BACKGROUND Approximately 75% of objective responses to anti–programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti–PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor–associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.)

SUMMARY Combined BRAF and MEK targeted therapy improves upon BRAF inhibitor (BRAFi) therapy but is still beset by acquired resistance. We show that melanomas acquire resistance to combined BRAF and MEK inhibition by augmenting or combining mechanisms of single-agent BRAFi resistance. These double-drug resistance-associated genetic configurations significantly altered molecular interactions underlying MAPK pathway reactivation. V600EBRAF, expressed at supra-physiological levels because of V600EBRAF ultra-amplification, dimerized with and activated CRAF. In addition, MEK mutants enhanced interaction with over-expressed V600EBRAF via a regulatory interface at R662 of V600EBRAF. Importantly, melanoma cell lines selected for resistance to BRAFi+MEKi, but not those to BRAFi alone, displayed robust drug addiction, providing a potentially exploitable therapeutic opportunity.

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up-or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.

) and (2ЈZ,3ЈE)-6-bromoindirubin-3Ј-oxime (BIO), two glycogen synthase kinase-3␤ (GSK3␤) inhibitors, promote survival of growth factor-deprived renal epithelial cells by activating the Wnt pathway. These studies demonstrate that Li ϩ and BIO activate Wnt signaling as indicated by the following changes: phosphorylation (inhibition) of GSK3␤; decreased phosphorylation of ␤-catenin (a GSK3␤ substrate); nuclear translocation of ␤-catenin; specific transcriptional activation of Tcf/catenin-responsive pTopflash constructs; and an increase in the expression of cyclin D1 (indicative of a promitogenic cell response). In addition, Li ϩ or BIO significantly increases the phosphorylation (activation) of Akt, an anti-apoptotic protein, and inhibits apoptosis (decreases both annexin-V staining and caspase-3 activation), during serum deprivation. Inhibition of phosphatidylinositol 3-kinase (responsible for Akt activation) either by wortmanin or LY-294002 prevented Li ϩ

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-⌬EEVD) or ATPase function (hsp70-⌬ATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-⌬EEVD) or the ATP hydrolytic domain (hsp70-⌬ATPase) failed to prevent mitochondrial AIF release. Although hsp70-⌬EEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-⌬ATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.Disruption of the outer mitochondrial membrane releases toxic proteins, including cytochrome c and apoptosis-inducing factor (AIF) 3 that activate caspase-dependent and -independent pathways responsible for apoptotic cell death. In a prior study in our laboratory, neither a pancaspase inhibitor nor a specific caspase 3 inhibitor completely prevented apoptosis in renal cells exposed to metabolic inhibitors (1), suggesting that at least a portion of the observed apoptosis is mediated by non-caspase-dependent factors including AIF. Nuclear translocation of AIF activates endogenous endonucleases leading to degradation of native DNA into 50-kilobase fragments, peripheral chromatin condensation, and nuclear shrinkage (2-4) that is sufficient to cause apoptosis (5, 6). AIF mediates apoptosis in multiple cell lines after diverse insults including ischemia (7-10), oxidant stress (11), ethanol (12), or p53 exposure (8).AIF is synthesized in the cytosol as a 67-kDa precursor protein that contains a mitochondrial-localizing sequence (13,14). After being imported into mitochondria, the mitochondrial-localizing sequence is cleaved, resulting in the accumulation of the mature, 57-kDa AIF protein (13). Under normal circumstances AIF may facilitate electron transport, since it exhibits robust oxidoreductase activity (14). AIF-mediated cell death is a two-step process that involves initial release from the intramembranous mitochondrial space into the cytosol followed by nuclear uptake and accumulation (5,13,15,16).hsp70, an inducible cytoprotectant protein, antagonizes apoptosis by interfering with multiple ch...