PROJECT SUMMARY/ABSTRACT Metastatic prostate cancer (PCa) remains incurable and lethal. Immunotherapy using immune checkpoint blockade (ICB) antibodies revolutionized cancer treatment, yet advanced PCa is refractory to ICB therapy. PCa has limited infiltration of effector cytotoxic T lymphocytes (CTLs), which may contribute to ICB resistance. However, the mechanism shaping the cold microenvironment of PCa is poorly understood. Recently, we identified chromatin effector PYGO2 in the 1q21.3 amplicon as an oncogenic and metastatic driver for PCa (Lu et al, 2018). PYGO2 binds to histone H3 tail methylated at lysine 4 (H3K4me) and modulates transcription. Our preliminary results suggest that PYGO2 is an emerging signaling hub to control immunosuppression and CTL infiltration in a Wnt/β-catenin-independent manner, yet the precise mechanism for PYGO2 function in PCa immune evasion is unclear. Moreover, the effect of targeting PYGO2 with selective inhibitors on PCa progression and response to immunotherapy is unknown. Therefore, there is a significant need to unravel the function, mechanism and targeting strategy of PYGO2 in order to enhance immunotherapy of PCa. Our central hypothesis is that PYGO2 is a major cancer cell-intrinsic mechanism to shape the immunosuppressive microenvironment in PCa, and ICB therapy can be significantly enhanced by PYGO2 inhibition using novel small molecule inhibitors. We propose to accomplish three Specific Aims: (Aim 1) Elucidate the epigenetic mechanism of PYGO2 function in the immunosuppression of PCa by integrating targeted and unbiased approaches. (Aim 2) Determine the synergistic anti-tumor effect on PCa progression and metastasis by combining PYGO2 ablation and ICB therapy. (Aim 3) Identify small molecule inhibitors of PYGO2 with anti- tumor activity as single agent and in combination with immunotherapy. Upon completion of the project, we expect to illuminate novel molecular and cellular mechanisms underlying the role of PYGO2 in immune evasion of the PCa tumor microenvironment, establish the key preclinical evidence on co-targeting PYGO2 and immune checkpoint pathways as a new strategy of effective combination immunotherapy, and identify selective PYGO2 inhibitors with potent anti-tumor activity when used as single agent or in combination with immunotherapy. Our results will advance understanding of how genetic changes in cancer cells (i.e. PYGO2 amplification) can shape the cold tumor microenvironment. Given the aberrant upregulation of PYGO2 in many cancer types, our studies are relevant to a large population of cancer patients. In the long term, we envision that the bench-to-bedside translation of our findings through development and clinical testing of the PYGO2 inhibitor(s) may accelerate the therapeutic application of the combined epigenetic modulation and immunotherapy to the curative treatment of PCa and other cancers.