ABSTRACT A commonly recognized mechanism of immune evasion in solid tumors is diminished MHC and antigen presentation. Most cases retain some MHC expression, and strategies for upregulation may enhance immunogenicity. Melanocyte development is regulated by the master transcription factor MITF which targets virtually all components of the pigmentation machinery. This pathway is vulnerable to protein aggregation (e.g., after UV/tanning). MITF appears to simultaneously target certain “quality control” factors that protect against misfolded species. We recently discovered MITF’s direct transcriptional regulation of a protease gene called DDI2 which we found to participate in ER “quality control.” Unexpectedly, among the proteins degraded downstream of DDI2, are MHC-I and MHC-II, leading to decreased antigen presentation. We believe this occurs because peptide-MHC complexes may be “mis-perceived” as unfolded due to thermodynamic features of peptide-MHC interactions. Importantly, we found that DDI2 suppression by gene knockout or drug, upregulates MHC-antigen levels, producing functional stimulation of antigen-specific T cell activation, and enhanced tumor killing both in vitro and in vivo (with anti-PD1) in multiple tumor types. The active sites of DDI2 and HIV-1 protease coincidentally bear striking homology, and two FDA approved HIV protease inhibitors also block DDI2 (multiple others do not). One of these, Nelfinavir (NF) is a well-tolerated drug, and potently phenocopies DDI2 knockout. The combination of highly defined human and mouse melanoma models, which include antigen-specific MHC- restricted matched CD8 and CD4 T cells, coupled to deep cellular, molecular, proteomic, immunophenotyping, and tumor treatment assays, permit us to propose the following studies: Specific Aim 1 expands our evidence that DDI2 suppression (knockout or drug) enhances anti-PD1 activity, by testing in complementary mouse models, combinations of DDI2-KO or NF with anti-CTLA4, anti-LAG3, and 3-way combinations together with anti-PD1, as these are FDA approved treatments. All will be scrutinized with detailed immunophenotyping, MHC/peptidomics, and other expression assays. Specific Aim 2 examines immunopeptidomics: isolation and sequencing of MHC-associated peptide antigens. This tests which antigenic peptides are increased by DDI2 suppression, as we hypothesize that DDI2 suppression rescues a more diverse peptide population, and may boost T cell diversity (also to be tested by TCRseq). Specific Aim 3 probes MITF’s demonstrated direct regulation of DDI2, predicting a pathway that links MITF to MHC-antigen levels and, thereby, to immune evasion, which would be a novel and druggable oncogenic mechanism. Specific Aim 4 dissects data from >200 single cell RNAseq of anti-PD1-resistant patients plus additional anti-PD1 sensitive ones, where we will determine DDI2 expression (RNA and protein) and use multiplex immunofluorescence to quantify MHC-I and -II levels— linked to microen...