Abstract An emerging body of literature has shown that nucleosomes, the fundamental subunit of chromatin, are destabilized in a broad array of common cancers. This destabilization was previously thought to require cancer-specific mutations in histone-encoding genes, called “oncohistones”. We identified a new mechanism by which cancers can acquire unstable nucleosomes, through upregulation of the nucleosome-poisoning histone H2A variant H2A.B. This variant is normally expressed in testis only, where it contributes to the unique chromatin environment in sperm. We compared the amino acid sequences of H2A.B and H2A and identified oncohistone features in the wildtype H2A.B sequence, suggesting that this is a readymade oncohistone sitting in our genomes. Consistent with this hypothesis, we identified H2A.B expression in 50% of diffuse large B-cell lymphomas and 5-10% of many other common malignancies but not in normal tissue outside of testis. We also found unique patterns of alternative splicing in H2A.B-expressing cancers, and these changes were previously reported in the context of nucleosome destabilization. Finally, we performed knockdown studies and found that H2A.B reduction impairs cancer cell growth in several different lymphoma cell lines, results that are supported by large-scale CRISPR studies. Together, these data substantially expand the number of cancers that may be driven by oncohistone biology. However, the contribution of H2A.B to oncogenesis is not known. This proposal will identify the effects of H2A.B on the transcriptome and epigenome in order to find new vulnerabilities in H2A.B-positive cancers. In aim 1, we employ RNA sequencing to identify changes in gene expression and alternative splicing in response to induction or reduction of H2A.B. In aim 2 we use chromatin profiling methods developed in the Henikoff lab to study the impact of H2A.B expression on the epigenome. In aim 3 we use data from aims 1 and 2 to identify vulnerabilities in H2A.B-positive cancers including investigation of aclarubicin, an anthracycline with similar efficacy but much more favorable side effect profile than doxorubicin. The experiments in this proposal will elucidate the role of H2A.B in cancer and identify novel therapeutic vulnerabilities.