Abstract 5841: Ceramide synthase-6 binding to CD95/Fas confers resistance to chemotherapy in T-cell acute lymphoblastic leukemia cells

Abstract

Abstract Ceramide synthase (CERS) produces ceramides, key intermediators in the biosynthesis of complex sphingolipids and play an important role in cell growth, differentiation, apoptosis, and senescence. Six isoforms of ceramide synthases (CERS1-6) with varying substrate specificity generate ceramides of diverse chain lengths. In normal tissues, CERS2, synthesizing ceramides with C24 acyl chain (C24-Cer), is highly expressed in most of the tissues while CerS6 generates C16-Cer with low and tissue-specific distribution. Our data showed that CERS6 and C16-Cer levels were significantly higher in acute lymphoblastic leukemia (ALL) cells in comparison to peripheral blood mononuclear cells and T-Lymphocytes derived from healthy human volunteers. The purpose of this study was to investigate the role of CERS6 in chemo resistance in T-ALL models. Stable knockdown/overexpression of CERS6 was achieved by lentiviral vector transduction. Ceramide levels were measured by LC/MS. Cytotoxicity of conventional and investigational anti-leukemia agents was evaluated in T- ALL cells with altered CERS6 levels using DIMSCAN assay. CERS6 knockdown significantly decreased C16-Cer by 4-fold (p<0.01) while CERS6 overexpression increased C16-Cer by 2-fold (p<0.05). CERS6 knockdown in CCRF-CEM cells increased their sensitivity to a pan BCL-2 inhibitor, ABT-737 as well as a glucocorticoid, dexamethasone (DXM). The percent survival of CCRF-CEM cells with CERS6 knockdown treated with ABT-737 (100nM) or DXM (100nM) was 5% and 2% at 72h while cells transduced with non-targeting shRNA showed survival of 87% and 33% (p<0.001). In CCRF-CEM cells with CERS6 overexpression, the percent survival, when treated with ABT-737 (300nM) or DXM (300nM) for 72h, was significantly higher relative to the cells transduced with vector control: 39% vs 2% for ABT-737 and 33% vs 0.4% for DXM, p<0.01. Higher cleavage of PARP, Caspase 3 and Caspase 8 was seen in ABT-737 or DXM treated cells with CERS6 knockdown, which was reversed by the overexpression of CERS6. Sensitivity to ABT-737 in CERS6 knockdown cells was significantly reduced by a Caspase 8 inhibitor, suggesting that CERS6 alters ALL cell sensitivity to chemotherapy via extrinsic pathway of apoptosis. Pull down of CERS6 in CCRF-CEM cells identified CD95/Fas, a death receptor leading to activation of extrinsic apoptotic pathway, as a CERS6 binding partner. In CERS6 knockdown cells, a higher level of FADD was detected in Fas pull-down samples when treated with ABT-737 compared with cells transduced with non-targeting shRNA, indicating CERS6 interferes Fas/FADD assembly in inducing apoptosis. Taken together, CERS6 modulation affected the sensitivity of T-ALL cells to chemotherapy by interfering with Fas/FADD assembly in extrinsic apoptotic pathway. CERS6 may serve as a biomarker in determining the efficacy of anticancer drugs acting via extrinsic apoptotic pathway in T-ALL. Citation Format: Dattesh U. Verlekar, Hwangeui Cho, Sung-Jen Wei, Min H. Kang. Ceramide synthase-6 binding to CD95/Fas confers resistance to chemotherapy in T-cell acute lymphoblastic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5841.