PROJECT 2 – SUMMARY Loss of the Y chromosome (LOY) is associated with increased risk of cancer development and death in men with several cancer types. In bladder cancer (BC), a tobacco related malignancy, LOY is seen in ~30% of tumors, yet the biological relevance of this is unknown. Here we address this Knowledge Gap with the Objective to define the molecular mechanisms responsible for LOY driven growth and progression and have Impact by translating these into effective interventions for bladder and other cancer types. Preliminary Data: We examined TCGA BC RNAseq dataset using an LOY gene signature we developed, and found low signature scores associated with reduced patient survival following surgery. To determine if LOY drives BC growth, we developed lineage related male murine cell lines with (Y+) or w/o (Y-) Y chromosome. Y- tumors grew faster than Y+ tumors in immunocompetent mice and cytometric profiling found the former to have increased immunosuppressive tumor-associated macrophages, CD8+ T cells with exhausted phenotype while latter had increased NK cells. In mice (Rag2-/-;Il2rg-/-) that are devoid of T, B and NK cells, Y+ and Y- tumors grew similarly. Transcriptional profiling found four Y chromosome genes differentially expressed in these tumor types, with chromatin organization genes Uty and Kdm5d being the only ones whose expression stratified outcome in TCGA BC patients. Thus, we genetically deleted these genes in Y+ cells and found enhanced tumor growth only in immunocompetent mice while their overexpression in Y- cells reduced their growth. Hence, we test the Hypothesis that Uty and Kdm5d loss enhance BC growth and progression via creation of an immunosuppressive tumor microenvironment in three Specific Aims. In Aim 1 we determine role of Uty and Kdm5d in tumor development and progression using full and urothelial specific conditional Kdm5d or Uty null mice be exposed to the chemical carcinogen BBN. With Project 3 we test the hypothesis that Uty and its X chromosome paralog Utx (aka, Kdm6a) have independent suppressive effects on BC formation and progression by crossing full and urothelial specific conditional Uty and Utx null mice and exposing them to BBN. In Aim 2 we define how Kdm5d and Uty suppress BC and the role of the immune system in this process using defined LOF functional mutants to define the regions on these proteins responsible for tumor growth suppression. With Project 1, we use spatial proteomics to examine immunological infiltrates in relation to mutant and WT Kdm5d and Uty cancer cell expression and various knockout mice or antibody depletion strategy to define which cellular components of the immune system drive tumor growth differences. In Aim 3 we examine ANPEP, the most upregulated gene in Y- tumors that is also a poor prognostic in BC patients and druggable target, as a candidate autosomal effector gene driving Y- tumor growth. We found Y- tumors are more responsive than Y+ tumors to anti-PD-1 and ANPEP depl...