PROJECT 3 – PROJECT SUMMARY Sex differences in bladder cancer (BC)—men being 3-5 times more likely to have BC than women—are a long- standing clinical observation. Such disparities persist across socioeconomic and geographic strata even when correcting for known hazards such as smoking and other environmental risks. Yet despite many years of research, the etiology of BC sex differences, especially as it relates to sex as a biological variable (SABV), remains ill-defined. Using “four-core genotypes” mice that consist of four instead of two sex types, we identified a prototypical sex-biasing tumor suppressor in females: histone lysine demethylase 6A (KDM6A, aka UTX). The X-linked KDM6A encodes a potent epigenetic regulator that expresses twice as high in females as in males due to its escape from X-chromosome inactivation. Consistently, somatic mutations of human KDM6A, frequently observed in BC patients of both sexes, are correlated with poor outcomes in females but not in males. We further showed that the X chromosome has both an independent and interactive sex-biasing effects with sex hormone pathways. Based on the published and preliminary findings, we hypothesize that sex differences in BC stems from effects of the sex chromosome linked epigenetic regulators (i.e., KDM6A and UTY) and effects of the sex hormones (i.e., androgens and estrogens), collectively shaping a sex specific epigenetic landscape in the bladder and sex specific immune tumor microenvironment. We will test this hypothesis with three aims: 1) compare the function of Y-linked UTY in males with its paralog X-linked KDM6A to determine whether UTY also plays a tumor suppressor role in vivo; 2) determine whether KDM6A, UTY, and sex hormones collectively shape the epigenetic landscape to drive sex differences in BC; 3) elucidate the mechanism through which KDM6A differentially modulates pro-tumorigenic immune microenvironment in the bladder. The long-term objectives of this proposal are two-fold: (1) identify key epigenetic and immune pathways that modulate sex-specific bladder tumorigenesis and (2) translate mechanistic discoveries to clinical improvement in BC screening and treatment for males and females.

PROJECT 3 – PROJECT NARRATIVE Bladder cancer is a highly sex-biased disease that disproportionately affects males, but the underlying mechanism remains largely undefined. In this Project, we aim to uncover the overlooked epigenetic basis of observed sex differences. We expect to identify sex-biased epigenetic pathways that can be used to develop future sex-specific bladder cancer screening and treatment to ultimately mitigate existing sex disparities.