Reversible LSD1 Inhibition Interferes with Global EWS/ETS Transcriptional Activity and Impedes Ewing Sarcoma Tumor Growth

Abstract

Purpose Ewing sarcoma is a pediatric bone tumor which 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 HCI-2509 in both EWS/FLI and EWS/ERG containing cell lines. We then evaluated morphological 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 HCI-2509 on tumorigenesis. Results HCI2509 caused a dramatic reversal of both the up- and down-regulated transcriptional profiles of EWS/FLI and EWS/ERG accompanied by the induction of apoptosis, and disruption of morphological 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.

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

“…In contrast to previous efforts to inhibit EWSR1-FLI1 activity that have capitalized on individual target gene expression or physical interactions (31)(32)(33)(34)(35)(36), the application of HT-FAIRE offered a strategy to identify therapeutics based on variation in chromatin accessibility, a universal genomic feature determined by the combined effects of transcriptional regulators and chromatin regulatory proteins (37). Indeed, FAIRE has been shown to detect specific chromatin changes resulting from treatment with anthracyclines that directly interact with DNA (38,39), thereby demonstrating the utility of this assay to explore the function of small molecule compounds.…”

Helicase CHD4 is an epigenetic coregulator of PAX3-FOXO1 in alveolar rhabdomyosarcoma

“…Further, we identified additional components of this complex in our initial proteomic screen, namely RBBP7, MTA2, GATAD2A, GATAD2B, HDAC1, and HDAC2, suggesting that PAX3-FOXO1 might indeed interact with the NuRD complex. It has been demonstrated that this remodeling complex is also required for EWS-FLI1-mediated transcriptional repression (50), and inhibition of LSD1 using small-molecule inhibitors impaired Ewing sarcoma cell viability (51). Hence, we next thought to identify components of the complex that might be necessary for PAX3-FOXO1 function.…”

Molecular genetics of Ewing sarcoma, model systems and finding novel (immuno-) therapeutic targets

“…Fusion of the N-terminal region of EWSR1, which contains a strong transcriptional activation domain [21,22] , and these DNA binding domains causes aberrant transcription of a multitude of genes. Additionally, EWSR1-ETS is known to affect epigenetic programs [23,24] , splicing [25,26] , and metabolic activity [27] of EWS. Oncogenic fusions between ETS genes and transcriptional activators are not unique to EWS: in 50%-70% of prostate cancers, similar chromosomal rearrangements are found [28] .…”

“…Targeting the fusion protein directly has proven to be challenging due to its flexibility, but progress has been made and a phase I clinical trial (NCT02657005) is ongoing with a small molecule inhibitor [108] . Aside from targeting the EWSR1-ETS fusion protein directly, inhibiting its dominant direct targets has been investigated as therapeutic alternative, as EWSR1-ETS is a key driver in the epigenetics [23,24] , transcription [109] , splicing [25,26] , and metabolic [27] reprogramming of EWSR1-ETS.…”