Abstract PO023: Uncovering hepatitis B virus-induced signaling changes in hepatocellular carcinoma
Authors: Turnham Rigney E , Lim Huat Chye , Ferret Lucille , Lo Katherine , Wang LeeAnn , Choi Alex , Gordan John D 
Published: September 2022

Abstract
Although precision medicine has revolutionized the treatment for other solid tumors, comprehensive genome profiling of hepatocellular carcinoma (HCC) has demonstrated that there are few oncogenic mutational drivers. HCC arises nearly universally in the context of co-morbid hepatitis, driven by hepatitis C virus or hepatitis B virus (HBV). Previous work has highlighted the effects of HBV protein X (HBx) on signaling and proliferation. However, it remains unclear how these signaling changes interact with the stress imposed by HBV replication. We hypothesize that HBV replication and maintenance of HBV proteins like HBx creates specific vulnerabilities that can be uncovered and exploited. We performed whole-genome clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening in two engineered, patient-derived HBV+ cell models, Hep3B and SNU-368, with cellular proliferation as the readout. A conditional induction of HBx was used to overexpress HBx in each cell model for the whole genome CRISPRi screen. Genes where HBx expression was significantly associated with a more negative dependency score were classified as HBV differential dependencies. 37 genes were identified as shared HBV-induced dependencies across both cell lines. Of these, 16 were also differentially expressed in HCC specimens with ongoing HBV replication vs. non-HBV expressing tumors collected in The Cancer Genome Atlas (TCGA), suggesting in vivo selective pressure to alter the expression of these genes. We focused on Zinc Fingers and Homeoboxes 2 (ZHX2) and methionine adenosyltransferase 2A (MAT2A). ZHX2 was identified as an HCC tumor suppressor, and data from TCGA showed differential gene expression of ZHX2 in HBV+HCC. Validation of ZHX2 sgRNA knockdown confirmed that ZHX2 knockdown preferentially reduced cell proliferation in the presence of HBx, consistent with an HBV-induced dependency. ZHX2 also can activate the HIF pathway; we identified alterations of HIF targets with ZHX2 knockdown and HBx expression. Similarly, MAT2A knockdown in HBV+HCC cell lines showed decreased cell proliferation with HBx expression. MAT2A is an emerging therapeutic target, with sensitivity to MAT2A inhibitors conferred by deletions in methylthioadenosine phosphorylase (MTAP), seen in 3% of HCC. Ongoing investigations include alteration of splicing and cell cycle changes with depletion of MAT2A in the presence of HBx. With small molecule inhibitors of MAT2A in clinical development, this finding may be therapeutically actionable. Thus, using functional genomics to interrogate HBV-associated gene dependencies has illuminated the biology of HCC and identified candidate therapeutic targets for pre-clinical validation.
Citation Format: Rigney E Turnham, Huat Chye Lim, Lucille Ferret, Katherine Lo, LeeAnn Wang, Alex Choi, John D Gordan. Uncovering hepatitis B virus-induced signaling changes in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO023.