PROJECT SUMMARY In the treatment of BRAFV600MUT melanoma, acquired resistance to MAPK inhibitor (MAPKi) limits its survival benefits. Currently, MAPKi therapy is unavailable to patients with non-BRAFV600MUT melanoma. To improve MAPKi therapy and to extend its application to more patients, we propose a project to develop two general concepts to render MAPKi therapy more effective. Both concepts aim to prevent acquired MAPKi resistance by targeting processes that are operative early on MAPKi therapy and that enable melanoma persisters to undergo clonal evolution. In prior publications, we and others have shown that the selective pressure of MAPKi elicits (i) a new round of genomic instability, leading to clonal diversification and selective expansion of clones that drive acquired resistance and (ii) a new round of immune editing and hence immune resistance, leading to selective expansion of clones that evade antitumor immunity (in particular cytotoxic T cells). We hypothesize that therapeutic strategies that address these adaptive mechanisms in residual tumor cells on MAPKi therapy can potentially prevent the seeds of acquired resistance. We expect that preventive approaches such as these, combined with the more durable efficacy of antitumor immunity, should be more effective than conventional approaches to target specific vulnerabilities of acquired resistance, due to extensive tumor heterogeneity and clonal evolution resulting from disease progression. In Aim 1, we build on our recent study showing that genomic instability such as chromothripsis gives rise to amplicons that are selected by MAPKi to cause acquired resistance. These amplicons may be intrachromosomal or extrachromosomal, the latter as circular ecDNAs. This finding led to further discovery that a combination to block these mechanisms of genomic instability may prevent acquired resistance and the design of a trial testing this combination as targeted therapy for NRASMUT melanoma. We hypothesize that the mechanisms by which MAPKi elicits de novo genomic instability represent combinatorial targets to prevent acquired resistance. These mechanisms involve the partial rupture of primary and micronuclear membranes, chromosome mis-segregation and DNA-damaging proteins that aberrantly access normally protected chromosomal DNAs. In Aim 2, we build on our recent published and unpublished studies showing that innate/adaptive immune resistance arises in persisters early on MAPKi or immune checkpoint blockade (ICB) therapy, which results in PD-L1 accumulation on the tumor surface. To diminish immune resistance, we identified a tool compound AK087 to degrade surface PD-L1, thereby improving the efficacy of MAPKi (and ICB) therapy in immune-competent murine tumor models. Here, we will dissect the mechanisms of action of AK087 to guide further the rational development of PD-L1 degraders as a general combinatorial strategy to improve the efficacy current melanoma therapies.