Abstract 3484: Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line

Abstract The nucleosome is the basic unit of chromatin, comprising 147 bp of DNA wrapped around an octamer of core histone proteins H2A, H2B, H3 and H4. Compact chromatin acts as a barrier to gene expression and is a key property governing DNA replication and gene expression. Analyzing over 46,000 in cancer patient samples in cBioPortal, we characterized mutations located in canonical histone genes and found several recurrent missense mutations. Foremost among the identified mutations was a glutamate to lysine missense mutation at amino acid 76 of histone H2B (H2B-E76K) that disrupts histone H2B-H4 interaction. Previously, we demonstrated that exogenous expression of H2B-E76K disrupted nucleosome stability, altered chromatin accessibility, and gene expression. We developed a new model utilizing CRISPR “knock-in” technology to integrate the E76K mutation into the endogenous H2BC4 gene of the lung epithelial cell line BEAS-2B to create a homozygous and heterozygous H2B-E76K cell line. Although the mutant histone only accounts for about 2-5% of total histone H2B, expression of H2B-E76K catalyzed distinct changes in cell migration, proliferation, colony formation and gene expression. CUT&RUN technology revealed mutant histones broadly incorporate throughout the genome. Gene set enrichment analysis (GSEA) revealed that H2B-E76K homozygous cells displayed expression changes similar to polycomb repressive complex 1 (PRC1) disruption or oncogenic KRAS expression. Using available datasets, we discovered that expression of H2B-E76K caused increased expression of PTN, PKIB, and CDH2, genes important in migration, epithelial-mesenchymal transition (EMT), and adhesion pathways. Furthermore, analysis of chromatin accessibility changes indicated that increased accessibility was significant in and around genes governing cell migration. Notably, H2B-E76K significantly increased heterogeneity of gene expression as measured using scRNA-seq. Together these results indicate that mutations disrupting histone structure may create millions of dysfunctional nucleosomes and new sites of “open” chromatin, resulting in loss of nucleosome-mediated gene repression and/or altered interactions with regulators of chromatin, fundamentally changing cell growth and migration properties to favor tumorigenesis. Citation Format: Kimberly Espinoza Pereira, Richard L. Bennett, Jixiu Shan, Jay Sarthy, Jonathan D. Licht. Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3484.