Overall Summary Tumor-microenvironment interactions underlie fundamental features of both tumorigenesis and therapeutics. Surviving a hostile environment represents profound selective pressure to which aggressive malignancies like melanoma evolve, in part because the cutaneous melanoma genome is typically riddled by numerous UV mutations which may be immunogenic. This competing renewal application presents projects born from collaborative and persistent efforts over the past 10 years (2 cycles) to understand these interactions and gradually move towards translatable opportunities for patients. Our productivity has been high, with potentially impactful studies proposed below based upon extensive preliminary data. The P01 collectively published 61 papers in the first 4 years of this cycle, 19 in Nature, Cell, Science, 33 in these journal “families” and 54 containing multiple P01 labs during the first 2 cycles. Building on deep study of the MITF oncoprotein, Project 1 discovered MITF regulation of DDI2 protease, a factor found to trigger destruction of MHC-antigen complexes, weakening immune recognition. DDI2 suppression (genetically or using an FDA approved inhibitor based on cross reactivity of DDI2’s active site) profoundly upregulates antigen presentation, T cell activation, tumor killing, and in vivo immune checkpoint inhibition in multiple syngeneic models. Project 2 has generated novel live-imaging technologies that visualize the in vivo cancer niche, which modulates tumor environment interactions, immunity, and signaling responses. Project 3 identified CARM1 from an in vivo screen for immune-tumor modulators. Carm1 is an epigenetic enzyme that negatively regulates T cell function and dendritic cell cross-presentation. To mechanistically probe and translationally develop these concepts further, the following Aims are proposed. Project 1 will test if DDI2 inhibition (knockout or drug) boosts only anti-PD1 or also anti-CTLA4, anti-LAG3 or triple combinations, with tumors subjected to detailed phenotyping. Immunopeptidomics will determine whether DDI2 inhibition enhances diversity of antigen-peptides presented by MHC, beyond overall MHC-antigen levels. DDI2 will be tested as a mediator of MITF-induced oncogenic immune evasion. And >200 human melanoma scRNAseq analyses will be examined to identify patients most likely to benefit from DDI2 targeting. Project 2 will utilize in vivo live imaging to examine immune interactions among cells upon activation of IFN, Wnt and TGFβ pathways, which are key to tumorigenesis and therapeutic responses. A novel in vivo cell-barcoding technology will also be deployed to understand clonality and cell function in the cancer niche. Project 3 will utilize highly defined genetic tools and detailed expression/epigenetic analyses to identify how CARM1 in T cells modulates an exhausted state, and how in dendritic cells CARM1 antagonizes antigen cross-presentation. The 3 projects all have detailed components that are inte...