The role of tumor cell-of-origin-specific PDL1 on tumorigenesis and tumor progression

ABSTRACT This F31-diversity proposal investigates the role of PDL1 in early melanoma progression using our novel cancer cell-of-origin autochthonous mouse model. Our overarching hypothesis is that tumor-intrinsic PDL1 signals promote early tumor progression by both immune and non-immune mechanisms. Our proposal will define novel cell-intrinsic PDL1 signals in the melanoma cell-of-origin and established melanomas providing the first model distinguishing bona-fide biologic tumor-intrinsic PDL1 signaling in the absence of potentially confounding mechanisms due to genetic PDL1KO of a previously PDL1-replete tumor cell. We will address this hypothesis with the following aims: Aim 1 Test the hypothesis that melanocyte-intrinsic PDL1 signals promote early melanoma progression and treatment sensitivities. We will investigate melanocyte-intrinsic PDL1 signals, downstream effectors, and contributions of UV exposure facilitating early tumor progression using the PDL1KO TNQ61R mouse model we created, and littermate controls, in which PDL1 is specifically deleted in melanocytes. Transcriptomic (RNA-seq) and proteomic (Luminex, RPPA) dynamics will be assessed as melanocytes progress from PDL1-null benign nevi to malignant melanomas defining when PDL1 emerges during carcinogenesis and determining the genetic and proteomic milieu governing PDL1 expression in vivo and in vitro and its consequences. PDL1 regulation of oncogenic signals (e.g., NRAS, BRAF, MEK, ERK, mTORC1) will be tested using small molecule inhibitors and in vitro CRISPR replacement of mutant NrasQ61R for WT Nras or BRAFV600E. PDL1 suppression of immunogenic STING signals following skin UV exposure will be explored in parallel and as immunogenicity mechanisms are defined, their contributions to de novo tumor growth and treatment sensitivities will be tested in Aim 2. Aim 2 Define TME factors in melanocyte-intrinsic PDL1-drive progression and treatment vulnerabilities. We will test melanocyte PDL1-dependent TME immune dynamics using our PDL1KO TNQ61R model by harvesting non-malignant skin and induced tumor over time, assessing immune populations as tumors progress with flow cytometry. Immunoblots, Luminex, and qRT-PCR will test immunogenic STING signals and chemokine production ex vivo and immune cell co-localization with tumor will be assessed by confocal imaging. Alternative immunogenic pathways known to affect treatment efficacy (e.g., pyroptosis, RIG-I/MAVS) will be studied in similar fashion. Studies of PDL1KO TNQ61R mice and derived cell lines with ICB, immune cell depletion, and immune deficient mice test PDL1-driven immune-dependent targetable treatment vulnerabilities. Immune outcomes of PDL1/Nras cross talk will be defined complementary to signaling studies in Aim 1.