ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFNγ)-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFNγR expression, tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor influences outcomes of ICB.

Loss of AMPKα2 promotes melanoma tumor growth and brain metastasis.

AMP-activated protein kinase (AMPK) is a critical cellular energy sensor at the interface of metabolism and cancer. However, the role of AMPK in carcinogenesis remains unclear. Here, through analysis of the TCGA melanoma dataset, we found that PRKAA2 gene that encodes the α2 subunit of AMPK is mutated in ∼9% of cutaneous melanomas, and these mutations tend to co-occur with NF1 mutations. Knockout of AMPKα2 promoted anchorage-independent growth of NF1-mutant melanoma cells, whereas ectopic expression of AMPKα2 inhibited their growth in soft agar assays. Moreover, loss of AMPKα2 accelerated tumor growth of NF1-mutant melanoma and enhanced their brain metastasis in immune-deficient mice. Our findings support that AMPKα2 serves as a tumor suppressor in NF1-mutant melanoma and suggest that AMPK could be a therapeutic target for treating melanoma brain metastasis.

Dasatinib Resensitizes MAPK Inhibitor Efficacy in Standard-of-Care Relapsed Melanomas.

Resistance to combination BRAF/MEK inhibitor (BRAFi/MEKi) therapy arises in nearly every patient with BRAFV600E/K melanoma, despite promising initial responses. Achieving cures in this expanding BRAFi/MEKi-resistant cohort represents one of the greatest challenges to the field; few experience additional durable benefit from immunotherapy and no alternative therapies exist. To better personalize therapy in cancer patients to address therapy relapse, umbrella trials have been initiated whereby genomic sequencing of a panel of potentially actionable targets guide therapy selection for patients; however, the superior efficacy of such approaches remains to be seen. We here test the robustness of the umbrella trial rationale by analyzing relationships between genomic status of a gene and the downstream consequences at the protein level of related pathway, which find poor relationships between mutations, copy number amplification, and protein level. To profile candidate therapeutic strategies that may offer clinical benefit in the context of acquired BRAFi/MEKi resistance, we established a repository of patient-derived xenograft models from heavily pretreated patients with resistance to BRAFi/MEKi and/or immunotherapy (R-PDX). With these R-PDXs, we executed in vivo compound repurposing screens using 11 FDA-approved agents from an NCI-portfolio with pan-RTK, non-RTK and/or PI3K-mTOR specificity. We identify dasatinib as capable of restoring BRAFi/MEKi antitumor efficacy in ~70% of R-PDX tested. A systems-biology analysis indicates elevated baseline protein expression of canonical drivers of therapy resistance (e.g., AXL, YAP, HSP70, phospho-AKT) as predictive of MAPKi/dasatinib sensitivity. We therefore propose that dasatinib-based MAPKi therapy may restore antitumor efficacy in patients that have relapsed to standard-of-care therapy by broadly targeting proteins critical in melanoma therapy escape. Further, we submit that this experimental PDX paradigm could potentially improve preclinical evaluation of therapeutic modalities and augment our ability to identify biomarker-defined patient subsets that may respond to a given clinical trial.

Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas.

BACKGROUND: Primary and posttreatment resistance to BRAFV600 mutation-targeting inhibitors leads to disease relapse in a majority of melanoma patients. In many instances, this resistance is promoted by upregulation of mitochondrial oxidative phosphorylation (OxPhos) in melanoma cells. We recently showed that a novel electron transport chain (ETC) complex I inhibitor, IACS-010759 (IACS), abolished OxPhos and significantly inhibited tumor growth of high-OxPhos, BRAF inhibitor (BRAFi)-resistant human melanomas. However, the inhibition was not uniform across different high OxPhos melanomas, and combination with BRAFi did not improve efficacy. METHODS: We performed a high-throughput unbiased combinatorial drug screen of clinically relevant small molecules to identify the most potent combination agent with IACS for inhibiting the growth of high-OxPhos, BRAFi-resistant melanomas. We performed bioenergetics and carbon-13 metabolite tracing to delineate the metabolic basis of sensitization of melanomas to the combination treatment. We performed xenograft tumor growth studies and Reverse-Phase Protein Array (RPPA)-based functional proteomics analysis of tumors from mice fed with regular or high-fat diet to evaluate in vivo molecular basis of sensitization to the combination treatment. RESULTS: A combinatorial drug screen and subsequent validation studies identified Atorvastatin (STN), a hydroxymethylglutaryl-coenzyme A reductase inhibitor (HMGCRi), as the most potent treatment combination with IACS to inhibit in vitro cell growth and induce tumor regression or stasis of some BRAFi-resistant melanomas. Bioenergetics analysis revealed a dependence on fatty acid metabolism in melanomas that responded to the combination treatment. RPPA analysis and carbon-13 tracing analysis in these melanoma cells showed that IACS treatment decreased metabolic fuel utilization for fatty acid metabolism, but increased substrate availability for activation of the mevalonate pathway by HMGCR, creating a dependence on this pathway. Functional proteomic analysis showed that IACS treatment inhibited MAPK but activated AKT pathway. Combination treatment with STN counteracted AKT activation. CONCLUSIONS: STN and other clinically approved HMGCRi could be promising combinatorial agents for improving the efficacy of ETC inhibitors like IACS in BRAFi-resistant melanomas.

Mechanism-Specific Pharmacodynamics of a Novel Complex-I Inhibitor Quantified by Imaging Reversal of Consumptive Hypoxia with [18F]FAZA PET In Vivo.

Tumors lack a well-regulated vascular supply of O2 and often fail to balance O2 supply and demand. Net O2 tension within many tumors may not only depend on O2 delivery but also depend strongly on O2 demand. Thus, tumor O2 consumption rates may influence tumor hypoxia up to true anoxia. Recent reports have shown that many human tumors in vivo depend primarily on oxidative phosphorylation (OxPhos), not glycolysis, for energy generation, providing a driver for consumptive hypoxia and an exploitable vulnerability. In this regard, IACS-010759 is a novel high affinity inhibitor of OxPhos targeting mitochondrial complex-I that has recently completed a Phase-I clinical trial in leukemia. However, in solid tumors, the effective translation of OxPhos inhibitors requires methods to monitor pharmacodynamics in vivo. Herein, 18F-fluoroazomycin arabinoside ([18F]FAZA), a 2-nitroimidazole-based hypoxia PET imaging agent, was combined with a rigorous test-retest imaging method for non-invasive quantification of the reversal of consumptive hypoxia in vivo as a mechanism-specific pharmacodynamic (PD) biomarker of target engagement for IACS-010759. Neither cell death nor loss of perfusion could account for the IACS-010759-induced decrease in [18F]FAZA retention. Notably, in an OxPhos-reliant melanoma tumor, a titration curve using [18F]FAZA PET retention in vivo yielded an IC50 for IACS-010759 (1.4 mg/kg) equivalent to analysis ex vivo. Pilot [18F]FAZA PET scans of a patient with grade IV glioblastoma yielded highly reproducible, high-contrast images of hypoxia in vivo as validated by CA-IX and GLUT-1 IHC ex vivo. Thus, [18F]FAZA PET imaging provided direct evidence for the presence of consumptive hypoxia in vivo, the capacity for targeted reversal of consumptive hypoxia through the inhibition of OxPhos, and a highly-coupled mechanism-specific PD biomarker ready for translation.

Increased Tumor Glycolysis Characterizes Immune Resistance to Adoptive T Cell Therapy.

Adoptive T cell therapy (ACT) produces durable responses in some cancer patients; however, most tumors are refractory to ACT and the molecular mechanisms underlying resistance are unclear. Using two independent approaches, we identified tumor glycolysis as a pathway associated with immune resistance in melanoma. Glycolysis-related genes were upregulated in melanoma and lung cancer patient samples poorly infiltrated by T cells. Overexpression of glycolysis-related molecules impaired T cell killing of tumor cells, whereas inhibition of glycolysis enhanced T cell-mediated antitumor immunity in vitro and in vivo. Moreover, glycolysis-related gene expression was higher in melanoma tissues from ACT-refractory patients, and tumor cells derived from these patients exhibited higher glycolytic activity. We identified reduced levels of IRF1 and CXCL10 immunostimulatory molecules in highly glycolytic melanoma cells. Our findings demonstrate that tumor glycolysis is associated with the efficacy of ACT and identify the glycolysis pathway as a candidate target for combinatorial therapeutic intervention.

ATG5 Mediates a Positive Feedback Loop between Wnt Signaling and Autophagy in Melanoma.

Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of ß-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low ß-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased ß-catenin. To define the physiologic relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing ß-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. Cancer Res; 77(21); 5873-85. ©2017 AACR.

Characterization of Human Cancer Cell Lines by Reverse-phase Protein Arrays.

Cancer cell lines are major model systems for mechanistic investigation and drug development. However, protein expression data linked to high-quality DNA, RNA, and drug-screening data have not been available across a large number of cancer cell lines. Using reverse-phase protein arrays, we measured expression levels of ∼230 key cancer-related proteins in >650 independent cell lines, many of which have publically available genomic, transcriptomic, and drug-screening data. Our dataset recapitulates the effects of mutated pathways on protein expression observed in patient samples, and demonstrates that proteins and particularly phosphoproteins provide information for predicting drug sensitivity that is not available from the corresponding mRNAs. We also developed a user-friendly bioinformatic resource, MCLP, to help serve the biomedical research community.

Inhibition of mTORC1/2 overcomes resistance to MAPK pathway inhibitors mediated by PGC1α and oxidative phosphorylation in melanoma.

Metabolic heterogeneity is a key factor in cancer pathogenesis. We found that a subset of BRAF- and NRAS-mutant human melanomas resistant to the MEK inhibitor selumetinib displayed increased oxidative phosphorylation (OxPhos) mediated by the transcriptional coactivator PGC1α. Notably, all selumetinib-resistant cells with elevated OxPhos could be resensitized by cotreatment with the mTORC1/2 inhibitor AZD8055, whereas this combination was ineffective in resistant cell lines with low OxPhos. In both BRAF- and NRAS-mutant melanoma cells, MEK inhibition increased MITF expression, which in turn elevated levels of PGC1α. In contrast, mTORC1/2 inhibition triggered cytoplasmic localization of MITF, decreasing PGC1α expression and inhibiting OxPhos. Analysis of tumor biopsies from patients with BRAF-mutant melanoma progressing on BRAF inhibitor ± MEK inhibitor revealed that PGC1α levels were elevated in approximately half of the resistant tumors. Overall, our findings highlight the significance of OxPhos in melanoma and suggest that combined targeting of the MAPK and mTORC pathways may offer an effective therapeutic strategy to treat melanomas with this metabolic phenotype.

Molecular profiling of patient-matched brain and extracranial melanoma metastases implicates the PI3K pathway as a therapeutic target.

PURPOSE: An improved understanding of the molecular pathogenesis of brain metastases, one of the most common and devastating complications of advanced melanoma, may identify and prioritize rational therapeutic approaches for this disease. In particular, the identification of molecular differences between brain and extracranial metastases would support the need for the development of organ-specific therapeutic approaches. EXPERIMENTAL DESIGN: Hotspot mutations, copy number variations (CNV), global mRNA expression patterns, and quantitative analysis of protein expression and activation by reverse-phase protein array (RPPA) analysis were evaluated in pairs of melanoma brain metastases and extracranial metastases from patients who had undergone surgical resection for both types of tumors. RESULTS: The status of 154 previously reported hotspot mutations, including driver mutations in BRAF and NRAS, were concordant in all evaluable patient-matched pairs of tumors. Overall patterns of CNV, mRNA expression, and protein expression were largely similar between the paired samples for individual patients. However, brain metastases demonstrated increased expression of several activation-specific protein markers in the PI3K/AKT pathway compared with the extracranial metastases. CONCLUSIONS: These results add to the understanding of the molecular characteristics of melanoma brain metastases and support the rationale for additional testing of the PI3K/AKT pathway as a therapeutic target in these highly aggressive tumors.

Resistance to BRAF inhibition in BRAF-mutant colon cancer can be overcome with PI3K inhibition or demethylating agents.

PURPOSE: Vemurafenib, a selective inhibitor of BRAF(V600), has shown significant activity in BRAF(V600) melanoma but not in less than 10% of metastatic BRAF(V600) colorectal cancers (CRC), suggesting that studies of the unique hypermethylated phenotype and concurrent oncogenic activation of BRAF(mut) CRC may provide combinatorial strategies. EXPERIMENTAL DESIGN: We conducted comparative proteomic analysis of BRAF(V600E) melanoma and CRC cell lines, followed by correlation of phosphoinositide 3-kinase (PI3K) pathway activation and sensitivity to the vemurafenib analogue PLX4720. Pharmacologic inhibitors and siRNA were used in combination with PLX4720 to inhibit PI3K and methyltransferase in cell lines and murine models. RESULTS: Compared with melanoma, CRC lines show higher levels of PI3K/AKT pathway activation. CRC cell lines with mutations in PTEN or PIK3CA were less sensitive to growth inhibition by PLX4720 (P = 0.03), and knockdown of PTEN expression in sensitive CRC cells reduced growth inhibition by the drug. Combined treatment of PLX4720 with PI3K inhibitors caused synergistic growth inhibition in BRAF-mutant CRC cells with both primary and secondary resistance. In addition, methyltransferase inhibition was synergistic with PLX4720 and decreased AKT activation. In vivo, PLX4720 combined with either inhibitors of AKT or methyltransferase showed greater tumor growth inhibition than PLX4720 alone. Clones with acquired resistance to PLX4720 in vitro showed PI3K/AKT activation with EGF receptor (EGFR) or KRAS amplification. CONCLUSIONS: We show that activation of the PI3K/AKT pathway is a mechanism of both innate and acquired resistance to BRAF inhibitors in BRAF(V600E) CRC and suggest combinatorial approaches to improve outcomes in this poor prognosis subset of patients.

Role and therapeutic potential of PI3K-mTOR signaling in de novo resistance to BRAF inhibition.

BRAF inhibition is highly active in BRAF-mutant melanoma, but the degree and duration of responses is quite variable. Improved understanding of the mechanisms of de novo resistance may lead to rational therapeutic strategies with improved efficacy. Proteomic analysis of BRAF-mutant, PTEN-wild-type human melanoma cell lines treated with PLX4720 demonstrated that sensitive and de novo resistant lines exhibit similar RAS-RAF-MEK-ERK pathway inhibition, but the resistant cells exhibited durable activation of S6 and P70S6K. Treatment with the mTOR inhibitor rapamycin blocked activation of P70S6K and S6, but it also increased activation of AKT and failed to induce cell death. Combined treatment with rapamycin and PX-866, a PI3K inhibitor, blocked the activation of S6 and AKT and resulted in marked cell death when combined with PLX4720. The results support the rationale for combined targeting of BRAF and the PI3K-AKT pathways and illustrate how target selection will be critical to such strategies.

Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY-142886) in Braf-mutant human cutaneous melanoma cells.

The majority of melanomas show constitutive activation of the RAS-RAF-MAP/ERK kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway. AZD6244 is a selective MEK1/2 inhibitor that markedly reduces tumor P-MAPK levels, but it produces few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the phosphoinositide 3-kinase (PI3K)-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines showed AZD6244-induced activation of AKT. In contrast, sensitive cell lines showed AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or insulin-like growth factor I receptor blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells and the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients.

Activity of dasatinib against L576P KIT mutant melanoma: molecular, cellular, and clinical correlates.

Point mutations in the KIT receptor tyrosine kinase gene have recently been identified in mucosal, acral lentiginous, and chronically sun-damaged melanomas. We have identified the first human melanoma cell line with an endogenous L576P mutation, the most common KIT mutation in melanoma ( approximately 30-40%). In vitro testing showed that the cell viability of the L576P mutant cell line was not reduced by imatinib, nilotinib, or sorafenib small molecule KIT inhibitors effective in nonmelanoma cells with other KIT mutations. However, the viability of the mutant cells was reduced by dasatinib at concentrations as low as 10 nM (P = 0.004). Molecular modeling studies found that the L576P mutation induces structural changes in KIT that reduce the affinity for imatinib (DeltaDeltaGbind = -2.52 kcal/mol) but not for dasatinib (DeltaDeltaGbind = +0.32 kcal/mol). Two metastatic melanoma patients with the L576P KIT mutation were treated with dasatinib, including one patient previously treated with imatinib. Both patients had marked reduction (>50%) and elimination of tumor F18-fluorodeoxyglucose (FDG)-avidity by positron emission tomography (PET) imaging after dasatinib treatment. These data support the selective inhibitory effect of dasatinib against cells harboring the most common KIT mutation in melanoma, and thus has therapeutic implications for acrallentiginous, chronic sun-damaged, and mucosal melanomas.

Parthenolide promotes the ubiquitination of MDM2 and activates p53 cellular functions.

MDM2 belongs to a class of ring-finger domain-containing ubiquitin ligases that mediate the proteasomal degradation of numerous proteins, including themselves. Arguably, the most important substrate of MDM2 is p53, which controls cell cycle progression and apoptosis. MDM2 and p53 are parts of a feedback regulatory loop whose perturbations are often present in cancer and are targets for anticancer drug development. We found that the natural product, small-molecule anti-inflammatory agent parthenolide (PN), which is actively being investigated as a potential therapeutic for many human cancers, induces ubiquitination of MDM2 in treated cells, resulting in the activation of p53 and other MDM2-regulated tumor-suppressor proteins. Using cells with functional gene deletions and small interfering RNA knockdown studies, we found that these effects required the DNA damage transducer ataxia telangiectasia mutated. The effects of PN on tumor suppressor activation were comparable with that of nutlin-3a, a recently developed small molecule that was designed to interfere with the interaction between MDM2 and p53 but does not promote MDM2 ubiquitination. Our study illustrates an alternative approach for controlling MDM2 and p53 activities and identifies an additional critically important cancer pathway affected by PN.

Parthenolide specifically depletes histone deacetylase 1 protein and induces cell death through ataxia telangiectasia mutated.

Histone deacetylases (HDACs), enzymes involved in chromatin remodeling, are promising targets for anticancer drug development. Several HDAC inhibitors (HDACi) are in clinical trials. One limitation of present HDACi is their nonspecificity, affecting many HDACs with similar effectiveness. We have identified a small molecule, the sesquiterpene lactone parthenolide (PN), which specifically depletes HDAC1 protein without affecting other class I/II HDACs. HDAC1 depletion occurred through proteasomal degradation and resulted in transcriptional consequences comparable to those observed with pan-HDACi. Surprisingly, HDAC1 depletion did not occur through the inflammation mediator IKK2, a known PN target and regulator of HDAC1. Rather, PN promoted HDAC1 depletion and cell death through the DNA-damage-transducer ataxia telangiectasia mutated. Our study suggests that modulating cellular HDAC protein levels with small molecules provides an alternative approach to specific HDAC inhibition and effective cancer treatment.

Depletion of histone deacetylase protein: a common consequence of inflammatory cytokine signaling?

The dynamics of histone acetylation and deacetylation have long been known to influence gene expression by cellular signaling pathways. However, the mechanisms that regulate histone acetyl transferases (HATs) and histone deacetylases (HDACs) by these pathways have only recently become the focus of scientific investigation, spurred by increasing knowledge that HDACs can promote cancer growth. We recently reported that pro-inflammatory signals such as tumor necrosis factor alpha (TNFalpha) induce HDAC1 ubiquitination and proteasomal degradation through the IkappaB kinase IKKbeta. The resulting depletion of cellular HDAC1 levels lead to a consequent depletion of HDAC1 associated with the CDKN1A gene promoter and increased expression of its protein product, p21(WAF1/CIP1). This phenomenon heralds a unique mechanism of HDAC regulation that modulates the pro-inflammatory activity of TNFalpha and other cytokines at the level of gene expression. Here we discuss the current knowledge of pro-inflammatory cytokine-induced regulation of gene expression, emphasizing the involvement of HDAC1, and its possible implications in inflammation, cancer, and their therapy.