S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability of cancer stem cells.
Strekalova Elena,Malin Dmitry,Weisenhorn Erin M M,Russell Jason D,Hoelper Dominik,Jain Aayushi,Coon Joshua J,Lewis Peter W,Cryns Vincent L
Breast cancer research and treatment
PURPOSE:Many transformed cells and embryonic stem cells are dependent on the biosynthesis of the universal methyl-donor S-adenosylmethionine (SAM) from methionine by the enzyme MAT2A to maintain their epigenome. We hypothesized that cancer stem cells (CSCs) rely on SAM biosynthesis and that the combination of methionine depletion and MAT2A inhibition would eradicate CSCs. METHODS:Human triple (ER/PR/HER2)-negative breast carcinoma (TNBC) cell lines were cultured as CSC-enriched mammospheres in control or methionine-free media. MAT2A was inhibited with siRNAs or cycloleucine. The effects of methionine restriction and/or MAT2A inhibition on the formation of mammospheres, the expression of CSC markers (CD44/C24), MAT2A and CSC transcriptional regulators, apoptosis induction and histone modifications were determined. A murine model of metastatic TNBC was utilized to evaluate the effects of dietary methionine restriction, MAT2A inhibition and the combination. RESULTS:Methionine restriction inhibited mammosphere formation and reduced the CD44/C24 CSC population; these effects were partly rescued by SAM. Methionine depletion induced MAT2A expression (mRNA and protein) and sensitized CSCs to inhibition of MAT2A (siRNAs or cycloleucine). Cycloleucine enhanced the effects of methionine depletion on H3K4me3 demethylation and suppression of Sox9 expression. Dietary methionine restriction induced MAT2A expression in mammary tumors, and the combination of methionine restriction and cycloleucine was more effective than either alone at suppressing primary and lung metastatic tumor burden in a murine TNBC model. CONCLUSIONS:Our findings point to SAM biosynthesis as a unique metabolic vulnerability of CSCs that can be targeted by combining methionine depletion with MAT2A inhibition to eradicate drug-resistant CSCs.
Methionine is a metabolic dependency of tumor-initiating cells.
Wang Zhenxun,Yip Lian Yee,Lee Jia Hui Jane,Wu Zhengwei,Chew Hui Yi,Chong Pooi Kiat William,Teo Chin Chye,Ang Heather Yin-Kuan,Peh Kai Lay Esther,Yuan Ju,Ma Siming,Choo Li Shi Kimberly,Basri Nurhidayah,Jiang Xia,Yu Qiang,Hillmer Axel M,Lim Wan Teck,Lim Tony Kiat Hon,Takano Angela,Tan Eng Huat,Tan Daniel Shao Weng,Ho Ying Swan,Lim Bing,Tam Wai Leong
Understanding cellular metabolism holds immense potential for developing new classes of therapeutics that target metabolic pathways in cancer. Metabolic pathways are altered in bulk neoplastic cells in comparison to normal tissues. However, carcinoma cells within tumors are heterogeneous, and tumor-initiating cells (TICs) are important therapeutic targets that have remained metabolically uncharacterized. To understand their metabolic alterations, we performed metabolomics and metabolite tracing analyses, which revealed that TICs have highly elevated methionine cycle activity and transmethylation rates that are driven by MAT2A. High methionine cycle activity causes methionine consumption to far outstrip its regeneration, leading to addiction to exogenous methionine. Pharmacological inhibition of the methionine cycle, even transiently, is sufficient to cripple the tumor-initiating capability of these cells. Methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Methionine cycle enzymes are also enriched in other tumor types, and MAT2A expression impinges upon the sensitivity of certain cancer cells to therapeutic inhibition.
Expression of Notch Gene and Its Impact on Survival of Patients with Resectable Non-small Cell Lung Cancer.
Chen Chung-Yu,Chen Ying-Yin,Hsieh Min-Shu,Ho Chao-Chi,Chen Kuan-Yu,Shih Jin-Yuan,Yu Chong-Jen
Journal of Cancer
Notch signaling has been demonstrated to frequently participate in the process of lung carcinogenesis. This study aimed to search Notch expression in non-small cell lung cancer (NSCLC) and its impact on survival. From 2001 to 2011, patients with diagnosis of NSCLC who received surgical resection were included. The expression of Notch gene was assessed by real-time polymerase chain reaction (RT-PCR). Clinical characteristics, histological types, disease stages, and outcomes were analyzed. Ninety-seven patients with NSCLC being explored the expression of Notch gene (Notch1 - 4). Seventy-five patients (77.3%) were adenocarcinoma. Patients with adenocarcinoma had higher expression of Notch2 than other histology types ( < 0.001). Otherwise, patients with squamous cell carcinoma had relative higher expression of Notch1 and Notch3 expression ( = 0.014 and = 0.032, respectively). Notch2 expression increased associated with patients with more advanced lung cancer stage. Patients who had cancer recurrence also had higher Notch2 expression ( = 0.008). The patient group with lung adenocarcinoma of both high Notch1 and Notch3 expression had a shorter median disease-free survival (DFS) (both high v.s both low: DFS, median, 7.2 v.s 25.3 months, = 0.03). However, the expression of Notch gene had no impact on overall survival. Patients with lung adenocarcinoma had higher Notch2 expression. Patients with higher Notch2 expression also had higher rate of cancer recurrence. Both higher Notch1 and Notch3 expression was associated with poor prognosis in lung adenocarcinoma.
High NOTCH activity induces radiation resistance in non small cell lung cancer.
Theys Jan,Yahyanejad Sanaz,Habets Roger,Span Paul,Dubois Ludwig,Paesmans Kim,Kattenbeld Bo,Cleutjens Jack,Groot Arjan J,Schuurbiers Olga C J,Lambin Philippe,Bussink Jan,Vooijs Marc
Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology
BACKGROUND AND PURPOSE:Patients with advanced NSCLC have survival rates <15%. The NOTCH pathway plays an important role during lung development and physiology but is often deregulated in lung cancer, making it a potential therapeutic target. We investigated NOTCH signaling in NSCLC and hypothesized that high NOTCH activity contributes to radiation resistance. MATERIALS AND METHODS:NOTCH signaling in NSCLC patient samples was investigated using quantitative RT-PCR. H460 NSCLC cells with either high or blocked NOTCH activity were generated and their radiation sensitivity monitored using clonogenic assays. In vivo, xenograft tumors were irradiated and response assessed using growth delay. Microenvironmental parameters were analyzed by immunohistochemistry. RESULTS:Patients with high NOTCH activity in tumors showed significantly worse disease-free survival. In vitro, NOTCH activity did not affect the proliferation or intrinsic radiosensitivity of NSCLC cells. In contrast, xenografts with blocked NOTCH activity grew slower than wild type tumors. Tumors with high NOTCH activity grew significantly faster, were more hypoxic and showed a radioresistant phenotype. CONCLUSIONS:We demonstrate an important role for NOTCH in tumor growth and correlate high NOTCH activity with poor prognosis and radioresistance. Blocking NOTCH activity in NSCLC might be a promising intervention to improve outcome after radiotherapy.
Baicalein suppresses non small cell lung cancer cell proliferation, invasion and Notch signaling pathway.
Su Guangfeng,Chen Hao,Sun Xinhua
Cancer biomarkers : section A of Disease markers
BACKGROUND:Baicalein is an important Chinese herbal medicine and has multiple pharmacological activities. However, the biological mechanisms of the anti-tumor effects of Baicalein on non small cell lung cancer (NSCLC) still need to be understood. METHODS:Human NSCLC A549 and H1299 cells were pretreated with Baicalein or DMSO. Cells viability and transwell cell invasion assays were performed to assess cell proliferation and invasion. QRT-PCR assay was used to analyze mRNA expression levels of Twist1, E-cadhertin, Vimentin, Notch1 and hes-1. Western blot analysis was also performed to determine protein expression. RESULTS:In the study, we found that Baicalein had a significantly inhibited effect on proliferation ability of A549 and H1299 cells. Cells treated with Baicalein showed a down-regulated expression of CyclinD1 and CDK1 in A549 and H1299 cells. Furthermore, we found that Baicalein significantly inhibited cell invasion and Epithelial-Mesenchymal Transition (EMT) by up-regulating the mRNA and protein expression of E-cadherin and down-regulated the Twist1 and Vimentin expression, Moreover, Treatment of Baicalein down-regulated Notch1 and hes-1 expression in A549 and H1299 cells, which indicated that Baicalein could suppress the Notch signaling pathway. CONCLUSION:Our studies suggest that Baicalein may be a potential phytochemical flavonoid for therapeutics of NSCLC and serve as a molecular target for NSCLC.
Meta-analysis reveals the correlation of Notch signaling with non-small cell lung cancer progression and prognosis.
Yuan Xun,Wu Hua,Xu Hanxiao,Han Na,Chu Qian,Yu Shiying,Chen Yuan,Wu Kongming
Various studies have assessed the clinicopathological and prognostic value of Notch1 and Notch3 expression in Non-small cell lung cancer (NSCLC), but their results remain controversial. This meta-analysis was conducted to address the above issues by using a total of 19 studies involving 3663 patients. The correlations between Notch1 and Notch3 expression and clinicopathological features and NSCLC prognosis were analyzed. The meta-analysis indicated that higher expression of Notch1 was associated with greater possibility of lymph node metastasis and higher TNM stages. Moreover, patients with Notch1 overexpression and Notch3 overexpression showed significantly poor overall survival (Notch1: HR, 1.29; 95% CI, 1.06-1.57, p = 0.468 and I(2) = 0.0%; Notch3: HR, 1.57; 95%CI, 1.04-2.36, p = 0.445 and I(2) = 0.0%). Furthermore, there are statistically significant association between overall survival of NSCLC patients and the expression of Notch signaling ligand DLL3 and target gene HES1. Our meta-analysis supports that Notch signaling is a valuable bio-marker to predict progression and targeting Notch signaling could benefit subpopulation of NSCLC patients.
Epidermal growth factor receptor status and Notch inhibition in non-small cell lung cancer cells.
Giannopoulou Efstathia,Nikolakopoulos Achilleas,Kotsirilou Dimitra,Lampropoulou Angeliki,Raftopoulou Sofia,Papadimitriou Evangelia,Theocharis Achilleas D,Makatsoris Thomas,Fasseas Konstantinos,Kalofonos Haralabos P
Journal of biomedical science
BACKGROUND:Notch may behave as an oncogene or a tumor suppressor gene in lung cancer cells. Notch receptor undergoes cleavage by enzymes, including γ-secretase, generating the active Notch intracellular domain (NICD). The aim of the present study was to investigate the effect of DAPT, a γ-secretase inhibitor, in non-small cell lung cancer (NSCLC) cells, as well as the impact of epidermal growth factor (EGF) that is over-expressed by NSCLC cells, on Notch signaling. H23, A549, H661 and HCC827 human NSCLC cell lines were used, expressing various NICD and EGF receptor (EGFR) protein levels. RESULTS:DAPT decreased the number of H661 cells in a concentration-dependent manner, while it had a small effect on H23 and A549 cells and no effect on HCC827 cells that carry mutated EGFR. Notch inhibition did not affect the stimulatory effect of EGF on cell proliferation, while EGF prevented DAPT-induced NICD decrease in H23 and H661 cells. The type of cell death induced by DAPT seems to depend on the cell type. CONCLUSIONS:Our data indicate that inhibition of Notch cleavage may not affect cell number in the presence of EGFR mutations and that EGFR may affect Notch signalling suggesting that a dual inhibition of these pathways might be promising in NSCLC.
Non-small-cell lung carcinoma: role of the Notch signaling pathway.
Barse Levi,Bocchetta Maurizio
Lung Cancer (Auckland, N.Z.)
Notch signaling plays a pivotal role during embryogenesis. It regulates three fundamental processes: lateral inhibition, boundary formation, and lineage specification. During post-natal life, Notch receptors and ligands control critical cell fate decisions both in compartments that are undergoing differentiation and in pluripotent progenitor cells. First recognized as a potent oncogene in certain lymphoblastic leukemias and mesothelium-derived tissue, the role of Notch signaling in epithelial, solid tumors has been far more controversial. The overall consequence of Notch signaling and which form of the Notch receptor drives malignancy in humans is deeply debated. Most likely, this is due to the high degree of context-dependent effects of Notch signaling. More recently, it has been discovered that Notch (especially Notch-1) can exert different, even opposite effects in the same tissue under differing microenvironmental conditions. Further complicating the understanding of Notch receptors is the recently discovered role for non-canonical Notch signaling. Additionally, the most frequent Notch signaling antagonists used in biological systems have been inhibitors of the transmembrane protease complex γ-secretase, which itself processes a plethora of class one transmembrane proteins and thus cannot be considered a Notch-specific upstream regulator. Here we review the available empirical evidence gathered in recent years concerning Notch receptors and ligands in non-small-cell lung carcinoma (NSCLC). Although an overview of the field reveals seemingly contradicting results, we propose that Notch signaling can be exploited as a therapeutic target in NSCLC and represents a promising complement to the current arsenal utilized to combat this malignancy, particularly in targeting NSCLC tissues under specific environmental conditions, such as hypoxia.
Gambogic Acid Induces Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells by Suppressing Notch Signaling.
Zhu Minghua,Wang Minjie,Jiang Yinfang,Wu Hao,Lu Guirong,Shi Wei,Cong Degang,Song Shaohui,Liu Keyuan,Wang Hao
Medical science monitor : international medical journal of experimental and clinical research
BACKGROUND Activation of Notch signaling was found to be associated with cancer. Gambogic acid (GA) was reported to be an anti-cancer agent. This study investigated the anti-cancer effect of GA on human non-small cell lung cancer (NSCLC) cells. Involvement of the Notch pathway was also studied. MATERIAL AND METHODS GA at 0, 0.5, 0.75, and 1.0 μmol/l was used to incubate A549 and SPC-A1 cells. MTT assay was used to determine the cell viability. TUNEL assay was used to detect the apoptosis. Western blotting was used to evaluate protein expression levels, protein phosphorylation levels, and nuclear translocation levels. RESULTS Notch signaling pathway was activated in NSCLC cells. GA treatment significantly inhibited NSCLC cell viability and increased cell apoptosis. GA treatment significantly decreased the expression levels of DLL1, DLL3, DLL4, Jagged1, Jagged2, Bcl2, and PK3K, inhibited NICD nuclear translocation and Akt phosphorylation, and increased expression level of active caspase3. CONCLUSIONS GA inhibited NSCLC cell viability by inducing apoptosis. Inhibition of the Notch signaling pathway was the mechanism involved in the anti-proliferation effect of GA on NSCLC.
NOTCH decoys that selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth.
Kangsamaksin Thaned,Murtomaki Aino,Kofler Natalie M,Cuervo Henar,Chaudhri Reyhaan A,Tattersall Ian W,Rosenstiel Paul E,Shawber Carrie J,Kitajewski Jan
UNLABELLED:A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)-class and JAG-class ligand-receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG-NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11-13 decoy interfered with DLL1-DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG-NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. SIGNIFICANCE:This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.
Activation of notch 3/c-MYC/CHOP axis regulates apoptosis and promotes sensitivity of lung cancer cells to mTOR inhibitor everolimus.
Li Ting,Xu Xiao-Huang,Guo Xia,Yuan Tao,Tang Zheng-Hai,Jiang Xiao-Ming,Xu Yu-Lian,Zhang Le-Le,Chen Xiuping,Zhu Hong,Shi Jia-Jie,Lu Jin-Jian
The mammalian target of rapamycin (mTOR) pathway converges diverse environmental cues to support the lung cancer growth and survival. However, the mTOR-targeted mono-therapy does not achieve expected therapeutic effect. Here, we revealed that fangchinoline (FCL), an active alkaloid that purified from the traditional Chinese medicine Stephania tetrandra S. Moore, enhanced the anti-lung cancer effect of mTOR inhibitor everolimus (EVE). The combination of EVE and FCL was effective to activate Notch 3, and subsequently evoked its downstream target c-MYC. The blockage of Notch 3 signal by the molecular inhibitor of γ-secretase or siRNA of Notch 3 reduced the c-MYC expression and attenuated the combinational efficacy of EVE and FCL on cell apoptosis and proliferation. Moreover, the c-MYC could bind to the C/EBP homologous protein (CHOP) promoter and facilitate CHOP transcription. The conditional genetic deletion of CHOP reduced the apoptosis on lung cancer cells to the same degree as blockage of Notch 3/c-MYC axis, providing further evidence for that the Notch 3/c-MYC axis regulates the transcription of CHOP and finally induces apoptosis upon co-treatment of FCL and EVE in lung cancer cells. Overall, our findings, to the best of our knowledge, firstly link CHOP to Notch 3/c-MYC axis-dependent apoptosis and provide the Notch 3/c-MYC/CHOP activation as a promising strategy for mTOR-targeted combination therapy in lung cancer treatment.
NK cells and ILCs in tumor immunotherapy.
Sivori Simona,Pende Daniela,Quatrini Linda,Pietra Gabriella,Della Chiesa Mariella,Vacca Paola,Tumino Nicola,Moretta Francesca,Mingari Maria Cristina,Locatelli Franco,Moretta Lorenzo
Molecular aspects of medicine
Cells of the innate immunity play an important role in tumor immunotherapy. Thus, NK cells can control tumor growth and metastatic spread. Thanks to their strong cytolytic activity against tumors, different approaches have been developed for exploiting/harnessing their function in patients with leukemia or solid tumors. Pioneering trials were based on the adoptive transfer of autologous NK cell-enriched cell populations that were expanded in vitro and co-infused with IL-2. Although relevant results were obtained in patients with advanced melanoma, the effect was mostly limited to certain metastatic localizations, particularly to the lung. In addition, the severe IL-2-related toxicity and the preferential IL-2-induced expansion of Treg limited this type of approach. This limitation may be overcome by the use of IL-15, particularly of modified IL-15 molecules to improve its half-life and optimize the biological effects. Other approaches to harness NK cell function include stimulation via TLR, the use of bi- and tri-specific NK cell engagers (BiKE and TriKE) linking activating NK receptors (e.g. CD16) to tumor-associated antigens and even incorporating an IL-15 moiety (TriKE). As recently shown, in tumor patients, NK cells may also express inhibitory checkpoints, primarily PD-1. Accordingly, the therapeutic use of checkpoint inhibitors may unleash NK cells against PD-L1 tumors. This effect may be predominant and crucial in tumors that have lost HLA cl-I expression, thus resulting "invisible" to T lymphocytes. Additional approaches in which NK cells may represent an important tool for cancer therapy, are to exploit the unique properties of the "adaptive" NK cells. These CD57 NKG2C cells, despite their mature stage and a potent cytolytic activity, maintain a strong proliferating capacity. This property revealed to be crucial in hematopoietic stem cell transplantation (HSCT), particularly in the haplo-HSCT setting, to cure high-risk leukemias. T depleted haplo-HSCT (e.g. from one of the parents) allowed to save the life of thousands of patients lacking a HLA-compatible donor. In this setting, NK cells have been shown to play an essential role against leukemia cells and infections. Another major advance is represented by chimeric antigen receptor (CAR)-engineered NK cells. CAR-NK, different from CAR-T cells, may be obtained from allogeneic donors since they do not cause GvHD. Accordingly, they may represent "off-the-shelf" products to promptly treat tumor patients, with affordable costs. Different from NK cells, helper ILC (ILC1, ILC2 and ILC3), the innate counterpart of T helper cell subsets, remain rather ambiguous with respect to their anti-tumor activity. A possible exception is represented by a subset of ILC3: their frequency in peri-tumoral tissues in patients with NSCLC directly correlates with a better prognosis, possibly reflecting their ability to contribute to the organization of tertiary lymphoid structures, an important site of T cell-mediated anti-tumor responses. It is conceivable that innate immunity may significantly contribute to the major advances that immunotherapy has ensured and will continue to ensure to the cure of cancer.
Combining Immunotherapy and Chemotherapy for Non-Small Cell Lung Cancer.
Judd Julia,Borghaei Hossein
Thoracic surgery clinics
Over the past year, the combination of platinum-based chemotherapy and immunotherapy has become the standard of care for patients with metastatic non-small-cell lung cancer with any programmed death ligand 1 tumor proportion score. There is preclinical evidence demonstrating potential synergistic immunomodulation with combination therapy by enhancing immune-mediated tumor death and by disrupting the immunosuppressive tumor microenvironment that prevents immune detection. This potential synergy or complementary activity has been demonstrated in clinical trials showing improved and durable responses with chemo-immunotherapy.
Mesothelin-targeted second generation CAR-T cells inhibit growth of mesothelin-expressing tumors .
Ye Lin,Lou Yuqing,Lu Liming,Fan Xiaohong
Experimental and therapeutic medicine
Non-small cell lung cancer (NSCLC) and mesothelioma are renowned for being diagnosed at a late stage and poor prognosis. Although surgery, chemotherapy, and radiotherapy have yielded successful outcomes, the improvement on the survival rate of NSCLC and mesothelioma have been less marked. Recently, adoptive immunotherapy, particularly chimeric antigen receptor T (CAR-T) cell therapy demonstrated promise for improving the survival of acute lymphoblastic leukemia with minimum toxicity. However, its application in solid tumors still warrants in-depth investigations and multiple consistent trial results, particularly in eliminating 'off-tumor' toxicity. To explore CAR-T therapy in NSCLC and mesothelioma, second-generation CAR-T cells were constructed targeting mesothelin (MSLN), which is abundant in NSCLC and mesothelioma but is under expressed in normal tissues. The second-generation design incorporated co-stimulatory CD28 and 4-1BB signaling domains to enhance the proliferation. Following the successful analysis of CAR-T cells by flow cytometry, cytotoxicity experiments were performed using the LDH kit to verify the killing effect of CAR-T cells on target cells. Otherwise, the killing tumor activity of MSLN CAR-T cells was verified by constructing a mouse model using tumor-derived cells from patients to inoculate the mice. When the effector-to-target ratio is >0.5:1, CAR-T MSLN cells exhibited significantly higher ability to kill tumor cells than T cells. In experiments, mice whose tail vein was injected with CAR-T MSLN cells demonstrated significantly slower tumor growth. Without continuous administration, both groups became gradually synchronized in growth of tumor size, which suggests that the persistence of CAR-T cells is an important issue in preclinical studies.
Chimeric antigen receptor T cells targeting PD-L1 suppress tumor growth.
Qin Le,Zhao Ruocong,Chen Dongmei,Wei Xinru,Wu Qiting,Long Youguo,Jiang Zhiwu,Li Yangqiu,Wu Haipeng,Zhang Xuchao,Wu Yilong,Cui Shuzhong,Wei Wei,Yao Huihui,Liu Zixia,Cao Su,Yao Yao,Zhang Zhenfeng,Li Peng
Background:Chimeric antigen receptor T cells (CAR-T cells) therapy has been well recognized for treating B cell-derived malignancy. However, the efficacy of CAR-T cells against solid tumors remains dissatisfactory, partially due to the heterogeneity of solid tumors and T cell exhaustion in tumor microenvironment. PD-L1 is up-regulated in multiple solid tumors, resulting in T cell exhaustion upon binding to its receptor PD-1. Methods:Here, we designed a dominant-negative form of PD-1, dPD1z, a vector containing the extracellular and transmembrane regions of human PD-1, and a CAR vector against PD-L1, CARPD-L1z, a vector employs a high-affinity single-chain variable fragment (scFv) against human PD-L1. These two vectors shared the same intracellular structure, including 4-1BB and TLR2 co-stimulatory domains, and the CD3ζ signaling domain. Results:dPD1z T and CARPD-L1z T cells efficiently lysed PD-L1 tumor cells and had enhanced cytokine secretion in vitro and suppressed the growth of non-small cell lung cancer (NSCLC), gastric cancer and hepatoma carcinoma in patient-derived xenograft (PDX). However, the combination of anti-mesothelin CAR-T cells (CARMSLNz T) with dPD1z T or CARPD-L1z T cells did not repress tumor growth synergistically in PDX, as CARMSLNz T cells upregulated PD-L1 expression upon activation and were subsequently attacked by dPD1z T or CARPD-L1z T cells. Conclusions:In conclusion, we demonstrate CAR-T cells targeting PD-L1 were effective for suppressing the growth of multiple types of solid tumors in PDX models though their safety needs to be carefully examined.
Chimeric antigen receptor T cell targeting EGFRvIII for metastatic lung cancer therapy.
Zhang Zhao,Jiang Jun,Wu Xiaodong,Zhang Mengyao,Luo Dan,Zhang Renyu,Li Shiyou,He Youwen,Bian Huijie,Chen Zhinan
Frontiers of medicine
Lung cancer is the most common incident cancer and the leading cause of cancer death. In recent years, the development of tumor immunotherapy especially chimeric antigen receptor T (CAR-T) cell has shown a promising future. Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation expressed in various types of tumors and has been detected in non-small cell lung cancer with a mutation rate of 10%. Thus, EGFRvIII is a potential antigen for targeted lung cancer therapy. In this study, CAR vectors were constructed and transfected into virus-packaging cells. Then, activated T cells were infected with retrovirus harvested from stable virus-producing single clone cell lines. CAR expression on the surfaces of the T cells was detected by flow cytometry and Western blot. The function of CAR-T targeting EGFRvIII was then evaluated. The EGFRvIII-CAR vector was successfully constructed and confirmed by DNA sequencing. A stable virus-producing cell line was produced from a single clone by limited dilution. The culture conditions for the cell line, including cell density, temperature, and culture medium were optimized. After infection with retrovirus, CAR was expressed on more than 90% of the T cells. The proliferation of CAR-T cells were induced by cytokine and specific antigen in vitro. More importantly, EGFRvIII-CART specifically and efficiently recognized and killed A549-EGFRvIII cells with an effector/target ratio of 10:1 by expressing and releasing cytokines, including perforin, granzyme B, IFN-γ, and TNF-α. The in vivo study indicated that the metastasis of A549-EGFRvIII cells in mice were inhibited by EGFRvIII-CART cells, and the survival of the mice was significantly prolonged with no serious side effects. EGFRvIII-CART showed significantly efficient antitumor activity against lung cancer cells expressing EGFRvIII in vivo and in vitro. Therefore, CAR-T targeting EGFRvIII is a potential therapeutic strategy in preventing recurrence and metastasis of lung cancer after surgery.
Chimeric antigen receptor (CAR)-T-cell therapy in non-small-cell lung cancer (NSCLC): current status and future perspectives.
Qu Jingjing,Mei Quanhui,Chen Lijun,Zhou Jianying
Cancer immunology, immunotherapy : CII
There has been a rapid progress in developing genetically engineered T cells in recent years both in basic and clinical cancer studies. Chimeric antigen receptor (CAR)-T cells exert an immune response against various cancers, including the non-small-cell lung cancer (NSCLC). As novel agents of immunotherapy, CAR-T cells show great promise for NSCLC. However, targeting specific antigens in NSCLC with engineered CAR-T cells is complicated because of a lack of tumor-specific antigens, the immunosuppressive tumor microenvironment, low levels of infiltration of CAR-T cells into tumor tissue, and tumor antigen escape. Meanwhile, the clinical application of CAR-T cells remains limited due to the cases of on-target/off-tumor and neurological toxicity, as well as cytokine release syndrome. Hence, optimal CAR-T-cell design against NSCLC is urgently needed. In this review, we describe the basic structure and generation of CAR-T cells and summarize the common tumor-associated antigens targeted in clinical trials on CAR-T-cell therapy for NSCLC, as well as point out current challenges and novel strategies. Although many obstacles remain, the new/next generation of CARs show much promise. Taken together, research on CAR-T cells for the treatment of NSCLC is underway and has yielded promising preliminary results both in basic and pre-clinical medicine. More pre-clinical experiments and clinical trials are, therefore, warranted.
Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer.
Feng Kaichao,Guo Yelei,Dai Hanren,Wang Yao,Li Xiang,Jia Hejin,Han Weidong
Science China. Life sciences
The successes achieved by chimeric antigen receptor-modified T (CAR-T) cells in hematological malignancies raised the possibility of their use in non-small lung cancer (NSCLC). In this phase I clinical study (NCT01869166), patients with epidermal growth factor receptor (EGFR)-positive (>50% expression), relapsed/refractory NSCLC received escalating doses of EGFR-targeted CAR-T cell infusions. The EGFR-targeted CAR-T cells were generated from peripheral blood after a 10 to 13-day in vitro expansion. Serum cytokines in peripheral blood and copy numbers of CAR-EGFR transgene in peripheral blood and in tissue biopsy were monitored periodically. Clinical responses were evaluated with RECIST1.1 and immune- related response criteria, and adverse events were graded with CTCAE 4.0. The EGFR-targeted CAR-T cell infusions were well-tolerated without severe toxicity. Of 11 evaluable patients, two patients obtained partial response and five had stable disease for two to eight months. The median dose of transfused CAR(+) T cells was 0.97×10(7) cells kg(-1) (interquartile range (IQR), 0.45 to 1.09×10(7) cells kg(-1)). Pathological eradication of EGFR positive tumor cells after EGFR-targeted CAR-T cell treatment can be observed in tumor biopsies, along with the CAR-EGFR gene detected in tumor-infiltrating T cells in all four biopsied patients. The EGFR-targeted CAR-T cell therapy is safe and feasible for EGFR-positive advanced relapsed/refractory NSCLC.
Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice.
Li He,Huang Yao,Jiang Du-Qing,Cui Lian-Zhen,He Zhou,Wang Chao,Zhang Zhi-Wei,Zhu Hai-Li,Ding Yong-Mei,Li Lin-Fang,Li Qiang,Jin Hua-Jun,Qian Qi-Jun
Cell death & disease
Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.
Pretreatment Neutrophil-to-Lymphocyte Ratio (NLR) May Predict the Outcomes of Advanced Non-small-cell Lung Cancer (NSCLC) Patients Treated With Immune Checkpoint Inhibitors (ICIs).
Li Ye,Zhang Zhibo,Hu Yi,Yan Xiang,Song Qi,Wang Guoqiang,Chen Runzhe,Jiao Shunchang,Wang Jinliang
Frontiers in oncology
Recent studies have demonstrated the predictive value of pretreatment neutrophil-to-lymphocyte ratio (NLR) in advanced cancers; however, the role of NLR in patients with advanced non-small-cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs) remained to be explored. Thus, we aimed to investigate whether pretreatment NLR was associated with the outcomes of advanced NSCLC patients treated with ICIs. A comprehensive literature research was first conducted in PubMed, the Cochrane Central Library, and Embase for studies that evaluated the association between pretreatment NLR and survival of advanced NSCLC patients with ICIs treatment. We then conducted a retrospective study in Chinese People's Liberation Army (PLA) General Hospital (Beijing, China) to validate these findings. A total of 17 eligible studies with 2,106 patients were included in our meta-analysis, of which, 12 studies reported progression-free survival (PFS), and 13 studies reported overall survival (OS). The pooled results showed that high pretreatment NLR was significantly associated with poorer PFS (HR = 1.44, 95% CI 1.26-1.65; < 0.001) and OS (HR = 2.86, 95% CI 2.11-3.87; < 0.001) compared with those with low pretreatment NLR. Subgroup analysis demonstrated that the association between baseline NLR and PFS remained significant except that the cut-off value of NLR was 3 (HR = 1.48, 95% CI 0.93-2.37; = 0.098) and region of Asia (HR = 1.55, 95% CI 1.00-2.39; = 0.051). These results were further validated in our retrospective study that patients with pretreatment NLR ≥ 6.0 had shorter PFS (median: 5.0 vs. 9.1 months, HR = 1.39; 95% CI 1.01-1.91; = 0.02) and OS (median: 10.0 vs. 17.3 months, HR = 1.71; 95% CI 1.18-2.46; < 0.001) compared with those with NLR < 6.0. The associations between NLR and survival were consistent in subgroup analysis stratified by age, gender, ECOG PS, histology, stage, smoking history, treatment, and prior lines of therapy. Dynamics of NLR (dNLR) that increased ≥3.0 from baseline was also significantly associated with worse PFS (median: 3.1 vs. 9.1 months; = 0.01) and OS (median: 6.8 vs. 17.0 months; < 0.0001). Our study demonstrates that pretreatment NLR and dNLR from baseline are associated with the outcomes of advanced NSCLC patients treated with ICIs; however, it warrants further prospective studies.
Dualistic Role of BARD1 in Cancer.
Cimmino Flora,Formicola Daniela,Capasso Mario
BRCA1 Associated RING Domain 1 (BARD1) encodes a protein which interacts with the N-terminal region of BRCA1 in vivo and in vitro. The full length (FL) BARD1 mRNA includes 11 exons and encodes a protein comprising of six domains (N-terminal RING-finger domain, three Ankyrin repeats and two C-terminal BRCT domains) with different functions. Emerging data suggest that BARD1 can have both tumor-suppressor gene and oncogene functions in tumor initiation and progression. Indeed, whereas FL BARD1 protein acts as tumor-suppressor with and without BRCA1 interactions, aberrant splice variants of BARD1 have been detected in various cancers and have been shown to play an oncogenic role. Further evidence for a dualistic role came with the identification of BARD1 as a neuroblastoma predisposition gene in our genome wide association study which has demonstrated that single nucleotide polymorphisms in BARD1 can correlate with risk or can protect against cancer based on their association with the expression of FL and splice variants of BARD1. This review is an overview of how BARD1 functions in tumorigenesis with opposite effects in various types of cancer.
Met inhibition revokes IFNγ-induction of PD-1 ligands in MET-amplified tumours.
Martin Valentina,Chiriaco Cristina,Modica Chiara,Acquadro Anna,Cortese Marco,Galimi Francesco,Perera Timothy,Gammaitoni Loretta,Aglietta Massimo,Comoglio Paolo M,Vigna Elisa,Sangiolo Dario
British journal of cancer
BACKGROUND:Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours. METHODS:PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy. RESULTS:PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression. CONCLUSIONS:These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.
EGFR-TKI resistance promotes immune escape in lung cancer via increased PD-L1 expression.
Peng Shunli,Wang Rong,Zhang Xiaojuan,Ma Yueyun,Zhong Longhui,Li Ke,Nishiyama Akihiro,Arai Sachiko,Yano Seiji,Wang Wei
BACKGROUND:The ATLANTIC trial reported that higher PD-L1 expression in tumors was involved in a higher objective response in patients with EGFR/ALK non-small cell lung cancer (NSCLC), indicating the possibility of anti-PD-1/PD-L1 therapy as a third-line (or later) treatment for advanced NSCLC. Therefore, the determination of status and regulatory mechanisms of PD-L1 in EGFR mutant NSCLC before and after acquired EGFR-TKIs resistance are meaningful. METHODS:The correlation among PD-L1, c-MET, and HGF was analyzed based on TCGA datasheets and paired NSCLC specimens before and after acquired EGFR-TKI resistance. EGFR-TKI resistant NSCLC cells with three well-known mechanisms, c-MET amplification, hepatocyte growth factor (HGF), and EGFR-T790M, were investigated to determinate PD-L1 expression status and immune escape ability. PD-L1-deleted EGFR-TKIs sensitive and resistant cells were used to evaluate the immune escape ability of tumors in mice xenograft models. RESULTS:Positive correlations were found among PD-L1, c-MET, and HGF, based on TCGA datasheets and paired NSCLC specimens. Moreover, the above three resistant mechanisms increased PD-L1 expression and attenuated activation and cytotoxicity of lymphocytes in vitro and in vivo, and downregulation of PD-L1 partially restored the cytotoxicity of lymphocytes. Both MAPK and PI3K pathways were involved in the three types of resistance mechanism-induced PD-L1 overexpression, whereas the NF-kappa B pathway was only involved in T790M-induced PD-L1 expression. CONCLUSIONS:HGF, MET-amplification, and EGFR-T790M upregulate PD-L1 expression in NSCLC and promote the immune escape of tumor cells through different mechanisms.
Immunotherapy Combined with Chemotherapy as a Promising Therapy for a EGFR Exon 19 Deletion with MET Amplification Patient with Non-Small-Cell Lung Cancer: A Case Report.
Ni QingTao,Pan Chi,Dai ShengBin,Wang Peng
OncoTargets and therapy
Advanced non-small-cell lung cancer (NSCLC) patients with EGFR exon 19 deletion often get benefits from the treatment of tyrosine kinase inhibitors (TKI). In the same way, the NSCLC patients with mesenchymal-to-epithelial transition (MET) amplification get benefits from crizotinib. The treatment becomes extremely difficult for the patients with both EGFR exon 19 deletion and MET amplification, after failure of first-line TKI. An advanced NSCLC patient with EGFR exon 19 deletion was treated with TKI. However, the disease recurred after four months. MET amplification was found after biopsy again. The patient was treated with the combination of crizotinib, while the disease recurred after eight months. The patient was treated by pembrolizumab and pemetrexed + carboplatin chemotherapy as salvage therapy. The therapeutic effect has been remarkable up to present. In conclusion, immunotherapy combined with chemotherapy could be a promising therapy for the NSCLC patients with both EGFR exon 19 deletion and MET amplification after the failure of first-line TKI treatment. Thus, further insights into the variant genes contribute to NSCLC treatment.
Correlation of gene amplification and mutation with PD-L1 expression in non-small cell lung cancer.
Albitar Maher,Sudarsanam Sucha,Ma Wanlong,Jiang Shiping,Chen Wayne,Funari Vincent,Blocker Forrest,Agersborg Sally
Background:The role of amplification in lung cancer, particularly in relation to checkpoint inhibition and WT, has not been fully explored. In this study, we correlated PD-L1 expression with amplification and , , or mutation in primary lung cancer. Methods:In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for amplification by FISH with a /centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and , , and mutations were tested using next generation sequencing. Results:Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with mutation (P=0.0003), and positively correlated with mutation (P=0.0001) and amplification (P=0.004). Patients with mutations had significantly higher amplification (P=0.007), and were more likely (P=0.0002) to be wild type. There was no correlation between mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and with co-mutation (P=0.0002). A cut-off for the ratio of : centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as amplified. Conclusions:This data suggests that in lung cancer both and play direct roles in regulating PD-L1 opposing . Moreover, and co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification.
Durable responses to immunotherapy of non-small cell lung cancers harboring MET exon-14-skipping mutation: A series of 6 cases.
Mayenga Marie,Assié Jean-Baptiste,Monnet Isabelle,Massiani Marie-Ange,Tabeze Laure,Friard Sylvie,Fraboulet Séverine,Métivier Anne-Cécile,Chouaïd Christos,Zemoura Leïla,Longchampt Elisabeth,Callens Céline,Melaabi Samia,Couderc Louis-Jean,Doubre Hélène
Lung cancer (Amsterdam, Netherlands)
INTRODUCTION:About 2-3% of non-small-cell lung cancers (NSCLCs) harbor MET exon-14-skipping (METex14) mutations. Efficacy of the MET-inhibitor crizotinib has been reported, but progression-free survival (PFS) was very short. Immune-checkpoint inhibitors (ICIs) have become a cornerstone of NSCLC treatment but appear to be less effective in non-smokers and against tumors exhibiting oncogenic addiction. We describe 6 remarkable (PFS exceeding 18 months) and durable responses to ICIs of NSCLCs harboring a METex14 mutation. METHODS:Each patient's clinical and biological characteristics, and tumor responses after ICIs were examined. Complete tumor-DNA sequencing was available after starting second-line ICIs, which followed first-line chemotherapy. Tumor-cell programmed cell-death protein-1 ligand-1 (PD-L1) expression on tumor cells was evaluated using antibody clone E1L3N (Cell Signaling Technology). RESULTS:Among 25 patients with METex14-mutated NSCLCs, 13 of whom were ICI-treated, 6 had prolonged responses: 5 women, 1 man; 57-80 years old; 3 never-smokers, 1 ex-smoker and 2 smokers; 5 adenocarcinomas, 1 sarcomatoid carcinoma; 5 received nivolumab, 1 pembrolizumab. No EGFR, BRAF or KRAS mutations (only 1 minority KRAS mutation), or ALK or ROS translocations were detected. No concurrent MET amplification was observed. Tumor-mutation burden was low (<10 mutations/Mb) in 3 tested tumors. Four partial and 2 complete responses were obtained during the first 3 months for 5 patients, while pseudoprogression was initially observed in 1. Tolerance was excellent, with only 1 grade-3 immune-related adverse event. Response was maintained for 18-49 months. CONCLUSION:ICIs could be considered to treat patients whose NSCLCs harbor a METex14 mutation. More biological marker data are needed to identify which patients are most likely to benefit from ICIs.
Elucidation of the relationships of MET protein expression and gene copy number status with PD-L1 expression and the immune microenvironment in non-small cell lung cancer.
Yoshimura Katsuhiro,Inoue Yusuke,Tsuchiya Kazuo,Karayama Masato,Yamada Hidetaka,Iwashita Yuji,Kawase Akikazu,Tanahashi Masayuki,Ogawa Hiroshi,Inui Naoki,Funai Kazuhito,Shinmura Kazuya,Niwa Hiroshi,Suda Takafumi,Sugimura Haruhiko
Lung cancer (Amsterdam, Netherlands)
OBJECTIVES:Alterations in the MET gene, such as mutations and high-level amplification, are important drivers of non-small cell lung cancer (NSCLC). The efficacy of immune checkpoint inhibitors (ICIs) in lung cancer with MET abnormalities is unclear. We evaluate the potential relationship between MET alterations and the tumor immune microenvironment and PD-1/PD-L1 axis. MATERIAL AND METHODS:MET and phospho-MET protein expression were assessed in 622 resected NSCLC specimens. MET amplification was assessed by fluorescence in-situ hybridization in 272 tumors. PD-L1 expression was evaluated by immunohistochemistry. CD8+, Foxp3+, CD45RO, and PD-1+ tumor-infiltrating lymphocytes (TILs) in the tumor nest and surrounding stroma were profiled. Associations with MET alterations were explored. RESULTS:The cohort comprised 425 male patients (68.3 %), 184 never-smokers (29.6 %), and 408 adenocarcinoma (ADC) patients (65.6 %). Median age was 68 years. MET alteration was observed mainly in ADCs (18.9 % MET-positive, 3.9 % phospho-MET-positive, and 15.1 % with MET amplification). PD-L1 expression was significantly increased in MET-altered ADCs (P < 0.001 for MET; P = 0.002 for phospho-MET; P = 0.019 for MET amplification). Most TIL subset numbers in the tumor nest were significantly increased in MET-altered tumors. Only MET amplification was independently associated with tumoral CD8 + TILs. Three of the six patients responded to ICI treatment; two of them showed MET overexpression and an increase in MET copy number. CONCLUSION:MET-altered tumors showed significantly stronger PD-L1 expression and more abundant tumoral TILs than non-MET-altered tumors. Among the MET alterations assessed, MET amplification was particularly implicated in the inflamed microenvironment, suggesting that MET-amplified tumors might respond to ICIs.
Beyond Tumor PD-L1: Emerging Genomic Biomarkers for Checkpoint Inhibitor Immunotherapy.
Lagos Galina G,Izar Benjamin,Rizvi Naiyer A
American Society of Clinical Oncology educational book. American Society of Clinical Oncology. Annual Meeting
Despite the success of immune checkpoint blockade as a strategy for activating an antitumor immune response and promoting cancer regression, only a subset of patients have durable clinical benefit. Efforts are ongoing to identify robust biomarkers that can effectively predict treatment response to immune checkpoint inhibitors (ICIs). Although PD-L1 expression is useful for stratifying patients, it is an imperfect tool. Comprehensive next-generation sequencing platforms that are readily used in clinical practice to identify a tumor's potentially actionable genetic alterations also reveal tumor genomic features, including tumor mutation burden (TMB), that may impact the response to ICIs. High TMB enhances tumor immunogenicity through increased numbers of tumor neoantigens that may promote an immune response. Defective DNA repair, leading to microsatellite instability, is an endogenous mechanism for increased tumor TMB that augments response to anti-PD-1 blockade. Alternatively, DNA damage from exogenous factors is responsible for high TMB seen in melanoma, lung cancer, and urothelial carcinoma, among tumor subtypes with higher response rates to ICIs. In this review, we summarize data supporting the use of TMB as a biomarker as well as its known limitations. We also highlight specific tumor suppressor genes and oncogenes that are under investigation as biomarkers for ICI response and resistance. Efforts are ongoing to delineate which genomic tumor characteristics can eventually be utilized in clinical practice to ascertain the benefit of ICIs for an individual patient.
Characterization of tumor mutation burden, PD-L1 and DNA repair genes to assess relationship to immune checkpoint inhibitors response in metastatic renal cell carcinoma.
Labriola Matthew Kyle,Zhu Jason,Gupta Rajan,McCall Shannon,Jackson Jennifer,Kong Eric F,White James R,Cerqueira Gustavo,Gerding Kelly,Simmons John K,George Daniel,Zhang Tian
Journal for immunotherapy of cancer
BACKGROUND:Immune checkpoint inhibitors (ICIs) have expanded treatment options for metastatic renal cell carcinoma (mRCC); however, there are limited predictive biomarkers for response to ICIs in this indication, with programmed death-ligand 1 (PD-L1) status demonstrating little predictive utility in mRCC. While predictive of ICI response in other tumor types, the utility of tumor mutation burden (TMB) in mRCC is unclear. Here, we assess TMB, loss of antigen presentation genes and PD-L1 status correlated with outcomes to ICI treatment in mRCC. METHODS:Tumor samples from 34 patients with mRCC treated with ICI therapy at Duke Cancer Institute were retrospectively evaluated using Personal Genome Diagnostics elio tissue complete (RUO version), a tumor genomic profiling assay for somatic variants, TMB, microsatellite status and genomic status of antigen presentation genes. Tumor samples were also analyzed with the Dako 28-8 PD-L1 immunohistochemistry assay. Deidentified clinical information was extracted from the medical record, and tumor response was evaluated based on the Response Evaluation Criteria In Solid Tumors (RECIST) V.1.1 criteria. RESULTS:Patients were stratified by overall response following ICI therapy and designated as progressive disease (PD; n=18) or disease control groups (DC; n=16). TMB scores ranged from 0.36 to 12.24 mutations/Mb (mean 2.83 mutations/Mb) with no significant difference between the PD and DC groups (3.01 vs 2.63 mutations/Mb, respectively; p=0.7682). Interestingly, 33% of PD patients displayed loss of heterozygosity of major histocompatibility complex class I genes (LOH-MHC) vs 6% of DC patients. Nine of 34 samples were PD-L1-positive (4 in the PD group; 5 in the DC group), suggesting no correlation between PD-L1 expression and response to ICI therapy. Notably, the DC group displayed an enrichment of mutations in DNA repair genes (p=0.04), with 68.8% exhibiting at least one mutated homologous recombination repair (HRR)-related gene compared with only 38.9% of the PD group (p=0.03). CONCLUSIONS:Overall, neither TMB nor PD-L1 correlated with ICI response and TMB was not significantly associated with PD-L1 expression. The higher incidence of LOH-MHC in PD group suggests that loss of antigen presentation may restrict response to ICIs. Separately, enrichment of HRR gene mutations in the DC group suggests potential utility in predicting ICI response and a potential therapeutic target, warranting future studies.
Efficacy of immunotherapy in lung cancer with co-occurring mutations in NOTCH and homologous repair genes.
Mazzotta Marco,Filetti Marco,Occhipinti Mario,Marinelli Daniele,Scalera Stefano,Terrenato Irene,Sperati Francesca,Pallocca Matteo,Rizzo Francesco,Gelibter Alain,Botticelli Andrea,Scafetta Giorgia,Di Napoli Arianna,Krasniqi Eriseld,Pizzuti Laura,Barba Maddalena,Carpano Silvia,Vici Patrizia,Fanciulli Maurizio,De Nicola Francesca,Ciuffreda Ludovica,Goeman Frauke,De Maria Ruggero,Vecchione Andrea,Giusti Raffaele,Ciliberto Gennaro,Marchetti Paolo,Maugeri-Saccà Marcello
Journal for immunotherapy of cancer
BACKGROUND:Immune checkpoint inhibitors (ICIs) provide significant survival benefits in non-small cell lung cancer (NSCLC). Nevertheless, while some patients obtain a prolonged benefit, a non-negligible fraction of patients experiences an ultrarapid disease progression. Identifying specific molecular backgrounds predicting opposite outcomes is instrumental to optimize the use of these agents in clinical practice. METHODS:We carried out an observational study with prospective design envisioning targeted next-generation sequencing (NGS) with an approved assay in 55 patients with metastatic NSCLC (Rome cohort), of whom 35 were treated with ICIs. Data from three clinically comparable datasets were collected and combined into a metadataset containing 779 patients. The datasets were related to the Memorial Sloan Kettering Cancer Center (MSKCC) cohort (tissue-based NGS) and the randomized phase II and III POPLAR and OAK trials (blood-based NGS). RESULTS:In patients treated with ICIs in the Rome cohort, co-occurring mutations in NOTCH1-3 and homologous repair (HR) genes were associated with durable clinical benefit. Using the MSKCC/POPLAR/OAK metadaset, we confirmed the relationship between the NOTCH/HR signature and longer progression-free survival (PFS) in ICI-treated patients (multivariate Cox: HR 0.51, 95% CI 0.34 to 0.76, p=0.001). The NOTCH/HR genomic predictor was also associated with longer survival (log-rank p=0.008), despite patients whose tumors carried the NOTCH/HR signature had higher metastatic burden as compared with their negative counterpart. Finally, we observed that this genomic predictor was also associated with longer survival in patients with other tumor types treated with ICIs (n=1311, log-rank p=0.002). CONCLUSIONS:Co-occurring mutations in the NOTCH and HR pathways are associated with increased efficacy of immunotherapy in advanced NSCLC. This genomic predictor deserves further investigation to fully assess its potential in informing therapeutic decisions.
Comutations in DNA Damage Response Pathways Serve as Potential Biomarkers for Immune Checkpoint Blockade.
Wang Zhijie,Zhao Jing,Wang Guoqiang,Zhang Fan,Zhang Zemin,Zhang Fan,Zhang Yuzi,Dong Hua,Zhao Xiaochen,Duan Jianchun,Bai Hua,Tian Yanhua,Wan Rui,Han Miao,Cao Yan,Xiong Lei,Liu Li,Wang Shuhang,Cai Shangli,Mok Tony S K,Wang Jie
Biomarkers such as programmed death receptor 1 ligand (PD-L1) expression, tumor mutational burden (TMB), and high microsatellite instability are potentially applicable to predict the efficacy of immune checkpoint blockade (ICB). However, several challenges such as defining the cut-off value, test platform uniformity, and low frequencies limit their broad clinical application. Here we identify comutations in the DNA damage response (DDR) pathways of homologous recombination repair and mismatch repair (HRR-MMR) or HRR and base excision repair (HRR-BER; defined as co-mut) that are associated with increased TMB and neoantigen load and increased levels of immune gene expression signatures. In four public clinical cohorts, co-mut patients presented a higher objective response rate and a longer progression-free survival or overall survival than co-mut patients. Overall, identification of DDR comutations in HRR-MMR or HRR-BER as predictors of response to ICB provides a potentially convenient approach for future clinical practice. Identification of comutations in specific DDR pathways as predictors of superior survival outcomes in response to immune checkpoint blockade provide a clinically convenient approach for estimation of tumor mutational burden and delivery of ICB therapy. .
The Notch Inhibitors Isolated from Nerium indicum.
Arai Midori A,Akamine Ryuta,Hayashi Narumi,Koyano Takashi,Kowithayakorn Thaworn,Ishibashi Masami
Journal of natural products
Notch signaling plays a crucial role in differentiation and cell maintenance, but once aberrantly activated, it contributes to cancer progression. Notch inhibitors were isolated from plant extracts and tested using an originally constructed cell-based assay system. We isolated eight compounds from Nerium indicum that showed inhibition of the Notch signaling pathway. HES1 and HES5 are target genes of the Notch signaling pathway, and oleandrin (1) decreased the protein levels of HES1 and HES5 in assay cells. Oleandrin (1) showed potent cytotoxicity against HPB-ALL cells and decreased HES1 and the Notch intracellular domain in these cells. The main mechanism of action of 1 appears to be inhibition of Notch signaling by acceleration of Notch intracellular domain degradation.
Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma.
Bousquet Mur Emilie,Bernardo Sara,Papon Laura,Mancini Maicol,Fabbrizio Eric,Goussard Marion,Ferrer Irene,Giry Anais,Quantin Xavier,Pujol Jean-Louis,Calvayrac Olivier,Moll Herwig P,Glasson Yaël,Pirot Nelly,Turtoi Andrei,Cañamero Marta,Wong Kwok-Kin,Yarden Yosef,Casanova Emilio,Soria Jean-Charles,Colinge Jacques,Siebel Christian W,Mazieres Julien,Favre Gilles,Paz-Ares Luis,Maraver Antonio
The Journal of clinical investigation
EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.
Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor-initiating cell frequency.
Hu Shi,Fu Wenyan,Li Tian,Yuan Qingning,Wang Feifei,Lv Gaojian,Lv Yuanyuan,Fan Xiaoyan,Shen Yafeng,Lin Fangxing,Tang Ying,Ye Xuting,Yang Yongji,Lei Changhai
Science translational medicine
Epidermal growth factor receptor (EGFR) blockade and radiation are efficacious in the treatment of cancer, but resistance is commonly reported. Studies have suggested that dysregulation of Notch signaling and enrichment of the cancer stem cell population underlie these treatment challenges. Our data show that dual targeting of EGFR and Notch2/3 receptors with antibody CT16 not only inhibited signaling mediated by these receptors but also showed a strong anti-stem cell effect both in vitro and in vivo. Treatment with CT16 prevented acquired resistance to EGFR inhibitors and radiation in non-small cell lung cancer (NSCLC) cell line models and patient-derived xenograft tumors. CT16 also had a superior radiosensitizing impact compared with EGFR inhibitors. CT16 in combination with radiation had a larger antitumor effect than the combination of radiation with EGFR inhibitors or tarextumab. Mechanistically, CT16 treatment inhibits the stem cell-like subpopulation, which has a high mesenchymal gene expression and DNA repair activity, and reduces tumor-initiating cell frequency. This finding highlights the capacity of a combined blockade of EGFR and Notch signaling to augment the response to radiation and suggests that CT16 may achieve clinical efficacy when combined with radiation in NSCLC treatment.
Rethinking Gamma-secretase Inhibitors for Treatment of Non-small-Cell Lung Cancer: Is Notch the Target?
Pine Sharon R
Clinical cancer research : an official journal of the American Association for Cancer Research
Lung cancer is the leading cause of cancer-related deaths among men and women. γ-Secretase inhibitors, a class of small-molecule compounds that target the Notch pathway, have been tested to treat non-small-cell lung cancer (NSCLC) in preclinical and clinical trials. Although γ-secretase inhibitors elicit a response in some tumors as single agents and sensitize NSCLC to cytotoxic and targeted therapies, they have not yet been approved for NSCLC therapy. We discuss our recently published preclinical study using the γ-secretase inhibitor AL101, formerly BMS906024, on cell lines and PDX models of NSCLC, primarily lung adenocarcinoma. We propose that Notch pathway mutations may not be the most suitable biomarker for predicting NSCLC response to γ-secretase inhibitors. γ-Secretases have over 100 known γ-secretase cleavage substrates. Many of the γ-secretase substrates are directly involved in carcinogenesis or tumor progression, and are ideal candidates to be the "on-target" biomarkers for γ-secretase inhibitors. We propose the need to systematically test the γ-secretase and other targets as potential biomarkers for sensitivity before continuing clinical trials. Now that we have entered the postgenome/transcriptome era, this goal is easily attainable. Discovery of the biomarker(s) that predict sensitivity to γ-secretase inhibitors would guide selection of the responder population that is most likely to benefit and move the compounds closer to approval for therapeutic use in NSCLC.
Isolation and evaluation of cardenolides from Lansium domesticum as Notch inhibitors.
Tsuchiya Anna,Makita Yoshinori,Koyano Takashi,Kowithayakorn Thaworn,Ishibashi Masami,Arai Midori A
Journal of natural medicines
Since Notch signaling plays important roles in cell proliferation and differentiation, aberrant activation of this signaling contributes to cancer progression. In neural stem cells, Notch signaling inhibits differentiation by activating HES1 expression. Therefore, Notch signaling inhibitors may be candidates for new anticancer drugs or have applications in neural regenerative medicine. In this study, six naturally occurring Notch inhibitors were isolated from the methanol (MeOH) extract of Lansium domesticum using our novel cell-based assay. Hongherin (2), a cardiac glycoside, demonstrated potent Notch inhibitory activity with an IC of 0.62 μM and was found to be cytotoxic in HPB-ALL human T cell acute lymphoblastic leukemia cells. Hongherin (2) also induced the differentiation of C17.2 neural stem cells to neurons, causing a 65% increase in differentiation compared to the control. Mechanistically, hongherin (2) reduced the amount of Notch1 (full length) and mastermind-like protein (MAML). This indicates that hongherin (2) inhibits Notch signaling through a dual mechanism involving the reduction of both Notch1 and MAML protein levels.
Identification of Deleterious Mutation as Novel Predictor to Efficacious Immunotherapy in NSCLC.
Zhang Kai,Hong Xiaohua,Song Zhengbo,Xu Yu,Li Chengcheng,Wang Guoqiang,Zhang Yuzi,Zhao Xiaochen,Zhao Zhengyi,Zhao Jing,Huang Mengli,Huang Depei,Qi Chuang,Gao Chan,Cai Shangli,Gu Feifei,Hu Yue,Xu Chunwei,Wang Wenxian,Lou Zhenkun,Zhang Yong,Liu Li
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:NOTCH signaling is associated with tumorigenesis, mutagenesis, and immune tolerance in non-small cell lung cancer (NSCLC), indicating its association with the clinical benefit of immune checkpoint inhibitors (ICI). We hypothesized that mutation in NSCLC might be a robust predictor of immunotherapeutic efficacy. EXPERIMENTAL DESIGN:Multiple-dimensional data including genomic, transcriptomic, and clinical data from cohorts of NSCLC internal and public cohorts involving immunotherapeutic patients were analyzed. Polymorphism Phenotyping v2 (PolyPhen-2) system was performed to determine deleterious mutation (del- ). Further investigation on molecular mechanism was performed in The Cancer Genome Atlas (TCGA) data via CIBERSORT and gene set enrichment analysis. RESULTS:Our 3DMed cohort ( = 58) and other four cohorts (Rizvi, POPLAR/OAK, Van Allen, and MSKCC; = 1,499) uncovered marked correlation between mutation and better ICI outcomes in population, including objective response rate (2.20-fold, = 0.001), progression-free survival [HR, 0.61; 95% confidence interval (CI), 0.46-0.81; = 0.001], and overall survival (HR, 0.56; 95% CI, 0.32-0.96; = 0.035). Del- exhibited better predictive function than non-deleterious mutation, potentially via greater transcription of genes related to DNA damage response and immune activation. Del- was not linked with prognosis in TCGA cohorts and chemotherapeutic response, but was independently associated with immunotherapeutic benefit, delineating the predictive, but not prognostic, utility of del- . CONCLUSIONS:This work distinguishes del- as a potential predictor to favorable ICI response in NSCLC, highlighting the importance of genomic profiling in immunotherapy. More importantly, our results unravel a possibility of personalized combination immunotherapy as adding NOTCH inhibitor to ICI regimen in NSCLC, for the optimization of ICI treatment in clinical practice.