Abstract 577: Deciphering the mechanism of the menin-MLL complex dependency in HCC

Abstract

Abstract Liver cancer is the third leading cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the most common primary liver cancer (~90%). The 5-year survival rate of patients with advanced HCC is ~18% due to the late prognosis and the moderate clinical benefit of available systemic therapy options. Mutations in chromatin regulators were detected in ~50% of liver cancer. Since many chromatin regulators have been shown a dependency in various cancer types and can modulate therapy response, we focused on identifying new therapeutic targets of HCC among epigenetic modifiers and evaluating them as potential targets for future drug development. We constructed a CRISPR library of 6000 gRNAs targeting 737 genes involved in chromatin-mediated gene regulation. Using the epigenome-focused CRISPR/Cas9 screening in two-dimensional and three-dimensional settings in multiple liver cancer cell lines (HepG2, HLF, PLC/PRF5), we identified a list of epigenetic regulators as potential new targets in HCC. Two of them, MEN1 and ASH2L, are core subunits of the menin-MLL complex mediating H3K4 trimethylation (me3). We validated dependencies of HCC on MEN1 and ASH2L by performing a negative selection CRISPR/Cas9 competitive growth assay in HLF and PLC/PRF5 cell lines. Moreover, treatment of HCC cell lines (HLF, PLC/PRF5, HepG2) with recently developed menin inhibitor SNDX-5613 (revumenib) revealed a dose-dependent reduction in cell proliferation. To determine transcriptional changes associated with disruption of the menin-MLL complex, we performed RNA sequencing of HLF and PLC/PRF5 cells following either inhibition with 5µM SNDX-5613 for 4 days or knockout of the MEN1 or ASH2L gene. We observed the upregulation of KRAS and the oncogenic signature of TGFβ signaling pathways in all menin-MLL complex disrupting conditions. We did not detect global changes in H3K4me3 levels upon the menin inhibition or MEN1 or ASH2L knockouts assessed by immunoblotting. To explore the mechanism of the antiproliferative effect of the menin inhibition on HCC further, we evaluated changes in the binding of menin to chromatin and H3K4me3 levels in HCC cells by performing CUT&RUN following treatment of HLF cells with 5µM SNDX-5613 for 4 days. Menin inhibition led to a drastic removal of menin from the chromatin, while H3K4me3 levels were only mildly decreased. We identified 8765 genes near promoter regions with menin binding and H3K4me3 mark, of which 1952 genes were near regions with simultaneous loss of menin and decreased H3K4me3 levels upon menin inhibition. Integration of our genomics data revealed a list of 30 genes, which are downregulated upon menin inhibition in both HLF and PLC/PRF5 cells (FC≥1.5) and the direct targets of the menin-MLL complex in HLF cells, suggesting their potential role in HCC cell survival. Altogether, we anticipate that menin and ASH2L serve as promising targets and represent an appealing therapeutic strategy for HCC treatment. Citation Format: Margarita M. Dzama, Peyton C. Kuhlers, Jesse R. Raab. Deciphering the mechanism of the menin-MLL complex dependency in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 577.