PROJECT 1 ABSTRACT Mutation-targeted small molecular inhibitors and immune checkpoint blockade or ICB (anti-PD-1 and -CTLA-4 antibodies) have extended the quality and quantity of life for patients with advanced BRAFMUT melanoma. Beyond ICB, which is ineffective in up to 60% of patients, patients with advanced NRASMUT melanoma have no standard- of-care options. Due to the rapid onset of resistance, MEK inhibitor (MEKi) monotherapy has limited clinical activity against NRASMUT melanoma, with the potential exception of those melanoma previously exposed to ICB (the NEMO trial). Here, we propose to design MEKi-based combinatorial-sequential regimens against advanced NRASMUT melanoma. First, we will test the concept of priming MEKi responsiveness via anti-PD-1/L1 pretreatment (regardless of anti-PD-1/L1 sensitivity). We will test this concept as well as dissect the mechanisms of action and innate/acquired resistance in human subjects (by conducting a phase II investigator-initiated trial) and in syngeneic melanoma models. Second, we will test the concept of combining a next-generation RAF inhibitor (RAFi) with a MEKi to overcome and to prevent MEKi resistance. These studies will be conducted in human melanoma cell lines, patient-derived xenografts (PDXs), and syngeneic models. Third, we will test the concept of overcoming MEKi resistance by pharmacologically exploiting a hallmark vulnerability of resistant tumors: MEKi-addiction or tumor cell death induced by MEKi withdrawal. Specifically, to induce the regression of a diverse array of NRASMUT MEKi-resistant PDX models, we will test one strategy involving sequencing from a MEKi to a poly-ADP ribose polymerase inhibitor (PARPi) or another strategy involving rotations between MEKi and PARPi. We will also test whether these sequencing/rotational strategies, by inducing various extents of immunogenic cell death and/or innate immunity, would sensitize NRASMUT melanoma to combined anti-PD-1/L1 therapy. In these clinical and preclinical studies, we will derive multi-omic data and evaluate candidate pathways to identify predictive biomarkers. In addition to bulk tumor-based, multi-omic analysis (exome, genome, transcriptome, methylome, chromatin accessibility, proteome, TCR clonotypes) of longitudinal tumor samples, we will also dissect the single-cell (scRNA-seq, CyTOF) evolution of NRASMUT melanoma to identify additional combinatorial-sequential targets to overcome and then to prevent resistance. These studies require a close collaboration with Projects 2 and 3 and Cores A-C at levels of shared technical expertise, reagents/models and preliminary data, and this collaboration is grounded on a 10-year track record of joint publications that have resulted in deep insights into clinically relevant melanoma biology, multiple clinical trials and even approved therapies. The combination of oncology and dermatology expertise (Ribas and Lo) and laboratories with complementary approaches (Ribas, Lo, Graeber) promises ...