PROJECT SUMMARY Significance: By examination of cancer patient genomic datasets, through the cBioPortal, we identified a new class of recurrent mutations in genes encoding histones H2A, H2B, H3 and H4. This supports an underlying premise, supported by rigorous publications from many groups including ours, that mutations of gene regulatory machinery, including histones are oncogenic drivers of human cancer. Many of these mutations affect residues of the histone folds important for the interactions among histones required for integrity of the histone octamer. Others may affect histone interactions with the chromatin modeling machinery. Histone fold mutations are found in 4% of lung (commonest tumor in US) and colorectal tumors, 6% of head and neck and 12% of bladder cancer. Histone mutations are subclonal. Tumors with histone mutations were significantly more likely to harbor mutations of oncogenic signaling molecules and tumor suppressive epigenetic regulators, suggesting that histone mutations augment the effect of other oncogenic lesions. Preliminary data: We characterized a recurrent mutation that changed amino acid 76 of histone H2B from glutamic acid to lysine (H2BE76K). This mutation disrupts the interaction of the H2B/H2A dimer with the H3/H4 tetramer, preventing formation of the histone octamer. Our Cancer Discovery paper showed that H2BE76K inhibits formation of nucleosomes in vitro and in yeast. Expression of H2BE76K in human cells disrupts chromatin structure, activates gene expression, promotes cell growth, and cooperates with PI3KCA to transform breast epithelial cells. We hypothesize that mutations that disrupt histone structure create millions of dysfunctional nucleosomes. These may deregulate gene expression and drive cancer development by creating new sites of “open” chromatin with loss of nucleosome-mediated gene repression, as well as by altered interactions with regulators of chromatin and gene expression. Approach: We will use cell and mouse models and epigenome profiling to characterize the oncogenic mechanisms of histone mutations, particularly in the transformation of lung cells, through three specific aims: 1) Elucidate mechanisms by which histone fold mutations alter cell growth, gene expression and chromatin structure. 2) Determine how histone fold mutations cooperate with co-occurring oncogenes and tumor suppressive chromatin regulators in cell and animal models. 3) Characterize the effects of histone mutations on tumor heterogeneity and response to therapy. Novelty: This proposal explores a new class of cancer-driving mutations to uncover novel mechanisms of oncogenesis by chromatin disruption. We will use the latest versions of gene editing to engineer histone mutations into cells, analyze tumor cells with advanced technologies for genome wide analysis of chromatin states and create new animal models. Impact: We will focus on lung cancer, where we estimate up to 10,000 patients/year will have tumors with histone mutat...