Abstract A14: Therapeutic targeting of KDM1A/LSD1 in Ewing sarcoma

Abstract

Abstract Objective: Multiagent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common solid bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/LSD1 (Lysine Specific Demethylase 1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P=0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). Methods: The antiproliferative effects of SP-2509 was determined through Cell Titre Glo assays following 72 hours of treatment in a comprehensive panel of 17 Ewing sarcoma cell lines with varying STAG2/TP53 mutational status and basal KDM1A expression levels. RNA-seq analysis of six Ewing sarcoma cell lines -/+ SP-2509 treatment (2μM) was also conducted. Results: We report that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (72hr IC50 range 81nM-1593nM); in contrast, resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (144hr IC50>300μM). Although TP53/STAG2 mutational status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B (mammalian homologue of KDM1A) following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity (R2=0.562). Indeed, shRNA-mediated knockdown of KDM1B significantly reduced the cytotoxic effects of SP-2509 (4.3-fold IC50 increase) only in hypersensitive cell lines. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum (ER) stress pathway, and that hypersensitive cell lines (IC50<300nM) share similar transcriptomic profiles. In addition, ETS1/ HIST1H2BM were specifically induced/repressed, respectively, following SP-2509 treatment only in our hypersensitive cell lines. Finally, we demonstrate that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Conclusion: Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Citation Format: Kathleen I. Pishas, Sunil Sharma, Stephen L. Lessnick. Therapeutic targeting of KDM1A/LSD1 in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A14.