Combinations of BRAF inhibitors and MEK inhibitors (BRAFi + MEKi) are FDAapproved to treat BRAF V600E/K-mutant melanoma. Effi cacy of BRAFi + MEKi associates with cancer cell death and alterations in the tumor immune microenvironment; however, the links are poorly understood. We show that BRAFi + MEKi caused durable melanoma regression in an immunemediated manner. BRAFi + MEKi treatment promoted cleavage of gasdermin E (GSDME) and release of HMGB1, markers of pyroptotic cell death. GSDME-defi cient melanoma showed defective HMGB1 release, reduced tumor-associated T cell and activated dendritic cell infi ltrates in response to BRAFi + MEKi, and more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi-resistant disease lacked pyroptosis markers and showed decreased intratumoral T-cell infi ltration but was sensitive to pyroptosis-inducing chemotherapy. These data implicate BRAFi + MEKi-induced pyroptosis in antitumor immune responses and highlight new therapeutic strategies for resistant melanoma. SIGNIFICANCE: Targeted inhibitors and immune checkpoint agents have advanced the care of patients with melanoma; however, detailed knowledge of the intersection between these two research areas is lacking. We describe a molecular mechanism of targeted inhibitor regulation of an immune-stimulatory form of cell death and provide a proof-of-principle salvage therapy concept for inhibitor-resistant melanoma. See related commentary by Smalley, p. 176.

Background Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through HSC activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood. Methods Expression of pri-miR17-92 and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b and 92 were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from tissue culture media and plasma of rodent models and patients with ALD. miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. Results Pro-fibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD were similar to those seen in a rodent model of alcoholic injury and in vitro. Conclusions Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights to the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

Epigenetic agents such as bromodomain and extra‐terminal region inhibitors (BETi) slow tumor growth via tumor intrinsic alterations; however, their effects on antitumor immunity remain unclear. A recent advance is the development of next‐generation BETi that are potent and display a favorable half‐life. Here, we tested the BETi, PLX51107, for immune‐based effects on tumor growth in BRAF V600E melanoma syngeneic models. PLX51107 delayed melanoma tumor growth and increased activated, proliferating, and functional CD8+ T cells in tumors leading to CD8+ T‐cell‐mediated tumor growth delay. PLX51107 decreased Cox2 expression, increased dendritic cells, and lowered PD‐L1, FasL, and IDO‐1 expression in the tumor microenvironment. Importantly, PLX51107 delayed the growth of tumors that progressed on anti‐PD‐1 therapy; a response associated with decreased Cox2 levels, decreased PD‐L1 expression on non‐immune cells, and increased intratumoral CD8+ T cells. Thus, next‐generation BETi represent a potential first‐line and secondary treatment strategy for metastatic melanoma by eliciting effects, at least in part, on antitumor CD8+ T cells.

BackgroundIn Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure.ResultsThe knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits.ConclusionsWe demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

◥Concurrent MEK and CDK4/6 inhibition shows promise in clinical trials for patients with advanced-stage mutant BRAF/NRAS solid tumors. The effects of CDK4/6 inhibitor (CDK4/6i) in combination with BRAF/MEK-targeting agents on the tumor immune microenvironment are unclear, especially in melanoma, for which immune checkpoint inhibitors are effective in approximately 50% of patients.Here, we show that patients progressing on CDK4/6i/MEK pathway inhibitor combinations exhibit T-cell exclusion. We found that MEK and CDK4/6 targeting was more effective at delaying regrowth of mutant BRAF melanoma in immunocompetent versus immune-deficient mice. Although MEK inhibitor (MEKi) treatment increased tumor immunogenicity and intratumoral recruitment of CD8 þ T cells, the main effect of CDK4/6i alone and in combination with MEKi was increased expression of CD137L, a T-cell costimulatory molecule on immune cells. Depletion of CD8 þ T cells or blockade of the CD137 ligand-receptor interaction reduced time to regrowth of melanomas in the context of treatment with CDK4/6i plus MEKi treatment in vivo. Together, our data outline an antitumor immunebased mechanism and show the efficacy of targeting both the MEK pathway and CDK4/6.