Given the modest efficacy of current sarcoma therapeutic modalities, there is a major unmet clinical need for the translation of novel agents to tackle the burden of Ewing sarcoma (pediatric and young adolescent bone cancer). We have recently shown that therapeutic targeting of LSD1, a highly expressed lysine specific demethylase, with the reversible small molecule inhibitor SP-2509, comprehensively disrupts the global transcriptional function of EWS-FLI, and significantly impairs the growth of Ewing sarcoma cell lines both in vitro and in vivo. Prior to clinical trial evaluation, the goal of this study was to elucidate mechanisms of Ewing sarcoma SP-2509 drug resistance.
In order to investigate SP-2509 resistance, A673 Ewing sarcoma cells were cultured in escalating concentrations of SP-2509 for 8 months. Cell Titer Glo analysis revealed that these SP-2509 drug resistant (DR) cells were 54.9 fold more resistant to SP-2509 compared to parental A673 cells (72hr IC50 0.138μM versus 7.586μM). SP-2509 DR cells also exhibited a significant decrease in their ability to form anchorage-independent colonies in soft agar. Whole exome (250X coverage) and RNA seq analysis of parental and SP-2509 DR A673 cells revealed 25 drug induced mutations, with 88% of the whole cell population harboring a Glu53* stop-gain mutation in MRPL45 (mitochondrial ribosomal protein L45). In addition, SP-2509 DR cells displayed a distinct transcriptomic profile, with 3250/2376 genes significantly up/down regulated (≥1.5 fold) in SP-2509 DR cells compared to parental controls. KEGG Pathway analysis revealed that the genes were highly enriched for hepatic stellate cell activation/interferon signaling and cholesterol biosynthesis respectively. Lastly, SP-2509 DR cells were shown to be equally sensitive to etoposide, resistant to vincristine/doxorubicin and surprisingly more sensitive to the HDAC inhibitors vorinostat and entinostat compared to parental A673 cells.
Together, our preliminary findings provide key insights into the mechanisms of SP-2509 drug resistance, information of which can be used to find combinatorial agents that can potentially be used to circumvent resistant clones to achieve maximal therapeutic effect.
Citation Format: Kathleen I. Pishas, Sunil Sharma, Stephen L. Lessnick. Mechanisms of Ewing sarcoma resistance to LSD1 inhibition [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 910.