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    Whole-exome and targeted sequencing identify ROBO1 and ROBO2 mutations as progression-related drivers in myelodysplastic syndromes. Xu Feng,Wu Ling-Yun,Chang Chun-Kang,He Qi,Zhang Zheng,Liu Li,Shi Wen-Hui,Guo Juan,Zhu Yang,Zhao You-Shan,Gu Shu-Cheng,Fei Cheng-Ming,Wu Dong,Zhou Li-Yu,Su Ji-Ying,Song Lu-Xi,Xiao Chao,Li Xiao Nature communications The progressive mechanism underlying myelodysplastic syndrome remains unknown. Here we identify ROBO1 and ROBO2 as novel progression-related somatic mutations using whole-exome and targeted sequencing in 6 of 16 (37.5%) paired MDS patients with disease progression. Further deep sequencing detects 20 (10.4%) patients with ROBO mutations in a cohort of 193 MDS patients. In addition, copy number loss and loss of heterogeneity (LOH) of ROBO1 and ROBO2 are frequently observed in patients with progression or carrying ROBO mutations. In in vitro experiments, overexpression of ROBO1 or ROBO2 produces anti-proliferative and pro-apoptotic effects in leukaemia cells. However, this effect was lost in ROBO mutants and ROBO-SLIT2 signalling is impaired. Multivariate analysis shows that ROBO mutations are independent factors for predicting poor survival. These findings demonstrate a novel contribution of ROBO mutations to the pathogenesis of MDS and highlight a key role for ROBO-SLIT2 signalling in MDS disease progression. 10.1038/ncomms9806
    Impact of iron overload and potential benefit from iron chelation in low-risk myelodysplastic syndrome. Shenoy Niraj,Vallumsetla Nishanth,Rachmilewitz Eliezer,Verma Amit,Ginzburg Yelena Blood Myelodysplastic syndromes (MDSs) are a group of heterogeneous clonal bone marrow disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, and potential for malignant transformation. Lower/intermediate-risk MDSs are associated with longer survival and high red blood cell (RBC) transfusion requirements resulting in secondary iron overload. Recent data suggest that markers of iron overload portend a relatively poor prognosis, and retrospective analysis demonstrates that iron chelation therapy is associated with prolonged survival in transfusion-dependent MDS patients. New data provide concrete evidence of iron's adverse effects on erythroid precursors in vitro and in vivo. Renewed interest in the iron field was heralded by the discovery of hepcidin, the main serum peptide hormone negative regulator of body iron. Evidence from β-thalassemia suggests that regulation of hepcidin by erythropoiesis dominates regulation by iron. Because iron overload develops in some MDS patients who do not require RBC transfusions, the suppressive effect of ineffective erythropoiesis on hepcidin may also play a role in iron overload. We anticipate that additional novel tools for measuring iron overload and a molecular-mechanism-driven description of MDS subtypes will provide a deeper understanding of how iron metabolism and erythropoiesis intersect in MDSs and improve clinical management of this patient population. 10.1182/blood-2014-03-563221
    CD34 megakaryocytes and megakaryocytic fragments in myelodysplastic syndrome. Chen Yan,Chang Hong Blood 10.1182/blood-2017-02-769240
    Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome. Colla Simona,Ong Derrick Sek Tong,Ogoti Yamini,Marchesini Matteo,Mistry Nipun A,Clise-Dwyer Karen,Ang Sonny A,Storti Paola,Viale Andrea,Giuliani Nicola,Ruisaard Kathryn,Ganan Gomez Irene,Bristow Christopher A,Estecio Marcos,Weksberg David C,Ho Yan Wing,Hu Baoli,Genovese Giannicola,Pettazzoni Piergiorgio,Multani Asha S,Jiang Shan,Hua Sujun,Ryan Michael C,Carugo Alessandro,Nezi Luigi,Wei Yue,Yang Hui,D'Anca Marianna,Zhang Li,Gaddis Sarah,Gong Ting,Horner James W,Heffernan Timothy P,Jones Philip,Cooper Laurence J N,Liang Han,Kantarjian Hagop,Wang Y Alan,Chin Lynda,Bueso-Ramos Carlos,Garcia-Manero Guillermo,DePinho Ronald A Cancer cell Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation. 10.1016/j.ccell.2015.04.007
    Myelodysplastic Syndrome Splicing Factor Mutations Induce R-Loops. Cancer discovery Mutations in and trigger replication stress and ATR activation via R-loop formation. 10.1158/2159-8290.CD-RW2018-025
    Luspatercept for the treatment of anaemia in patients with lower-risk myelodysplastic syndromes (PACE-MDS): a multicentre, open-label phase 2 dose-finding study with long-term extension study. Platzbecker Uwe,Germing Ulrich,Götze Katharina S,Kiewe Philipp,Mayer Karin,Chromik Jörg,Radsak Markus,Wolff Thomas,Zhang Xiaosha,Laadem Abderrahmane,Sherman Matthew L,Attie Kenneth M,Giagounidis Aristoteles The Lancet. Oncology BACKGROUND:Myelodysplastic syndromes are characterised by ineffective erythropoiesis. Luspatercept (ACE-536) is a novel fusion protein that blocks transforming growth factor beta (TGF β) superfamily inhibitors of erythropoiesis, giving rise to a promising new investigative therapy. We aimed to assess the safety and efficacy of luspatercept in patients with anaemia due to lower-risk myelodysplastic syndromes. METHODS:In this phase 2, multicentre, open-label, dose-finding study (PACE-MDS), with long-term extension, eligible patients were aged 18 years or older, had International Prognostic Scoring System-defined low or intermediate 1 risk myelodysplastic syndromes or non-proliferative chronic myelomonocytic leukaemia (white blood cell count <13 000/μL), and had anaemia with or without red blood cell transfusion support. Enrolled patients were classified as having low transfusion burden, defined as requiring less than 4 red blood cell units in the 8 weeks before treatment (and baseline haemoglobin <10 g/dL), or high transfusion burden, defined as requiring 4 or more red blood cell units in the 8 weeks before treatment. Patients received luspatercept subcutaneously once every 21 days at dose concentrations ranging from 0·125 mg/kg to 1·75 mg/kg bodyweight for five doses (over a maximum of 12 weeks). Patients in the expansion cohort were treated with 1·0 mg/kg luspatercept; dose titration up to 1·75 mg/kg was allowed, and patients could be treated with luspatercept for a maximum of 5 years. Patients in the base study were assessed for response and safety after 12 weeks in order to be considered for enrolment into the extension study. The primary endpoint was the proportion of patients achieving modified International Working Group-defined haematological improvement-erythroid (HI-E), defined as a haemoglobin concentration increase of 1·5 g/dL or higher from baseline for 14 days or longer in low transfusion burden patients, and a reduction in red blood cell transfusion of 4 or more red blood cell units or a 50% or higher reduction in red blood cell units over 8 weeks versus pre-treatment transfusion burden in high transfusion burden patients. Patient data were subcategorised by: luspatercept dose concentrations (0·125-0·5 mg/kg vs 0·75-1·75 mg/kg); pre-study transfusion burden (high transfusion burden vs low transfusion burden, defined as ≥4 vs <4 red blood cell units per 8 weeks); pre-study serum erythropoietin concentration (<200 IU/L, 200-500 IU/L, and >500 IU/L); presence of 15% or more ring sideroblasts; and presence of SF3B1 mutations. Efficacy analyses were carried out on the efficacy evaluable and intention-to-treat populations. This trial is currently ongoing. This study is registered with ClinicalTrials.gov, numbers NCT01749514 and NCT02268383. FINDINGS:Between Jan 21, 2013, and Feb 12, 2015, 58 patients with myelodysplastic syndromes were enrolled in the 12 week base study at nine treatment centres in Germany; 27 patients were enrolled in the dose-escalation cohorts (0·125-1·75 mg/kg) and 31 patients in the expansion cohort (1·0-1·75 mg/kg). 32 (63% [95% CI 48-76]) of 51 patients receiving higher dose luspatercept concentrations (0·75-1·75 mg/kg) achieved HI-E versus two (22% [95% CI 3-60]) of nine receiving lower dose concentrations (0·125-0·5 mg/kg). Three treatment-related grade 3 adverse events occurred in one patient each: myalgia (one [2%]), increased blast cell count (one [2%]), and general physical health deterioration (one [2%]). Two of these treatment-related grade 3 adverse events were reversible serious grade 3 adverse events: one patient (2%) had myalgia and one patient (2%) had general physical health deterioration. INTERPRETATION:Luspatercept was well tolerated and effective for the treatment of anaemia in lower-risk myelodysplastic syndromes and so could therefore provide a novel therapeutic approach for the treatment of anaemia associated with lower-risk myelodysplastic syndromes; further studies are ongoing. FUNDING:Acceleron Pharma. 10.1016/S1470-2045(17)30615-0
    Granulocyte Colony-Stimulating Factors in Therapy-Related Myelodysplastic Syndrome and Acute Myeloid Leukemia. Kaplan Henry G,Malmgren Judith A,Calip Gregory S JAMA oncology 10.1001/jamaoncol.2019.0737
    PAK1 is a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome. Pandolfi Ashley,Stanley Robert F,Yu Yiting,Bartholdy Boris,Pendurti Gopichand,Gritsman Kira,Boultwood Jacqueline,Chernoff Jonathan,Verma Amit,Steidl Ulrich Blood Poor clinical outcome of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) has been attributed to failure of current chemotherapeutic regimens to target leukemic stem cells. We recently identified p21-activated kinase (PAK1) as a downstream effector molecule of H2.0-like homeobox (HLX), a gene functionally relevant for AML pathogenesis. In this study, we find that inhibition of PAK1 activity by small molecule inhibitors or by RNA interference leads to profound leukemia inhibitory effects both in vitro and in vivo. Inhibition of PAK1 induces differentiation and apoptosis of AML cells through downregulation of the MYC oncogene and a core network of MYC target genes. Importantly, we find that inhibition of PAK1 inhibits primary human leukemic cells including immature leukemic stem cell-enriched populations. Moreover, we find that PAK1 upregulation occurs during disease progression and is relevant for patient survival in MDS. Our studies highlight PAK1 as a novel target in AML and MDS and support the use of PAK1 inhibitors as a therapeutic strategy in these diseases. 10.1182/blood-2014-12-618801
    Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice. Pang Wendy W,Czechowicz Agnieszka,Logan Aaron C,Bhardwaj Rashmi,Poyser Jessica,Park Christopher Y,Weissman Irving L,Shizuru Judith A Blood The myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering. 10.1182/blood-2018-06-858159
    Myelodysplastic syndrome with excess blasts-2 associated with erythroid predominance. Pourabdollah Maryam,Chang Hong Blood 10.1182/blood-2016-10-745471
    The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype. Basiorka Ashley A,McGraw Kathy L,Eksioglu Erika A,Chen Xianghong,Johnson Joseph,Zhang Ling,Zhang Qing,Irvine Brittany A,Cluzeau Thomas,Sallman David A,Padron Eric,Komrokji Rami,Sokol Lubomir,Coll Rebecca C,Robertson Avril A B,Cooper Matthew A,Cleveland John L,O'Neill Luke A,Wei Sheng,List Alan F Blood Despite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1β (IL-1β) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and β-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear β-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. 10.1182/blood-2016-07-730556
    Targeting of the bone marrow microenvironment improves outcome in a murine model of myelodysplastic syndrome. Balderman Sophia R,Li Allison J,Hoffman Corey M,Frisch Benjamin J,Goodman Alexandra N,LaMere Mark W,Georger Mary A,Evans Andrew G,Liesveld Jane L,Becker Michael W,Calvi Laura M Blood In vitro evidence suggests that the bone marrow microenvironment (BMME) is altered in myelodysplastic syndromes (MDSs). Here, we study the BMME in MDS in vivo using a transgenic murine model of MDS with hematopoietic expression of the translocation product NUP98-HOXD13 (NHD13). This model exhibits a prolonged period of cytopenias prior to transformation to leukemia and is therefore ideal to interrogate the role of the BMME in MDS. In this model, hematopoietic stem and progenitor cells (HSPCs) were decreased in NHD13 mice by flow cytometric analysis. The reduction in the total phenotypic HSPC pool in NHD13 mice was confirmed functionally with transplantation assays. Marrow microenvironmental cellular components of the NHD13 BMME were found to be abnormal, including increases in endothelial cells and in dysfunctional mesenchymal and osteoblastic populations, whereas megakaryocytes were decreased. Both CC chemokine ligand 3 and vascular endothelial growth factor, previously shown to be increased in human MDS, were increased in NHD13 mice. To assess whether the BMME contributes to disease progression in NHD13 mice, we performed transplantation of NHD13 marrow into NHD13 mice or their wild-type (WT) littermates. WT recipients as compared with NHD13 recipients of NHD13 marrow had a lower rate of the combined outcome of progression to leukemia and death. Moreover, hematopoietic function was superior in a WT BMME as compared with an NHD13 BMME. Our data therefore demonstrate a contributory role of the BMME to disease progression in MDS and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function and overall survival. 10.1182/blood-2015-06-653113
    Differential diagnosis of myelodysplastic syndrome: anemia associated with copper deficiency. Villalba Ana,Senent Leonor Blood 10.1182/blood-2017-11-818013
    MSI2 is required for maintaining activated myelodysplastic syndrome stem cells. Taggart James,Ho Tzu-Chieh,Amin Elianna,Xu Haiming,Barlowe Trevor S,Perez Alexendar R,Durham Benjamin H,Tivnan Patrick,Okabe Rachel,Chow Arthur,Vu Ly,Park Sun Mi,Prieto Camila,Famulare Christopher,Patel Minal,Lengner Christopher J,Verma Amit,Roboz Gail,Guzman Monica,Klimek Virginia M,Abdel-Wahab Omar,Leslie Christina,Nimer Stephen D,Kharas Michael G Nature communications Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease. 10.1038/ncomms10739
    Characterization and targeting of malignant stem cells in patients with advanced myelodysplastic syndromes. Stevens Brett M,Khan Nabilah,D'Alessandro Angelo,Nemkov Travis,Winters Amanda,Jones Courtney L,Zhang Wei,Pollyea Daniel A,Jordan Craig T Nature communications Myelodysplastic syndrome (MDS) is a chronic hematologic disorder that frequently evolves to more aggressive stages and in some cases leads to acute myeloid leukemia (AML). MDS arises from mutations in hematopoietic stem cells (HSCs). Thus, to define optimal therapies, it is essential to understand molecular events driving HSC pathogenesis. In this study, we report that during evolution of MDS, malignant HSCs activate distinct cellular programs that render such cells susceptible to therapeutic intervention. Specifically, metabolic analyses of the MDS stem cell compartment show a profound activation of protein synthesis machinery and increased oxidative phosphorylation. Pharmacological targeting of protein synthesis and oxidative phosphorylation demonstrated potent and selective eradication of MDS stem cells in primary human patient specimens. Taken together, our findings indicate that MDS stem cells are reliant on specific metabolic events and that such properties can be targeted prior to the onset of clinically significant AML, during antecedent MDS. 10.1038/s41467-018-05984-x
    SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Malcovati Luca,Karimi Mohsen,Papaemmanuil Elli,Ambaglio Ilaria,Jädersten Martin,Jansson Monika,Elena Chiara,Gallì Anna,Walldin Gunilla,Della Porta Matteo G,Raaschou-Jensen Klas,Travaglino Erica,Kallenbach Klaus,Pietra Daniela,Ljungström Viktor,Conte Simona,Boveri Emanuela,Invernizzi Rosangela,Rosenquist Richard,Campbell Peter J,Cazzola Mario,Hellström Lindberg Eva Blood Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated erythroid dysplasia and 15% or more bone marrow ring sideroblasts. Ring sideroblasts are found also in other MDS subtypes, such as refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS). A high prevalence of somatic mutations of SF3B1 was reported in these conditions. To identify mutation patterns that affect disease phenotype and clinical outcome, we performed a comprehensive mutation analysis in 293 patients with myeloid neoplasm and 1% or more ring sideroblasts. SF3B1 mutations were detected in 129 of 159 cases (81%) of RARS or RCMD-RS. Among other patients with ring sideroblasts, lower prevalence of SF3B1 mutations and higher prevalence of mutations in other splicing factor genes were observed (P < .001). In multivariable analyses, patients with SF3B1 mutations showed significantly better overall survival (hazard ratio [HR], .37; P = .003) and lower cumulative incidence of disease progression (HR = 0.31; P = .018) compared with SF3B1-unmutated cases. The independent prognostic value of SF3B1 mutation was retained in MDS without excess blasts, as well as in sideroblastic categories (RARS and RCMD-RS). Among SF3B1-mutated patients, coexisting mutations in DNA methylation genes were associated with multilineage dysplasia (P = .015) but had no effect on clinical outcome. TP53 mutations were frequently detected in patients without SF3B1 mutation, and were associated with poor outcome. Thus, SF3B1 mutation identifies a distinct MDS subtype that is unlikely to develop detrimental subclonal mutations and is characterized by indolent clinical course and favorable outcome. 10.1182/blood-2015-03-633537
    Revisiting the case for genetically engineered mouse models in human myelodysplastic syndrome research. Zhou Ting,Kinney Marsha C,Scott Linda M,Zinkel Sandra S,Rebel Vivienne I Blood Much-needed attention has been given of late to diseases specifically associated with an expanding elderly population. Myelodysplastic syndrome (MDS), a hematopoietic stem cell-based blood disease, is one of these. The lack of clear understanding of the molecular mechanisms underlying the pathogenesis of this disease has hampered the development of efficacious therapies, especially in the presence of comorbidities. Mouse models could potentially provide new insights into this disease, although primary human MDS cells grow poorly in xenografted mice. This makes genetically engineered murine models a more attractive proposition, although this approach is not without complications. In particular, it is unclear if or how myelodysplasia (abnormal blood cell morphology), a key MDS feature in humans, presents in murine cells. Here, we evaluate the histopathologic features of wild-type mice and 23 mouse models with verified myelodysplasia. We find that certain features indicative of myelodysplasia in humans, such as Howell-Jolly bodies and low neutrophilic granularity, are commonplace in healthy mice, whereas other features are similarly abnormal in humans and mice. Quantitative hematopoietic parameters, such as blood cell counts, are required to distinguish between MDS and related diseases. We provide data that mouse models of MDS can be genetically engineered and faithfully recapitulate human disease. 10.1182/blood-2015-01-624239
    Clinical Implications of Genetic Mutations in Myelodysplastic Syndrome. Kennedy James A,Ebert Benjamin L Journal of clinical oncology : official journal of the American Society of Clinical Oncology Myelodysplastic syndrome (MDS) is clonal disorder characterized by ineffective hematopoiesis and a tendency to evolve into acute myeloid leukemia (AML). Genetic studies have enabled the identification of a set of recurrently mutated genes central to the pathogenesis of MDS, which can be organized into a limited number of cellular processes, including RNA splicing, epigenetic and traditional transcriptional regulation, and signal transduction. The sequential accumulation of mutations drives disease evolution from asymptomatic clonal hematopoiesis to frank MDS, and, ultimately, to secondary AML. This detailed understanding of the molecular landscape of MDS, coupled with the emergence of cost- and time-effective methodologies for DNA sequencing has led to the introduction of genetic studies into the clinical realm. Here, we review recent advances in our genetic understanding of MDS, with a particular focus on the emerging role for mutational data in clinical management as a potential tool to assist in diagnosis, risk stratification, and therapeutic decision-making. 10.1200/JCO.2016.71.0806
    Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level. Chen Jiahao,Kao Yun-Ruei,Sun Daqian,Todorova Tihomira I,Reynolds David,Narayanagari Swathi-Rao,Montagna Cristina,Will Britta,Verma Amit,Steidl Ulrich Nature medicine Myelodysplastic syndromes (MDS) frequently progress to acute myeloid leukemia (AML); however, the cells leading to malignant transformation have not been directly elucidated. As progression of MDS to AML in humans provides a biological system to determine the cellular origins and mechanisms of neoplastic transformation, we studied highly fractionated stem cell populations in longitudinal samples of patients with MDS who progressed to AML. Targeted deep sequencing combined with single-cell sequencing of sorted cell populations revealed that stem cells at the MDS stage, including immunophenotypically and functionally defined pre-MDS stem cells (pre-MDS-SC), had a significantly higher subclonal complexity compared to blast cells and contained a large number of aging-related variants. Single-cell targeted resequencing of highly fractionated stem cells revealed a pattern of nonlinear, parallel clonal evolution, with distinct subclones within pre-MDS-SC and MDS-SC contributing to generation of MDS blasts or progression to AML, respectively. Furthermore, phenotypically aberrant stem cell clones expanded during transformation and stem cell subclones that were not detectable in MDS blasts became dominant upon AML progression. These results reveal a crucial role of diverse stem cell compartments during MDS progression to AML and have implications for current bulk cell-focused precision oncology approaches, both in MDS and possibly other cancers that evolve from premalignant conditions, that may miss pre-existing rare aberrant stem cells that drive disease progression and leukemic transformation. 10.1038/s41591-018-0267-4
    A calcium- and calpain-dependent pathway determines the response to lenalidomide in myelodysplastic syndromes. Fang Jing,Liu Xiaona,Bolanos Lyndsey,Barker Brenden,Rigolino Carmela,Cortelezzi Agostino,Oliva Esther N,Cuzzola Maria,Grimes H Leighton,Fontanillo Celia,Komurov Kakajan,MacBeth Kyle,Starczynowski Daniel T Nature medicine Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway. 10.1038/nm.4127
    A variant erythroferrone disrupts iron homeostasis in -mutated myelodysplastic syndrome. Bondu Sabrina,Alary Anne-Sophie,Lefèvre Carine,Houy Alexandre,Jung Grace,Lefebvre Thibaud,Rombaut David,Boussaid Ismael,Bousta Abderrahmane,Guillonneau François,Perrier Prunelle,Alsafadi Samar,Wassef Michel,Margueron Raphaël,Rousseau Alice,Droin Nathalie,Cagnard Nicolas,Kaltenbach Sophie,Winter Susann,Kubasch Anne-Sophie,Bouscary Didier,Santini Valeria,Toma Andrea,Hunault Mathilde,Stamatoullas Aspasia,Gyan Emmanuel,Cluzeau Thomas,Platzbecker Uwe,Adès Lionel,Puy Hervé,Stern Marc-Henri,Karim Zoubida,Mayeux Patrick,Nemeth Elizabeta,Park Sophie,Ganz Tomas,Kautz Léon,Kosmider Olivier,Fontenay Michaëla Science translational medicine Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the splicing factor gene. Patients with MDS with mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative transcript in patients with MDS with the mutation. Induction of this transcript in primary -mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an gene mutation than in patients with wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with -mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant transcript that was restricted to -mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis. 10.1126/scitranslmed.aav5467
    SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function. Sun Jie,He Xin,Zhu Yinghui,Ding Zonghui,Dong Haojie,Feng Yimei,Du Juan,Wang Hanying,Wu Xiwei,Zhang Lei,Yu Xiaochun,Lin Allen,McDonald Tinisha,Zhao Dandan,Wu Herman,Hua Wei-Kai,Zhang Bin,Feng Lifeng,Tohyama Kaoru,Bhatia Ravi,Oberdoerffer Philipp,Chung Yang Jo,Aplan Peter D,Boultwood Jacqueline,Pellagatti Andrea,Khaled Samer,Kortylewski Marcin,Pichiorri Flavia,Kuo Ya-Huei,Carlesso Nadia,Marcucci Guido,Jin Hongchuan,Li Ling Cell stem cell Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs. 10.1016/j.stem.2018.07.018
    CSNK1A1 mutations and isolated del(5q) abnormality in myelodysplastic syndrome: a retrospective mutational analysis. Smith Alexander E,Kulasekararaj Austin G,Jiang Jie,Mian Syed,Mohamedali Azim,Gaken Joop,Ireland Robin,Czepulkowski Barbara,Best Steven,Mufti Ghulam J The Lancet. Haematology BACKGROUND:A mechanism for clonal growth advantage in isolated del(5q) disease remains elusive. CSNK1A1 resides on the critically deleted region, and deletion of this gene has been shown in mouse knockout and transplantation studies to produce some characteristics of bone marrow failure, including a proliferative advantage. We aimed to establish the frequency, nature, and clinical association of CSNK1A1 mutations in patients with myelodysplastic syndrome and associated myeloid neoplasms. METHODS:Between June 1, 2004, and May 31, 2014, in King's College (London, UK), we did whole-exome sequencing of five patients with isolated del(5q) followed by targeted screening for CSNK1A1 mutations and 20 myelodysplastic syndrome-associated mutations in 245 additional patients with myeloid neoplasms. All patients met present WHO diagnostic criteria for myelodysplastic syndrome and other related myeloid neoplasms. FINDINGS:39 (16%) of 250 patients with myeloid neoplasms had isolated del(5q), of whom seven (18%) had CSNK1A1 mutations. All these mutations were missense and presented in a highly conserved region that is implicated in ATP catalysis. Serial sampling and response to lenalidomide treatment showed that CSNK1A1 mutations were highly associated with the del(5q) clone. Only one patient with a CSNK1A1 mutation showed complete cytogenetic response to lenalidomide. Four (57%) of the seven patients carrying a CSNK1A1 mutation showed disease progression coupled with an increase in mutant allele burden (all four were on lenalidomide). We detected coexisting myelodysplastic syndrome-related gene mutations in patients with CSNK1A1 mutations, including TP53. INTERPRETATION:Similar to the effect of TP53 mutations on progression of del(5q) abnormality, mutant CSNK1A1 also gives rise to a poor prognosis in del(5q) abnormality, for which a coupled increase in P53 activation is suggested. CSNK1A1 mutations in del(5q) disease are important in the context of therapeutic manipulation and need incorporation into future prospective studies. FUNDING:Leukaemia and Lymphoma Research. 10.1016/S2352-3026(15)00050-2
    Clonal evolution in myelodysplastic syndromes. da Silva-Coelho Pedro,Kroeze Leonie I,Yoshida Kenichi,Koorenhof-Scheele Theresia N,Knops Ruth,van de Locht Louis T,de Graaf Aniek O,Massop Marion,Sandmann Sarah,Dugas Martin,Stevens-Kroef Marian J,Cermak Jaroslav,Shiraishi Yuichi,Chiba Kenichi,Tanaka Hiroko,Miyano Satoru,de Witte Theo,Blijlevens Nicole M A,Muus Petra,Huls Gerwin,van der Reijden Bert A,Ogawa Seishi,Jansen Joop H Nature communications Cancer development is a dynamic process during which the successive accumulation of mutations results in cells with increasingly malignant characteristics. Here, we show the clonal evolution pattern in myelodysplastic syndrome (MDS) patients receiving supportive care, with or without lenalidomide (follow-up 2.5-11 years). Whole-exome and targeted deep sequencing at multiple time points during the disease course reveals that both linear and branched evolutionary patterns occur with and without disease-modifying treatment. The application of disease-modifying therapy may create an evolutionary bottleneck after which more complex MDS, but also unrelated clones of haematopoietic cells, may emerge. In addition, subclones that acquired an additional mutation associated with treatment resistance (TP53) or disease progression (NRAS, KRAS) may be detected months before clinical changes become apparent. Monitoring the genetic landscape during the disease may help to guide treatment decisions. 10.1038/ncomms15099
    Prevalence, clinical characteristics, and prognosis of GATA2-related myelodysplastic syndromes in children and adolescents. Wlodarski Marcin W,Hirabayashi Shinsuke,Pastor Victor,Starý Jan,Hasle Henrik,Masetti Riccardo,Dworzak Michael,Schmugge Markus,van den Heuvel-Eibrink Marry,Ussowicz Marek,De Moerloose Barbara,Catala Albert,Smith Owen P,Sedlacek Petr,Lankester Arjan C,Zecca Marco,Bordon Victoria,Matthes-Martin Susanne,Abrahamsson Jonas,Kühl Jörn Sven,Sykora Karl-Walter,Albert Michael H,Przychodzien Bartlomiej,Maciejewski Jaroslaw P,Schwarz Stephan,Göhring Gudrun,Schlegelberger Brigitte,Cseh Annámaria,Noellke Peter,Yoshimi Ayami,Locatelli Franco,Baumann Irith,Strahm Brigitte,Niemeyer Charlotte M, Blood Germline GATA2 mutations cause cellular deficiencies with high propensity for myeloid disease. We investigated 426 children and adolescents with primary myelodysplastic syndrome (MDS) and 82 cases with secondary MDS enrolled in 2 consecutive prospective studies of the European Working Group of MDS in Childhood (EWOG-MDS) conducted in Germany over a period of 15 years. Germline GATA2 mutations accounted for 15% of advanced and 7% of all primary MDS cases, but were absent in children with MDS secondary to therapy or acquired aplastic anemia. Mutation carriers were older at diagnosis and more likely to present with monosomy 7 and advanced disease compared with wild-type cases. For stratified analysis according to karyotype, 108 additional primary MDS patients registered with EWOG-MDS were studied. Overall, we identified 57 MDS patients with germline GATA2 mutations. GATA2 mutations were highly prevalent among patients with monosomy 7 (37%, all ages) reaching its peak in adolescence (72% of adolescents with monosomy 7). Unexpectedly, monocytosis was more frequent in GATA2-mutated patients. However, when adjusted for the selection bias from monosomy 7, mutational status had no effect on the hematologic phenotype. Finally, overall survival and outcome of hematopoietic stem cell transplantation (HSCT) were not influenced by mutational status. This study identifies GATA2 mutations as the most common germline defect predisposing to pediatric MDS with a very high prevalence in adolescents with monosomy 7. GATA2 mutations do not confer poor prognosis in childhood MDS. However, the high risk for progression to advanced disease must guide decision-making toward timely HSCT. 10.1182/blood-2015-09-669937
    Assessment of ASC specks as a putative biomarker of pyroptosis in myelodysplastic syndromes: an observational cohort study. Basiorka Ashley A,McGraw Kathy L,Abbas-Aghababazadeh Farnoosh,McLemore Amy F,Vincelette Nicole D,Ward Grace A,Eksioglu Erika A,Sallman David A,Ali Najla Al,Padron Eric,Pinilla-Ibarz Javier,Komrokji Rami,Masala Erico,Santini Valeria,Kosmider Olivier,Fontenay Michaela,Fenaux Pierre,Sokol Lubomir,Wei Sheng,Fridley Brooke,List Alan F The Lancet. Haematology BACKGROUND:NLRP3 inflammasome-directed pyroptotic cell death drives ineffective haemopoiesis in myelodysplastic syndromes. During inflammasome assembly, the apoptosis-associated speck-like protein containing a CARD (PYCARD, commonly known as ASC) adaptor protein polymerises into large, filamentous clusters termed ASC specks that are released upon cytolysis. Specks are resistant to proteolytic degradation because of their prion-like structure, and therefore might serve as a biomarker for pyroptotic cell death in myelodysplastic syndromes. METHODS:This observational cohort study was done at the H Lee Moffitt Cancer Center (Tampa, FL, USA). Patients with myelodysplastic syndromes, healthy controls, and patients with non-myelodysplastic syndrome haematological cancers or type 2 diabetes were recruited. We used confocal and electron microscopy to visualise, and flow cytometry to quantify, ASC specks in peripheral blood and bone marrow plasma samples. Speck percentages were compared by t test or ANOVA, correlations were assessed by Spearman's rank correlation coefficient, and biomarker efficiency was assessed by receiver operating characteristics and area under the curve (AUC) analysis. FINDINGS:Between Jan 1, 2005, and Jan 12, 2017, we obtained samples from 177 patients with myelodysplastic syndromes and 29 healthy controls for the discovery cohort, and 113 patients with myelodysplastic syndromes and 31 healthy controls for the validation cohort. We also obtained samples from 22 patients with del(5q) myelodysplastic syndromes, 230 patients with non-myelodysplastic syndrome haematological cancers and 23 patients with type 2 diabetes. After adjustment for glucose concentration, the log-transformed mean percentage of peripheral blood plasma-derived ASC specks was significantly higher in the 177 patients with myelodysplastic syndromes versus the 29 age-matched, healthy donors (-0·41 [SD 0·49] vs -0·67 [0·59], p=0·034). The percentages of ASC specks in samples from patients with myelodysplastic syndromes were significantly greater than those in samples from individuals with every other haematological cancer studied (all p<0·05) except myelofibrosis (p=0·19). The findings were confirmed in the independent validation cohort (p<0·0001). Peripheral blood plasma danger-associated molecular pattern protein S100-A8 and protein S100-A9 concentrations from 144 patients with myelodysplastic syndromes from the discovery cohort directly correlated with ASC speck percentage (r=0·4, p<0·0001 for S100-A8 and r=0·2, p=0·017 for S100-A9). Patients with at least two somatic gene mutations had a significantly greater mean percentage of peripheral blood plasma ASC specks than patients with one or no mutation (-0·22 [SD 0·63] vs -0·53 [0·44], p=0·008). The percentage of plasma ASC specks was a robust marker for pyroptosis in myelodysplastic syndromes (AUC=0·888), in which a cutoff of 0·80 maximised sensitivity at 0·84 (95% CI 0·65-0·91) and specificity at 0·87 (0·58-0·97). INTERPRETATION:Our results underscore the pathobiological relevance of ASC specks and suggest that ASC specks are a sensitive and specific candidate plasma biomarker that provides an index of medullary pyroptotic cell death and ineffective haemopoiesis in patients with myelodysplastic syndromes. FUNDING:T32 Training Grant (NIH/NCI 5T32 CA115308-08), Edward P Evans Foundation, The Taub Foundation Grants Program, the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the H Lee Moffitt Cancer Center and Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (P30-CA076292). 10.1016/S2352-3026(18)30109-1