Abstract 212: Disruption of the menin-MLL complex impedes HCC maintenance and increases sensitivity to NF-Y loss

Most patients with hepatocellular carcinoma (HCC) are diagnosed at advanced stages, and there are only a few moderately effective systemic therapies available. Despite the decreased mortality rate for most cancer types over the last decade, liver cancer, with HCC being the prevailing type (∼90%), showed an increase, making this disease the third most common cancer-related cause of death. Since ∼50% liver cancer samples carry mutations in epigenetic modifiers and the imbalance in the transcriptional landscape can be potentially restored using epigenetic drugs, we focused on identifying new therapeutic targets of HCC among chromatin modifiers and understanding their role in HCC maintenance. Using our epigenome-focused CRISPR library of 6000 gRNAs targeting 737 chromatin-related genes, we performed screenings in two- (2D) and three-dimensional (3D) cultures using multiple HCC cell lines (HLF and PLC/PRF5). Our screen identified several potential new targets essential for HCC survival, including several core subunits of the H3K4me3-mediating menin-MLL complex (MEN1, ASH2L, KMT2A). We validated HCC dependency on these targets by performing a negative selection CRISPR/Cas9 competitive growth assay in HLF and PLC/PRF5 cell lines. Moreover, treatment of HCC cell lines with the next-generation menin inhibitor SNDX5613 in 2D and 3D settings revealed a dose-dependent reduction in cell proliferation. We assessed the changes in the chromatin binding profile of menin (MEN1) and MLL (KMT2A) as well as changes in H3K4me3 levels upon treatment with 5µM SNDX5613 by CUT&RUN. While menin peaks showed a ∼90% reduction upon treatment, MLL and H3K4me3 peaks exhibited a ∼3- and ∼2-fold reduction, respectively. Next, we evaluated transcriptional changes associated with disrupting the menin-MLL complex by performing RNA sequencing of HCC cells following either MEN1 gene knockout or treatment with 5µM SNDX5613. Integrated CUT&RUN and RNA-seq data revealed genes involved in the PI3K/AKT signaling pathway, whose independent knockouts affected HCC survival. ATAC sequencing of HLF cells treated with 5µM SNDX5613 revealed a connection between closed genomic sites and loss of menin-MLL complex binding, while newly accessible chromatin was enriched for NF-Y transcriptional factor motifs and were independent of menin binding. Loss of NFYA or NFYB significantly affected cell growth, supporting their role in HCC survival. The combined NFYB knockout and SNDX5613 treatment showed a significantly decreased HCC cell proliferation compared to a single menin inhibition. We propose inhibition of the menin-MLL interaction disrupts HCC homeostasis through changes in the expression of direct target genes within the PI3K/AKT pathway and alters chromatin accessibility, creating sensitivity to NFYB gene loss. We believe targeting core subunits of the menin-MLL complex represents an appealing therapeutic strategy for HCC treatment. Citation Format: Margarita M. Dzama, Peyton C. Kuhlers, Jesse R. Raab. Disruption of the menin-MLL complex impedes HCC maintenance and increases sensitivity to NF-Y loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 212.