Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N 6 -methyladenosine (m 6 A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m 6 A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m 6 A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m 6 A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.

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Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.

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.

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Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal–regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1–mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.

Melanoma cells are highly resistant to anoikis, a form of apoptosis induced in nonadherent/inappropriate adhesion conditions. Depleting B-RAF or the prosurvival Bcl-2 family protein Mcl-1 renders mutant B-RAF melanoma cells susceptible to anoikis. In this study, we examined the effect of targeting B-RAF on the survival of primary stage melanoma cells cultured in three-dimensional type I collagen gels, which partially mimics the dermal microenvironment. Depletion/inhibition of B-RAF with small interfering RNA or the mutant B-RAF inhibitor, PLX4720, induced apoptosis of mutant B-RAF melanoma cells in three-dimensional collagen. Apoptosis was dependent on two upregulated BH3-only proteins, Bim-EL and Bmf, and was inhibited by ectopic Mcl-1 expression. Akt3 activation has been associated with the survival of melanoma cells. Mutant B-RAF melanoma cells ectopically expressing a constitutively activated form of Akt3 or endogenously expressing mutant Akt3 were protected from apoptosis induced by B-RAF knockdown or PLX4720 treatment. Furthermore, intrinsically resistant metastatic melanoma cells displayed elevated Akt phosphorylation in three-dimensional collagen and were rendered susceptible to PLX4720 by Akt3 knockdown. Importantly, myristylated Akt3 prevented B-RAF targeting-induced upregulation of Bim-EL and Bmf in three-dimensional collagen and partially protected Mcl-1-depleted cells from apoptosis. These findings delineate how mutant B-RAF protects melanoma cells from apoptosis and provide insight into possible resistance mechanisms to B-RAF inhibitors.

Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2–Sos complex. Since Grb2–Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a β1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.