Background: Cancer immunotherapy is a major breakthrough for many patients with advanced cancer. However, benefits are still limited to a subset of patients, and we need to better understand the mechanisms of response and resistance to improve therapeutic efficacy. We have identified loss of function (LoF) mutations in JAK1 or JAK2 (immediate downstream signaling molecule of interferon receptor) that are associated with resistance to PD-1 blockade. We also found these mutations in human melanoma cell lines by screening PD- L1 expression with IFN-g treatment (48 cell lines). Tumors harbor LoF in JAK1/2, completely lost PD-L1 expression. Interestingly, one of them harbors no mutation with an active signaling pathway, yet lost PD-L1 expression. We explored why some human cancer cells lost adaptive PD-L1 expression even with intact interferon signaling and hypothesized that the epigenetic perturbation is mediating this phenotype. With this approach, we observed reduced PD-L1 expression with ATRX siRNA which was further tested with in vivo mouse models. In vivo mouse experiments with ATRX KO MC38 cells, anti-PD-1 antibody therapy produced either accelerated tumor growth or no effect. The current study is designed to understand the mechanism of resistance mediated by loss of ATRX in cancer immunotherapy. Objective/hypothesis: ATRX, a SWI/SNF-like chromatin remodeler is modulating the accessibility of interferon responsive genes that are associated with immunotherapy response. Specific aims: I have two aims for this study. The first aim is to interrogate the mechanisms of immune evasion with loss of ATRX using various tools to probe epigenetic state. The second aim is to establish in vivo tumor growth with ATRX KO using various murine cancer models. Study design: Aim 1. Subaim1) Generate ATRX KO B16 cells followed by Assess epigenetics state with IFN-g stimulation (both MC38 and B16 ATRX KO clones) using ChIP/ATAC-seq. Subaim 2) Correlate genomic studies (ChIP/ATAC) with Chromatin-Associated RNA-sequence (ChAR). Subaim 3) Assess the impact of epigenetic modifiers in IFN-g response in ATRX KO clones (using various epigenetic modifiers, such as HDAC inhibitor, demethylating agents and EZH2 inhibitor). Subaim 4) Coculture assay with murine T cells with ATRX wild-type parent cells and KO clones. Aim 2. Subaim 1) In vivo experiments with MC38 and B16 models with ATRX KO. Subaim2) Kras mutant murine lung cancer cell line models with ATRX KO. Subaim 3) ATRX KO in lung cancer and melanoma genetically engineered mouse models using sleeping beauty transposase vector system. Relevant to Military health: Improving treatment of many types of advanced cancers is critically important to the health of Veterans. Many Veterans suffer from significant morbidity when they are diagnosed with cancer that limits their therapeutic options. Immunotherapy is generally well tolerated and has a significant potential for durable response, however, the benefit is limited to a subset...