCTION:Idelalisib is approved for the treatment of relapsed chronic lymphocytic leukemia together with Rituximab and for monotherapy of follicular B-cell non-Hodgkin's lymphoma and small lymphocytic lymphoma. It is a potent and selective phosphatidylinositol 3-kinase-δ (PI3K-δ) inhibitor. PI3K-δ primarily is expressed in B-cells and prevents effectively proliferation in malignant B-cells. METHODS:We provide a detailed report on treatment history and photo documentation of acute adverse effects of radiation therapy with simultaneous Idelalisib medication in one case of B-CLL. Radiosensitivity tests were performed for the index patient under Idelalisib and after the addition of Idelalisib to healthy individuals' blood. Radiosensitivity in human lymphocytes was analyzed with a three color in situ hybridization assay. Primary skin fibroblasts were studied after a treatment with Idelalisib for apoptosis, necrosis and cell cycle using flow cytometry. DNA double-strand break repair was analyzed by γH2AX immunostaining. RESULTS:The index patient presented a strong grade 2 radiodermatitis and grade 3 mucositis after irradiation with 20 Gy and a simultaneous intake of Idelalisib. Irradiations without Idelalisib medication were well tolerated and resulted in not more than grade 1 radiodermatitis. The index patient under Idelalisib had a radiosensitivity of 0.62 B/M which is in the range of clearly radiosensitive patients. A combined treatment of lymphocytes with 2 Gy and 10 nmol/l Idelalisib showed a tendency to an increased radiosensitivity. We found a clear increase of apoptosis as a result of the combined treatment in the Idelalisib dose range of 1 to 100 nmol/l compared to solely irradiated cells or solely Idelalisib treated cells (p = 0.05). CONCLUSION:A combined Idelalisib radiotherapy treatment has an increased risk of side effects. However, combined therapy seems to be feasible when patients are monitored closely. 10.1186/s13014-017-0827-7
    Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells. Modi Prexy,Balakrishnan Kumudha,Yang Qingshan,Wierda William G,Keating Michael J,Gandhi Varsha Oncotarget Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL. 10.18632/oncotarget.15180
    Management of adverse events associated with idelalisib treatment: expert panel opinion. Coutré Steven E,Barrientos Jacqueline C,Brown Jennifer R,de Vos Sven,Furman Richard R,Keating Michael J,Li Daniel,O'Brien Susan M,Pagel John M,Poleski Martin H,Sharman Jeff P,Yao Nai-Shun,Zelenetz Andrew D Leukemia & lymphoma Idelalisib is a first-in-class selective, oral, phosphatidylinositol 3-kinase delta (PI3Kδ) inhibitor approved for the treatment of several types of blood cancer. Idelalisib has demonstrated significant efficacy and a tolerable safety profile in clinical trials. However, the US prescribing information contains a black box warning for fatal and/or severe diarrhea or colitis, hepatotoxicity, pneumonitis and intestinal perforation. An expert panel was convened to review the pathology of these treatment-emergent adverse events (TEAEs) to propose key management tools for patients receiving idelalisib therapy. This article provides an overview of idelalisib TEAEs reported in clinical trials, and a summary of the panel's recommendations for identification and management of idelalisib treatment-emergent diarrhea or colitis as well as a discussion of transaminitis and pneumonitis. For idelalisib-related diarrhea or colitis (including unresolved grade 2 and grade ≥ 3), after exclusion of infectious causes, the panel recommends individualized treatment with budesonide or oral or intravenous steroid therapy. 10.3109/10428194.2015.1022770
    Idelalisib- a PI3Kδ targeting agent for B-cell malignancies. Hewett Yvonne G,Uprety Dipesh,Shah Binay K Journal of oncology pharmacy practice : official publication of the International Society of Oncology Pharmacy Practitioners Idelalisib, the first in-class phosphotidlyinositol 3-kinase delta (PI3Kδ) inhibitor, was approved by the US Food and Drug Administration in July 2014. It simultaneously received breakthrough therapy designation in combination with rituximab for the treatment of relapsed chronic lymphocytic leukemia (CLL) as well as accelerated approval as monotherapy for the treatment of relapsed follicular lymphoma and relapsed small lymphocytic lymphoma. In a pivotal phase III study of 220 patients with relapsed CLL, the overall response rate of patients who received rituximab plus idelalisib was 81%. The median progression-free survival (PFS) was 5 months with rituximab plus placebo group, but was not reached in the idelalisib arm. At 24 weeks, the PFS in patients receiving idelalisib was 93%. In a phase II trial of 125 patients with relapsed or refractory indolent non-Hodgkin lymphoma who received idelalisib 150 mg twice daily, the response rate was 57%. Complete response was seen in 6% of patients. The median duration of response was 12.5 months, and median PFS was 11 months. Idelalisib is a promising new therapy for relapsed indolent B-cell malignancies. 10.1177/1078155215572933
    Idelalisib: a review of its use in chronic lymphocytic leukaemia and indolent non-Hodgkin's lymphoma. Keating Gillian M Targeted oncology Idelalisib (Zydelig®) is a first-in-class, orally administered, phosphatidylinositol 3-kinase-δ inhibitor that was recently approved for the treatment of relapsed chronic lymphocytic leukaemia (CLL), relapsed follicular B-cell non-Hodgkin's lymphoma (NHL) and relapsed small lymphocytic lymphoma (SLL) in the USA and for the treatment of CLL and refractory follicular lymphoma in the EU. In a pivotal phase III trial in patients with relapsed CLL who were not able to receive cytotoxic agents, recipients of idelalisib plus rituximab had significantly improved progression-free survival, overall survival, overall response and lymph node response, compared with recipients of placebo plus rituximab. In a pivotal phase II trial, idelalisib monotherapy was effective in patients with relapsed indolent NHL who were refractory to rituximab and an alkylating agent, including in the subgroups of patients with follicular lymphoma or SLL. Oral idelalisib had a generally manageable adverse event profile, although episodes of serious/fatal diarrhoea or colitis, hepatotoxicity, pneumonitis and intestinal perforation were reported. In conclusion, idelalisib represents an important advance in the treatment of relapsed CLL and relapsed indolent NHL. 10.1007/s11523-015-0359-8
    Long-Term Ibrutinib Therapy Reverses CD8 T Cell Exhaustion in B Cell Chronic Lymphocytic Leukaemia. Parry Helen M,Mirajkar Nikhil,Cutmore Natasha,Zuo Jianmin,Long Heather,Kwok Marwan,Oldrieve Ceri,Hudson Chris,Stankovic Tatjana,Paneesha Shankara,Kelly Melanie,Begum Jusnara,McSkeane Tina,Pratt Guy,Moss Paul Frontiers in immunology Chronic Lymphocytic Leukaemia (CLL) is associated with immune suppression and susceptibility to infection. CD8 T cell numbers are increased and demonstrate elevated expression of PD-1 and impaired function. The mechanisms driving these features of exhaustion are uncertain but are likely to include chronic immune recognition of tumor and/or infectious agents. We investigated the number, phenotype and function of total and virus-specific CD8+ T cells in 65 patients with CLL and 14 patients undergoing long-term ibrutinib therapy (median 21 months). Ibrutinib substantially reduced the number of both CD3+ T cells and CD8+ T cells. Importantly, this was associated with a reduction in PD-1 expression on CD8+ T cells (median 28 vs. 24%; = 0.042) and 3.5 fold increase in cytokine production following mitogen stimulation. The influence of ibrutinib on antigen-specific CD8+ T cell function was assessed by HLA-peptide tetramers and revealed increased IFNγ and TNFα cytokine responses following stimulation with CMV or EBV peptides together with a 55% reduction in the frequency of "inflated" virus-specific CD8+ T cells. These findings reveal that long-term ibrutinib therapy is associated with substantial reversal of T cell exhaustion in B-CLL and is likely to contribute to the reduced infection risk seen in association with this agent. 10.3389/fimmu.2019.02832
    Proliferating Transitory T Cells with an Effector-like Transcriptional Signature Emerge from PD-1 Stem-like CD8 T Cells during Chronic Infection. Hudson William H,Gensheimer Julia,Hashimoto Masao,Wieland Andreas,Valanparambil Rajesh M,Li Peng,Lin Jian-Xin,Konieczny Bogumila T,Im Se Jin,Freeman Gordon J,Leonard Warren J,Kissick Haydn T,Ahmed Rafi Immunity T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1 Tcf-1 CD8 T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1 CD8 T cells initially differentiated into a transitory population of CD101Tim3 cells that later converted into CD101 Tim3 cells. Recently generated CD101Tim3 cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101Tim3 CD8 T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy. 10.1016/j.immuni.2019.11.002
    Increased frequency of CD4 PD-1 HLA-DR T cells is associated with disease progression in CLL. Elston Lauren,Fegan Chris,Hills Robert,Hashimdeen Shaikh S,Walsby Elisabeth,Henley Peter,Pepper Chris,Man Stephen British journal of haematology Chronic lymphocytic leukaemia (CLL) patients often have abnormal expansions of CD4 and CD8 T cells and this can be associated with progressive disease. To characterise the key T-cell populations involved in this phenomenon, we used flow cytometry and 11 phenotypic markers to study 74 CLL patients and 14 controls. T cells of CLL patients were more phenotypically complex than those of healthy controls with significant increases in the frequencies of CD4 and CD8 memory T cells expressing exhaustion-, activation- and senescence-associated markers. Multivariate analysis of 111 different T-cell subsets showed that high frequencies of four subsets (three CD8 and one CD4) were associated with shorter progression-free survival. The most significant association was with CD4 HLA-DR PD-1 T cells, and patients could be stratified into high- and low-risk groups based on the frequency of these T cells. The expansion of this CD4 subset could not be accounted for by age, cytomegalovirus infection or increases in Treg cells. Overall, these results highlight two relatively simple biomarkers, percentage CD8 and percentage CD4 PD-1 HLA-DR T cells, which can be used to risk-stratify CLL patients, independent of other tumour-associated markers. They also provide further evidence for the pivotal role of T cells in modulating the pathology of CLL. 10.1111/bjh.16260
    Prognostic and clinicopathological significance of PD-1/PD-L1 expression in the tumor microenvironment and neoplastic cells for lymphoma. International immunopharmacology BACKGROUND:Recently, unprecedented clinical efficacy was observed during treatment of many solid tumors because of the introduction of programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) immune checkpoint inhibitors. Preliminary clinical data indicates that checkpoint inhibition also represents a promising therapeutic strategy for certain lymphoid malignancies. However, PD-1/PD-L1 expression levels on neoplastic cells and in the tumor microenvironment vary among subtypes and their prognostic implications remain uncertain. MAIN BODY:Here, we review the clinicopathological significance of PD-1/PD-L1 expression in lymphomas. Increased infiltration of PD-1+ tumor-infiltrating lymphocytes (TILs) is a favorable prognostic factor in diffuse large B-cell lymphoma (DLBCL) but not in Hodgkin's lymphoma (HL). Higher numbers of PD-1+ TILs were observed in follicular lymphoma (FL) than in other subtypes of B-cell lymphoma; however, its prognostic significance remains controversial. Infiltration of PD-L1+ immune cells showed a trend toward better overall survival in nasal natural killer (NK)/T-cell lymphoma and adult T-cell leukemia/lymphoma, more likely to be classified as activated macrophages and dendritic cells in microenvironment but its biological effect is not clarified. Peripheral PD-1+ T cells could be detected in blood samples from DLBCL and chronic lymphocytic leukemia (CLL) and correlated with disease progression and poor prognosis. PD-1+ neoplastic T cells were more frequently observed in cutaneous T-cell lymphoma, including Sézary syndrome and mycosis fungoides, which may be involved in the progression of epithelial-derived T lymphoma. Studies on PD-L1 expression in neoplastic cells mostly focused on DLBCL. PD-L1+ neoplastic cells were observed only in a small subset of DLBCL, mainly associated with activated B cell (ABC) subtypes and Epstein-Barr virus (EBV) positivity; however, its prognostic role remains controversial. In either T or B lymphoma, elevated serum or plasma levels of soluble PD-L1 represent adverse prognostic factors. Notably, in clinical trials of classical HL, the frequency of 9p24.1 chromosome alterations increases the abundance of PD-1 ligand expression, appearing to predict responses to anti-PD-1/PD-L1 therapy. The cytogenetic alterations affecting chromosome 9p24.1 including the CIITA rearrangement were also frequently observed in certain specific subtypes of large B-cell lymphomas. CONCLUSIONS:The clinical roles of PD-1/PD-L1 expression vary between subtypes of lymphoma. Future studies should delineate the prognostic and predictive roles of PD-1 and PD-L1 expression. 10.1016/j.intimp.2019.105999
    Epigenetic strategies synergize with PD-L1/PD-1 targeted cancer immunotherapies to enhance antitumor responses. Chen Xi,Pan Xiaohui,Zhang Wenxin,Guo Hongjie,Cheng Shuyuan,He Qiaojun,Yang Bo,Ding Ling Acta pharmaceutica Sinica. B Immunotherapy strategies targeting the programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) pathway in clinical treatments have achieved remarkable success in treating multiple types of cancer. However, owing to the heterogeneity of tumors and individual immune systems, PD-L1/PD-1 blockade still shows slow response rates in controlling malignancies in many patients. Accumulating evidence has shown that an effective response to anti-PD-L1/anti-PD-1 therapy requires establishing an integrated immune cycle. Damage in any step of the immune cycle is one of the most important causes of immunotherapy failure. Impairments in the immune cycle can be restored by epigenetic modification, including reprogramming the environment of tumor-associated immunity, eliciting an immune response by increasing the presentation of tumor antigens, and by regulating T cell trafficking and reactivation. Thus, a rational combination of PD-L1/PD-1 blockade and epigenetic agents may offer great potential to retrain the immune system and to improve clinical outcomes of checkpoint blockade therapy. 10.1016/j.apsb.2019.09.006
    Trabectedin Reveals a Strategy of Immunomodulation in Chronic Lymphocytic Leukemia. Banerjee Priyanka,Zhang Ronghua,Ivan Cristina,Galletti Giovanni,Clise-Dwyer Karen,Barbaglio Federica,Scarfò Lydia,Aracil Miguel,Klein Christian,Wierda William,Plunkett William,Caligaris-Cappio Federico,Gandhi Varsha,Keating Michael J,Bertilaccio Maria Teresa S Cancer immunology research Chronic lymphocytic leukemia (CLL) is a B-cell neoplasia characterized by protumor immune dysregulation involving nonmalignant cells of the microenvironment, including T lymphocytes and tumor-associated myeloid cells. Although therapeutic agents have improved treatment options for CLL, many patients still fail to respond. Some patients also show immunosuppression. We have investigated trabectedin, a marine-derived compound with cytotoxic activity on macrophages in solid tumors. Here, we demonstrate that trabectedin induces apoptosis of human primary leukemic cells and also selected myeloid and lymphoid immunosuppressive cells, mainly through the TRAIL/TNF pathway. Trabectedin modulates transcription and translation of IL6, CCL2, and IFNα in myeloid cells and FOXP3 in regulatory T cells. Human memory CD8 T cells downregulate PD-1 and, along with monocytes, exert antitumor function. In xenograft and immunocompetent CLL mouse models, trabectedin has antileukemic effects and antitumor impact on the myeloid and lymphoid cells compartment. It depletes myeloid-derived suppressor cells and tumor-associated macrophages and increases memory T cells. Trabectedin also blocks the PD-1/PD-L1 axis by targeting PD-L1 CLL cells, PD-L1 monocytes/macrophages, and PD-1 T cells. Thus, trabectedin behaves as an immunomodulatory drug with potentially attractive therapeutic value in the subversion of the protumor microenvironment and in overcoming chemoimmune resistance. 10.1158/2326-6066.CIR-19-0152
    Clonal replacement of tumor-specific T cells following PD-1 blockade. Yost Kathryn E,Satpathy Ansuman T,Wells Daniel K,Qi Yanyan,Wang Chunlin,Kageyama Robin,McNamara Katherine L,Granja Jeffrey M,Sarin Kavita Y,Brown Ryanne A,Gupta Rohit K,Curtis Christina,Bucktrout Samantha L,Davis Mark M,Chang Anne Lynn S,Chang Howard Y Nature medicine Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of patients with cancer. However, whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells remains unclear. Here we performed paired single-cell RNA and T cell receptor sequencing on 79,046 cells from site-matched tumors from patients with basal or squamous cell carcinoma before and after anti-PD-1 therapy. Tracking T cell receptor clones and transcriptional phenotypes revealed coupling of tumor recognition, clonal expansion and T cell dysfunction marked by clonal expansion of CD8CD39 T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, the expansion of T cell clones did not derive from pre-existing tumor-infiltrating T lymphocytes; instead, the expanded clones consisted of novel clonotypes that had not previously been observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8 T cells and evident in patients with basal or squamous cell carcinoma. These results demonstrate that pre-existing tumor-specific T cells may have limited reinvigoration capacity, and that the T cell response to checkpoint blockade derives from a distinct repertoire of T cell clones that may have just recently entered the tumor. 10.1038/s41591-019-0522-3
    Nanoenabled Modulation of Acidic Tumor Microenvironment Reverses Anergy of Infiltrating T Cells and Potentiates Anti-PD-1 Therapy. Zhang Yu-Xue,Zhao Yang-Yang,Shen Jizhou,Sun Xun,Liu Yi,Liu Hang,Wang Yucai,Wang Jun Nano letters While tumor-infiltrating cytotoxic T lymphocytes play a critical role in controlling tumor development, they are generally impotent in an acidic tumor microenvironment. Systemic treatment to neutralize tumor acidity thus holds promise for the reversal of the anergic state of T cells and the improvement of T cell-associated immunotherapy. Herein, we report a proof-of-concept of RNAi nanoparticle-mediated therapeutic reversion of tumor acidity to restore the antitumor functions of T cells and potentiate the checkpoint blockade therapy. Our strategy utilized an in vivo optimized vesicular cationic lipid-assisted nanoparticle, as opposed to its micellar counterpart, to mediate systematic knockdown of lactate dehydrogenase A (LDHA) in tumor cells. The treatment resulted in the reprogramming of pyruvate metabolism, a reduction of the production of lactate, and the neutralization of the tumor pH. In immunocompetent syngeneic melanoma and breast tumor models, neutralization of tumor acidity increased infiltration with CD8 T and NK cells, decreased the number of immunosuppressive T cells, and thus significantly inhibited the growth of tumors. Furthermore, the restoration of tumoral pH potentiated checkpoint inhibition therapy using the antibody of programmed cell death protein 1 (PD-1). However, in immunodeficient B6/ Rag1 and NOG mice, the same treatment failed to control tumor growth, further proving that the attenuation of tumor growth by tumor acidity modulation was attributable to the activation of tumor-infiltrating immune cells. 10.1021/acs.nanolett.8b04296
    Do PD-1 and PD-L2 expressions have prognostic impact in hematologic malignancies? Korkmaz Serdal,Erdem Selahattin,Akay Ebru,Taşdemir Erdem Arzu,Karaman Hatice,Keklik Muzaffer Turkish journal of medical sciences Background/aim:PD-1 (programmed death-1) is an immune checkpoint receptor that modulates T-cell activity in peripheral tissues via interaction with its ligands, PD-L1 (programmed death-ligand 1) and PD-L2 (programmed death-ligand 2). Tumor cells upregulate PD-L1 or PD-L2 to inhibit this T lymphocyte attack. Our goal was to determine the PD-1 and PD-L2 expression rates of various hematologic malignancies, and evaluate whether PD-1 and PD-L2 expressions have an impact on prognosis. Materials and methods:For this purpose, pretreatment bone marrow biopsy specimens of 83 patients [42 multiple myeloma (MM), 21 acute leukemia, and 20 chronic lymphocytic leukemia (CLL)] were stained with monoclonal antibody immunostains of PD-1 and PD-L2. Results:As a result, the overall expression rate of PD-1 was 26.2%, 4.8%, and 60% in patients with MM, acute leukemia, and CLL, respectively, whereas the PD-L2 expression rate was 61.9%, 14.3%, and 10% in patients with MM, acute leukemia, and CLL, respectively. Conclusion:Finally, we concluded that the role of the PD-1 pathway can be demonstrated by immunohistochemistry (IHC). Since we evaluated whether there is a correlation between the (IHC) results and survival of patients with MM, acute leukemia, and CLL, we could not demonstrate meaningful evidence that these markers have an impact on prognosis. 10.3906/sag-1706-194
    CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia. Lewinsky Hadas,Barak Avital F,Huber Victoria,Kramer Matthias P,Radomir Lihi,Sever Lital,Orr Irit,Mirkin Vita,Dezorella Nili,Shapiro Mika,Cohen Yosef,Shvidel Lev,Seiffert Martina,Herishanu Yair,Becker-Herman Shirly,Shachar Idit The Journal of clinical investigation Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression. 10.1172/JCI96610
    PD-1 Expression in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) and Large B-cell Richter Transformation (DLBCL-RT): A Characteristic Feature of DLBCL-RT and Potential Surrogate Marker for Clonal Relatedness. He Rong,Ding Wei,Viswanatha David S,Chen Dong,Shi Min,Van Dyke Daniel,Tian Shulan,Dao Linda N,Parikh Sameer A,Shanafelt Tait D,Call Timothy G,Ansell Stephen M,Leis Jose F,Mai Ming,Hanson Curtis A,Rech Karen L The American journal of surgical pathology Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a low-grade B-cell neoplasm and ∼2% to 9% patients develop an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (Richter transformation, DLBCL-RT). Programmed death-1 (PD-1) pathway plays a crucial role in tumor host immunity evasion and its blockade has emerged as an effective anti-cancer immunotherapy. PD-L1 and PD-1 expression has shown predictive value in anti-PD cancer immunotherapy; however, it has not been well documented in CLL/SLL and DLBCL-RT. We evaluated PD-1 and PD-L1 expression by immunohistochemistry in 39 CLL/SLL, 15 DLBCL-RT, and 26 other DLBCL. In CLL/SLL, neoplastic B-cell PD-1 expression was weak and restricted to prolymphocytes/paraimmunoblasts within proliferation centers (PCs) and accentuated PCs of all sizes. Neoplastic B-cell PD-1 expression was highly prevalent and demonstrated increased intensity in DLBCL-RT, but in contrast was only rarely seen in other DLBCL (12/15 vs. 1/26; P<0.0001). An excellent correlation (90% concordance) was observed between neoplastic B-cell PD-1 immunohistochemistry positivity and molecularly defined CLL/SLL clonal relatedness in DLBCL-RT. PD-L1 expression was observed on the neoplastic B cells in rare DLBCL-RT and other DLBCL cases (1/15 vs. 1/26; P>0.05) as well as background histiocytes and dendritic cells. Overall survival of DLBCL-RT was significantly inferior to that of the other DLBCL (median, 16.9 vs. 106.1 mo; P=0.002). Our findings suggest a biological continuum from prolymphocytes/paraimmunoblasts in CLL/SLL PCs to the neoplastic B-cells in DLBCL-RT. The characteristic PD-1 expression in DLBCL-RT makes it a potential surrogate marker for determining clonal relatedness to CLL/SLL, which may have important prognostic and therapeutic implications. 10.1097/PAS.0000000000001077
    Elevated levels of follicular T helper cells and their association with therapeutic effects in patients with chronic lymphocytic leukaemia. Qiu Liannv,Zhou Yonglie,Yu Qinhua,Zheng Sujie,Wang Zhenni,Huang Qiang Immunology letters Chronic lymphocytic leukaemia (CLL) is characterized by an abnormal expansion of mature B cells with variable progression. Follicular T helper (Tfh) cells help B cells differentiate into plasma cells or long-lived memory B cells in germinal centres (GCs). However, the role of Tfh cells in CLL is poorly understand, and whether it plays a critical role in disease progression in vivo is lacking. In this study, we investigate the dynamic change of circulating Tfh cells in peripheral blood from patients with CLL during the treatment periods to evaluate their utility to predict disease progression. Our findings revealed the expansion of circulating CD4CXCR5, CD4ICOS, CD4PD-1 and CD4CXCR5ICOSPD-1 (Tfh) cells but lower serum IL-21 levels and CD4 T cell polarization not only to Tfh2 subtypes but also to Tfh17 subtypes in patients with CLL at pretreatment compared to patients with monoclonal B cell lymphocytosis (MBL) and healthy individuals, especially in those with advanced stage, which indicate these Tfh cells could be employed as a novel indicator for disease progression. Moreover, we observed significant correlations of Tfh17 and immunoglobulin heavy chain variable region (IGHV) mutation. Importantly, significantly decreased CD4ICOS, CD4PD-1 and Tfh cells were found after effective treatments, whereas a significantly high CD4ICOS, CD4PD-1 and Tfh cells were still found in those with progressive disease after treatments, suggesting that circulating CD4ICOS, CD4PD-1, Tfh cells could predict therapeutic effects. 10.1016/j.imlet.2018.03.002
    Dual PD1/LAG3 immune checkpoint blockade limits tumor development in a murine model of chronic lymphocytic leukemia. Wierz Marina,Pierson Sandrine,Guyonnet Léa,Viry Elodie,Lequeux Audrey,Oudin Anaïs,Niclou Simone P,Ollert Markus,Berchem Guy,Janji Bassam,Guérin Coralie,Paggetti Jerome,Moussay Etienne Blood 10.1182/blood-2017-06-792267
    PD-1 /PD-L1 checkpoint in hematological malignancies. Annibali O,Crescenzi A,Tomarchio V,Pagano A,Bianchi A,Grifoni A,Avvisati G Leukemia research Programmed cell death protein 1 (PD-1), is a cell surface receptor with an important role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1/PDL1 axis represents a checkpoint to control immune responses and it is often used as a mechanism of immune escaping by cancers and infectious diseases. Many data demonstrate its important role in solid tumors and report emerging evidences in lymphoproliferative disorders. In this review, we summarized the available data on the role of PD-1/PD-L1 checkpoint in lymphoproliferative diseases and the therapeutics use of monoclonal blocking antibodies. 10.1016/j.leukres.2018.01.014
    The diverse functions of the PD1 inhibitory pathway. Sharpe Arlene H,Pauken Kristen E Nature reviews. Immunology T cell activation is a highly regulated process involving peptide-MHC engagement of the T cell receptor and positive costimulatory signals. Upon activation, coinhibitory 'checkpoints', including programmed cell death protein 1 (PD1), become induced to regulate T cells. PD1 has an essential role in balancing protective immunity and immunopathology, homeostasis and tolerance. However, during responses to chronic pathogens and tumours, PD1 expression can limit protective immunity. Recently developed PD1 pathway inhibitors have revolutionized cancer treatment for some patients, but the majority of patients do not show complete responses, and adverse events have been noted. This Review discusses the diverse roles of the PD1 pathway in regulating immune responses and how this knowledge can improve cancer immunotherapy as well as restore and/or maintain tolerance during autoimmunity and transplantation. 10.1038/nri.2017.108
    PD-1 expression and clinical PD-1 blockade in B-cell lymphomas. Xu-Monette Zijun Y,Zhou Jianfeng,Young Ken H Blood Programmed cell death protein 1 (PD-1) blockade targeting the PD-1 immune checkpoint has demonstrated unprecedented clinical efficacy in the treatment of advanced cancers including hematologic malignancies. This article reviews the landscape of PD-1/programmed death-ligand 1 (PD-L1) expression and current PD-1 blockade immunotherapy trials in B-cell lymphomas. Most notably, in relapsed/refractory classical Hodgkin lymphoma, which frequently has increased PD-1 tumor-infiltrating T cells, 9p24.1 genetic alteration, and high PD-L1 expression, anti-PD-1 monotherapy has demonstrated remarkable objective response rates (ORRs) of 65% to 87% and durable disease control in phase 1/2 clinical trials. The median duration of response was 16 months in a phase 2 trial. PD-1 blockade has also shown promise in a phase 1 trial of nivolumab in relapsed/refractory B-cell non-Hodgkin lymphomas, including follicular lymphoma, which often displays abundant PD-1 expression on intratumoral T cells, and diffuse large B-cell lymphoma, which variably expresses PD-1 and PD-L1. In primary mediastinal large B-cell lymphoma, which frequently has 9p24.1 alterations, the ORR was 35% in a phase 2 trial of pembrolizumab. In contrast, the ORR with pembrolizumab was 0% in relapsed chronic lymphocytic leukemia (CLL) and 44% in CLL with Richter transformation in a phase 2 trial. T cells from CLL patients have elevated PD-1 expression; CLL PD-1 T cells can exhibit a pseudo-exhaustion or a replicative senescence phenotype. PD-1 expression was also found in marginal zone lymphoma but not in mantle cell lymphoma, although currently anti-PD-1 clinical trial data are not available. Mechanisms and predictive biomarkers for PD-1 blockade immunotherapy, treatment-related adverse events, hyperprogression, and combination therapies are discussed in the context of B-cell lymphomas. 10.1182/blood-2017-07-740993
    Ibrutinib modulates the immunosuppressive CLL microenvironment through STAT3-mediated suppression of regulatory B-cell function and inhibition of the PD-1/PD-L1 pathway. Kondo K,Shaim H,Thompson P A,Burger J A,Keating M,Estrov Z,Harris D,Kim E,Ferrajoli A,Daher M,Basar R,Muftuoglu M,Imahashi N,Alsuliman A,Sobieski C,Gokdemir E,Wierda W,Jain N,Liu E,Shpall E J,Rezvani K Leukemia Ibrutinib, a covalent inhibitor of Bruton Tyrosine Kinase (BTK), is approved for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). Besides directly inhibiting BTK, ibrutinib possesses immunomodulatory properties through targeting multiple signaling pathways. Understanding how this ancillary property of ibrutinib modifies the CLL microenvironment is crucial for further exploration of immune responses in this disease and devising future combination therapies. Here, we investigated the mechanisms underlying the immunomodulatory properties of ibrutinib. In peripheral blood samples collected prospectively from CLL patients treated with ibrutinib monotherapy, we observed selective and durable downregulation of PD-L1 on CLL cells by 3 months post-treatment. Further analysis showed that this effect was mediated through inhibition of the constitutively active signal transducer and activator of transcription 3 (STAT3) in CLL cells. Similar downregulation of PD-1 was observed in CD4+ and CD8+ T cells. We also demonstrated reduced interleukin (IL)-10 production by CLL cells in patients receiving ibrutinib, which was also linked to suppression of STAT3 phosphorylation. Taken together, these findings provide a mechanistic basis for immunomodulation by ibrutinib through inhibition of the STAT3 pathway, critical in inducing and sustaining tumor immune tolerance. The data also merit testing of combination treatments combining ibrutinib with agents capable of augmenting its immunomodulatory effects. 10.1038/leu.2017.304
    Upregulation of Galectin-9 and PD-L1 Immune Checkpoints Molecules in Patients with Chronic Lymphocytic Leukemia Taghiloo Saeid,Allahmoradi Esmaeil,Ebadi Reza,Tehrani Mohsen,Hosseini-Khah Zahra,Janbabaei Ghasem,Shekarriz Ramin,Asgarian-Omran Hossein Asian Pacific journal of cancer prevention : APJCP Background: Deviation of host immune response by engagement of inhibitory receptors is one of the well-knownmechanisms of tumor cells for immune evasion and survival. PD-1/PD-L1 and Tim-3/Gal-9 axes are two majorpathways in this area which their contribution has been documented in a variety of malignancies. In this study, Gal-9and PD-L1 expression was investigated in leukemic cells from patients with Chronic Lymphocytic Leukemia (CLL).Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 25 untreated CLL patients and 15 sex- andage-matched healthy controls. CLL patients were classified into different clinical stages based on the Rai staging system.Total RNA was extracted from all samples and applied for cDNA synthesis. Relative expression of Gal-9 and PD-L1mRNA was determined by Real-Time PCR using β-actin as a housekeeping gene. Results: Gal-9 and PD-L1 mRNA wassignificantly more expressed in CLL patients compared to healthy controls (p<0.0001 and p=0.005, respectively). CLLpatients in advanced clinical stages showed higher expression of Gal-9 and PD-L1 in comparison to patients in earlyclinical stages (p<0.0001 and p=0.004, respectively). Conclusion: Our promising results regarding over-expression ofGal-9 and PD-L1 in CLL patients call future complementary studies to more evaluate and confirm these pathways forimmunotherapy approaches of this malignancy. Upregulation of both Gal-9 and PD-L1 in CLL patients with advancedclinical stages introduces them as useful prognostic biomarkers for disease progression. 10.22034/APJCP.2017.18.8.2269
    PD-1/PD-L1 inhibitors in haematological malignancies: update 2017. Jelinek Tomas,Mihalyova Jana,Kascak Michal,Duras Juraj,Hajek Roman Immunology The introduction of PD-1/PD-L1 pathway inhibitors is an important landmark in solid oncology with unprecedented practice-changing activity in various types of solid tumours. Among haematological malignancies, PD-1/PD-L1 inhibitors have been successful, so far, only in the treatment of classical Hodgkin lymphoma, which typically exhibits an over-expression of PD-1 ligands (PD-L1, PD-L2) due to alterations in chromosome 9p24.1. Such positive outcomes led to the US Food and Drug Administration approval of nivolumab use in relapsed Hodgkin lymphoma in 2016 as the first haematological indication. Although the results in other lymphoid malignancies have not been so striking, blockade of the PD-1/PD-L1 axis has led to meaningful responses in other lymphoma types such as diffuse large B-cell lymphoma, follicular lymphoma or several T-cell lymphomas. Monotherapy with PD-1/PD-L1 inhibitors in chronic lymphocytic leukaemia and multiple myeloma has been unsatisfactory, suggesting that a combinational approach with other synergistic drugs is needed. In the case of multiple myeloma, immunomodulatory agents together with corticosteroids represent the most promising combinations. Among myeloid malignancies, the anti-PD-1 monoclonal antibodies are examined dominantly in acute myeloid leukaemia and myelodysplastic syndromes in combination with potentially synergistic hypomethylating drugs such as 5-azacitidine, resulting in promising outcomes that warrant further investigation. We have described all available clinical results of PD-1/PD-L1 inhibitors in haematological malignancies and discussed related toxicities, as well as highlighted crucial preclinical studies in this review. 10.1111/imm.12788
    De Novo Epigenetic Programs Inhibit PD-1 Blockade-Mediated T Cell Rejuvenation. Ghoneim Hazem E,Fan Yiping,Moustaki Ardiana,Abdelsamed Hossam A,Dash Pradyot,Dogra Pranay,Carter Robert,Awad Walid,Neale Geoff,Thomas Paul G,Youngblood Ben Cell Immune-checkpoint-blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA-methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells, and approaches to reverse these programs improved T cell responses and tumor control during ICB. These data establish de novo DNA-methylation programming as a regulator of T cell exhaustion and barrier of ICB-mediated T cell rejuvenation. 10.1016/j.cell.2017.06.007
    Frequency and functional characterization of exhausted CD8 T cells in chronic lymphocytic leukemia. Taghiloo Saeid,Allahmoradi Esmaeil,Tehrani Mohsen,Hossein-Nataj Hadi,Shekarriz Ramin,Janbabaei Ghasem,Abediankenari Saeid,Asgarian-Omran Hossein European journal of haematology OBJECTIVES:The phenotypic and functional properties of Tim-3 /PD-1 /CD8 cells as exhausted T cells were investigated in chronic lymphocytic leukemia (CLL). METHODS:Frequency of CD8 /Tim-3 /PD-1 exhausted cells was determined by flow cytometry. For functional analysis, magnetic beads-isolated CD8 T cells were stimulated with PHA and PMA/ionocymin to assess their proliferative responses and cytokine production by MTT and ELISA, respectively. Cytotoxic activity of isolated CD8 T cells was determined using CD107a degranulation assay. RESULTS:The proportion of exhausted CD8 T cells was significantly higher in CLL compared to controls. Isolated CD8 T cells from CLL showed functional defects in proliferation, degranulation, and cytokines production. While IL-2, TNF-α, and IFN-γ were significantly lower in CLL patients, IL-10 was higher in the patients group. Patients with progressive clinical stages showed higher frequency and dysfunction of exhausted CD8 T cells. CONCLUSION:Targeting immune inhibitory receptors to restore the function of tumor surrounding T cells could be helpful for immunotherapy of CLL. 10.1111/ejh.12880
    T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers. Palma Marzia,Gentilcore Giusy,Heimersson Kia,Mozaffari Fariba,Näsman-Glaser Barbro,Young Emma,Rosenquist Richard,Hansson Lotta,Österborg Anders,Mellstedt Håkan Haematologica Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3 cells and the CD8 subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4 and CD8 cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients (=0.0003 and =0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4 and CD8 subsets, with a significantly higher PD-1 expression. Higher numbers of CD4 and CD8 cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67) and activated (CD69) CD4 and CD8 cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls (<0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways. 10.3324/haematol.2016.151100
    The PD-1/PD-L1 axis contributes to immune metabolic dysfunctions of monocytes in chronic lymphocytic leukemia. Qorraj M,Bruns H,Böttcher M,Weigand L,Saul D,Mackensen A,Jitschin R,Mougiakakos D Leukemia Immune dysfunctions in chronic lymphocytic leukemia (CLL) contribute to tumor immune escape and attenuate immune-based therapies. Monocytes/macrophages represent key components of cancer immune surveillance and effectors for antibody-mediated antitumor effects. Monocytes display an altered subset composition in CLL. Moreover, we find a changed metabolic phenotype: glucose uptake, glucose transporters and expression of glycolytic molecules are reduced. Our data establish a link between glycolytic competence and monocyte-mediated phagocytosis of tumor cells. Furthermore, we report that CLL monocytes express Bruton's tyrosine kinase (BTK). Our observations suggest that using BTK inhibitors in CLL might further aggravate the observed immune metabolic defects in monocytes. Triggering the programmed cell death-1 (PD-1) checkpoint on monocytes hampers glycolysis, phagocytosis and BTK signaling. Conversely, disrupting PD-1/PD-L1 signaling reverses these immune metabolic dysfunctions. Taken together, our findings imply a novel metabolic interplay between CLL cells and monocytes and that blocking PD-1/PD-L1 might restore metabolic together with antitumor activity of CLL monocytes/macrophages. 10.1038/leu.2016.214
    Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality. Saha Asim,O'Connor Roddy S,Thangavelu Govindarajan,Lovitch Scott B,Dandamudi Durga Bhavani,Wilson Caleph B,Vincent Benjamin G,Tkachev Victor,Pawlicki Jan M,Furlan Scott N,Kean Leslie S,Aoyama Kazutoshi,Taylor Patricia A,Panoskaltsis-Mortari Angela,Foncea Rocio,Ranganathan Parvathi,Devine Steven M,Burrill Joel S,Guo Lili,Sacristan Catarina,Snyder Nathaniel W,Blair Ian A,Milone Michael C,Dustin Michael L,Riley James L,Bernlohr David A,Murphy William J,Fife Brian T,Munn David H,Miller Jeffrey S,Serody Jonathan S,Freeman Gordon J,Sharpe Arlene H,Turka Laurence A,Blazar Bruce R The Journal of clinical investigation Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD. 10.1172/JCI85796
    PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion. Tinoco Roberto,Carrette Florent,Barraza Monique L,Otero Dennis C,Magaña Jonathan,Bosenberg Marcus W,Swain Susan L,Bradley Linda M Immunity Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment. 10.1016/j.immuni.2016.04.015
    MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade. Ebert Peter J R,Cheung Jeanne,Yang Yagai,McNamara Erin,Hong Rebecca,Moskalenko Marina,Gould Stephen E,Maecker Heather,Irving Bryan A,Kim Jeong M,Belvin Marcia,Mellman Ira Immunity Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy. 10.1016/j.immuni.2016.01.024
    Mechanisms of PD-L1/PD-1-mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model. McClanahan Fabienne,Riches John C,Miller Shaun,Day William P,Kotsiou Eleni,Neuberg Donna,Croce Carlo M,Capasso Melania,Gribben John G Blood T-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eμ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3(+)CD8(+) T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1(+) T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8(+) T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity. 10.1182/blood-2015-02-626754
    Chronic lymphocytic leukaemia induces an exhausted T cell phenotype in the TCL1 transgenic mouse model. Gassner Franz J,Zaborsky Nadja,Catakovic Kemal,Rebhandl Stefan,Huemer Michael,Egle Alexander,Hartmann Tanja N,Greil Richard,Geisberger Roland British journal of haematology Although chronic lymphocytic leukaemia (CLL) is a B cell malignancy, earlier studies have indicated a role of T cells in tumour growth and disease progression. In particular, the functional silencing of antigen-experienced T cells, called T cell exhaustion, has become implicated in immune evasion in CLL. In this study, we tested whether T cell exhaustion is recapitulated in the TCL1(tg) mouse model for CLL. We show that T cells express high levels of the inhibitory exhaustion markers programmed cell death 1 (PDCD1, also termed PD-1) and lymphocyte-activation gene 3 (LAG3), whereas CLL cells express high levels of CD274 (also termed PD-ligand 1). In addition, the fraction of exhausted T cells increases with CLL progression. Finally, we demonstrate that exhausted T cells are reinvigorated towards CLL cytotoxicity by inhibition of PDCD1/CD274 interaction in vivo. These results suggest that T cell exhaustion contributes to CLL pathogenesis and that interference with PDCD1/CD274 signalling holds high potential for therapeutic approaches. 10.1111/bjh.13467
    PD-L1 checkpoint blockade prevents immune dysfunction and leukemia development in a mouse model of chronic lymphocytic leukemia. McClanahan Fabienne,Hanna Bola,Miller Shaun,Clear Andrew James,Lichter Peter,Gribben John G,Seiffert Martina Blood Blockade of the programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint augments antitumor immunity and induces durable responses in patients with solid cancers, but data on clinical efficacy in leukemias are sparse. Chronic lymphocytic leukemia (CLL) is associated with a tumor-supportive microenvironment and a dysfunctional immune system, as shown by "exhausted" T cells, defective immunologic synapse formation, and immunosuppressive myeloid cells. These defects involve aberrant expression of PD-L1 and are closely mirrored in the Eµ-TCL1 mouse model for CLL. In this study, we treated mice after adoptive transfer of Eµ-TCL1 CLL with PD-L1-blocking antibodies, which prevented CLL development and was accompanied by a reactivation of immune effector functions. This included restoration of mature macrophages and major histocompatibility complex class II-expressing dendritic cells and prevention of aberrant and exhaustion-like T-cell phenotypes. In addition, PD-L1 blockade restored CD8 T-cell cytotoxicity and immune synapse formation and normalized T-cell cytokines and proliferation ex vivo and in vivo. Our data demonstrate that early PD-L1 blockade effectively corrects leukemia-induced immune dysfunction and thus prevents CLL development in mice. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies with CLL patients, ideally in combination with novel compounds to help eliminate CLL. 10.1182/blood-2015-01-622936
    Inhibition of bromodomain and extra-terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia. Carrà Giovanna,Nicoli Paolo,Lingua Marcello Francesco,Maffeo Beatrice,Cartellà Antonio,Circosta Paola,Brancaccio Mara,Parvis Guido,Gaidano Valentina,Guerrasio Angelo,Saglio Giuseppe,Taulli Riccardo,Morotti Alessandro Journal of cellular and molecular medicine The development of drugs able to target BTK, PI3k-delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non-recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19 lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax-resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first-line therapy in combination with venetoclax and as second-line therapy, after the emergence of venetoclax-resistant clones. 10.1111/jcmm.14857
    Bayesian Population Model of the Pharmacokinetics of Venetoclax in Combination with Rituximab in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia: Results from the Phase III MURANO Study. Deng Rong,Gibiansky Leonid,Lu Tong,Agarwal Priya,Ding Hao,Li Xiaobin,Kshirsagar Smita,Lu Dan,Li Chunze,Girish Sandhya,Wang Jue,Boyer Michelle,Humphrey Kathryn,Freise Kevin J,Salem Ahmed Hamed,Seymour John F,Kater Arnon P,Miles Dale Clinical pharmacokinetics BACKGROUND:Venetoclax is a selective B-cell lymphoma-2 (BCL-2) inhibitor approved for use as monotherapy or with rituximab in patients with chronic lymphocytic leukemia (CLL). The objectives of the current analysis of observed data from adult patients randomized to venetoclax-rituximab in the phase III MURANO study were to characterize venetoclax pharmacokinetics (PKs) using a Bayesian approach, evaluate whether a previously developed population PK model for venetoclax can describe the PKs of venetoclax when administered with rituximab, and to determine post hoc estimates of PK parameters for the exposure-response analysis. METHODS:Parameter estimates and uncertainty estimated by a population PK model were used as priors. Additional covariate effects (CLL risk status, geographic region, and 17p deletion [del(17p)] status) were added to the model. The updated model was used to describe venetoclax PKs after repeated dosing in combination with rituximab, and to determine post hoc estimates of PK parameters for exposure-response analysis. RESULTS:The PK analysis included 600 quantifiable venetoclax PK samples from 182 patients in the MURANO study. Model evaluation using standard diagnostic plots, visual predictive checks, and normalized prediction distribution error plots indicated no model deficiencies. There was no significant relationship between venetoclax apparent clearance (CL/F) and bodyweight, age, sex, mild and moderate hepatic and renal impairment, or coadministration of weak cytochrome P450 3A inhibitors. The chromosomal abnormality del(17p) and CLL risk status had no apparent effect on the PKs of venetoclax. A minimal increase in venetoclax CL/F (approximately 7%) was observed after coadministration with rituximab. CL/F was 30% lower in patients from Central and Eastern Europe (n = 60) or Asia (n = 4) compared with other regions (95% confidence interval [CI] 21-39%). Apparent central volume of distribution was 30% lower (95% CI 22-38%) in females (n = 56) compared with males (n = 126). No clinically significant impact of region or sex was observed on key safety and efficacy outcomes. CONCLUSIONS:The Bayesian model successfully characterized venetoclax PKs over time and confirmed key covariates affecting PKs in the MURANO study. The model was deemed appropriate for further use in simulations and for generating individual patient PK parameters for subsequent exposure-response evaluation. 10.1007/s40262-019-00788-8
    Acquisition of the recurrent Gly101Val mutation in confers resistance to venetoclax in patients with progressive chronic lymphocytic leukemia (). Weiss Jonathan,Peifer Martin,Herling Carmen D,Frenzel Lukas P,Hallek Michael Haematologica 10.3324/haematol.2019.232835
    Specific interactions of BCL-2 family proteins mediate sensitivity to BH3-mimetics in diffuse large B-cell lymphoma. Smith Victoria M,Dietz Anna,Henz Kristina,Bruecher Daniela,Jackson Ross,Kowald Lisa,van Wijk Sjoerd J L,Jayne Sandrine,Macip Salvador,Fulda Simone,Dyer Martin J S,Vogler Meike Haematologica The BCL-2-specific inhibitor, ABT-199 (venetoclax) has exhibited remarkable clinical activity in nearly all cases of chronic lymphocytic leukemia. In contrast, responses are usually much less in diffuse large B-cell lymphoma (DLBCL), despite high level expression of BCL-2 in over 40% of cases, indicating that co-expression of related anti-apoptotic BCL-2 family proteins may limit the activity of ABT-199. We have investigated the roles of BCL-2 proteins in DLBCL cells using a panel of specific BCL-2 homology 3 (BH3)-mimetics and identified subgroups of these cells that exhibited marked and specific dependency on either BCL-2, BCL-X or MCL-1 for survival. Dependency was associated with selective sequestration of the pro-apoptotic proteins BIM, BAX and BAK by the specific anti-apoptotic BCL-2 protein which was important for cellular survival. Sensitivity to BH3-mimetics was independent of genetic alterations involving the BCL-2 family and only partially correlated with protein expression levels. Treatment with ABT-199 displaced BAX and BIM from BCL-2, subsequently leading to BAK activation and apoptosis. In contrast, apoptosis induced by inhibiting BCL-X with A1331852 was associated with a displacement of both BAX and BAK from BCL-X and occurred independently of BIM. Finally, the MCL-1 inhibitor S63845 induced mainly BAX-dependent apoptosis mediated by a displacement of BAK, BIM and NOXA from MCL-1. In conclusion, our study indicates that in DLBCL, the heterogeneous response to BH3-mimetics is mediated by selective interactions between BAX, BAK and anti-apoptotic BCL-2 proteins. 10.3324/haematol.2019.220525
    Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Guièze Romain,Liu Vivian M,Rosebrock Daniel,Jourdain Alexis A,Hernández-Sánchez María,Martinez Zurita Aina,Sun Jing,Ten Hacken Elisa,Baranowski Kaitlyn,Thompson Philip A,Heo Jin-Mi,Cartun Zachary,Aygün Ozan,Iorgulescu J Bryan,Zhang Wandi,Notarangelo Giulia,Livitz Dimitri,Li Shuqiang,Davids Matthew S,Biran Anat,Fernandes Stacey M,Brown Jennifer R,Lako Ana,Ciantra Zoe B,Lawlor Matthew A,Keskin Derin B,Udeshi Namrata D,Wierda William G,Livak Kenneth J,Letai Anthony G,Neuberg Donna,Harper J Wade,Carr Steven A,Piccioni Federica,Ott Christopher J,Leshchiner Ignaty,Johannessen Cory M,Doench John,Mootha Vamsi K,Getz Gad,Wu Catherine J Cancer cell Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance. 10.1016/j.ccell.2019.08.005
    [Ibrutinib combined with CAR-T cells in the treatment of del (17p) chronic lymphocytic leukemia with BCL-2 inhibitor resistance: a case report and literature review]. Gong J J,Yin Q S,Li M J,Ai H,Wang Q,Chen L,Wei X D,Song Y P Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi To improve the knowledge and experience of ibrutinib combined with CAR-T cells in the treatment of high-risk chronic lymphoblastic leukemia (CLL) patients or small lymphocytic lymphoma (SLL) with TP53 gene aberration. One case of del (17p) CLL patients with BCL-2 inhibitor resistance was treated with ibrutinib combined with CAR-T cells, successfully bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) , and the relative literatures were reviewed. The patient was a young female with superficial lymph node enlarging at the beginning of the onset. Lymph node biopsy was confirmed as small lymphocytic lymphoma (SLL) without del (17p) . The disease progressed rapidly to CLL/SLL with del (17p) and bone marrow hematopoietic failure 2 years later. Firstly, the patient was treated with BCL-2 inhibitor (Venetoclax) , and the enlarged lymph nodes shrank significantly 2 months later. After 3 months, the disease progressed rapidly. The spleen was enlarged to 16 cm below the ribs, the neck lymph nodes was rapidly enlarged, and the superior vena cava syndrome appeared, which were mainly attributed to venetoclax resistance; so BTK inhibitor (ibrutinib) was used continuously after venetoclax discontinuation. Partial remission (PR) was achieved without lymphocytosis after 2 months, then ibrutinib was combined with CAR-T cells targeting CD19 antigen. Grade 1 of cytokine release syndrome (CRS) appeared after CAR-T cells infusion, and the complete remission (CR) was achieved after 1 month both in bone marrow and peripheral blood, with minimal residual disease (MRD) negative, then bridging allo-HSCT after 2 months of combined therapy. CLL/SLL patients with TP53 aberration have poor prognosis because of rapid progression, drug resistance, etc. Ibrutinib combined with CAR-T cell therapy can quickly achieved complete remission. 10.3760/cma.j.issn.0253-2727.2019.09.008
    Targeting BCL2 in Chronic Lymphocytic Leukemia and Other Hematologic Malignancies. Yalniz Fevzi F,Wierda William G Drugs Apoptosis, the process of programmed cell death, occurs normally during development and aging. Members of the B-cell lymphoma 2 (BCL2) family of proteins are central regulators of apoptosis, and resistance to apoptosis is one of the hallmarks of cancer. Targeting the apoptotic pathway via BCL2 inhibitors has been considered a promising treatment strategy in the past decade. Initial efforts with small molecule BH3 mimetics such as ABT-737 and ABT-263 (navitoclax) pioneered the development of the first-in-class Food and Drug Administration (FDA)-approved oral BCL2 inhibitor, venetoclax. Venetoclax was approved for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia, and is now being studied in a number of hematologic malignancies. Several other inhibitors targeting different BCL2 family members are now in early stages of development. 10.1007/s40265-019-01163-4
    Venetoclax, the first BCL-2 inhibitor for use in patients with chronic lymphocytic leukemia. Seymour John Clinical advances in hematology & oncology : H&O
    Apoptotic resistance in chronic lymphocytic leukemia and therapeutic perspectives. Bagacean Cristina,Tomuleasa Ciprian,Tempescul Adrian,Grewal Ravnit,Brooks Wesley H,Berthou Christian,Renaudineau Yves Critical reviews in clinical laboratory sciences Increased resistance to apoptosis represents a key oncogenic mechanism in chronic lymphocytic leukemia (CLL) that has been attributed to the upregulation of the anti-apoptotic B cell lymphoma 2 (Bcl-2) family members. Such an observation was associated with the development of molecules inhibiting Bcl-2 activity, and among them, BH3-mimetics represent a novel class of therapeutic compounds. In 2016, venetoclax became the first approved oral inhibitor of Bcl-2, and it has been used with success in patients with CLL who present with a 17p deletion or mutations and in those who have received at least one prior therapy. However, its mechanism for controlling relapses, and its optimal use in terms of duration and combinations with other drugs, remain unknown. Therefore, this review focuses on the mechanisms controlling apoptosis, CLL B cell strategies to prevent apoptosis including in response to BH3-mimetics, and arguments supporting the use of BH3-mimetics in association with other therapies in order to limit compensatory mechanisms. 10.1080/10408363.2019.1600468
    New Quinoline-Based Heterocycles as Anticancer Agents Targeting Bcl-2. Hamdy Rania,Elseginy Samia A,Ziedan Noha I,Jones Arwyn T,Westwell Andrew D Molecules (Basel, Switzerland) The Bcl-2 protein has been studied as an anticancer drug target in recent years, due to its gatekeeper role in resisting programmed cancer cell death (apoptosis), and the design of BH3 domain mimetics has led to the clinical approval of Venetoclax (ABT-199) for the treatment of chronic lymphocytic leukaemia. In this work we extend our previous studies on the discovery of indole-based heterocycles as Bcl-2 inhibitors, to the identification of quinolin-4-yl based oxadiazole and triazole analogues. Target compounds were readily synthesized via a common aryl-substituted quinolin-4-carbonyl--arylhydrazine-1-carbothioamide () intermediate, through simple variation of the basic cyclisation conditions. Some of the quinoline-based oxadiazole analogues (e.g. compound ) were found to exhibit sub-micromolar anti-proliferative activity in Bcl-2-expressing cancer cell lines, and sub-micromolar IC activity within a Bcl2-Bim peptide ELISA assay. The Bcl-2 targeted anticancer activity of was further rationalised via computational molecular modelling, offering possibilities to extend this work into the design of further potent and selective Bcl-2 inhibitory heteroaromatics with therapeutic potential. 10.3390/molecules24071274
    BCL-2 Inhibitors, Present and Future. Ryan Christine E,Davids Matthew S Cancer journal (Sudbury, Mass.) The members of the B-cell leukemia/lymphoma-2 (BCL-2) family of proteins are key regulators of the intrinsic apoptotic pathway; dysregulation of this pathway leads to pathologic survival of cancer cells. B-cell leukemia/lymphoma-2 had long been viewed as a promising target for the treatment of several hematologic malignancies, specifically chronic lymphocytic leukemia (CLL), yet for many years the development of a drug to successfully target this protein remained elusive. The approval of the BCL-2 inhibitor venetoclax for relapsed/refractory del(17p) CLL in 2016 represented the culmination of decades of molecular and clinical research and has paved the way for new combination therapy regimens in CLL, including the venetoclax + rituximab regimen approved for relapsed/refractory CLL in 2018 and the venetoclax + obinutuzumab regimen approved for frontline CLL treatment in 2019. Here, we provide an overview of the mechanism of action of BCL-2 inhibition, the role of this approach in the current treatment paradigm of CLL, and an in-depth focus on the clinical trials in CLL involving venetoclax. Additionally, we review key areas of active research including the integration of minimal residual disease as a marker of clinical efficacy in current clinical trials as well as the emergence of venetoclax resistance mechanisms and potential strategies to overcome this resistance. Given the success of venetoclax in the clinical setting thus far, it is likely that BCL-2 inhibition will take on an increasingly important role in the treatment of CLL going forward. 10.1097/PPO.0000000000000408
    [Chronic Lymphocytic Leukemia]. Schwarb Heike,Heim Dominik Therapeutische Umschau. Revue therapeutique Chronic Lymphocytic Leukemia CLL is an indolent B-cell lymphoma characterized by a leukemic course. It is clinically and biologically very heterogeneous. The disease is preceded by a condition called monoclonal B lymphocytosis (MBL). The treatment of CLL has changed dramatically in recent years. Chemoimmunotherapy is being replaced by new molecularly targeted therapeutic modalities like B cell receptor inhibitors and BCL-2 regulators of programmed cell death. When to initiate therapy and what "new" drug combinations to use in which line of treatment are issues currently addressed in clinical trials. 10.1024/0040-5930/a001129
    BH3-Mimetic Drugs: Blazing the Trail for New Cancer Medicines. Merino Delphine,Kelly Gemma L,Lessene Guillaume,Wei Andrew H,Roberts Andrew W,Strasser Andreas Cancer cell Defects in apoptotic cell death can promote cancer and impair responses of malignant cells to anti-cancer therapy. Pro-survival BCL-2 proteins prevent apoptosis by keeping the cell death effectors, BAX and BAK, in check. The BH3-only proteins initiate apoptosis by neutralizing the pro-survival BCL-2 proteins. Structural analysis and medicinal chemistry led to the development of small-molecule drugs that mimic the function of the BH3-only proteins to kill cancer cells. The BCL-2 inhibitor venetoclax has been approved for treatment of refractory chronic lymphocytic leukemia and this drug and inhibitors of pro-survival MCL-1 and BCL-XL are being tested in diverse malignancies. 10.1016/j.ccell.2018.11.004
    Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia. Blombery Piers,Anderson Mary Ann,Gong Jia-Nan,Thijssen Rachel,Birkinshaw Richard W,Thompson Ella R,Teh Charis E,Nguyen Tamia,Xu Zhen,Flensburg Christoffer,Lew Thomas E,Majewski Ian J,Gray Daniel H D,Westerman David A,Tam Constantine S,Seymour John F,Czabotar Peter E,Huang David C S,Roberts Andrew W Cancer discovery The BCL2 inhibitor venetoclax induces high rates of durable remission in patients with previously treated chronic lymphocytic leukemia (CLL). However, despite continuous daily treatment, leukemia recurs in most patients. To investigate the mechanisms of secondary resistance, we analyzed paired pre-venetoclax and progression samples from 15 patients with CLL progression enrolled on venetoclax clinical trials. The novel Gly101Val mutation in BCL2 was identified at progression in 7 patients, but not at study entry. It was first detectable after 19 to 42 months of therapy, and its emergence anticipated clinical disease progression by many months. Gly101Val reduces the affinity of BCL2 for venetoclax by ∼180-fold in surface plasmon resonance assays, thereby preventing the drug from displacing proapoptotic mediators from BCL2 in cells and conferring acquired resistance in cell lines and primary patient cells. This mutation provides new insights into the pathobiology of venetoclax resistance and provides a potential biomarker of impending clinical relapse. SIGNIFICANCE: Why CLL recurs in patients who achieve remission with the BCL2 inhibitor venetoclax has been unknown. We provide the first description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is sufficient to confer resistance... 10.1158/2159-8290.CD-18-1119
    Constitutive IP signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP receptor disruptor BIRD-2. Bittremieux Mart,La Rovere Rita M,Akl Haidar,Martines Claudio,Welkenhuyzen Kirsten,Dubron Kathia,Baes Myriam,Janssens Ann,Vandenberghe Peter,Laurenti Luca,Rietdorf Katja,Morciano Giampaolo,Pinton Paolo,Mikoshiba Katsuhiko,Bootman Martin D,Efremov Dimitar G,De Smedt Humbert,Parys Jan B,Bultynck Geert Cell death and differentiation Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP) receptor (IPR)-mediated Ca-signaling. A peptide tool (Bcl-2/IPR Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IPRs by targeting Bcl-2's BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IPR2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IPR2-expression levels, but also on constitutive IP signaling, downstream of the tonically active B-cell receptor. The basal Ca level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP production. This constitutive IP signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP signaling using a lower U73122 concentration (1 µM) or expression of an IP sponge suppressed both BIRD-2-induced Ca elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IPR activity. BIRD-2 seems to switch constitutive IP signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy. 10.1038/s41418-018-0142-3
    Venetoclax for patients with chronic lymphocytic leukemia who progressed during or after idelalisib therapy. Coutre Steven,Choi Michael,Furman Richard R,Eradat Herbert,Heffner Leonard,Jones Jeffrey A,Chyla Brenda,Zhou Lang,Agarwal Suresh,Waskiewicz Tina,Verdugo Maria,Humerickhouse Rod A,Potluri Jalaja,Wierda William G,Davids Matthew S Blood B-cell receptor pathway inhibitors (BCRis) have transformed treatment of chronic lymphocytic leukemia (CLL); however, the efficacy of therapies for patients whose disease is refractory to/relapses after (R/R) BCRis is unknown. Venetoclax is a selective, orally bioavailable BCL-2 inhibitor with activity in patients with CLL, including those who are heavily pretreated or have 17p deletion. This phase 2 study prospectively evaluated venetoclax in patients with R/R CLL after ibrutinib or idelalisib; here we report on patients who received idelalisib as the last BCRi before enrollment. Venetoclax was initiated at 20 mg daily, followed by intrapatient ramp-up to 400 mg daily. Primary objectives included efficacy (objective response rate [ORR]) and safety of venetoclax. The study enrolled 36 patients who previously received idelalisib (ORR, 67% [24/36]); 2 patients achieved complete remission, and 1 had complete remission with incomplete bone marrow recovery. Median progression-free survival (PFS) has not yet been reached; estimated 12-month PFS was 79%. The most common adverse events (AEs; all grades) were neutropenia (56%), diarrhea (42%), upper respiratory tract infection (39%), thrombocytopenia (36%), nausea (31%), fatigue (28%), cough (22%), rash (22%), and anemia (22%). Grade 3 or 4 AEs were primarily hematologic (neutropenia [50%], thrombocytopenia [25%], and anemia [17%]). No patients experienced tumor lysis syndrome. Venetoclax demonstrated promising clinical activity and favorable tolerability in patients with CLL whose disease progressed during or after idelalisib therapy. This trial was registered at www.clinicaltrials.gov as #NCT02141282. 10.1182/blood-2017-06-788133
    BCL2 and miR-15/16: from gene discovery to treatment. Pekarsky Yuri,Balatti Veronica,Croce Carlo M Cell death and differentiation In 1984, we investigated the t(14;18) chromosomal translocations that frequently occur in patients with follicular lymphoma. We first identified a locus on chromosome 18 involved in these translocations with the chromosome 14 containing the immunoglobulin heavy chain locus. Within this region on chromosome 18, we then discovered a gene that we called BCL2, which was activated by the translocations. Since that time, many studies determined that BCL2 is one of the most important oncogenes involved in cancer by inhibiting apoptosis. In 2002, we studied 13q deletions in chronic lymphocytic leukemia (CLL) and found that the microRNA cluster miR-15a/miR-16-1 (miR-15/16) is deleted by 13q deletions. In 2005, we discovered that miR-15/16 function as tumor suppressors by directly targeting BCL2. Thus the loss of two negative regulators of BCL2 expression results in overexpression of BCL2. Very recently, a specific BCL2 inhibitor ABT-199 (Venetoclax) was developed and approved by FDA for CLL treatment. Thus it took 32 years from fundamental discovery of a critical oncogene to the development of a drug capable to cure CLL. In this review, we discuss the discovery, functions and clinical relevance of miR-15/16 and BCL2. 10.1038/cdd.2017.159
    Targeting BCL-2 in B-cell lymphomas. Davids Matthew S Blood The B-cell leukemia/lymphoma-2 (BCL-2) family of proteins governs the intrinsic pathway of mitochondrial apoptosis. Dysregulation of BCL-2 has long been known to be a crucial part of the pathophysiology of B-cell lymphomas; however, several early attempts to target this pathway therapeutically were unsuccessful because of toxicity, lack of efficacy, or both. Recently, a highly potent and selective oral BCL-2 antagonist, venetoclax, was approved in chronic lymphocytic leukemia, where it has proven to be highly active, even in patients with high-risk del(17p) disease. Venetoclax has also demonstrated efficacy in other B-cell non-Hodgkin lymphoma subtypes, in particular mantle cell lymphoma and follicular lymphoma. Here, I review the history of targeting BCL-2 in B-cell lymphomas, and I discuss recent data on venetoclax used as monotherapy and in combination with monoclonal antibodies, chemotherapy, and other novel agents. I also discuss how genomic and functional approaches such as BH3 profiling may allow us to prioritize novel-agent combinations for further study in clinical trials. These approaches may also help us to understand resistance mechanisms to BCL-2-selective therapy and how to overcome resistance. Finally, I provide my perspective on how to move BCL-2-directed therapies forward toward a goal of developing well-tolerated, time-limited combination regimens with curative potential for patients with B-cell lymphomas. 10.1182/blood-2017-04-737338
    The potential combination of BCL-2 inhibitors and ibrutinib as frontline therapy in chronic lymphocytic leukemia. Aw Andrew,Brown Jennifer R Leukemia & lymphoma The recent development of small molecule inhibitors targeted at the B-cell receptor (BCR) pathway and the anti-apoptotic protein BCL-2 has revolutionized the care of patients with chronic lymphocytic leukemia (CLL). While durable responses to the BCR inhibitor ibrutinib have been observed in both previously untreated and relapsed/refractory CLL patients, residual disease is common in patients treated with single-agent ibrutinib. Interest remains high in therapeutic combinations that may lead to better quality remissions. A potential partner to ibrutinib with a distinct mechanism of action that is likely to lead to deeper responses is the BCL-2 inhibitor venetoclax. Preclinical studies have suggested synergism between inhibitors of BCR and BCL-2 and have paved the way to the development of ongoing clinical trials aimed at evaluating the combination of ibrutinib with venetoclax in CLL patients. 10.1080/10428194.2017.1312387
    BCL-2 as a therapeutic target in chronic lymphocytic leukemia. Daniel Catherine,Mato Anthony R Clinical advances in hematology & oncology : H&O Venetoclax (formerly ABT-199) was recently approved in the United States for the treatment of patients who have relapsed or refractory chronic lymphocytic leukemia (CLL) with the 17p deletion. Venetoclax has demonstrated marked activity as monotherapy as well as in combination with cytotoxic chemotherapies, B-cell receptor inhibitors, and anti-CD20 monoclonal antibodies across the spectrum of CLL. The potency of venetoclax has been associated with a unique ability to induce deep (minimal residual disease-negative) complete remissions that appear to be durable. Its toxicity profile includes manageable hematologic toxicities, as well as the potential for tumor lysis syndrome. Here, we review the BCL-2 pathway and the mechanism of action of BCL-2 inhibitors, the activity and safety profile of venetoclax, and the practical application of venetoclax in the management of patients with CLL.
    Bruton's tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia. Deng J,Isik E,Fernandes S M,Brown J R,Letai A,Davids M S Leukemia Although the BTK inhibitor ibrutinib has transformed the management of patients with chronic lymphocytic leukemia (CLL), it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL-2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition. 10.1038/leu.2017.32
    Development of a flow cytometric method for quantification of BCL-2 family members in chronic lymphocytic leukemia and correlation with sensitivity to BCL-2 family inhibitors. Smith Morey L,Chyla Brenda,McKeegan Evelyn,Tahir Stephen K Cytometry. Part B, Clinical cytometry BACKGROUND:We have developed a quantitative fluorescence cytometry (QFCM) method that can be used to measure BCL-2 family member proteins in cell lines and clinical samples. We described the validation of antibodies, methods development and application of the assay. METHOD:We characterized and validated antibodies to BCL-2, BCL-X , and MCL-1 in cell lines to confirm specificity for flow cytometry. Each protein was measured in a panel of leukemia/lymphoma cell lines and B-cells from chronic lymphocytic leukemia (CLL) patients treated with the BCL-2/BCL-X inhibitor navitoclax. The cellular activity of various BCL-2 family member inhibitors alone and in combination was determined to demonstrate utility of our assay to correlate protein levels with efficacy. RESULTS:We identified antibodies that were highly specific for each protein. The expression profile in cell lines as determined by molecules of equivalent soluble fluorochrome was comparable to western blot. Using our assay, BCL-2, BCL-X , and MCL-1 protein levels were shown to correlate with response to BCL-2 family inhibitors in vitro and could be measured in clinical samples. CONCLUSIONS:This method can quantify BCL-2 family members in a specific, highly reproducible and sensitive fashion, and requires fewer cells compared to western blot. It is particularly useful for identifying BCL-2, BCL-X , and MCL-1 protein levels in a specific cell population within a heterogeneous population like those collected from CLL patients. These data show that our QFCM method can be used to facilitate the quantification and evaluation of biomarkers predictive of response in patients treated with BCL-2 family member inhibitors. © 2016 International Clinical Cytometry Society. 10.1002/cyto.b.21383
    Clinical significance of bax/bcl-2 ratio in chronic lymphocytic leukemia. Del Principe Maria Ilaria,Dal Bo Michele,Bittolo Tamara,Buccisano Francesco,Rossi Francesca Maria,Zucchetto Antonella,Rossi Davide,Bomben Riccardo,Maurillo Luca,Cefalo Mariagiovanna,De Santis Giovanna,Venditti Adriano,Gaidano Gianluca,Amadori Sergio,de Fabritiis Paolo,Gattei Valter,Del Poeta Giovanni Haematologica In chronic lymphocytic leukemia the balance between the pro-apoptotic and anti-apoptotic members of the bcl-2 family is involved in the pathogenesis, chemorefractoriness and clinical outcome. Moreover, the recently proposed anti-bcl-2 molecules, such as ABT-199, have emphasized the potential role of of bcl-2 family proteins in the context of target therapies. We investigated bax/bcl-2 ratio by flow cytometry in 502 patients and identified a cut off of 1.50 to correlate bax/bcl-2 ratio with well-established clinical and biological prognosticators. Bax/bcl-2 was 1.50 or over in 263 patients (52%) with chronic lymphocytic leukemia. Higher bax/bcl-2 was associated with low Rai stage, lymphocyte doubling time over 12 months, beta-2 microglobulin less than 2.2 mg/dL, soluble CD23 less than 70 U/mL and a low risk cytogenetic profile (P<0.0001). On the other hand, lower bax/bcl-2 was correlated with unmutated IGHV (P<0.0001), mutated NOTCH1 (P<0.0001) and mutated TP53 (P=0.00007). Significant shorter progression-free survival and overall survival were observed in patients with lower bax/bcl-2 (P<0.0001). Moreover, within IGHV unmutated (168 patients) and TP53 mutated (37 patients) subgroups, higher bax/bcl-2 identified cases with significant longer PFS (P=0.00002 and P=0.039). In multivariate analysis of progression-free survival and overall survival, bax/bcl-2 was an independent prognostic factor (P=0.0002 and P=0.002). In conclusion, we defined the prognostic power of bax/bcl-2 ratio, as determined by a flow cytometric approach, and highlighted a correlation with chemoresistance and outcome in chronic lymphocytic leukemia. Finally, the recently proposed new therapies employing bcl-2 inhibitors prompted the potential use of bax/bcl-2 ratio to identify patients putatively resistant to these molecules. 10.3324/haematol.2015.131854
    Cirmtuzumab blocks Wnt5a/ROR1 stimulation of NF-κB to repress autocrine STAT3 activation in chronic lymphocytic leukemia. Chen Yun,Chen Liguang,Yu Jian,Ghia Emanuela M,Choi Michael Y,Zhang Ling,Zhang Suping,Sanchez-Lopez Elsa,Widhopf George F,Messer Karen,Rassenti Laura Z,Jamieson Catriona,Kipps Thomas J Blood Coculture of nurse-like cells (NLCs) with chronic lymphocytic leukemia (CLL) cells induced leukemia cell phosphorylation of STAT3 (pSTAT3), which could be blocked by anti-Wnt5a antibodies or the anti-ROR1 monoclonal antibody, cirmtuzumab. Time-course studies revealed Wnt5a could induce activation of NF-κB within 30 minutes, but required more than 3 hours to induce pSTAT3. Culture of isolated CLL cells for 24 hours revealed Wnt5a-induced expression of interleukin 6 (IL-6), IL-8, CCL2, CCL3, CCL4, and CXCL1, which in turn could induce pSTAT3 in unstimulated CLL cells within 30 minutes. We found that Wnt5a could induce CLL cell expression of NF-κB target genes, including IL-6, and that this effect could be blocked by cirmtuzumab or drugs that inhibit NF-κB. Examination of CLL cells and plasma collected from patients treated with cirmtuzumab revealed reduced levels of phosphorylated p65 and diminished expression of NF-κB and STAT3 target genes in CLL cells, as well as lower plasma levels of IL-6, in the samples after therapy. Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling contributes to CLL cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pSTAT3. As such, this study demonstrates that cirmtuzumab can inhibit leukemia cell activation of both NF-κB and STAT3 in patients with CLL. 10.1182/blood.2019001366
    ROR1-targeted delivery of miR-29b induces cell cycle arrest and therapeutic benefit in vivo in a CLL mouse model. Chiang Chi-Ling,Goswami Swagata,Frissora Frank W,Xie Zhiliang,Yan Pearlly S,Bundschuh Ralf,Walker Logan A,Huang Xiaomeng,Mani Rajeswaran,Mo Xiaokui M,Baskar Sivasubramanian,Rader Christoph,Phelps Mitch A,Marcucci Guido,Byrd John C,Lee L James,Muthusamy Natarajan Blood Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1 CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eμ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1 B-cell malignancies. 10.1182/blood.2018882290
    Notch2 controls non-autonomous Wnt-signalling in chronic lymphocytic leukaemia. Mangolini Maurizio,Götte Frederik,Moore Andrew,Ammon Tim,Oelsner Madlen,Lutzny-Geier Gloria,Klein-Hitpass Ludger,Williamson James C,Lehner Paul J,Dürig Jan,Möllmann Michael,Rásó-Barnett Lívia,Hughes Katherine,Santoro Antonella,Méndez-Ferrer Simón,Oostendorp Robert A J,Zimber-Strobl Ursula,Peschel Christian,Hodson Daniel J,Schmidt-Supprian Marc,Ringshausen Ingo Nature communications The Wnt signalling pathway, one of the core de-regulated pathways in chronic lymphocytic leukaemia (CLL), is activated in only a subset of patients through somatic mutations. Here we describe alternative, microenvironment-dependent mechanisms of Wnt activation in malignant B cells. We show that tumour cells specifically induce Notch2 activity in mesenchymal stromal cells (MSCs) required for the transcription of the complement factor C1q. MSC-derived C1q in turn inhibits Gsk3-β mediated degradation of β-catenin in CLL cells. Additionally, stromal Notch2 activity regulates N-cadherin expression in CLL cells, which interacts with and further stabilises β-catenin. Together, these stroma Notch2-dependent mechanisms induce strong activation of canonical Wnt signalling in CLL cells. Pharmacological inhibition of the Wnt pathway impairs microenvironment-mediated survival of tumour cells. Similarly, inhibition of Notch signalling diminishes survival of stroma-protected CLL cells in vitro and disease engraftment in vivo. Notch2 activation in the microenvironment is a pre-requisite for the activation of canonical Wnt signalling in tumour cells. 10.1038/s41467-018-06069-5
    Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia. Choi Michael Y,Widhopf George F,Ghia Emanuela M,Kidwell Reilly L,Hasan Md Kamrul,Yu Jian,Rassenti Laura Z,Chen Liguang,Chen Yun,Pittman Emily,Pu Minya,Messer Karen,Prussak Charles E,Castro Januario E,Jamieson Catriona,Kipps Thomas J Cell stem cell Cirmtuzumab is a humanized monoclonal antibody (mAb) that targets ROR1, an oncoembryonic orphan receptor for Wnt5a found on cancer stem cells (CSCs). Aberrant expression of ROR1 is seen in many malignancies and has been linked to Rho-GTPase activation and cancer stem cell self-renewal. For patients with chronic lymphocytic leukemia (CLL), self-renewing, neoplastic B cells express ROR1 in 95% of cases. High-level leukemia cell expression of ROR1 is associated with an unfavorable prognosis. We conducted a phase 1 study involving 26 patients with progressive, relapsed, or refractory CLL. Patients received four biweekly infusions, with doses ranging from 0.015 to 20 mg/kg. Cirmtuzumab had a long plasma half-life and did not have dose-limiting toxicity. Inhibition of ROR1 signaling was observed, including decreased activation of RhoA and HS1. Transcriptome analyses showed that therapy inhibited CLL stemness gene expression signatures in vivo. Cirmtuzumab is safe and effective at inhibiting tumor cell ROR1 signaling in patients with CLL. 10.1016/j.stem.2018.05.018
    Wnt5a induces ROR1 to recruit DOCK2 to activate Rac1/2 in chronic lymphocytic leukemia. Hasan Md Kamrul,Yu Jian,Widhopf George F,Rassenti Laura Z,Chen Liguang,Shen Zhouxin,Briggs Steven P,Neuberg Donna S,Kipps Thomas J Blood Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on chronic lymphocytic leukemia (CLL) that can serve as a receptor for Wnt5a, which can promote leukemia cell migration, proliferation, and survival. We found Wnt5a could induce ROR1 to complex with DOCK2 (dedicator of cytokinesis 2) and induce activation of Rac1/2; these effects could be blocked by cirmtuzumab, a humanized anti-ROR1 monoclonal antibody. We find that silencing DOCK2 specifically impaired the capacity of Wnt5a to induce activation of Rac1/2 or enhance CLL cell proliferation. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for Wnt5a to induce ROR1 to complex with DOCK2 and activate Rac1/2 in the CLL cell-line MEC1. We introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at potential binding sites for the Src-homology 3 domain of DOCK2. In contrast to wild-type ROR1, or other ROR1 P→A variants, ROR1 was unable to recruit DOCK2 in response to Wnt5a. Moreover, unlike MEC1 cells transfected with wild-type ROR1 or ROR1 with P→A substitutions at positions 784, 826, or 841, MEC1 cells transfected to express ROR1 did not have a growth advantage over MEC1 cells that do not express ROR1. This study reveals that the recruitment of DOCK2 may be critical for the capacity of Wnt5a to enhance CLL proliferation, which may contribute to the observed increased tendency for disease progression in patients who have CLL cells that express high levels of ROR1. 10.1182/blood-2017-12-819383
    Casein kinase 1 is a therapeutic target in chronic lymphocytic leukemia. Janovska Pavlina,Verner Jan,Kohoutek Jiri,Bryjova Lenka,Gregorova Michaela,Dzimkova Marta,Skabrahova Hana,Radaszkiewicz Tomasz,Ovesna Petra,Vondalova Blanarova Olga,Nemcova Tereza,Hoferova Zuzana,Vasickova Katerina,Smyckova Lucie,Egle Alexander,Pavlova Sarka,Poppova Lucie,Plevova Karla,Pospisilova Sarka,Bryja Vitezslav Blood Casein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1-induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition. 10.1182/blood-2017-05-786947
    dysregulation to identify therapeutic target combinations for chronic lymphocytic leukemia. Rassenti Laura Z,Balatti Veronica,Ghia Emanuela M,Palamarchuk Alexey,Tomasello Luisa,Fadda Paolo,Pekarsky Yuri,Widhopf George F,Kipps Thomas J,Croce Carlo M Proceedings of the National Academy of Sciences of the United States of America Loss of is the most common genetic lesion in chronic lymphocytic leukemia (CLL), promoting overexpression of , which factors in leukemia pathogenesis. Indeed, an inhibitor of Bcl2, venetoclcax, is highly active in the treatment of patients with CLL. However, single-agent venetoclcax fails to eradicate minimal residual disease in most patients. Accordingly, we were interested in other genes that may be regulated by , which may target other drivers in CLL. We found that targets , which encodes an onco-embryonic surface protein expressed on the CLL cells of over 90% of patients, but not on virtually all normal postpartum tissues. CLL with high-level expression of ROR1 also have high-level expression of Bcl2, but low-to-negligible Moreover, CLL cases with high-level ROR1 have deletion(s) at the chromosomal location of the genes encoding (13q14) more frequently than cases with low-to-negligible ROR1, implying that deletion of may promote overexpression of , in addition to ROR1 is a receptor for Wnt5a, which can promote leukemia-cell proliferation and survival, and can be targeted by cirmtuzumab, a humanized anti-ROR1 mAb. We find that this mAb can enhance the in vitro cytotoxic activity of venetoclcax for CLL cells with high-level expression of ROR1, indicating that combining these agents, which target ROR1 and Bcl2, may have additive, if not synergistic, activity in patients with this disease. 10.1073/pnas.1708264114
    Wnt5a induces ROR1 to associate with 14-3-3ζ for enhanced chemotaxis and proliferation of chronic lymphocytic leukemia cells. Yu J,Chen L,Chen Y,Hasan M K,Ghia E M,Zhang L,Wu R,Rassenti L Z,Widhopf G F,Shen Z,Briggs S P,Kipps T J Leukemia Wnt5a can activate Rho GTPases in chronic lymphocytic leukemia (CLL) cells by inducing the recruitment of ARHGEF2 to ROR1. Mass spectrometry on immune precipitates of Wnt5a-activated ROR1 identified 14-3-3ζ, which was confirmed by co-immunoprecipitation. The capacity of Wnt5a to induce ROR1 to complex with 14-3-3ζ could be blocked in CLL cells by treatment with cirmtuzumab, a humanized mAb targeting ROR1. Silencing 14-3-3ζ via small interfering RNA impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance in vitro exchange activity of ARHGEF2 and (3) induce activation of RhoA and Rac1 in CLL cells. Furthermore, CRISPR/Cas9 deletion of 14-3-3ζ in ROR1-negative CLL cell-line MEC1, and in MEC1 cells transfected to express ROR1 (MEC1-ROR1), demonstrated that 14-3-3ζ was necessary for the growth/engraftment advantage of MEC1-ROR1 over MEC1 cells. We identified a binding motif (RSPSSAS) in ROR1 for 14-3-3ζ. Site-directed mutagenesis of ROR1 demonstrated that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2 to ROR1, and activation of RhoA and Rac1. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1 signaling, leading to enhanced CLL migration and proliferation. 10.1038/leu.2017.132
    Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells. Hasan M K,Yu J,Chen L,Cui Bing,Widhopf Ii G F,Rassenti L,Shen Z,Briggs S P,Kipps T J Leukemia ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1 mutants, ROR1 had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1 to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration. 10.1038/leu.2017.133
    Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation. Yu Jian,Chen Liguang,Cui Bing,Widhopf George F,Shen Zhouxin,Wu Rongrong,Zhang Ling,Zhang Suping,Briggs Steven P,Kipps Thomas J The Journal of clinical investigation Evolutionarily conserved receptor tyrosine kinase–like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocytic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation. 10.1172/JCI83535
    Insight into the role of Wnt5a-induced signaling in normal and cancer cells. Endo Mitsuharu,Nishita Michiru,Fujii Masanori,Minami Yasuhiro International review of cell and molecular biology Wnt5a is involved in the activation of noncanonical Wnt signaling, including planar cell polarity (PCP) and Wnt-Ca(2+) pathways. The Ror-family of receptor tyrosine kinases is composed of Ror1 and Ror2 in mammals. Ror2 acts as a receptor or coreceptor for Wnt5a and regulates Wnt5a-induced activation of PCP pathway, and Wnt5a-Ror2 axis indeed plays critical roles in the developmental morphogenesis by regulating cell polarity and migration. Furthermore, Wnt5a-Ror2 axis is constitutively activated in cancer cells and confers highly motile and invasive properties on cancer cells through the expression of matrix metalloproteinase genes and enhanced formation of invadopodia. Meanwhile, Wnt5a also exhibits a tumor-suppressive function in certain cancers, including breast and colorectal carcinomas. Thus, it is of great importance to understand the respective molecular mechanisms governing Wnt5a-mediated tumor-progressive and tumor-suppressive functions, in order to develop novel and proper diagnostic and therapeutic strategies targeting Wnt5a signaling for human cancers. 10.1016/bs.ircmb.2014.10.003
    Ublituximab and umbralisib in relapsed/refractory B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia. Lunning Matthew,Vose Julie,Nastoupil Loretta,Fowler Nathan,Burger Jan A,Wierda William G,Schreeder Marshall T,Siddiqi Tanya,Flowers Christopher R,Cohen Jonathon B,Sportelli Peter,Miskin Hari P,Weiss Michael S,O'Brien Susan Blood Targeting both CD20 and phosphatidylinositol 3-kinase (PI3K), a protein that is critically involved in B-cell maturation, could be an efficacious strategy for treating B-cell malignancies. The safety of the next-generation compounds umbralisib, a PI3K-δ inhibitor, plus ublituximab, an anti-CD20 monoclonal antibody (combination referred to as U2), was evaluated in patients with chronic lymphocytic lymphoma (CLL) or non-Hodgkin lymphoma (NHL) in this phase 1/1b study. Phase 1 dose escalation was performed with a 3 + 3 design to establish the maximum tolerated dose. In this portion, ublituximab was given intravenously (NHL, 900 mg; CLL, 600 or 900 mg) for 12 cycles. Umbralisib was given orally once daily at 800 or 1200 mg (initial formulation) or 400 to 1200 mg (micronized formulation) in the phase 1 dose escalation portion, and at 800 to 1200 mg in the phase 1b portion until progression, toxicity, or study removal. The maximum tolerated dose was not reached in either the CLL or NHL cohort, and only 1 dose-limiting toxicity was observed. U2 had low instances of grade 3 or higher diarrhea (8%), pneumonia (8%), or hepatic toxicity (4%). Treatment discontinuation due to adverse events occurred in 13% of patients, and umbralisib dose reductions occurred in 15% of patients. The overall response rate for all patients was 46% with 17% complete responses. The median duration of response was 20 months (95% confidence interval, 11.3-not reached). U2 was well tolerated, and no new safety signals were observed over single-agent umbralisib. Preliminary efficacy with this combination is promising and warrants further investigation. This study was registered at www.clinicaltrials.gov as #NCT02006485. 10.1182/blood.2019002118
    Duvelisib for CLL/SLL and follicular non-Hodgkin lymphoma. Patel Krish,Danilov Alexey V,Pagel John M Blood Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and follicular lymphoma (FL) represent indolent malignancies characterized by multiple episodes of relapse. Therapy has centered on agents that largely target the B-cell receptor pathway. Duvelisib is a second-generation oral inhibitor of phosphoinositide-3 kinase, downstream of the B-cell receptor pathway, approved in the United States for relapsed CLL/SLL and FL. Duvelisib represents a highly active agent, and ongoing investigations, including fixed-duration drug combinations and alternative dosing schedules, are aimed at reducing immune-mediated toxicities. 10.1182/blood.2019001795
    STIM1 at the plasma membrane as a new target in progressive chronic lymphocytic leukemia. Debant Marjolaine,Burgos Miguel,Hemon Patrice,Buscaglia Paul,Fali Tinhinane,Melayah Sarra,Le Goux Nelig,Vandier Christophe,Potier-Cartereau Marie,Pers Jacques-Olivier,Tempescul Adrian,Berthou Christian,Bagacean Cristina,Mignen Olivier,Renaudineau Yves Journal for immunotherapy of cancer BACKGROUND:Dysregulation in calcium (Ca) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca pathway has been associated with disease progression, the importance of the newly described constitutive Ca entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study. METHODS:An extensive analysis of the Ca entry (CE) pathway in CLL B cells was performed including constitutive Ca entry, basal Ca levels, and store operated Ca entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients. RESULTS:Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca concentrations; (iii) independent from the BCR-PLCγ2-InsPR (SOCE) Ca signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1); and (vi) blocked when using a mAb targeting STIM1. Next, we further established an association between an elevated expression of STIM1 and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1 CLL subgroup. CONCLUSIONS:These data establish the critical role of a newly discovered BCR independent Ca entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane. 10.1186/s40425-019-0591-3
    IGF1R as druggable target mediating PI3K-δ inhibitor resistance in a murine model of chronic lymphocytic leukemia. Scheffold Annika,Jebaraj Billy Michael Chelliah,Tausch Eugen,Bloehdorn Johannes,Ghia Paolo,Yahiaoui Anella,Dolnik Anna,Blätte Tamara Jacqueline,Bullinger Lars,Dheenadayalan Rashmi Priyadharshini,Li Li,Schneider Christof,Chen Shih-Shih,Chiorazzi Nicholas,Dietrich Sascha,Seiffert Martina,Tannheimer Stacey,Döhner Hartmut,Mertens Daniel,Stilgenbauer Stephan Blood Targeted therapy is revolutionizing the treatment of cancers, but resistance evolves against these therapies and derogates their success. The phosphatidylinositol 3-kinase delta (PI3K-δ) inhibitor idelalisib has been approved for treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, but the mechanisms conferring resistance in a subset of patients are unknown. Here, we modeled resistance to PI3K-δ inhibitor in vivo using a serial tumor transfer and treatment scheme in mice. Whole-exome sequencing did not identify any recurrent mutation explaining resistance to PI3K-δ inhibitor. In the murine model, resistance to PI3K-δ inhibitor occurred as a result of a signaling switch mediated by consistent and functionally relevant activation of insulin-like growth factor 1 receptor (IGF1R), resulting in enhanced MAPK signaling in the resistant tumors. Overexpression of in vitro demonstrated its prominent role in PI3K-δ inhibitor resistance. IGF1R upregulation in PI3K-δ inhibitor-resistant tumors was mediated by functional activation and enhanced nuclear localization of forkhead box protein O1 transcription factors and glycogen synthase kinase 3β. In human CLL, high expression was associated with trisomy 12. CLL cells from an idelalisib-treated patient showed decreased sensitivity to idelalisib in vitro concomitant with enhanced MAPK signaling and strong upregulation of IGF1R upon idelalisib exposure. Thus, our results highlight that alternative signaling cascades play a predominant role in the resistance and survival of cancer cells under PI3K-δ inhibition. We also demonstrate that these pathway alterations can serve as therapeutic targets, because inhibition of IGF1R offered efficacious salvage treatment of PI3K-δ inhibitor-resistant tumors in vitro and in vivo. 10.1182/blood.2018881029
    Umbralisib, a novel PI3Kδ and casein kinase-1ε inhibitor, in relapsed or refractory chronic lymphocytic leukaemia and lymphoma: an open-label, phase 1, dose-escalation, first-in-human study. Burris Howard A,Flinn Ian W,Patel Manish R,Fenske Timothy S,Deng Changchun,Brander Danielle M,Gutierrez Martin,Essell James H,Kuhn John G,Miskin Hari P,Sportelli Peter,Weiss Michael S,Vakkalanka Swaroop,Savona Michael R,O'Connor Owen A The Lancet. Oncology BACKGROUND:Umbralisib (TGR-1202) is a novel next-generation inhibitor of phosphatidylinositol 3-kinase (PI3K) isoform p110δ (PI3Kδ), which is structurally distinct from other PI3Kδ inhibitors and shows improved isoform selectivity. Umbralisib also uniquely inhibits casein kinase-1ε, a major regulator of protein translation. The aim of this first-in-human phase 1 study was to establish the safety and preliminary activity profile of umbralisib in patients with haematological malignancies. METHODS:We did an open-label, phase 1, dose-escalation study at seven clinics in the USA. We recruited patients aged at least 18 years with relapsed or refractory chronic lymphocytic leukaemia or small lymphocytic lymphoma, B-cell and T-cell non-Hodgkin lymphoma, or Hodgkin's lymphoma, who had received one or more previous lines of therapy, with measurable and assessable disease, and adequate organ system function. Patients self-administered an umbralisib oral tablet once per day in 28-day cycles, with dose escalation done in a traditional 3 + 3 design to establish safety and determine the maximum tolerated dose. In initial cohorts, patients took umbralisib in a fasting state at a starting dose of 50 mg, increasing to 100, 200, 400, 800, 1200, and 1800 mg until the maximum tolerated dose was reached, or the maximal dose cohort was accrued without a dose-limiting toxicity. Subsequent cohorts self-administered a micronised formulation of umbralisib tablet in a fed state at an initial dose of 200 mg, increased in increments to 400, 800, 1200, and 1800 mg until the maximum tolerated dose or the maximal dose level was accrued. In August, 2014, all patients still on study were transitioned to 800 mg of the micronised formulation and dosing of the initial formulation was discontinued. The primary endpoints of the study were investigator-assessed safety in all treated patients (the safety population), the maximum tolerated dose, and the pharmacokinetics of umbralisib. Secondary endpoints included preliminary assessments of anti-cancer activity (objective responses and duration of response). Follow-up stopped for a patient once they discontinued therapy. This study has been completed and is registered with ClinicalTrials.gov, number NCT01767766. FINDINGS:Between Jan 17, 2013, and Jan 14, 2016, we enrolled and treated 90 patients with umbralisib. The median duration of treatment and follow-up was 4·7 cycles (IQR 2·0-14·0) or 133 days (IQR 55-335). The most common treatment-emergent adverse events irrespective of causality were diarrhoea (in 39 [43%] of 90 patients), nausea (38 [42%]), and fatigue (28 [31%]). The most common grade 3 or 4 adverse events were neutropenia (in 12 [13%] patients), anaemia (eight [9%]) and thrombocytopenia (six [7%]). Serious adverse events considered at least possibly related to umbralisib occurred in seven patients: pneumonia in three (3%) patients, lung infection in one (1%), febrile neutropenia in one (1%), and colitis in two (2%), one of whom also had febrile neutropenia. The maximum tolerated dose was 1200 mg of the micronised formulation, with 800 mg of this formulation selected as the recommended phase 2 dose. Both cases of colitis occurred at above the recommended phase 2 dose. 33 (37%) of the 90 patients enrolled had an objective response to treatment with umbralisib. INTERPRETATION:Umbralisib was well tolerated and showed preliminary signs of activity in patients with relapsed or refractory haematological malignancies. The safety profile of umbralisib in this phase 1 study was distinct from that of other PI3Kδ inhibitors, with fewer occurrences of autoimmune-like toxicities such as colitis. These findings warrant further evaluation of this agent in this setting. FUNDING:TG Therapeutics. 10.1016/S1470-2045(18)30082-2
    The evolutionary landscape of chronic lymphocytic leukemia treated with ibrutinib targeted therapy. Landau Dan A,Sun Clare,Rosebrock Daniel,Herman Sarah E M,Fein Joshua,Sivina Mariela,Underbayev Chingiz,Liu Delong,Hoellenriegel Julia,Ravichandran Sarangan,Farooqui Mohammed Z H,Zhang Wandi,Cibulskis Carrie,Zviran Asaf,Neuberg Donna S,Livitz Dimitri,Bozic Ivana,Leshchiner Ignaty,Getz Gad,Burger Jan A,Wiestner Adrian,Wu Catherine J Nature communications Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts (change >0.1 in clonal cancer cell fraction, Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones. 10.1038/s41467-017-02329-y
    Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia. Minici Claudia,Gounari Maria,Übelhart Rudolf,Scarfò Lydia,Dühren-von Minden Marcus,Schneider Dunja,Tasdogan Alpaslan,Alkhatib Alabbas,Agathangelidis Andreas,Ntoufa Stavroula,Chiorazzi Nicholas,Jumaa Hassan,Stamatopoulos Kostas,Ghia Paolo,Degano Massimo Nature communications Cell-autonomous B-cell receptor (BcR)-mediated signalling is a hallmark feature of the neoplastic B lymphocytes in chronic lymphocytic leukaemia (CLL). Here we elucidate the structural basis of autonomous activation of CLL B cells, showing that BcR immunoglobulins initiate intracellular signalling through homotypic interactions between epitopes that are specific for each subgroup of patients with homogeneous clinicobiological profiles. The molecular details of the BcR-BcR interactions apparently dictate the clinical course of disease, with stronger affinities and longer half-lives in indolent cases, and weaker, short-lived contacts mediating the aggressive ones. The diversity of homotypic BcR contacts leading to cell-autonomous signalling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL and offers opportunities for innovative treatment strategies. 10.1038/ncomms15746
    S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression. Prieto Daniel,Sotelo Natalia,Seija Noé,Sernbo Sandra,Abreu Cecilia,Durán Rosario,Gil Magdalena,Sicco Estefanía,Irigoin Victoria,Oliver Carolina,Landoni Ana Inés,Gabus Raúl,Dighiero Guillermo,Oppezzo Pablo Blood Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL. 10.1182/blood-2017-02-769851
    Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells. Compagno Mara,Wang Qi,Pighi Chiara,Cheong Taek-Chin,Meng Fei-Long,Poggio Teresa,Yeap Leng-Siew,Karaca Elif,Blasco Rafael B,Langellotto Fernanda,Ambrogio Chiara,Voena Claudia,Wiestner Adrian,Kasar Siddha N,Brown Jennifer R,Sun Jing,Wu Catherine J,Gostissa Monica,Alt Frederick W,Chiarle Roberto Nature Activation-induced cytidine deaminase (AID) is a B-cell-specific enzyme that targets immunoglobulin genes to initiate class switch recombination and somatic hypermutation. In addition, through off-target activity, AID has a much broader effect on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in the development and progression of lymphoma. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation. The phosphatidylinositol 3-kinase δ (PI3Kδ) pathway regulates AID by suppressing its expression in B cells. Drugs for leukaemia or lymphoma therapy such as idelalisib, duvelisib and ibrutinib block PI3Kδ activity directly or indirectly, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both of these effects were completely abrogated in AID-deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumours in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IGH and AID off-target sites in human chronic lymphocytic leukaemia and mantle cell lymphoma cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased somatic hypermutation in AID off-targets. In summary, we show that PI3Kδ or Bruton's tyrosine kinase inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism. This effect should be carefully considered, as such inhibitors can be administered to patients for years. 10.1038/nature21406
    Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling. Myklebust June H,Brody Joshua,Kohrt Holbrook E,Kolstad Arne,Czerwinski Debra K,Wälchli Sébastien,Green Michael R,Trøen Gunhild,Liestøl Knut,Beiske Klaus,Houot Roch,Delabie Jan,Alizadeh Ash A,Irish Jonathan M,Levy Ronald Blood Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors. 10.1182/blood-2016-05-718494
    PI3Kδ inhibition elicits anti-leukemic effects through Bim-dependent apoptosis. Carter M J,Cox K L,Blakemore S J,Turaj A H,Oldham R J,Dahal L N,Tannheimer S,Forconi F,Packham G,Cragg M S Leukemia PI3Kδ plays pivotal roles in the maintenance, proliferation and survival of malignant B-lymphocytes. Although not curative, PI3Kδ inhibitors (PI3Kδi) demonstrate impressive clinical efficacy and, alongside other signaling inhibitors, are revolutionizing the treatment of hematological malignancies. However, only limited in vivo data are available regarding their mechanism of action. With the rising number of novel treatments, the challenge is to identify combinations that deliver curative regimes. A deeper understanding of the molecular mechanism is required to guide these selections. Currently, immunomodulation, inhibition of B-cell receptor signaling, chemokine/cytokine signaling and apoptosis represent potential therapeutic mechanisms for PI3Kδi. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced apoptosis in an in vivo model of chronic lymphocytic leukemia (CLL). In vitro, PI3Kδi-induced substantive apoptosis and disrupted microenvironment-derived signaling in murine (Eμ-Tcl1) and human (CLL) leukemia cells. Furthermore, PI3Kδi imparted significant therapeutic responses in Eμ-Tcl1-bearing animals and enhanced anti-CD20 monoclonal antibody therapy. Responses correlated with upregulation of the pro-apoptotic BH3-only protein Bim. Accordingly, Bim Eμ-Tcl1 Tg leukemias demonstrated resistance to PI3Kδi-induced apoptosis were refractory to PI3Kδi in vivo and failed to display combination efficacy with anti-CD20 monoclonal antibody therapy. Therefore, Bim-dependent apoptosis represents a key in vivo therapeutic mechanism for PI3Kδi, both alone and in combination therapy regimes. 10.1038/leu.2016.333
    The Dual Syk/JAK Inhibitor Cerdulatinib Antagonizes B-cell Receptor and Microenvironmental Signaling in Chronic Lymphocytic Leukemia. Blunt Matthew D,Koehrer Stefan,Dobson Rachel C,Larrayoz Marta,Wilmore Sarah,Hayman Alice,Parnell Jack,Smith Lindsay D,Davies Andrew,Johnson Peter W M,Conley Pamela B,Pandey Anjali,Strefford Jonathan C,Stevenson Freda K,Packham Graham,Forconi Francesco,Coffey Greg P,Burger Jan A,Steele Andrew J Clinical cancer research : an official journal of the American Association for Cancer Research B-cell receptor (BCR)-associated kinase inhibitors, such as ibrutinib, have revolutionized the treatment of chronic lymphocytic leukemia (CLL). However, these agents are not curative, and resistance is already emerging in a proportion of patients. IL4, expressed in CLL lymph nodes, can augment BCR signaling and reduce the effectiveness of BCR kinase inhibitors. Therefore, simultaneous targeting of the IL4- and BCR signaling pathways by cerdulatinib, a novel dual Syk/JAK inhibitor currently in clinical trials (NCT01994382), may improve treatment responses in patients. PBMCs from patients with CLL were treated with cerdulatinib alone or in combination with venetoclax. Cell death, chemokine, and cell signaling assay were performed and analyzed by flow cytometry, immunoblotting, q-PCR, and ELISA as indicated. At concentrations achievable in patients, cerdulatinib inhibited BCR- and IL4-induced downstream signaling in CLL cells using multiple readouts and prevented anti-IgM- and nurse-like cell (NLC)-mediated CCL3/CCL4 production. Cerdulatinib induced apoptosis of CLL cells, in a time- and concentration-dependent manner, and particularly in IGHV-unmutated samples with greater BCR signaling capacity and response to IL4, or samples expressing higher levels of sIgM, CD49d, or ZAP70 Cerdulatinib overcame anti-IgM, IL4/CD40L, or NLC-mediated protection by preventing upregulation of MCL-1 and BCL-X; however, BCL-2 expression was unaffected. Furthermore, in samples treated with IL4/CD40L, cerdulatinib synergized with venetoclax to induce greater apoptosis than either drug alone. Cerdulatinib is a promising therapeutic for the treatment of CLL either alone or in combination with venetoclax, with the potential to target critical survival pathways in this currently incurable disease. . 10.1158/1078-0432.CCR-16-1662
    A BH3 Mimetic for Killing Cancer Cells. Green Douglas R Cell Venetoclax is a BH3 mimetic approved for treating chronic lymphocytic leukemia. Cancer cells are resistant to apoptosis but "primed for death" by elevated BCL-2, which binds to pro-apoptotic proteins and holds them in check. Venetoclax releases this antagonism and is the first approved drug to target a protein-protein interaction. 10.1016/j.cell.2016.05.080
    Idelalisib given front-line for treatment of chronic lymphocytic leukemia causes frequent immune-mediated hepatotoxicity. Lampson Benjamin L,Kasar Siddha N,Matos Tiago R,Morgan Elizabeth A,Rassenti Laura,Davids Matthew S,Fisher David C,Freedman Arnold S,Jacobson Caron A,Armand Philippe,Abramson Jeremy S,Arnason Jon E,Kipps Thomas J,Fein Joshua,Fernandes Stacey,Hanna John,Ritz Jerome,Kim Haesook T,Brown Jennifer R Blood Idelalisib is a small-molecule inhibitor of PI3Kδ with demonstrated efficacy for the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL). To evaluate idelalisib as front-line therapy, we enrolled 24 subjects in a phase 2 study consisting of 2 months of idelalisib monotherapy followed by 6 months of combination therapy with idelalisib and the anti-CD20 antibody ofatumumab. After a median follow-up period of 14.7 months, hepatotoxicity was found to be a frequent and often severe adverse event. A total of 19 subjects (79%) experienced either grade ≥1 ALT or AST elevation during the study, and 13 subjects (54%) experienced grade ≥3 transaminitis. The median time to development of transaminitis was 28 days, occurring before ofatumumab introduction. Younger age and mutated immunoglobulin heavy chain status were significant risk factors for the development of hepatotoxicity. Multiple lines of evidence suggest that this hepatotoxicity was immune mediated. A lymphocytic infiltrate was seen on liver biopsy specimens taken from 2 subjects with transaminitis, and levels of the proinflammatory cytokines CCL-3 and CCL-4 were higher in subjects experiencing hepatotoxicity. All cases of transaminitis resolved either by holding the drug, initiating immunosuppressants, or both, and rates of recurrent toxicity were lower in patients taking steroids when idelalisib was reinitiated. A decrease in peripheral blood regulatory T cells was seen in patients experiencing toxicity on therapy, which is consistent with an immune-mediated mechanism. These results suggest that caution should be taken as drugs within this class are developed for CLL, particularly in younger patients who have not received prior disease-specific therapy. This study was registered at www.clinicaltrials.gov as #NCT02135133. 10.1182/blood-2016-03-707133
    Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia. Thijssen Rachel,Ter Burg Johanna,Garrick Brett,van Bochove Gregor G W,Brown Jennifer R,Fernandes Stacey M,Rodríguez María Solé,Michot Jean-Marie,Hallek Michael,Eichhorst Barbara,Reinhardt Hans Christian,Bendell Johanna,Derks Ingrid A M,van Kampen Roel J W,Hege Kristen,Kersten Marie José,Trowe Torsten,Filvaroff Ellen H,Eldering Eric,Kater Arnon P Blood Inhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity however to induce cell death as monotherapy and are unlikely to eradicate the disease. Acquired resistance to therapy in CLL is often caused by mutations in the response network being targeted, both for DNA damage or BCR signaling pathways. Thus, drugs with dual targeting capacity could offer improved therapeutic value. Here, the potency of CC-115, a novel inhibitor of mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK), was evaluated in primary CLL cells in vitro and in CLL patients. Combined TORK and DNA-PK inhibition in vitro resulted in caspase-dependent cell killing irrespective of p53, ATM, NOTCH1, or SF3B1 status. Proliferation induced by CD40(+) interleukin-21 stimulation was completely blocked by CC-115, and CD40-mediated resistance to fludarabine and venetoclax could be reverted by CC-115. BCR-mediated signaling was inhibited by CC-115 and also in CLL samples obtained from patients with acquired resistance to idelalisib treatment. Clinical efficacy of CC-115 was demonstrated in 8 patients with relapsed/refractory CLL/small lymphocytic lymphoma harboring ATM deletions/mutations; all but 1 patient had a decrease in lymphadenopathy, resulting in 1 IWCLL partial response (PR) and 3 PRs with lymphocytosis. In conclusion, these preclinical results, along with early promising clinical activity, suggest that CC-115 may be developed further for treatment of CLL. The trial was registered at www.clinicaltrials.gov as #NCT01353625. 10.1182/blood-2016-02-700328
    IL-4 rescues surface IgM expression in chronic lymphocytic leukemia. Guo Benchang,Zhang Lu,Chiorazzi Nicholas,Rothstein Thomas L Blood Chronic lymphocytic leukemia (CLL) cells express poor levels of surface immunoglobulin (sIg), and many are minimally activated or anergic in response to B-cell receptor (BCR) crosslinking in vitro. Paradoxically, CLL cells in patients are highly activated through BCR signaling and expand in proliferation centers, suggesting that the function of sIg signaling is rescued. Here, we find that, compared with normal naïve B cells, CLL cells express a low level of total CD79b protein but normal levels of CD79a and IgM protein. Association of both CD79a and CD79b to IgM is markedly reduced. We further find that interleukin-4 (IL-4) markedly rescues CD79b and sIgM protein in CLL samples. These changes significantly enhance signaling in response to BCR crosslinking. Furthermore, we find that these changes are more pronounced in immunoglobulin heavy chain variable (IGHV)-unmutated CLL cells than IGHV-mutated CLL cells. The results described herein reveal that reduced sIgM is due to low expression of total CD79b protein in CLL cells. IL-4 substantially restores CD79b protein expression, sIgM expression, and BCR signaling. 10.1182/blood-2015-11-682997
    BCR signaling inhibitors differ in their ability to overcome Mcl-1-mediated resistance of CLL B cells to ABT-199. Bojarczuk Kamil,Sasi Binu K,Gobessi Stefania,Innocenti Idanna,Pozzato Gabriele,Laurenti Luca,Efremov Dimitar G Blood The Bcl-2 antagonist ABT-199 (venetoclax) has demonstrated promising clinical activity in patients with chronic lymphocytic leukemia (CLL). ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cells in vitro but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xL and Bfl-1. In this study, we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199 resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk, and BimEL A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK, and phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with SYK inhibitors R406 and GS-9973 (entospletinib), whereas the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199. 10.1182/blood-2015-10-675009
    IL-4 enhances expression and function of surface IgM in CLL cells. Aguilar-Hernandez Maria M,Blunt Matthew D,Dobson Rachel,Yeomans Alison,Thirdborough Stephen,Larrayoz Marta,Smith Lindsay D,Linley Adam,Strefford Jonathan C,Davies Andrew,Johnson Peter M W,Savelyeva Natalia,Cragg Mark S,Forconi Francesco,Packham Graham,Stevenson Freda K,Steele Andrew J Blood Kinase inhibitors targeting the B-cell receptor (BCR) are now prominent in the treatment of chronic lymphocytic leukemia (CLL). We have focused here on interleukin 4 (IL-4), a cytokine that protects normal and malignant B cells from apoptosis and increases surface immunoglobulin M (sIgM) expression on murine splenic B cells. First, we have demonstrated that IL-4 treatment increased sIgM expression in vitro on peripheral blood B cells obtained from healthy individuals. In CLL, IL-4 target genes are overexpressed in cells purified from the lymph nodes of patients compared with cells derived from matched blood and bone marrow samples. As for normal B cells, IL-4 increased sIgM expression on CLL cells in vitro, especially in samples expressing unmutated V-genes. IL-4-induced sIgM expression was associated with increased receptor signalling activity, measured by anti-IgM-induced calcium mobilization, and with increased expression of CD79B messenger RNA and protein, and the "mature" glycoform of sIgM. Importantly, the ability of the BCR-associated kinase inhibitors idelalisib and ibrutinib, approved for treatment of CLL and other B-cell malignancies, to inhibit anti-IgM-induced signalling was reduced following IL-4 pretreatment in samples from the majority of patients. In contrast to stimulatory effects on sIgM, IL-4 decreased CXCR4 and CXCR5 expression; therefore, CLL cells, particularly within the progressive unmutated V-gene subset, may harness the ability of IL-4 to promote BCR signalling and B-cell retention within lymph nodes. Effects of IL-4 were mediated via JAK3/STAT6 and we propose a potential role for JAK inhibitors in combination with BCR kinase inhibitors for the treatment of CLL. 10.1182/blood-2015-11-682906
    Sprouty 2: a novel attenuator of B-cell receptor and MAPK-Erk signaling in CLL. Shukla Ashima,Rai Karan,Shukla Vipul,Chaturvedi Nagendra K,Bociek R Gregory,Pirruccello Samuel J,Band Hamid,Lu Runqing,Joshi Shantaram S Blood Clinical heterogeneity is a major barrier to effective treatment of chronic lymphocytic leukemia (CLL). Emerging evidence suggests that constitutive activation of various signaling pathways like mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-Erk) signaling plays a role in the heterogeneous clinical outcome of CLL patients. In this study, we have investigated the role of Sprouty (SPRY)2 as a negative regulator of receptor and nonreceptor tyrosine kinase signaling in the pathogenesis of CLL. We show that SPRY2 expression is significantly decreased in CLL cells, particularly from poor-prognosis patients compared with those from good-prognosis patients. Overexpression of SPRY2 in CLL cells from poor-prognosis patients increased their apoptosis. Conversely, downregulation of SPRY2 in CLL cells from good-prognosis patients resulted in increased proliferation. Furthermore, CLL cells with low SPRY2 expression grew more rapidly in a xenograft model of CLL. Strikingly, B-cell-specific transgenic overexpression of spry2 in mice led to a decrease in the frequency of B1 cells, the precursors of CLL cells in rodents. Mechanistically, we show that SPRY2 attenuates the B-cell receptor (BCR) and MAPK-Erk signaling by binding to and antagonizing the activities of RAF1, BRAF, and spleen tyrosine kinase (SYK) in normal B cells and CLL cells. We also show that SPRY2 is targeted by microRNA-21, which in turn leads to increased activity of Syk and Erk in CLL cells. Taken together, these results establish SPRY2 as a critical negative regulator of BCR-mediated MAPK-Erk signaling in CLL, thereby providing one of the molecular mechanisms to explain the clinical heterogeneity of CLL. 10.1182/blood-2015-09-669317
    Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib. Sun Clare,Tian Xin,Lee Yuh Shan,Gunti Sreenivasulu,Lipsky Andrew,Herman Sarah E M,Salem Dalia,Stetler-Stevenson Maryalice,Yuan Constance,Kardava Lela,Moir Susan,Maric Irina,Valdez Janet,Soto Susan,Marti Gerald E,Farooqui Mohammed Z,Notkins Abner L,Wiestner Adrian,Aue Georg Blood Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT015007330. 10.1182/blood-2015-04-639203
    The U1 spliceosomal RNA is recurrently mutated in multiple cancers. Shuai Shimin,Suzuki Hiromichi,Diaz-Navarro Ander,Nadeu Ferran,Kumar Sachin A,Gutierrez-Fernandez Ana,Delgado Julio,Pinyol Magda,López-Otín Carlos,Puente Xose S,Taylor Michael D,Campo Elías,Stein Lincoln D Nature Cancers are caused by genomic alterations known as drivers. Hundreds of drivers in coding genes are known but, to date, only a handful of noncoding drivers have been discovered-despite intensive searching. Attention has recently shifted to the role of altered RNA splicing in cancer; driver mutations that lead to transcriptome-wide aberrant splicing have been identified in multiple types of cancer, although these mutations have only been found in protein-coding splicing factors such as splicing factor 3b subunit 1 (SF3B1). By contrast, cancer-related alterations in the noncoding component of the spliceosome-a series of small nuclear RNAs (snRNAs)-have barely been studied, owing to the combined challenges of characterizing noncoding cancer drivers and the repetitive nature of snRNA genes. Here we report a highly recurrent A>C somatic mutation at the third base of U1 snRNA in several types of tumour. The primary function of U1 snRNA is to recognize the 5' splice site via base-pairing. This mutation changes the preferential A-U base-pairing between U1 snRNA and the 5' splice site to C-G base-pairing, and thus creates novel splice junctions and alters the splicing pattern of multiple genes-including known drivers of cancer. Clinically, the A>C mutation is associated with heavy alcohol use in patients with hepatocellular carcinoma, and with the aggressive subtype of chronic lymphocytic leukaemia with unmutated immunoglobulin heavy-chain variable regions. The mutation in U1 snRNA also independently confers an adverse prognosis to patients with chronic lymphocytic leukaemia. Our study demonstrates a noncoding driver in spliceosomal RNAs, reveals a mechanism of aberrant splicing in cancer and may represent a new target for treatment. Our findings also suggest that driver discovery should be extended to a wider range of genomic regions. 10.1038/s41586-019-1651-z
    Insight into genetic predisposition to chronic lymphocytic leukemia from integrative epigenomics. Speedy Helen E,Beekman Renée,Chapaprieta Vicente,Orlando Giulia,Law Philip J,Martín-García David,Gutiérrez-Abril Jesús,Catovsky Daniel,Beà Sílvia,Clot Guillem,Puiggròs Montserrat,Torrents David,Puente Xose S,Allan James M,López-Otín Carlos,Campo Elias,Houlston Richard S,Martín-Subero José I Nature communications Genome-wide association studies have provided evidence for inherited genetic predisposition to chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms underlying CLL risk we analyze chromatin accessibility, active regulatory elements marked by H3K27ac, and DNA methylation at 42 risk loci in up to 486 primary CLLs. We identify that risk loci are significantly enriched for active chromatin in CLL with evidence of being CLL-specific or differentially regulated in normal B-cell development. We then use in situ promoter capture Hi-C, in conjunction with gene expression data to reveal likely target genes of the risk loci. Candidate target genes are enriched for pathways related to B-cell development such as MYC and BCL2 signalling. At 14 loci the analysis highlights 63 variants as the probable functional basis of CLL risk. By integrating genetic and epigenetic information our analysis reveals novel insights into the relationship between inherited predisposition and the regulatory chromatin landscape of CLL. 10.1038/s41467-019-11582-2
    Activation of hedgehog signaling associates with early disease progression in chronic lymphocytic leukemia. Ghia Emanuela M,Rassenti Laura Z,Neuberg Donna S,Blanco Alejandro,Yousif Fouad,Smith Erin N,McPherson John D,Hudson Thomas J, ,Harismendy Olivier,Frazer Kelly A,Kipps Thomas J Blood Targeted sequencing of 103 leukemia-associated genes in leukemia cells from 841 treatment-naive patients with chronic lymphocytic leukemia (CLL) identified 89 (11%) patients as having CLL cells with mutations in genes encoding proteins that putatively are involved in hedgehog (Hh) signaling. Consistent with this finding, there was a significant association between the presence of these mutations and the expression of GLI1 (χ test, < .0001), reflecting activation of the Hh pathway. However, we discovered that 38% of cases without identified mutations also were GLI1 Patients with GLI1 CLL cells had a shorter median treatment-free survival than patients with CLL cells lacking expression of GLI1 independent of IGHV mutation status. We found that GANT61, a small molecule that can inhibit GLI1, was highly cytotoxic for GLI1 CLL cells relative to that of CLL cells without GLI1. Collectively, this study shows that a large proportion of patients have CLL cells with activated Hh signaling, which is associated with early disease progression and enhanced sensitivity to inhibition of GLI1. 10.1182/blood-2018-09-873695
    Anti-tumour immunity controlled through mRNA mA methylation and YTHDF1 in dendritic cells. Han Dali,Liu Jun,Chen Chuanyuan,Dong Lihui,Liu Yi,Chang Renbao,Huang Xiaona,Liu Yuanyuan,Wang Jianying,Dougherty Urszula,Bissonnette Marc B,Shen Bin,Weichselbaum Ralph R,Xu Meng Michelle,He Chuan Nature There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response. Here we show that durable neoantigen-specific immunity is regulated by mRNA N-methyadenosine (mA) methylation through the mA-binding protein YTHDF1. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8 T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8 T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by mA and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1 mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy. 10.1038/s41586-019-0916-x
    A Murine Model of Chronic Lymphocytic Leukemia Based on B Cell-Restricted Expression of Sf3b1 Mutation and Atm Deletion. Yin Shanye,Gambe Rutendo G,Sun Jing,Martinez Aina Zurita,Cartun Zachary J,Regis Fara Faye D,Wan Youzhong,Fan Jean,Brooks Angela N,Herman Sarah E M,Ten Hacken Elisa,Taylor-Weiner Amaro,Rassenti Laura Z,Ghia Emanuela M,Kipps Thomas J,Obeng Esther A,Cibulskis Carrie L,Neuberg Donna,Campagna Dean R,Fleming Mark D,Ebert Benjamin L,Wiestner Adrian,Leshchiner Ignaty,DeCaprio James A,Getz Gad,Reed Robin,Carrasco Ruben D,Wu Catherine J,Wang Lili Cancer cell SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment. 10.1016/j.ccell.2018.12.013
    Creating novel translation inhibitors to target pro-survival proteins in chronic lymphocytic leukemia. Chen Rong,Zhu Mingzhao,Chaudhari Rajan R,Robles Omar,Chen Yuling,Skillern Wesley,Qin Qun,Wierda William G,Zhang Shuxing,Hull Kenneth G,Romo Daniel,Plunkett William Leukemia The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of PatA analogs and identified three new leads with potent inhibition of translation. They exhibited less plasma protein binding and increased cytotoxic potency toward CLL cells than DMDAPatA, with greater selectivity towards CLL cells over normal lymphocytes. Computer modeling analysis correlated their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL. 10.1038/s41375-018-0364-x
    17p deletion strongly influences rituximab elimination in chronic lymphocytic leukemia. Bagacean Cristina,Tempescul Adrian,Ternant David,Banet Anne,Douet-Guilbert Nathalie,Bordron Anne,Bendaoud Boutahar,Saad Hussam,Zdrenghea Mihnea,Berthou Christian,Paintaud Gilles,Renaudineau Yves Journal for immunotherapy of cancer Chronic lymphocytic leukemia (CLL) is the most common type of leukemia and the anti-CD20 monoclonal antibody, rituximab, represents the therapeutic gold standard for more than 2 decades in this pathology, when used in combination with chemotherapy. However, some patients experience treatment resistance or rapid relapses, and in particular, those harboring a 17p/TP53 deletion (del(17p)). This resistance could be explained by a chemo-resistance, but it could also result from the direct impact of del(17p) on the pharmacokinetics of rituximab, which represents the aim of the present study. Accordingly, 44 CLL patients were included in the study, and among them 9 presented a del(17p). Next, a total of 233 rituximab sera were selected for a pharmacokinetic study and analyzed in a two-compartment model showing important differences when del(17p) CLL patients were compared with non-del(17p) patients treated with rituximab and chemotherapy: (1) clearance of rituximab was faster; (2) central volume of rituximab distribution V1 (peripheral blood) was reduced while peripheral volume V2 (lymphoid organs and tissues) was increased; and (3) the rate of rituximab elimination (Kout) was faster. In contrast, the group with a better prognosis harboring isolated del(13q) presented a slower rate of elimination (Kout). Pharmacokinetic parameters were independent from the other factors tested such as age, sex, chemotherapy regimen (fludarabine/cyclophosphamide versus bendamustine), IGHV mutational status, and FCGR3A 158VF status. In conclusion, this study provides an additional argument to consider that del(17p) is effective not only to control chemoresistance but also monoclonal antibody activity, based on higher rituximab turnover. 10.1186/s40425-019-0509-0
    A retinoic acid-dependent stroma-leukemia crosstalk promotes chronic lymphocytic leukemia progression. Farinello Diego,Wozińska Monika,Lenti Elisa,Genovese Luca,Bianchessi Silvia,Migliori Edoardo,Sacchetti Nicolò,di Lillo Alessia,Bertilaccio Maria Teresa Sabrina,de Lalla Claudia,Valsecchi Roberta,Gleave Sabrina Bascones,Lligé David,Scielzo Cristina,Mauri Laura,Ciampa Maria Grazia,Scarfò Lydia,Bernardi Rosa,Lazarevic Dejan,Gonzalez-Farre Blanca,Bongiovanni Lucia,Campo Elias,Cerutti Andrea,Ponzoni Maurilio,Pattini Linda,Caligaris-Cappio Federico,Ghia Paolo,Brendolan Andrea Nature communications In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression. 10.1038/s41467-018-04150-7
    Chronic lymphocytic leukemia and mantle cell lymphoma: crossroads of genetic and microenvironment interactions. Puente Xose S,Jares Pedro,Campo Elias Blood Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are 2 well-defined entities that diverge in their basic pathogenic mechanisms and clinical evolution but they share epidemiological characteristics, cells of origin, molecular alterations, and clinical features that differ from other lymphoid neoplasms. CLL and MCL are classically considered indolent and aggressive neoplasms, respectively. However, the clinical evolution of both tumors is very heterogeneous, with subsets of patients having stable disease for a long time whereas others require immediate intervention. Both CLL and MCL include 2 major molecular subtypes that seem to derive from antigen-experienced CD5 B cells that retain a naive or memory-like epigenetic signature and carry a variable load of immunoglobulin heavy-chain variable region somatic mutations from truly unmutated to highly mutated, respectively. These 2 subtypes of tumors differ in their molecular pathways, genomic alterations, and clinical behavior, being more aggressive in naive-like than memory-like-derived tumors in both CLL and MCL. The pathogenesis of the 2 entities integrates the relevant influence of B-cell receptor signaling, tumor cell microenvironment interactions, genomic alterations, and epigenome modifications that configure the evolution of the tumors and offer new possibilities for therapeutic intervention. This review will focus on the similarities and differences of these 2 tumors based on recent studies that are enhancing the understanding of their pathogenesis and creating solid bases for new management strategies. 10.1182/blood-2017-10-764373
    Low expression confers redox hypersensitivity and identifies an indolent clinical behavior in CLL. Cavallini Chiara,Chignola Roberto,Dando Ilaria,Perbellini Omar,Mimiola Elda,Lovato Ornella,Laudanna Carlo,Pizzolo Giovanni,Donadelli Massimo,Scupoli Maria Teresa Blood B-cell receptor (BCR) signaling is a key determinant of variable clinical behavior and a target for therapeutic interventions in chronic lymphocytic leukemia (CLL). Endogenously produced HO is thought to fine-tune the BCR signaling by reversibly inhibiting phosphatases. However, little is known about how CLL cells sense and respond to such redox cues and what effect they have on CLL. We characterized the response of BCR signaling proteins to exogenous HO in cells from patients with CLL, using phosphospecific flow cytometry. Exogenous HO in the absence of BCR engagement induced a signaling response of BCR proteins that was higher in CLL with favorable prognostic parameters and an indolent clinical course. We identified low expression as a possible mechanism accounting for redox signaling hypersensitivity. Decreased catalase could cause an escalated accumulation of exogenous HO in leukemic cells with a consequent greater inhibition of phosphatases and an increase of redox signaling sensitivity. Moreover, lower levels of were significantly associated with a slower progression of the disease. In leukemic cells characterized by redox hypersensitivity, we also documented an elevated accumulation of ROS and an increased mitochondrial amount. Taken together, our data identified redox sensitivity and metabolic profiles that are linked to differential clinical behavior in CLL. This study advances our understanding of the redox and signaling heterogeneity of CLL and provides the rationale for the development of therapies targeting redox pathways in CLL. 10.1182/blood-2017-08-800466
    Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence. Cutrona Giovanna,Tripodo Claudio,Matis Serena,Recchia Anna Grazia,Massucco Carlotta,Fabbi Marina,Colombo Monica,Emionite Laura,Sangaletti Sabina,Gulino Alessandro,Reverberi Daniele,Massara Rosanna,Boccardo Simona,de Totero Daniela,Salvi Sandra,Cilli Michele,Pellicanò Mariavaleria,Manzoni Martina,Fabris Sonia,Airoldi Irma,Valdora Francesca,Ferrini Silvano,Gentile Massimo,Vigna Ernesto,Bossio Sabrina,De Stefano Laura,Palummo Angela,Iaquinta Giovanni,Cardillo Martina,Zupo Simonetta,Cerruti Giannamaria,Ibatici Adalberto,Neri Antonino,Fais Franco,Ferrarini Manlio,Morabito Fortunato Science translational medicine Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rβ1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rβ1 chain when cocultured with activated T cells or CD40L cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies. 10.1126/scitranslmed.aal1571
    Microenvironment-induced CD44v6 promotes early disease progression in chronic lymphocytic leukemia. Gutjahr Julia C,Szenes Eva,Tschech Lisa,Asslaber Daniela,Schlederer Michaela,Roos Simone,Yu Xiaobing,Girbl Tamara,Sternberg Christina,Egle Alexander,Aberger Fritz,Alon Ronen,Kenner Lukas,Greil Richard,Orian-Rousseau Veronique,Hartmann Tanja N Blood Chronic lymphocytic leukemia (CLL) outgrowth depends on signals from the microenvironment. We have previously found that in vitro reconstitution of this microenvironment induces specific variant isoforms of the adhesion molecule CD44, which confer human CLL with high affinity to hyaluronan (HA). Here, we determined the in vivo contribution of standard CD44 and its variants to leukemic B-cell homing and proliferation in Tcl1 transgenic mice with a B-cell-specific CD44 deficiency. In these mice, leukemia onset was delayed and leukemic infiltration of spleen, liver, and lungs, but not of bone marrow, was decreased. Competitive transplantation revealed that CLL homing to spleen and bone marrow required functional CD44. Notably, enrichment of CD44v6 variants particularly in spleen enhanced CLL engraftment and proliferation, along with increased HA binding. We recapitulated CD44v6 induction in the human disease and revealed the involvement of MAPK and NF-κB signaling upon CD40 ligand and B-cell receptor stimulation by in vitro inhibition experiments and chromatin immunoprecipitation assays. The investigation of downstream signaling after CD44v6-HA engagement uncovered the activation of extracellular signal-regulated kinase and p65. Consequently, anti-CD44v6 treatment reduced leukemic cell proliferation in vitro in human and mouse, confirming the general nature of the findings. In summary, we propose a CD44-NF-κB-CD44v6 circuit in CLL, allowing tumor cells to gain HA binding capacity and supporting their proliferation. 10.1182/blood-2017-08-802462
    SnapShot: Chronic Lymphocytic Leukemia. Ten Hacken Elisa,Guièze Romain,Wu Catherine J Cancer cell Despite the recent advances in the therapeutic management of Chronic Lymphocytic Leukemia (CLL) patients, this common B cell malignancy still remains incurable. This SnapShot provides an overview of CLL biology and therapy, with a focus on genetics and microenvironmental interactions, which contribute to disease progression and therapy resistance. To view this SnapShot, open or download the PDF. 10.1016/j.ccell.2017.10.015
    USP7 inhibition alters homologous recombination repair and targets CLL cells independently of ATM/p53 functional status. Agathanggelou Angelo,Smith Edward,Davies Nicholas J,Kwok Marwan,Zlatanou Anastasia,Oldreive Ceri E,Mao Jingwen,Da Costa David,Yadollahi Sina,Perry Tracey,Kearns Pamela,Skowronska Anna,Yates Elliot,Parry Helen,Hillmen Peter,Reverdy Celine,Delansorne Remi,Paneesha Shankara,Pratt Guy,Moss Paul,Taylor A Malcolm R,Stewart Grant S,Stankovic Tatjana Blood The role of deubiquitylase ubiquitin-specific protease 7 (USP7) in the regulation of the p53-dependent DNA damage response (DDR) pathway is well established. Whereas previous studies have mostly focused on the mechanisms underlying how USP7 directly controls p53 stability, we recently showed that USP7 modulates the stability of the DNA damage responsive E3 ubiquitin ligase RAD18. This suggests that targeting USP7 may have therapeutic potential even in tumors with defective p53 or ibrutinib resistance. To test this hypothesis, we studied the effect of USP7 inhibition in chronic lymphocytic leukemia (CLL) where the ataxia telangiectasia mutated (ATM)-p53 pathway is inactivated with relatively high frequency, leading to treatment resistance and poor clinical outcome. We demonstrate that USP7 is upregulated in CLL cells, and its loss or inhibition disrupts homologous recombination repair (HRR). Consequently, USP7 inhibition induces significant tumor-cell killing independently of ATM and p53 through the accumulation of genotoxic levels of DNA damage. Moreover, USP7 inhibition sensitized p53-defective, chemotherapy-resistant CLL cells to clinically achievable doses of HRR-inducing chemotherapeutic agents in vitro and in vivo in a murine xenograft model. Together, these results identify USP7 as a promising therapeutic target for the treatment of hematological malignancies with DDR defects, where ATM/p53-dependent apoptosis is compromised. 10.1182/blood-2016-12-758219
    Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo. Leanza Luigi,Romio Matteo,Becker Katrin Anne,Azzolini Michele,Trentin Livio,Managò Antonella,Venturini Elisa,Zaccagnino Angela,Mattarei Andrea,Carraretto Luca,Urbani Andrea,Kadow Stephanie,Biasutto Lucia,Martini Veronica,Severin Filippo,Peruzzo Roberta,Trimarco Valentina,Egberts Jan-Hendrik,Hauser Charlotte,Visentin Andrea,Semenzato Gianpietro,Kalthoff Holger,Zoratti Mario,Gulbins Erich,Paradisi Cristina,Szabo Ildiko Cancer cell The potassium channel Kv1.3 is highly expressed in the mitochondria of various cancerous cells. Here we show that direct inhibition of Kv1.3 using two mitochondria-targeted inhibitors alters mitochondrial function and leads to reactive oxygen species (ROS)-mediated death of even chemoresistant cells independently of p53 status. These inhibitors killed 98% of ex vivo primary chronic B-lymphocytic leukemia tumor cells while sparing healthy B cells. In orthotopic mouse models of melanoma and pancreatic ductal adenocarcinoma, the compounds reduced tumor size by more than 90% and 60%, respectively, while sparing immune and cardiac functions. Our work provides direct evidence that specific pharmacological targeting of a mitochondrial potassium channel can lead to ROS-mediated selective apoptosis of cancer cells in vivo, without causing significant side effects. 10.1016/j.ccell.2017.03.003
    Genome-wide association analysis implicates dysregulation of immunity genes in chronic lymphocytic leukaemia. Nature communications Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223, P=5.04 × 10), 1q42.13 (rs41271473, P=1.06 × 10), 4q24 (rs71597109, P=1.37 × 10), 4q35.1 (rs57214277, P=3.69 × 10), 6p21.31 (rs3800461, P=1.97 × 10), 11q23.2 (rs61904987, P=2.64 × 10), 18q21.1 (rs1036935, P=3.27 × 10), 19p13.3 (rs7254272, P=4.67 × 10) and 22q13.33 (rs140522, P=2.70 × 10). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response. 10.1038/ncomms14175
    LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia. Nguyen Phuong-Hien,Fedorchenko Oleg,Rosen Natascha,Koch Maximilian,Barthel Romy,Winarski Tomasz,Florin Alexandra,Wunderlich F Thomas,Reinart Nina,Hallek Michael Cancer cell Survival of chronic lymphocytic leukemia (CLL) cells strictly depends on the support of an appropriate tumor microenvironment. Here, we demonstrate that LYN kinase is essential for CLL progression. Lyn deficiency results in a significantly reduced CLL burden in vivo. Loss of Lyn within leukemic cells reduces B cell receptor (BCR) signaling including BTK phosphorylation, but surprisingly does not affect leukemic cell expansion. Instead, syngeneic CLL transplantation of CLL cells into Lyn- or Btk-deficient recipients results in a strongly delayed leukemic progression and prolonged survival. Moreover, Lyn deficiency in macrophages hinders nursing functions for CLL cells, which is mediated by direct contact rather than secretion of soluble factors. Taken together, LYN and BTK seem essential for the formation of a microenvironment supporting leukemic growth. 10.1016/j.ccell.2016.09.007
    Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration. Pasikowska Marta,Walsby Elisabeth,Apollonio Benedetta,Cuthill Kirsty,Phillips Elizabeth,Coulter Eve,Longhi Maria Serena,Ma Yun,Yallop Deborah,Barber Linda D,Patten Piers,Fegan Chris,Ramsay Alan G,Pepper Chris,Devereux Stephen,Buggins Andrea G S Blood Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4β1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells. 10.1182/blood-2016-01-683128
    HIF-1α regulates the interaction of chronic lymphocytic leukemia cells with the tumor microenvironment. Valsecchi Roberta,Coltella Nadia,Belloni Daniela,Ponente Manfredi,Ten Hacken Elisa,Scielzo Cristina,Scarfò Lydia,Bertilaccio Maria Teresa Sabrina,Brambilla Paola,Lenti Elisa,Martinelli Boneschi Filippo,Brendolan Andrea,Ferrero Elisabetta,Ferrarini Marina,Ghia Paolo,Tonon Giovanni,Ponzoni Maurilio,Caligaris-Cappio Federico,Bernardi Rosa Blood Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis. 10.1182/blood-2015-07-657056
    ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells. Kwok Marwan,Davies Nicholas,Agathanggelou Angelo,Smith Edward,Oldreive Ceri,Petermann Eva,Stewart Grant,Brown Jeff,Lau Alan,Pratt Guy,Parry Helen,Taylor Malcolm,Moss Paul,Hillmen Peter,Stankovic Tatjana Blood TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation. 10.1182/blood-2015-05-644872
    Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts. Paggetti Jerome,Haderk Franziska,Seiffert Martina,Janji Bassam,Distler Ute,Ammerlaan Wim,Kim Yeoun Jin,Adam Julien,Lichter Peter,Solary Eric,Berchem Guy,Moussay Etienne Blood Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. 10.1182/blood-2014-12-618025
    Microenvironmental interleukin-6 suppresses toll-like receptor signaling in human leukemia cells through miR-17/19A. Li Yanmei,Shi Yonghong,McCaw Lindsay,Li You-Jun,Zhu Fang,Gorczynski Reg,Duncan Gordon S,Yang Burton,Ben-David Yaacov,Spaner David E Blood The regulation of toll-like receptor (TLR) signaling in a tumor microenvironment is poorly understood despite its importance in cancer biology. To address this problem, TLR7-responses of chronic lymphocytic leukemia (CLL) cells were studied in the presence and absence of a human stromal cell-line derived from a leukemic spleen. CLL cells alone produced high levels of tumor necrosis factor (TNF)-α and proliferated in response to TLR7-agonists. A signal transducer and activator of transcription 3 -activating stromal factor, identified as interleukin (IL)-6, was found to upregulate microRNA (miR)-17 and miR-19a, target TLR7 and TNFA messenger RNA, and induce a state of tolerance to TLR7-agonists in CLL cells. Overexpression of the miR-17-92 cluster tolerized CLL cells directly and miR-17 and miR-19a antagomiRs restored TLR7-signaling. Inhibition of IL-6 signaling with antibodies or small-molecule Janus kinase inhibitors reversed tolerization and increased TLR7-stimulated CLL cell numbers in vitro and in NOD-SCIDγc (null) mice. These results suggest IL-6 can act as tumor suppressor in CLL by inhibiting TLR-signaling. 10.1182/blood-2014-12-618678
    SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients. Chen Yu-Chen Enya,Burgess Melinda,Mapp Sally,Mollee Peter,Gill Devinder,Blumenthal Antje,Saunders Nicholas A Frontiers in immunology Targeted antibody therapies improve outcomes for chronic lymphocytic leukemia (CLL) patients. However, resistance often develops. We have previously shown that resistance to therapeutic antibodies, by monocyte derived macrophages (referred to as nurse like cells, NLCs), from CLL patients is characterized by suppression of antibody dependent phagocytosis (ADP). The mechanism(s) contributing to the muted ADP responses remain unresolved. In this regard, an innate immune checkpoint was recently described that uses the CD47:SIRPα axis to suppress phagocytic responses by macrophages. In this study we examine whether the SIRPα axis regulates ADP responses to the anti-CD20 antibody, obinutuzumab, by NLCs. Using siRNA depletion strategies we show that SIRPα is a suppressor of ADP responses. Moreover, we show that this innate immune checkpoint contributes to the resistance phenotype in NLCs derived from CLL patients. Finally, we show that SIRPα suppression is mediated the phosphatase, Shp1, which in turn suppresses SYK-dependent activation of ADP. Thus, we identify a druggable pathway that could be exploited to enhance sensitivity to existing therapeutic antibodies used in CLL. This is the first study to show that activation of the CD47:SIRPα innate immune checkpoint contributes to ADP resistance in NLCs from CLL patients. 10.3389/fimmu.2020.610523
    Beclin-1 as a neutrophil-specific immune checkpoint. Su Yu-Lin,Kortylewski Marcin The Journal of clinical investigation Neutrophils are early wound healing and inflammation regulators that, due to functional plasticity, can adopt either pro- or antitumor functions. Until recently, beclin-1 was a protein known mainly for its role as a critical regulator of autophagy. In this issue of the JCI, Tan et al. describe the effects of the beclin-1 conditional myeloid cell-specific deletion in mice, in which immunostimulation resulted in hypersensitive neutrophils. The chronic proinflammatory effect of these neutrophils triggered spontaneous B cell malignancies to develop. Such tumorigenic effects were mediated primarily by IL-21 and CD40 signaling, leading to the upregulation of tolerogenic molecules, such as IL-10 and PD-L1. The authors went on to examine samples derived from patient lymphoid malignancies and showed that beclin-1 expression in neutrophils positively correlated with pre-B cell leukemia/lymphoma. Overall, the study provides an elegant model for neutrophil-driven carcinogenesis and identifies potential targets for immunotherapy of B cell malignancies. 10.1172/JCI132534
    Chronic stimulation drives human NK cell dysfunction and epigenetic reprograming. Merino Aimee,Zhang Bin,Dougherty Philip,Luo Xianghua,Wang Jinhua,Blazar Bruce R,Miller Jeffrey S,Cichocki Frank The Journal of clinical investigation A population of Natural Killer (NK) cells expressing the activating receptor NKG2C and the maturation marker CD57 expands in response to human cytomegalovirus (HCMV) infection. CD3-CD56dimCD57+NKG2C+ NK cells are similar to CD8+ memory T cells with rapid and robust effector function upon re-stimulation, persistence, and epigenetic remodeling of the IFNG locus. Chronic antigen stimulation drives CD8+ memory T cell proliferation while also inducing genome-wide epigenetic reprograming and dysfunction. We hypothesized that chronic stimulation could similarly induce epigenetic reprograming and dysfunction in NK cells. Here we show that chronic stimulation of adaptive NK cells through NKG2C using plate-bound agonistic antibodies in combination with IL-15 drove robust proliferation and activation of CD3-CD56dimCD57+NKG2C+ NK cells while simultaneously inducing high expression of the checkpoint inhibitory receptors LAG-3 and PD-1. Marked induction of checkpoint inhibitory receptors was also observed on the surface of adaptive NK cells co-cultured with HCMV-infected endothelial cells. Chronically stimulated adaptive NK cells were dysfunctional when challenged with tumor targets. These cells exhibited a pattern of epigenetic reprograming, with genome-wide alterations in DNA methylation. Our study has important implications for cancer immunotherapy and suggest that exhausted NK cells could be targeted with inhibitory checkpoint receptor blockade. 10.1172/JCI125916
    Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting. Rotolo Antonia,Caputo Valentina S,Holubova Monika,Baxan Nicoleta,Dubois Olivier,Chaudhry Mohammed Suhail,Xiao Xiaolin,Goudevenou Katerina,Pitcher David S,Petevi Kyriaki,Kachramanoglou Carolina,Iles Sandra,Naresh Kikkeri,Maher John,Karadimitris Anastasios Cancer cell Chimeric antigen receptor anti-CD19 (CAR19)-T cell immunotherapy-induced clinical remissions in CD19 B cell lymphomas are often short lived. We tested whether CAR19-engineering of the CD1d-restricted invariant natural killer T (iNKT) cells would result in enhanced anti-lymphoma activity. CAR19-iNKT cells co-operatively activated by CD1d- and CAR19-CD19-dependent interactions are more effective than CAR19-T cells against CD1d-expressing lymphomas in vitro and in vivo. The swifter in vivo anti-lymphoma activity of CAR19-iNKT cells and their enhanced ability to eradicate brain lymphomas underpinned an improved tumor-free and overall survival. CD1D transcriptional de-repression by all-trans retinoic acid results in further enhanced cytotoxicity of CAR19-iNKT cells against CD19 chronic lymphocytic leukemia cells. Thus, iNKT cells are a highly efficient platform for CAR-based immunotherapy of lymphomas and possibly other CD1d-expressing cancers. 10.1016/j.ccell.2018.08.017
    Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity. Zhang Qing,Bi Jiacheng,Zheng Xiaodong,Chen Yongyan,Wang Hua,Wu Wenyong,Wang Zhengguang,Wu Qiang,Peng Hui,Wei Haiming,Sun Rui,Tian Zhigang Nature immunology Checkpoint blockade enhances effector T cell function and has elicited long-term remission in a subset of patients with a broad spectrum of cancers. TIGIT is a checkpoint receptor thought to be involved in mediating T cell exhaustion in tumors; however, the relevance of TIGIT to the dysfunction of natural killer (NK) cells remains poorly understood. Here we found that TIGIT, but not the other checkpoint molecules CTLA-4 and PD-1, was associated with NK cell exhaustion in tumor-bearing mice and patients with colon cancer. Blockade of TIGIT prevented NK cell exhaustion and promoted NK cell-dependent tumor immunity in several tumor-bearing mouse models. Furthermore, blockade of TIGIT resulted in potent tumor-specific T cell immunity in an NK cell-dependent manner, enhanced therapy with antibody to the PD-1 ligand PD-L1 and sustained memory immunity in tumor re-challenge models. This work demonstrates that TIGIT constitutes a previously unappreciated checkpoint in NK cells and that targeting TIGIT alone or in combination with other checkpoint receptors is a promising anti-cancer therapeutic strategy. 10.1038/s41590-018-0132-0
    PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse. Huang Yu,Chen Zhiying,Jang Joon Hee,Baig Mirza S,Bertolet Grant,Schroeder Casey,Huang Shengjian,Hu Qian,Zhao Yong,Lewis Dorothy E,Qin Lidong,Zhu Michael Xi,Liu Dongfang The Journal of allergy and clinical immunology BACKGROUND:The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. OBJECTIVE:We sought to determine the effect of PD-1 signaling on NK cells. METHODS:PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). RESULTS:PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. CONCLUSION:Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function. 10.1016/j.jaci.2018.02.050
    Invariant NKT cells contribute to chronic lymphocytic leukemia surveillance and prognosis. Gorini Francesca,Azzimonti Laura,Delfanti Gloria,Scarfò Lydia,Scielzo Cristina,Bertilaccio Maria Teresa,Ranghetti Pamela,Gulino Alessandro,Doglioni Claudio,Di Napoli Arianna,Capri Miriam,Franceschi Claudio,Caligaris-Cappio Federico,Ghia Paolo,Bellone Matteo,Dellabona Paolo,Casorati Giulia,de Lalla Claudia Blood Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant CD5 B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance. We investigated the impact of iNKT cells in the natural history of the disease in the Eμ-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients. We found that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression. 10.1182/blood-2016-11-751065
    Macrophages confer survival signals via CCR1-dependent translational MCL-1 induction in chronic lymphocytic leukemia. van Attekum M H A,Terpstra S,Slinger E,von Lindern M,Moerland P D,Jongejan A,Kater A P,Eldering E Oncogene Protective interactions with bystander cells in micro-environmental niches, such as lymph nodes (LNs), contribute to survival and therapy resistance of chronic lymphocytic leukemia (CLL) cells. This is caused by a shift in expression of B-cell lymphoma 2 (BCL-2) family members. Pro-survival proteins B-cell lymphoma-extra large (BCL-X), BCL-2-related protein A1 (BFL-1) and myeloid leukemia cell differentiation protein 1 (MCL-1) are upregulated by LN-residing T cells through CD40L interaction, presumably via nuclear factor (NF)-κB signaling. Macrophages (Mφs) also reside in the LN, and are assumed to provide important supportive functions for CLL cells. However, if and how Mφs are able to induce survival is incompletely known. We first established that Mφs induced survival because of an exclusive upregulation of MCL-1. Next, we investigated the mechanism underlying MCL-1 induction by Mφs in comparison with CD40L. Genome-wide expression profiling of in vitro Mφ- and CD40L-stimulated CLL cells indicated activation of the phosphoinositide 3-kinase (PI3K)-V-Akt murine thymoma viral oncogene homolog (AKT)-mammalian target of rapamycin (mTOR) pathway, which was confirmed in ex vivo CLL LN material. Inhibition of PI3K-AKT-mTOR signaling abrogated MCL-1 upregulation and survival by Mφs, as well as CD40 stimulation. MCL-1 can be regulated at multiple levels, and we established that AKT leads to increased MCL-1 translation, but does not affect MCL-1 transcription or protein stabilization. Furthermore, among Mφ-secreted factors that could activate AKT, we found that induction of MCL-1 and survival critically depended on C-C motif chemokine receptor-1 (CCR1). In conclusion, this study indicates that two distinct micro-environmental factors, CD40L and Mφs, signal via CCR1 to induce AKT activation resulting in translational stabilization of MCL-1, and hence can contribute to CLL cell survival. 10.1038/onc.2016.515
    CLL-cells induce IDOhi CD14+HLA-DRlo myeloid-derived suppressor cells that inhibit T-cell responses and promote TRegs. Jitschin Regina,Braun Martina,Büttner Maike,Dettmer-Wilde Katja,Bricks Juliane,Berger Jana,Eckart Michael J,Krause Stefan W,Oefner Peter J,Le Blanc Katarina,Mackensen Andreas,Mougiakakos Dimitrios Blood Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population that shares certain characteristics including an aberrant myeloid phenotype and the ability to suppress T cells. MDSCs have been predominantly studied in malignant diseases and findings suggest involvement in tumor-associated immune suppression. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults. Immune defects occur already at early disease stages and impact the clinical course. We assessed presence, frequency, association to other immune parameters, and functional properties of circulating CD14(+) cells lacking HLA-DR expression (HLA-DR(lo)) in patients with untreated CLL. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14(+)HLA-DR(lo) cells was significantly increased in CLL patients. Furthermore, MDSCs suppressed in vitro T-cell activation and induced suppressive regulatory T cells (TRegs). The MDSC-mediated modulation of T cells could be attributed to their increased indoleamine 2,3-dioxygenase (IDO) activity. CLL cells induced IDO(hi) MDSCs from healthy donor monocytes suggesting bidirectional crosstalk between CLL-cells, MDSCs, and TRegs. Overall, we identified a MDSC population that expands in CLL. The exact mechanisms responsible for such accumulation remain to be elucidated and it will be of interest to test whether antagonizing suppressive functions of CLL MDSCs could represent a mean for enhancing immune responses. 10.1182/blood-2013-12-546416
    B-cell-specific IRF4 deletion accelerates chronic lymphocytic leukemia development by enhanced tumor immune evasion. Asslaber Daniela,Qi Yuan,Maeding Nicole,Steiner Markus,Denk Ursula,Höpner Jan Philip,Hartmann Tanja Nicole,Zaborsky Nadja,Greil Richard,Egle Alexander Blood Chronic lymphocytic leukemia (CLL) is a heterogenous disease that is highly dependent on a cross talk of CLL cells with the microenvironment, in particular with T cells. T cells derived from CLL patients or murine CLL models are skewed to an antigen-experienced T-cell subset, indicating a certain degree of antitumor recognition, but they are also exhausted, preventing an effective antitumor immune response. Here we describe a novel mechanism of CLL tumor immune evasion that is independent of T-cell exhaustion, using B-cell-specific deletion of the transcription factor IRF4 (interferon regulatory factor 4) in Tcl-1 transgenic mice developing a murine CLL highly similar to the human disease. We show enhanced CLL disease progression in IRF4-deficient Tcl-1 tg mice, associated with a severe downregulation of genes involved in T-cell activation, including genes involved in antigen processing/presentation and T-cell costimulation, which massively reduced T-cell subset skewing and exhaustion. We found a strong analogy in the human disease, with inferior prognosis of CLL patients with low IRF4 expression in independent CLL patient cohorts, failed T-cell skewing to antigen-experienced subsets, decreased costimulation capacity, and downregulation of genes involved in T-cell activation. These results have therapeutic relevance because our findings on molecular mechanisms of immune privilege may be responsible for the failure of immune-therapeutic strategies in CLL and may lead to improved targeting in the future. 10.1182/blood.2019000973
    TIM3 comes of age as an inhibitory receptor. Wolf Yochai,Anderson Ana C,Kuchroo Vijay K Nature reviews. Immunology T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a member of the TIM family, was originally identified as a receptor expressed on interferon-γ-producing CD4 and CD8 T cells. Initial data indicated that TIM3 functioned as a 'co-inhibitory' or 'checkpoint' receptor, but due to the lack of a definable inhibitory signalling motif, it was also suggested that TIM3 might act as a co-stimulatory receptor. Recent studies have shown that TIM3 is part of a module that contains multiple co-inhibitory receptors (checkpoint receptors), which are co-expressed and co-regulated on dysfunctional or 'exhausted' T cells in chronic viral infections and cancer. Furthermore, co-blockade of TIM3 and programmed cell death 1 (PD1) can result in tumour regression in preclinical models and can improve anticancer T cell responses in patients with advanced cancers. Here, we highlight the developments in understanding TIM3 biology, including novel ligand identification and the discovery of loss-of-function mutations associated with human disease. In addition, we summarize emerging data from human clinical trials showing that TIM3 indeed acts as a 'checkpoint' receptor and that inhibition of TIM3 enhances the antitumour effect of PD1 blockade. 10.1038/s41577-019-0224-6
    Defining 'T cell exhaustion'. Blank Christian U,Haining W Nicholas,Held Werner,Hogan Patrick G,Kallies Axel,Lugli Enrico,Lynn Rachel C,Philip Mary,Rao Anjana,Restifo Nicholas P,Schietinger Andrea,Schumacher Ton N,Schwartzberg Pamela L,Sharpe Arlene H,Speiser Daniel E,Wherry E John,Youngblood Benjamin A,Zehn Dietmar Nature reviews. Immunology 'T cell exhaustion' is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1 and the self-renewing TCF1 population from which they derive. These TCF1 cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations. 10.1038/s41577-019-0221-9
    PTPN2 regulates the generation of exhausted CD8 T cell subpopulations and restrains tumor immunity. Nature immunology CD8 T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6 progenitor exhausted and Tim-3 terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8 T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3 cells without altering Slamf6 numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8 T cells enhanced Tim-3 anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3CD8 T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target. 10.1038/s41590-019-0480-4
    TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection. Alfei Francesca,Kanev Kristiyan,Hofmann Maike,Wu Ming,Ghoneim Hazem E,Roelli Patrick,Utzschneider Daniel T,von Hoesslin Madlaina,Cullen Jolie G,Fan Yiping,Eisenberg Vasyl,Wohlleber Dirk,Steiger Katja,Merkler Doron,Delorenzi Mauro,Knolle Percy A,Cohen Cyrille J,Thimme Robert,Youngblood Benjamin,Zehn Dietmar Nature Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential. This process, known as T cell exhaustion or dysfunction, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1). The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1 self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology. 10.1038/s41586-019-1326-9
    Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients. Stock Sophia,Übelhart Rudolf,Schubert Maria-Luisa,Fan Fuli,He Bailin,Hoffmann Jean-Marc,Wang Lei,Wang Sanmei,Gong Wenjie,Neuber Brigitte,Hückelhoven-Krauss Angela,Gern Ulrike,Christ Christiane,Hexel Monika,Schmitt Anita,Schmidt Patrick,Krauss Jürgen,Jäger Dirk,Müller-Tidow Carsten,Dreger Peter,Schmitt Michael,Sellner Leopold International journal of cancer Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted. 10.1002/ijc.32201
    A functional subset of CD8 T cells during chronic exhaustion is defined by SIRPα expression. Myers Lara M,Tal Michal Caspi,Torrez Dulgeroff Laughing Bear,Carmody Aaron B,Messer Ronald J,Gulati Gunsagar,Yiu Ying Ying,Staron Matthew M,Angel Cesar Lopez,Sinha Rahul,Markovic Maxim,Pham Edward A,Fram Benjamin,Ahmed Aijaz,Newman Aaron M,Glenn Jeffrey S,Davis Mark M,Kaech Susan M,Weissman Irving L,Hasenkrug Kim J Nature communications Prolonged exposure of CD8 T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8 T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα CD8 T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα cells that actively proliferate, transcribe IFNγ and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPα, CD47, are more susceptible to CD8 T cell-killing in vivo. SIRPα CD8 T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8 T cells during chronic infection expands the cytotoxic subset of SIRPα CD8 T cells. 10.1038/s41467-019-08637-9
    Safety and activity of ibrutinib in combination with nivolumab in patients with relapsed non-Hodgkin lymphoma or chronic lymphocytic leukaemia: a phase 1/2a study. Younes Anas,Brody Joshua,Carpio Cecilia,Lopez-Guillermo Armando,Ben-Yehuda Dina,Ferhanoglu Burhan,Nagler Arnon,Ozcan Muhit,Avivi Irit,Bosch Francesc,Caballero Barrigón Maria Dolores,Hellmann Andrzej,Kuss Bryone,Ma David D F,Demirkan Fatih,Yağci Münci,Horowitz Netanel A,Marlton Paula,Cordoba Raul,Wrobel Tomasz,Buglio Daniela,Streit Michael,Hodkinson Brendan P,Schaffer Michael,Alvarez John,Ceulemans Rob,Balasubramanian Sriram,de Jong Jan,Wang Shean-Sheng,Fourneau Nele,Jurczak Wojciech The Lancet. Haematology BACKGROUND:Preclinical studies have shown synergistic antitumour effects between ibrutinib and immune-checkpoint blockade. The aim of this study was to assess the safety and activity of ibrutinib in combination with nivolumab in patients with relapsed or refractory B-cell malignant diseases. METHODS:We did a two-part, open-label, phase 1/2a study at 21 hospitals in Australia, Israel, Poland, Spain, Turkey, and the USA. The primary objective of part A (dose escalation) was to assess the safety of daily oral ibrutinib (420 mg or 560 mg) in combination with intravenous nivolumab (3 mg/kg every 2 weeks) to ascertain a recommended phase 2 dose in patients with relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, or diffuse large B-cell lymphoma. Dose optimisation was investigated using a modified toxicity probability interval design. The primary objective of the part B expansion phase was to establish the preliminary activity (the proportion of patients who achieved an overall response) of the combination of ibrutinib and nivolumab in four cohorts: relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, diffuse large B-cell lymphoma, and Richter's transformation. All participants who received at least one dose of treatment were included in the primary analysis and analyses were done by disease cohort. This trial is registered with ClinicalTrials.gov, number NCT02329847. The trial is ongoing. FINDINGS:Between March 12, 2015, and April 11, 2017, 144 patients were enrolled in the study. Three patients died before receiving study treatment; thus, 141 patients were included in the analysis, 14 in part A and 127 in part B. One dose-limiting toxicity (grade 3 hyperbilirubinaemia) was reported at the 420 mg dose in the diffuse large B-cell lymphoma cohort, which resolved after 5 days. The combination of ibrutinib and nivolumab led to overall responses in 22 (61%) of 36 patients with high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma, 13 (33%) of 40 patients with follicular lymphoma, 16 (36%) of 45 patients with diffuse large B-cell lymphoma, and 13 (65%) of 20 patients with Richter's transformation. The most common all-grade adverse events were diarrhoea (47 [33%] of 141 patients), neutropenia (44 [31%]), and fatigue (37 [26%]). 11 (8%) of 141 patients had adverse events leading to death; none were reported as drug-related. The most common grade 3-4 adverse events were neutropenia (40 [28%] of 141 patients) and anaemia (32 [23%]). The incidence of grade 3-4 neutropenia ranged from eight (18%) of 45 patients with diffuse large B-cell lymphoma to 19 (53%) of 36 patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma; incidence of grade 3-4 anaemia ranged from five (13%) of 40 patients with follicular lymphoma to seven (35%) of 20 patients with Richter's transformation. The most common serious adverse events included anaemia (six [4%] of 141 patients) and pneumonia (five [4%]). The most common grade 3-4 immune-related adverse events were rash (11 [8%] of 141 patients) and increased alanine aminotransferase (three [2%]). INTERPRETATION:The combination of ibrutinib and nivolumab had an acceptable safety profile and preliminary activity was similar to that reported with single-agent ibrutinib in chronic lymphocytic leukaemia or small lymphocytic lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. The clinical response in patients with Richter's transformation was promising and supports further clinical assessment. FUNDING:Janssen R&D. 10.1016/S2352-3026(18)30217-5
    PI3K p110δ inactivation antagonizes chronic lymphocytic leukemia and reverses T cell immune suppression. Dong Shuai,Harrington Bonnie K,Hu Eileen Y,Greene Joseph T,Lehman Amy M,Tran Minh,Wasmuth Ronni L,Long Meixiao,Muthusamy Natarajan,Brown Jennifer R,Johnson Amy J,Byrd John C The Journal of clinical investigation Targeted therapy with small molecules directed at essential survival pathways in leukemia represents a major advance, including the phosphatidylinositol-3'-kinase (PI3K) p110δ inhibitor idelalisib. Here, we found that genetic inactivation of p110δ (p110δD910A/D910A) in the Eμ-TCL1 murine chronic lymphocytic leukemia (CLL) model impaired B cell receptor signaling and B cell migration, and significantly delayed leukemia pathogenesis. Regardless of TCL1 expression, p110δ inactivation led to rectal prolapse in mice resembling autoimmune colitis in patients receiving idelalisib. Moreover, we showed that p110δ inactivation in the microenvironment protected against CLL and acute myeloid leukemia. After receiving higher numbers of TCL1 leukemia cells, half of p110δD910A/D910A mice spontaneously recovered from high disease burden and resisted leukemia rechallenge. Despite disease resistance, p110δD910A/D910A mice exhibited compromised CD4+ and CD8+ T cell response, and depletion of CD4+ or CD8+ T cells restored leukemia. Interestingly, p110δD910A/D910A mice showed significantly impaired Treg expansion that associated with disease clearance. Reconstitution of p110δD910A/D910A mice with p110δWT/WT Tregs reversed leukemia resistance. Our findings suggest that p110δ inhibitors may have direct antileukemic and indirect immune-activating effects, further supporting that p110δ blockade may have a broader immune-modulatory role in types of leukemia that are not sensitive to p110δ inhibition. 10.1172/JCI99386
    A Portrait of CXCR5 Follicular Cytotoxic CD8 T cells. Yu Di,Ye Lilin Trends in immunology CD8 T cells differentiate into multiple effector and memory subsets to carry out immune clearance of infected and cancerous cells and provide long-term protection. Recent research identified a CXCR5Tcf1Tim-3 subset that localizes in, or proximal to, B cell follicles in secondary lymphoid organs of mice, non-human primates, and humans, hereby termed follicular cytotoxic T (T) cells. With remarkable similarity to follicular helper T (T) cells, T differentiation is dependent on transcription factors E2A, Bcl6, and Tcf1, but inhibited by other regulators, including Blimp1, Id2, and Id3. This review summarizes the phenotype, function, and differentiation of this new subset. Owing to its follicular location and self-renewal capability, we propose immunotherapeutic strategies to target T cells to potentially treat certain cancers and chronic infections such as HIV-1. 10.1016/j.it.2018.10.002
    Biology and regulation of IL-2: from molecular mechanisms to human therapy. Spolski Rosanne,Li Peng,Leonard Warren J Nature reviews. Immunology IL-2 was first identified as a growth factor capable of driving the expansion of activated human T cell populations. In the more than 40 years since its discovery, a tremendous amount has been learned regarding the mechanisms that regulate the expression of both IL-2 and its cell surface receptor, its mechanisms of signalling and its range of biological actions. More recently, the mechanisms by which IL-2 regulates CD4 T cell differentiation and function have been elucidated. IL-2 also regulates the effector and memory responses of CD8 T cells, and the loss of IL-2 or responsiveness to IL-2 at least in part explains the exhausted phenotype that occurs during chronic viral infections and in tumour responses. These basic mechanistic studies have led to the therapeutic ability to manipulate the action of IL-2 on regulatory T (T) cells for the treatment of autoimmune disease and on CD8 T cells for immunotherapy of cancer. IL-2 can have either positive or deleterious effects, and we discuss here recent ideas and approaches for manipulating the actions and overall net effects of IL-2 in disease settings, including cancer. 10.1038/s41577-018-0046-y
    A cloning and expression system to probe T-cell receptor specificity and assess functional avidity to neoantigens. Hu Zhuting,Anandappa Annabelle J,Sun Jing,Kim Jintaek,Leet Donna E,Bozym David J,Chen Christina,Williams Louise,Shukla Sachet A,Zhang Wandi,Tabbaa Diana,Steelman Scott,Olive Oriol,Livak Kenneth J,Kishi Hiroyuki,Muraguchi Atsushi,Guleria Indira,Stevens Jonathan,Lane William J,Burkhardt Ute E,Fritsch Edward F,Neuberg Donna,Ott Patrick A,Keskin Derin B,Hacohen Nir,Wu Catherine J Blood Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent antitumor immune responses and provide strong motivation for improving our understanding of antigen-T-cell receptor (TCR) interactions. Advances in single-cell sequencing technologies have opened the door for detailed investigation of the TCR repertoire, providing paired information from TCRα and TCRβ, which together determine specificity. However, a need remains for efficient methods to assess the specificity of discovered TCRs. We developed a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. Here, we first demonstrate the system's capacity to identify viral-antigen-specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then apply this system to identify neoantigen-specific TCR sequences from patients with melanoma treated with personalized neoantigen vaccines and characterize functional avidity of neoantigen-specific TCRs. Furthermore, we use a neoantigen-prediction pipeline to show that an insertion-deletion mutation in a putative chronic lymphocytic leukemia (CLL) driver gives rise to an immunogenic neoantigen mut- and use this approach to identify the mut--specific TCR sequence. This approach provides a means to identify and express TCRs, and then rapidly assess antigen specificity and functional avidity of a reconstructed TCR, which can be applied for monitoring antigen-specific T-cell responses, and potentially for guiding the design of effective T-cell-based immunotherapies. 10.1182/blood-2018-04-843763
    CEACAM1 promotes CD8 T cell responses and improves control of a chronic viral infection. Khairnar Vishal,Duhan Vikas,Patil Ashwini M,Zhou Fan,Bhat Hilal,Thoens Christine,Sharma Piyush,Adomati Tom,Friendrich Sarah-Kim,Bezgovsek Judith,Dreesen Janine D,Wennemuth Gunther,Westendorf Astrid M,Zelinskyy Gennadiy,Dittmer Ulf,Hardt Cornelia,Timm Jörg,Göthert Joachim R,Lang Philipp A,Singer Bernhard B,Lang Karl S Nature communications Dysfunction of CD8 T cells can lead to the development of chronic viral infection. Identifying mechanisms responsible for such T cell dysfunction is therefore of great importance to understand how to prevent persistent viral infection. Here we show using lymphocytic choriomeningitis virus (LCMV) infection that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is fundamental for recruiting lymphocyte-specific protein kinase (Lck) into the T cell receptor complex to form an efficient immunological synapse. CEACAM1 is essential for activation of CD8 T cells, and the absence of CEACAM1 on virus-specific CD8 T cells limits the antiviral CD8 T cell response. Treatment with anti-CEACAM1 antibody stabilizes Lck in the immunological synapse, prevents CD8 T cell exhaustion, and improves control of virus infection in vivo. Treatment of human virus-specific CD8 T cells with anti-CEACAM1 antibody similarly enhances their proliferation. We conclude that CEACAM1 is an important regulator of virus-specific CD8 T cell functions in mice and humans and represents a promising therapeutic target for modulating CD8 T cells. 10.1038/s41467-018-04832-2
    Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Fraietta Joseph A,Lacey Simon F,Orlando Elena J,Pruteanu-Malinici Iulian,Gohil Mercy,Lundh Stefan,Boesteanu Alina C,Wang Yan,O'Connor Roddy S,Hwang Wei-Ting,Pequignot Edward,Ambrose David E,Zhang Changfeng,Wilcox Nicholas,Bedoya Felipe,Dorfmeier Corin,Chen Fang,Tian Lifeng,Parakandi Harit,Gupta Minnal,Young Regina M,Johnson F Brad,Kulikovskaya Irina,Liu Li,Xu Jun,Kassim Sadik H,Davis Megan M,Levine Bruce L,Frey Noelle V,Siegel Donald L,Huang Alexander C,Wherry E John,Bitter Hans,Brogdon Jennifer L,Porter David L,June Carl H,Melenhorst J Joseph Nature medicine Tolerance to self-antigens prevents the elimination of cancer by the immune system. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27CD45ROCD8 T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27PD-1CD8 CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies. 10.1038/s41591-018-0010-1
    Regulatory T cells trigger effector T cell DNA damage and senescence caused by metabolic competition. Liu Xia,Mo Wei,Ye Jian,Li Lingyun,Zhang Yanping,Hsueh Eddy C,Hoft Daniel F,Peng Guangyong Nature communications Defining the suppressive mechanisms used by regulatory T (Treg) cells is critical for the development of effective strategies for treating tumors and chronic infections. The molecular processes that occur in responder T cells that are suppressed by Treg cells are unclear. Here we show that human Treg cells initiate DNA damage in effector T cells caused by metabolic competition during cross-talk, resulting in senescence and functional changes that are molecularly distinct from anergy and exhaustion. ERK1/2 and p38 signaling cooperate with STAT1 and STAT3 to control Treg-induced effector T-cell senescence. Human Treg-induced T-cell senescence can be prevented via inhibition of the DNA damage response and/or STAT signaling in T-cell adoptive transfer mouse models. These studies identify molecular mechanisms of human Treg cell suppression and indicate that targeting Treg-induced T-cell senescence is a checkpoint for immunotherapy against cancer and other diseases associated with Treg cells. 10.1038/s41467-017-02689-5
    Ibrutinib treatment improves T cell number and function in CLL patients. Long Meixiao,Beckwith Kyle,Do Priscilla,Mundy Bethany L,Gordon Amber,Lehman Amy M,Maddocks Kami J,Cheney Carolyn,Jones Jeffrey A,Flynn Joseph M,Andritsos Leslie A,Awan Farrukh,Fraietta Joseph A,June Carl H,Maus Marcela V,Woyach Jennifer A,Caligiuri Michael A,Johnson Amy J,Muthusamy Natarajan,Byrd John C The Journal of clinical investigation BACKGROUND:Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton's tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS:Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS:Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS:Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION:ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. FUNDING:The National Cancer Institute. 10.1172/JCI89756
    Pembrolizumab in patients with CLL and Richter transformation or with relapsed CLL. Ding Wei,LaPlant Betsy R,Call Timothy G,Parikh Sameer A,Leis Jose F,He Rong,Shanafelt Tait D,Sinha Sutapa,Le-Rademacher Jennifer,Feldman Andrew L,Habermann Thomas M,Witzig Thomas E,Wiseman Gregory A,Lin Yi,Asmus Erik,Nowakowski Grzegorz S,Conte Michael J,Bowen Deborah A,Aitken Casey N,Van Dyke Daniel L,Greipp Patricia T,Liu Xin,Wu Xiaosheng,Zhang Henan,Secreto Charla R,Tian Shulan,Braggio Esteban,Wellik Linda E,Micallef Ivana,Viswanatha David S,Yan Huihuang,Chanan-Khan Asher A,Kay Neil E,Dong Haidong,Ansell Stephen M Blood Chronic lymphocytic leukemia (CLL) patients progressed early on ibrutinib often develop Richter transformation (RT) with a short survival of about 4 months. Preclinical studies suggest that programmed death 1 (PD-1) pathway is critical to inhibit immune surveillance in CLL. This phase 2 study was designed to test the efficacy and safety of pembrolizumab, a humanized PD-1-blocking antibody, at a dose of 200 mg every 3 weeks in relapsed and transformed CLL. Twenty-five patients including 16 relapsed CLL and 9 RT (all proven diffuse large cell lymphoma) patients were enrolled, and 60% received prior ibrutinib. Objective responses were observed in 4 out of 9 RT patients (44%) and in 0 out of 16 CLL patients (0%). All responses were observed in RT patients who had progression after prior therapy with ibrutinib. After a median follow-up time of 11 months, the median overall survival in the RT cohort was 10.7 months, but was not reached in RT patients who progressed after prior ibrutinib. Treatment-related grade 3 or above adverse events were reported in 15 (60%) patients and were manageable. Analyses of pretreatment tumor specimens from available patients revealed increased expression of PD-ligand 1 (PD-L1) and a trend of increased expression in PD-1 in the tumor microenvironment in patients who had confirmed responses. Overall, pembrolizumab exhibited selective efficacy in CLL patients with RT. The results of this study are the first to demonstrate the benefit of PD-1 blockade in CLL patients with RT, and could change the landscape of therapy for RT patients if further validated. This trial was registered at www.clinicaltrials.gov as #NCT02332980. 10.1182/blood-2017-02-765685
    Chimeric antigen receptor T cells targeting Fc μ receptor selectively eliminate CLL cells while sparing healthy B cells. Faitschuk Elena,Hombach Andreas A,Frenzel Lukas P,Wendtner Clemens-Martin,Abken Hinrich Blood Adoptive cell therapy of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR)-modified T cells targeting CD19 induced lasting remission of this refractory disease in a number of patients. However, the treatment is associated with prolonged "on-target off-tumor" toxicities due to the targeted elimination of healthy B cells demanding more selectivity in targeting CLL cells. We identified the immunoglobulin M Fc receptor (FcμR), also known as the Fas apoptotic inhibitory molecule-3 or TOSO, as a target for a more selective treatment of CLL by CAR T cells. FcμR is highly and consistently expressed by CLL cells; only minor levels are detected on healthy B cells or other hematopoietic cells. T cells with a CAR specific for FcμR efficiently responded toward CLL cells, released a panel of proinflammatory cytokines and lytic factors, like soluble FasL and granzyme B, and eliminated the leukemic cells. In contrast to CD19 CAR T cells, anti-FcμR CAR T cells did not attack healthy B cells. T cells with anti-FcμR CAR delayed outgrowth of Mec-1-induced leukemia in a xenograft mouse model. T cells from CLL patients in various stages of the disease, modified by the anti-FcμR CAR, purged their autologous CLL cells in vitro without reducing the number of healthy B cells, which is the case with anti-CD19 CAR T cells. Compared with the currently used therapies, the data strongly imply a superior therapeutic index of anti-FcμR CAR T cells for the treatment of CLL. 10.1182/blood-2016-01-692046
    Clinical responses with T lymphocytes targeting malignancy-associated κ light chains. Ramos Carlos A,Savoldo Barbara,Torrano Vicky,Ballard Brandon,Zhang Huimin,Dakhova Olga,Liu Enli,Carrum George,Kamble Rammurti T,Gee Adrian P,Mei Zhuyong,Wu Meng-Fen,Liu Hao,Grilley Bambi,Rooney Cliona M,Brenner Malcolm K,Heslop Helen E,Dotti Gianpietro The Journal of clinical investigation BACKGROUND:Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan-B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment. METHODS:We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ+ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 108 to 2 × 108 κ.CARTs/m2). No other lymphodepletion was used. RESULTS:κ.CART expansion peaked 1-2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2-17 months. No toxicities attributable to κ.CARTs were observed. CONCLUSION:κ.CART infusion is feasible and safe and can lead to complete clinical responses. TRIAL REGISTRATION:ClinicalTrials.gov NCT00881920. FUNDING:National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018. 10.1172/JCI86000
    Extracellular vesicles released by CD40/IL-4-stimulated CLL cells confer altered functional properties to CD4+ T cells. Smallwood Dawn T,Apollonio Benedetta,Willimott Shaun,Lezina Larissa,Alharthi Afaf,Ambrose Ashley R,De Rossi Giulia,Ramsay Alan G,Wagner Simon D Blood The complex interplay between cancer cells, stromal cells, and immune cells in the tumor microenvironment (TME) regulates tumorigenesis and provides emerging targets for immunotherapies. Crosstalk between CD4(+) T cells and proliferating chronic lymphocytic leukemia (CLL) tumor B cells occurs within lymphoid tissue pseudofollicles, and investigating these interactions is essential to understand both disease pathogenesis and the effects of immunotherapy. Tumor-derived extracellular vesicle (EV) shedding is emerging as an important mode of intercellular communication in the TME. In order to characterize tumor EVs released in response to T-cell-derived TME signals, we performed microRNA (miRNA [miR]) profiling of EVs released from CLL cells stimulated with CD40 and interleukin-4 (IL-4). Our results reveal an enrichment of specific cellular miRNAs including miR-363 within EVs derived from CD40/IL-4-stimulated CLL cells compared with parental cell miRNA content and control EVs from unstimulated CLL cells. We demonstrate that autologous patient CD4(+) T cells internalize CLL-EVs containing miR-363 that targets the immunomodulatory molecule CD69. We further reveal that autologous CD4(+) T cells that are exposed to EVs from CD40/IL-4-stimulated CLL cells exhibit enhanced migration, immunological synapse signaling, and interactions with tumor cells. Knockdown of miR-363 in CLL cells prior to CD40/IL-4 stimulation prevented the ability of CLL-EVs to induce increased synapse signaling and confer altered functional properties to CD4(+) T cells. Taken together, these data reveal a novel role for CLL-EVs in modifying T-cell function that highlights unanticipated complexity of intercellular communication that may have implications for bidirectional CD4(+) T-cell:tumor interactions within the TME. 10.1182/blood-2015-11-682377
    Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor Induce Remissions of B-Cell Malignancies That Progress After Allogeneic Hematopoietic Stem-Cell Transplantation Without Causing Graft-Versus-Host Disease. Brudno Jennifer N,Somerville Robert P T,Shi Victoria,Rose Jeremy J,Halverson David C,Fowler Daniel H,Gea-Banacloche Juan C,Pavletic Steven Z,Hickstein Dennis D,Lu Tangying L,Feldman Steven A,Iwamoto Alexander T,Kurlander Roger,Maric Irina,Goy Andre,Hansen Brenna G,Wilder Jennifer S,Blacklock-Schuver Bazetta,Hakim Frances T,Rosenberg Steven A,Gress Ronald E,Kochenderfer James N Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Progressive malignancy is the leading cause of death after allogeneic hematopoietic stem-cell transplantation (alloHSCT). After alloHSCT, B-cell malignancies often are treated with unmanipulated donor lymphocyte infusions (DLIs) from the transplant donor. DLIs frequently are not effective at eradicating malignancy and often cause graft-versus-host disease, a potentially lethal immune response against normal recipient tissues. METHODS:We conducted a clinical trial of allogeneic T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Patients with B-cell malignancies that had progressed after alloHSCT received a single infusion of CAR T cells. No chemotherapy or other therapies were administered. The T cells were obtained from each recipient's alloHSCT donor. RESULTS:Eight of 20 treated patients obtained remission, which included six complete remissions (CRs) and two partial remissions. The response rate was highest for acute lymphoblastic leukemia, with four of five patients obtaining minimal residual disease-negative CR. Responses also occurred in chronic lymphocytic leukemia and lymphoma. The longest ongoing CR was more than 30 months in a patient with chronic lymphocytic leukemia. New-onset acute graft-versus-host disease after CAR T-cell infusion developed in none of the patients. Toxicities included fever, tachycardia, and hypotension. Peak blood CAR T-cell levels were higher in patients who obtained remissions than in those who did not. Programmed cell death protein-1 expression was significantly elevated on CAR T cells after infusion. Presence of blood B cells before CAR T-cell infusion was associated with higher postinfusion CAR T-cell levels. CONCLUSION:Allogeneic anti-CD19 CAR T cells can effectively treat B-cell malignancies that progress after alloHSCT. The findings point toward a future when antigen-specific T-cell therapies will play a central role in alloHSCT. 10.1200/JCO.2015.64.5929
    Novel role of prostate apoptosis response-4 tumor suppressor in B-cell chronic lymphocytic leukemia. McKenna Mary K,Noothi Sunil K,Alhakeem Sara S,Oben Karine Z,Greene Joseph T,Mani Rajeswaran,Perry Kathryn L,Collard James P,Rivas Jacqueline R,Hildebrandt Gerhard C,Fleischman Roger A,Durbin Eric B,Byrd John C,Wang Chi,Muthusamy Natarajan,Rangnekar Vivek M,Bondada Subbarao Blood Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is downregulated in many cancers including renal cell carcinoma, glioblastoma, endometrial, and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL-derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1-to-S cell-cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eμ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with US Food and Drug Administration (FDA)-approved drugs caused a decrease in Par-4 messenger RNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase, and Bruton tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel progrowth rather than proapoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR-signaling inhibitors. 10.1182/blood-2017-10-813931
    BRD4 Profiling Identifies Critical Chronic Lymphocytic Leukemia Oncogenic Circuits and Reveals Sensitivity to PLX51107, a Novel Structurally Distinct BET Inhibitor. Ozer Hatice Gulcin,El-Gamal Dalia,Powell Ben,Hing Zachary A,Blachly James S,Harrington Bonnie,Mitchell Shaneice,Grieselhuber Nicole R,Williams Katie,Lai Tzung-Huei,Alinari Lapo,Baiocchi Robert A,Brinton Lindsey,Baskin Elizabeth,Cannon Matthew,Beaver Larry,Goettl Virginia M,Lucas David M,Woyach Jennifer A,Sampath Deepa,Lehman Amy M,Yu Lianbo,Zhang Jiazhong,Ma Yan,Zhang Ying,Spevak Wayne,Shi Songyuan,Severson Paul,Shellooe Rafe,Carias Heidi,Tsang Garson,Dong Ken,Ewing Todd,Marimuthu Adhirai,Tantoy Christina,Walters Jason,Sanftner Laura,Rezaei Hamid,Nespi Marika,Matusow Bernice,Habets Gaston,Ibrahim Prabha,Zhang Chao,Mathé Ewy A,Bollag Gideon,Byrd John C,Lapalombella Rosa Cancer discovery Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel and pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers. To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. . 10.1158/2159-8290.CD-17-0902
    Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukemia cells from apoptosis. Abbaci A,Talbot H,Saada S,Gachard N,Abraham J,Jaccard A,Bordessoule D,Fauchais A L,Naves T,Jauberteau M O Oncogene B-cell chronic lymphocytic leukemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Overexpression of cell-surface G protein-coupled receptors drives cell transformation, and thus plays a critical role in malignancies. In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterization revealed that the NTSR2-TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand brain-derived neurotrophic factor (BDNF), which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2-TrkB interaction activates survival signaling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death, reflected by DNA fragmentation and Annexin V presentation. Together, our findings demonstrate that the NTSR2-TrkB interaction plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy. 10.1038/onc.2017.365
    Ectopic ILT3 controls BCR-dependent activation of Akt in B-cell chronic lymphocytic leukemia. Zurli Vanessa,Wimmer Giuliana,Cattaneo Francesca,Candi Veronica,Cencini Emanuele,Gozzetti Alessandro,Raspadori Donatella,Campoccia Giuseppe,Sanseviero Francesca,Bocchia Monica,Baldari Cosima Tatiana,Kabanova Anna Blood The high proportion of long-term nonprogressors among chronic lymphocytic leukemia (CLL) patients suggests the existence of a regulatory network that restrains the proliferation of tumor B cells. The identification of molecular determinants composing such network is hence fundamental for our understanding of CLL pathogenesis. Based on our previous finding establishing a deficiency in the signaling adaptor p66Shc in CLL cells, we undertook to identify unique phenotypic traits caused by this defect. Here we show that a lack of p66Shc shapes the transcriptional profile of CLL cells and leads to an upregulation of the surface receptor ILT3, the immunoglobulin-like transcript 3 that is normally found on myeloid cells. The ectopic expression of ILT3 in CLL was a distinctive feature of neoplastic B cells and hematopoietic stem cells, thus identifying ILT3 as a selective marker of malignancy in CLL and the first example of phenotypic continuity between mature CLL cells and their progenitors in the bone marrow. ILT3 expression in CLL was found to be driven by Deltex1, a suppressor of antigen receptor signaling in lymphocytes. Triggering of ILT3 inhibited the activation of Akt kinase upon B-cell receptor (BCR) stimulation. This effect was achieved through the dynamic coalescence of ILT3, BCRs, and phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 into inhibitory clusters at the cell surface. Collectively, our findings identify ILT3 as a signature molecule of p66Shc deficiency in CLL and indicate that ILT3 may functionally contribute to a regulatory network controlling tumor progression by suppressing the Akt pathway. 10.1182/blood-2017-03-775858
    Targeting BTK through microRNA in chronic lymphocytic leukemia. Bottoni Arianna,Rizzotto Lara,Lai Tzung-Huei,Liu Chaomei,Smith Lisa L,Mantel Rose,Reiff Sean,El-Gamal Dalia,Larkin Karilyn,Johnson Amy J,Lapalombella Rosa,Lehman Amy,Plunkett William,Byrd John C,Blachly James S,Woyach Jennifer A,Sampath Deepa Blood Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling. 10.1182/blood-2016-07-727750
    Integration of innate into adaptive immune responses in ZAP-70-positive chronic lymphocytic leukemia. Wagner Michaela,Oelsner Madlen,Moore Andrew,Götte Frederik,Kuhn Peer-Hendrik,Haferlach Torsten,Fiegl Michael,Bogner Christian,Baxter E Joanna,Peschel Christian,Follows George A,Ringshausen Ingo Blood The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL. 10.1182/blood-2015-05-646935
    Immune Response Dysfunction in Chronic Lymphocytic Leukemia: Dissecting Molecular Mechanisms and Microenvironmental Conditions. Arruga Francesca,Gyau Benjamin Baffour,Iannello Andrea,Vitale Nicoletta,Vaisitti Tiziana,Deaglio Silvia International journal of molecular sciences Representing the major cause of morbidity and mortality for chronic lymphocytic leukemia (CLL) patients, immunosuppression is a common feature of the disease. Effectors of the innate and the adaptive immune response show marked dysfunction and skewing towards the generation of a tolerant environment that favors disease expansion. Major deregulations are found in the T lymphocyte compartment, with inhibition of CD8 cytotoxic and CD4 activated effector T cells, replaced by exhausted and more tolerogenic subsets. Likewise, differentiation of monocytes towards a suppressive M2-like phenotype is induced at the expense of pro-inflammatory sub-populations. Thanks to their B-regulatory phenotype, leukemic cells play a central role in driving immunosuppression, progressively inhibiting immune responses. A number of signaling cascades triggered by soluble mediators and cell-cell contacts contribute to immunomodulation in CLL, fostered also by local environmental conditions, such as hypoxia and derived metabolic acidosis. Specifically, molecular pathways modulating T-cell activity in CLL, spanning from the best known cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death 1 (PD-1) to the emerging T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)/CD155 axes, are attracting increasing research interest and therapeutic relevance also in the CLL field. On the other hand, in the microenvironment, the B cell receptor (BCR), which is undoubtedly the master regulator of leukemic cell behavior, plays an important role in orchestrating immune responses, as well. Lastly, local conditions of hypoxia, typical of the lymphoid niche, have major effects both on CLL cells and on non-leukemic immune cells, partly mediated through adenosine signaling, for which novel specific inhibitors are currently under development. In summary, this review will provide an overview of the molecular and microenvironmental mechanisms that modify innate and adaptive immune responses of CLL patients, focusing attention on those that may have therapeutic implications. 10.3390/ijms21051825
    Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Burger Jan A,Wiestner Adrian Nature reviews. Cancer B cell receptor (BCR) signalling is crucial for normal B cell development and adaptive immunity. BCR signalling also supports the survival and growth of malignant B cells in patients with B cell leukaemias or lymphomas. The mechanism of BCR pathway activation in these diseases includes continuous BCR stimulation by microbial antigens or autoantigens present in the tissue microenvironment, activating mutations within the BCR complex or downstream signalling components and ligand-independent tonic BCR signalling. The most established agents targeting BCR signalling are Bruton tyrosine kinase (BTK) inhibitors and PI3K isoform-specific inhibitors, and their introduction into the clinic is rapidly changing how B cell malignancies are treated. B cells and BCR-related kinases, such as BTK, also play a role in the microenvironment of solid tumours, such as squamous cell carcinoma and pancreatic cancer, and therefore targeting B cells or BCR-related kinases may have anticancer activity beyond B cell malignancies. 10.1038/nrc.2017.121
    Evolution of CLL treatment - from chemoimmunotherapy to targeted and individualized therapy. Burger Jan A,O'Brien Susan Nature reviews. Clinical oncology During the past 5 years, a number of highly active novel agents, including kinase inhibitors targeting BTK or PI3Kδ, an antagonist of the antiapoptotic protein BCL-2, and new anti-CD20 monoclonal antibodies, have been added to the therapeutic armamentarium for patients with chronic lymphocytic leukaemia (CLL). In these exciting times, care is needed to optimally integrate these novel agents into the traditional treatment algorithm without overlooking or compromising the benefits of established treatments, especially chemoimmunotherapy. A more personalized approach to CLL therapy that takes into account individual risk factors, patient characteristics, and their treatment preferences is now possible. Herein, we discuss the biological basis for the novel therapeutic agents and outline not only the major advantages of these agents over traditional therapies but also their adverse effects and the rationale for continued use of older versus newer types of therapy for selected patients with CLL. We conclude by providing recommendations for an individualized therapy approach for different populations of patients with CLL. 10.1038/s41571-018-0037-8
    The molecular pathogenesis of chronic lymphocytic leukaemia. Fabbri Giulia,Dalla-Favera Riccardo Nature reviews. Cancer Recent investigations have provided an increasingly complete picture of the genetic landscape of chronic lymphocytic leukaemia (CLL). These analyses revealed that the CLL genome displays a high degree of heterogeneity between patients and within the same patient. In addition, they highlighted molecular mechanisms and functionally relevant biological programmes that may be important for the pathogenesis and therapeutic targeting of this disease. This Review focuses on recent insights into the understanding of CLL biology, with emphasis on the role of genetic lesions in the initiation and clinical progression of CLL. We also consider the translation of these findings into the development of risk-adapted and targeted therapeutic approaches. 10.1038/nrc.2016.8
    Chronic lymphocytic leukaemia. Kipps Thomas J,Stevenson Freda K,Wu Catherine J,Croce Carlo M,Packham Graham,Wierda William G,O'Brien Susan,Gribben John,Rai Kanti Nature reviews. Disease primers Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5 B cells that is characterized by the accumulation of small, mature-appearing lymphocytes in the blood, marrow and lymphoid tissues. Signalling via surface immunoglobulin, which constitutes the major part of the B cell receptor, and several genetic alterations play a part in CLL pathogenesis, in addition to interactions between CLL cells and other cell types, such as stromal cells, T cells and nurse-like cells in the lymph nodes. The clinical progression of CLL is heterogeneous and ranges from patients who require treatment soon after diagnosis to others who do not require therapy for many years, if at all. Several factors, including the immunoglobulin heavy-chain variable region gene (IGHV) mutational status, genomic changes, patient age and the presence of comorbidities, should be considered when defining the optimal management strategies, which include chemotherapy, chemoimmunotherapy and/or drugs targeting B cell receptor signalling or inhibitors of apoptosis, such as BCL-2. Research on the biology of CLL has profoundly enhanced our ability to identify patients who are at higher risk for disease progression and our capacity to treat patients with drugs that selectively target distinctive phenotypic or physiological features of CLL. How these and other advances have shaped our current understanding and treatment of patients with CLL is the subject of this Primer. 10.1038/nrdp.2016.96