PROJECT SUMMARY/ABSTRACT The long-term goals of this project are to define the mechanisms of resistance to immunotherapy and to develop effective therapies for patients with metastatic bladder cancer (BC). The overall objective of this proposal is to establish successful combination therapies for patients with a specific genomic subset of metastatic BC harboring homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene from the chromosome 9p21 region. Although novel immune checkpoint therapy (ICT), including anti-PD1/PD-L1, provides substantial benefits to patients with metastatic BC, response rates are usually modest at 15% to 25%. This is partly because this biologically heterogeneous cancer is still treated clinically as a uniform disease. Therefore, identification of specific genomic subtypes of BC that confer insensitivity to ICT may provide novel opportunities to improve clinical responses. We have confirmed that ~1/4 of BC contain homozygous deletion of MTAP (MTAPdef) from the 9p21region. The MTAP gene encodes for an essential enzyme to catalyze methylthioadenosine (MTA) in the salvage pathway for adenine synthesis. Tumor MTAPdef leads to both immunologic and metabolic consequences. Immunologically, tumor MTAPdef results in accumulation of its substrate MTA, which acts through the adenosine 2B receptor (A2BR) to inhibit IFN signaling and T cell function. Therefore, MTAPdef BC may foster a “cold” tumor immune microenvironment (TIME) unfavorable to ICT. Metabolically, tumor MTAPdef results in a lack of salvage pathway adenine synthesis; thus, MTAPdef BC should be very sensitive to the cytotoxic effects of anti-folate agents (e.g., pemetrexed), which effectively inhibit de novo adenine synthesis. This concept is confirmed by pre-clinical and clinical data to be presented. Importantly, our data also indicate that pemetrexed increases tumor immune cell infiltration and PD-L1 expression and thus may sensitize BC to ICT. Based on these data, we hypothesize that, by targeting the metabolic vulnerability of MTAPdef BC and directly modulating its tumor immune microenvironment, effective combination therapies can be established for MTAPdef BC. To test this hypothesis, we proposed two Specific Aims: (1) Define the immunological consequences of MTAPdef in BC; (2) Identify successful combination therapies specifically targeting MTAPdef BC. Patient-derived BC tissues, gene knockout and “rescue” mouse BC models, and samples from an IRB-approved clinical trial will be used to address these goals. At completion, we expect to establish the contribution of MTAPdef and/or loss of adjacent genes such as CDKN2A in the 9p21 region to the BC TIME. In addition, we will determine the extent of TIME modulation by pemetrexed +/- avelumab (anti-PD-L1) in relation to their therapeutic efficacy in patients with metastatic BC. Furthermore, we will define the preclinical therapeutic benefits of triple combination treatment with pemetrexed, anti-PD-L1, and A2...