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Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth.

ABSTRACT: 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.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.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.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.

SUBMITTER: Sankar S

PROVIDER: S-EPMC4155010 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth.

Sankar Savita S Theisen Emily R ER Bearss Jared J Mulvihill Timothy T Hoffman Laura M LM Sorna Venkataswamy V Beckerle Mary C MC Sharma Sunil S Lessnick Stephen L SL

Clinical cancer research : an official journal of the American Association for Cancer Research 20140624 17

<h4>Purpose</h4>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 ...[more]

PMID: 24963049

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Project description:The pathognomonic EWS/ETS fusion transcription factors drive Ewing sarcoma (EWS) by orchestrating an oncogenic transcription program. Therapeutic targeting of EWS/ETS has not been successful; therefore identifying mediators of the EWS/ETS function could offer new therapeutic targets. Here we describe the dependency of chromatin reader BET bromodomain proteins in EWS/ETS driven transcription and investigate the potential of BET inhibitors in treating this lethal cancer. Similar to EWS/ETS fusions, knockdown of BET proteins BRD2/3/4 severely impaired the oncogenic phenotype of EWS cells. Notably, EWS/FLI1 and EWS/ERG was found to be in a transcriptional complex consisting of BRD4. RNA-Seq analysis upon BRD4 knockdown or its pharmacologic inhibition by the BET inhibitor JQ1 revealed an attenuated EWS/ETS transcriptional signature. In contrast to other reports, JQ1 reduced proliferation, and induced apoptosis through MYC-independent mechanism without affecting EWS/ETS protein levels, which was further confirmed by depleting BET proteins using PROTAC-BET degrader (BETd). Interestingly, polycomb repressive complex 2 (PRC2) associated factor PHF19 was downregulated by JQ1/BETd or BRD4 knockdown in multiple EWS cells. ChIP-seq analysis revealed occupancy of EWS/FLI1 at a distal regulatory element of PHF19 and its subsequent knockdown resulted in downregulation of PHF19 expression. Furthermore, deletion of PHF19 by CRISPR-Cas9 system lead to a decreased tumorigenic phenotype and increased sensitivity to JQ1. Importantly, PHF19 expression was associated with worse prognosis of Ewing sarcoma patients. In vivo, JQ1 demonstrated anti-tumor efficacy in multiple mouse xenograft models of EWS. Together, these results indicate that EWS/ETS require BET epigenetic reader proteins for its transcriptional program including PHF19 expression, which can be mitigated by BET inhibitors. Moreover, this study provides a clear rationale for the clinical utility of BET inhibitors in treating Ewing sarcoma.

Dataset Information

Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth.

Publications

Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth.

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