Purpose: Ewing sarcoma is a pediatric bone tumor that absolutely relies on the transcriptional activity of the EWS/ETS family of fusion oncoproteins. While the most common fusion, EWS/FLI, utilizes lysine-specific demethylase 1 (LSD1) to repress critical tumor suppressors, small-molecule blockade of LSD1 has not yet been thoroughly explored as a therapeutic approach for Ewing sarcoma. We therefore evaluated the translational potential of potent and specific LSD1 inhibition with HCI2509 on the transcriptional program of both EWS/FLI and EWS/ERG as well as the downstream oncogenic phenotypes driven by EWS/ETS fusions in both in vitro and in vivo models of Ewing sarcoma.

Experimental Design: RNA-seq was used to compare the transcriptional profiles of EWS/FLI, EWS/ERG, and treatment with HCI2509 in both EWS/FLI- and EWS/ERG-containing cell lines. We then evaluated morphologic phenotypes of treated cells with immunofluorescence. The induction of apoptosis was evaluated using caspase-3/7 activation and TUNEL staining. Colony forming assays were used to test oncogenic transformation and xenograft studies with patient-derived cell lines were used to evaluate the effects of HCI2509 on tumorigenesis.

Results: HCI2509 caused a dramatic reversal of both the up- and downregulated transcriptional profiles of EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of morphologic and oncogenic phenotypes modulated by EWS/FLI. Importantly, HCI2509 displayed single-agent efficacy in multiple xenograft models.

Conclusions: These data support epigenetic modulation with HCI2509 as a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins. Clin Cancer Res; 20(17); 4584–97. ©2014 AACR.