Understanding PD-L1/L2 protein regulation, detection and signaling to predict melanoma therapeutic sensitivity

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PROJECT SUMMARY/ABSTRACT Modulators and effectors of anti-PD-1 responsiveness in cancer include T cell infiltration, an immune- suppressive microenvironment, tumor mutational burden, and tumor cell-intrinsic pathway (e.g., - catenin) alterations. Cell-surface PD-L1 level has been implicated as a baseline predictive marker of anti-PD-1 responsiveness, and cell-surface PD-L2 level may have predictive value independent of PD- L1. In melanoma, MAPK inhibitor therapy strongly induces PD-L1/L2 expression levels in tumor, stromal and immune cells, suggesting contributions to adaptive resistance. Induction of cell-surface PD-L1 is central to adaptive immune resistance and implicated as a mechanism of acquired anti-PD-1 resistance. Moreover, the regulation of surface PD-L1 protein stability has been implicated in tumor immune surveillance, where increased degradation augments tumor-specific T cell activity. We hypothesize that a better understanding of regulatory mechanisms controlling cell-surface PD- L1/L2 stability, clinical detection, and tumor cell-intrinsic pro-survival signaling could shed insights into melanoma immune evasion and therapeutic responsiveness. We will use proteomic approaches to dissect these processes and to explore the melanoma surface glycoproteome, the PD-L1/L2 interactome and the cytoplasmic signalosome in order to nominate mechanisms and/or markers of therapeutic responsiveness. We will interrogate iteratively clinical tumor samples and syngeneic mouse models of Braf, Nras and Nf1 mutant melanoma. These immune-competent models of melanoma are clinically relevant given their UV-induced high mutational burdens, dependence on CD8 T cells for therapeutic responses and capability for widespread metastases, including metastases to the brain. We will use cell-surface labeling of sialic acid-containing glycans to analyze the live cell surface glycoproteome, co-immunoprecipitation of PD-L1/L2 to enrich for interactomes, and APEX-based proteomic strategy to define in situ dynamic intracellular PD-L1/L2 neighborhood interactomes in response to PD-1 ligation or IFN treatment. We will address what regulate PD-L1/L2 ubiquitination, recycling and degradation, how glycosylation affects membrane PD-L1 immunohistochemical detection, whether deglycosylation improves prediction of anti-PD-1 responses at baseline and on- treatment, and how novel cytoplasmic motifs of PD-L1/L2 mediate PD-1-dependent tumor cell pro- survival signaling. Using proximity ligation assays on clinical melanoma, we will test whether PD-L1/L2 tumor cell-intrinsic signaling reduces therapy efficacy. These proteomic approaches should advance our understanding of therapy response patterns in metastatic melanoma and nominate predictive biomarkers and combinatorial targets.