Abstract 2967: ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A

Abstract

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers (>800,000 new cases each year worldwide) and a leading cause of cancer mortality (~700,000 deaths per year globally), with an average 5-year survival rate of 20~30%. The prevalence of HCC and the unmet clinical needs emphasize the urge for novel regimens for these malignancies. In this study, we performed an epigenetics-focused CRISPR interference (CRISPRi) library screen in the HepG2-dCas9-Krab cells (a total of 3,669 sgRNAs targeting 728 chromatin regulator genes at their transcription start sites) and identified ACTR5 as a novel essential gene in HCC. CRISPR depletion of ACTR5 led to arrested cell cycle and attenuated HCC tumor progression in vivo. Transcriptomic analysis revealed a significantly increased CDKN2A expression in the sgACTR5 transduced HCC cells, which led to the activation of Rb and repression of CDK6 and E2F signaling. Meanwhile, histone modification profiling identified loss of H3K9me2 and Hek9me3 at the CDKN2A promoters upon ACTR5 depletion, indicating the involvement of the epigenetic silencing of tumor suppressors (including CDKN2A) underlying the ACTR5-driven HCC progression. Mechanistically, we found that CRISPR targeting the ACTR5’s interacting protein IES6 (also known as INO80C) recapitulated the sgACTR5 phenotypes, highlighting the collaborative roles between these two partners. We then conducted a high-density CRISPR gene tiling scan using a sgRNA library that targets every NGG protospacer adjacent motif (PAM) within the ACTR5 (284 sgRNAs) and IES6 (90 sgRNAs) coding exons. This saturating mutagenesis screen revealed the requirement of 2 regions (G502-S519 and I552-C569) within ACTR5’s 2nd Actin-fold domain for IES6 interaction. Reciprocally, our unbiased screen identified a previously uncharacterized domain (T163-V187) in IES6’s C-terminal region to mediate the ACTR5-IES6 complex formation. To investigate the clinical impact of ACTR5, we examined the response of HCC cells to various inhibitors upon ACTR5 depletion. While sgACTR5 did not affect the cellular response to sorafenib (an FDA-approved RAF and VEGF inhibitor for HCC patients), we found that suppression of ACTR5 sensitized the HCC cells to ribociclib (a CDK6 inhibitor) and UNC0642 (an H3K9 methyltransferase G9a and GLP inhibitor). Based on these observations, we rationalized a combination treatment of ribociclib/UNC0642 that synergistically suppresses the proliferation of the HCC cells. In summary, our study revealed a pivotal role of ACTR5 (and its complex member IES6) in supporting the CDK6-driven cell cycle progression via an H3K9me2 H3K9me3-mediated silencing of CDKN2A. These notions can be exploited to develop novel combinational therapies against the difficult to cure liver cancers. Citation Format: Xiaobao Xu, Lu Yang, Anthony Chan, Sheela Pangeni Pokharel, Mingli Li, Qiao Liu, Nicole Mattson, Chun-wei Chen. ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2967.