MicroRNA‐125a promotes resistance to BRAF inhibitors through suppression of the intrinsic apoptotic pathway

Melanoma patients with BRAF^V600E‐mutant tumors display striking responses to BRAF inhibitors (BRAFi); however, almost all invariably relapse with drug‐resistant disease. Here, we report that microRNA‐125a (miR‐125a) expression is upregulated in human melanoma cells and patient tissues upon acquisition of BRAFi resistance. We show that miR‐125a induction confers resistance to BRAF^V600E melanoma cells to BRAFi by directly suppressing pro‐apoptotic components of the intrinsic apoptosis pathway, including BAK1 and MLK3. Apoptotic suppression and prolonged survival favor reactivation of the MAPK and AKT pathways by drug‐resistant melanoma cells. We demonstrate that miR‐125a inhibition suppresses the emergence of resistance to BRAFi and, in a subset of resistant melanoma cell lines, leads to partial drug resensitization. Finally, we show that miR‐125a upregulation is mediated by TGFβ signaling. In conclusion, the identification of this novel role for miR‐125a in BRAFi resistance exposes clinically relevant mechanisms of melanoma cell survival that can be exploited therapeutically.     

AMPK promotes survival of c‐Myc‐positive melanoma cells by suppressing oxidative stress

Although c‐Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras^Q61KINK4a^?/? mouse melanoma model to show that c‐Myc is essential for tumor initiation, maintenance, and metastasis. c‐Myc‐expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c‐Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c‐Myc‐positive melanoma cells activated and became dependent on the metabolic energy sensor AMP‐activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c‐Myc‐expressing melanoma cells, while AMPK activation protected against cell death of c‐Myc‐depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early‐stage human melanoma samples revealed an inverse correlation between C‐MYC and patient survival, suggesting that C‐MYC expression levels could serve as a prognostic marker for early‐stage disease.     

Metabolic imaging using hyperpolarized 13C‐pyruvate to assess sensitivity to the B‐Raf inhibitor vemurafenib in melanoma cells and xenografts

Nearly all melanoma patients with a BRAF‐activating mutation will develop resistance after an initial clinical benefit from BRAF inhibition (BRAFi). The aim of this work is to evaluate whether metabolic imaging using hyperpolarized (HP) ¹³C pyruvate can serve as a metabolic marker of early response to BRAFi in melanoma, by exploiting the metabolic effects of BRAFi. Mice bearing human melanoma xenografts were treated with the BRAFi vemurafenib or vehicle. In vivo HP ¹³C magnetic resonance spectroscopy was performed at baseline and 24 hours after treatment to evaluate changes in pyruvate‐to‐lactate conversion. Oxygen partial pressure was measured via electron paramagnetic resonance oximetry. Ex vivo qRT‐PCR, immunohistochemistry and WB analysis were performed on tumour samples collected at the same time‐points selected for in vivo experiments. Similar approaches were applied to evaluate the effect of BRAFi on sensitive and resistant melanoma cells in vitro, excluding the role of tumour microenvironment. BRAF inhibition induced a significant increase in the HP pyruvate‐to‐lactate conversion in vivo, followed by a reduction of hypoxia. Conversely, the conversion was inhibited in vitro, which was consistent with BRAFi‐mediated impairment of glycolysis. The paradoxical increase of pyruvate‐to‐lactate conversion in vivo suggests that such conversion is highly influenced by the tumour microenvironment.     

miR‐200c/Bmi1 axis and epithelial–mesenchymal transition contribute to acquired resistance to BRAF inhibitor treatment

Resistance to BRAF inhibitors (BRAFi) is one of the major challenges for targeted therapies for BRAF‐mutant melanomas. However, little is known about the role of microRNAs in conferring BRAFi resistance. Herein, we demonstrate that miR‐200c expression is significantly reduced whereas miR‐200c target genes including Bmi1, Zeb2, Tubb3, ABCG5, and MDR1 are significantly increased in melanomas that acquired BRAFi resistance compared to pretreatment tumor biopsies. Similar changes were observed in BRAFi‐resistant melanoma cell lines. Overexpression of miR‐200c or knock‐down of Bmi1 in resistant melanoma cells restores their sensitivities to BRAFi, leading to deactivation of the PI3K/AKT and MAPK signaling cascades, and acquisition of epithelial–mesenchymal transition‐like phenotypes, including upregulation of E‐cadherin, downregulation of N‐cadherin, and ABCG5 and MDR1 expression. Conversely, knock‐down of miR‐200c or overexpression of Bmi1 in BRAFi‐sensitive melanoma cells activates the PI3K/AKT and MAPK pathways, upregulates N‐cadherin, ABCG5, and MDR1 expression, and downregulates E‐cadherin expression, leading to BRAFi resistance. Together, our data identify miR‐200c as a critical signaling node in BRAFi‐resistant melanomas impacting the MAPK and PI3K/AKT pathways, suggesting miR‐200c as a potential therapeutic target for overcoming acquired BRAFi resistance.     

Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four ^V600BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R variants and worse outcomes in terms of both overall response rate (ORR; 59% versus 95%, P = 0.011 univariate, P = 0.028 multivariate analysis) and progression‐free survival (PFS) shorter than 6 months (72% versus 33%, P = 0.012 univariate, P = 0.027 multivariate analysis). No difference in overall survival (OS) was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase in vemurafenib IC50 in MC1R variant cell lines.     

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Receptor tyrosine kinases in cancer escape from BRAF inhibitors

The BRAF inhibitors (BRAFi) induce anti-tumor responses in nearly 60% of patients with advanced ^V600BRAF-mutant melanomas but only 5% of patients with ^V600BRAF-mutant colorectal carcinomas. Earlier studies of how a subset of melanoma that initially responds to BRAFi but later acquires drug resistance pointed to the importance of receptor tyrosine kinases (RTKs) in drug escape. In a pair of recent reports, this RTK-mediated mechanism of acquired BRAFi resistance in melanoma is re-surfacing in the context of innate or primary BRAFi resistance in ^V600BRAF-mutant colorectal carcinomas, suggesting potential upfront therapeutic strategies to prevent BRAFi resistance.^V600BRAF mutations are found in >50% of melanomas, nearly 100% of hairy cell leukemias but smaller subsets of more common human malignancies (e.g., colorectal, thyroid)¹. The in-human “druggability” of mutant BRAF has been best demonstrated in metastatic BRAF mutant melanomas using the novel small-molecule BRAF inhibitor (BRAFi) PLX4032/vemurafenib, producing survival benefits². Early clinical results of BRAFi in colorectal carcinoma, however, were disappointing, with only 5% of patients (1 of 21 patients) experiencing a partial response and 19% of patients (4 of 21 patients) experiencing minor responses³. This difference in the clinical results (melanoma vs. colorectal carcinoma) may relate less to their ontological origins but more to alternative states of a dynamic and plastic survival signaling network.The majority of BRAF mutant melanomas responds to BRAFi rapidly but acquires drug resistance within a median time of 6-7 months. The specific mechanisms of acquired BRAFi resistance are variegated but fall under two core pathways: 1) reactivation of RAF-MEK-ERK MAPK signaling, and 2) activation of MAPK-redundant signaling via the receptor tyrosine kinase (RTK)-PI3K-AKT pathway, which is parallel but interconnected to the MAPK pathway. MAPK reactivation can occur via NRAS activating mutations⁴, COT overexpression⁵, ^V600BRAF alternative splicing⁶, ^V600BRAF amplification⁷, and MEK1 activating mutation^8,9. MAPK-redundant signaling via RTK overexpression has been shown to result in AKT activation and RAS-CRAF-MEK signaling, bypassing mutant BRAF^4,10,11. The repertoire of RTK overexpressed appears restricted but shares a common pattern of PDGFRβ and EGFR overexpression, at least in melanoma cell lines with acquired resistance to vemurafenib⁴. It is unclear at present how this overexpression of a select number of wild-type RTKs contributes to the molecular details of survival pathway redundancy and cooperativity. Nevertheless, understanding how melanomas acquire BRAFi resistance via core pathways may shed key insights into mechanisms of innate BRAFi resistance in multiple malignancies. Hence, it came as not a complete surprise that a pair of papers published recently implicated RTKs in innate BRAFi resistance in colorectal cancer cell lines^12,13. Both studies pointed to EGFR activation and downstream signaling as a key component to innate BRAFi resistance, at least in a majority of colorectal carcinoma (CRC) cell lines examined.Corcoran et al.¹² showed that BRAF mutant CRC cell lines, in contrast to BRAF mutant melanoma cell lines, displayed innate resistance to growth inhibition by vemurafenib. An important clue implicating RTK involvement in innate vemurafenib resistance of BRAF mutant CRC cell lines came from the observation that p-ERK recovery occurred soon (hours to days) after vemurafenib treatment, unlike the kinetics of p-ERK recovery in BRAF mutant melanoma cell lines. This relatively rapid recovery of p-ERK post vemurafenib treatment in CRC cell lines is akin to that in melanoma cell lines with acquired BRAFi resistance driven by RTK overexpresion¹⁰. Corcoran et al. then traced this propensity for early p-ERK recovery to vemurafenib treatment (24 h)-dependent enhancement of (activated) RAS-GTP levels and MEK activity, parallel to elevated RAS-GTP levels in melanoma cell lines with RTK-driven, acquired BRAFi resistance⁴. In phospho-RTK arrays, they determined that the p-EGFR level (among others such as p-c-MET and p-IGF1R levels) was elevated in CRC cell lines relative to those in melanoma cells. Vemurafenib treatment (24 h) did not significantly enhance the p-EGFR level (but did elevate the p-IGFR1 level). Elevated p-EGFR levels in BRAF mutant CRC cell lines were correlated with elevated total EGFR levels (i.e., overexpressed compared with BRAF mutant melanoma cell lines). Thus, several observations correlated with innate BRAFi resistance in CRC cell lines: RTK (mostly consistently EGFR) overexpression (at baseline); upregulation of activation-associated phosphorylation of RTKs (at baseline); and upregulation of RAS-GTP levels (in response to BRAFi treatment). Curiously, although EGFR is highly phosphorylated at baseline, the RAS-GTP levels only rose in response to vemurafenib treatment.Corcoran et al. further showed that small-molecule EGFR inhibitors (EGFRi) could downregulate, partially or completely, the RAS-GTP level induced by vemurafenib treatment. The combination of vemurafenib (BRAFi) and gefitnib (EGFRi) could synergistically reduce p-ERK levels and the net growth inhibition of most but not all CRC cell lines studied, suggesting that survival in some CRC cell lines may also depend on other RTKs and downstream signaling (e.g., AKT). Consistently, the combination of vemurafenib and erlotinib (EGFRi) stabilized the growth of, but did not cause significant regression of, CRC xenografts. Simultaneous inhibition or genetic knockdown of multiple RTKs was not explored, leaving unresolved the issue of how multiple RTKs may potentially play cooperative survival roles at baseline or in response to kinase inhibitor therapy.Prahallad et al.¹³ also compared CRC and melanoma cell lines and showed that EGFR expression is generally higher in CRC cell lines. Vemurafenib treatment (6 h) of the WiDr CRC cell line led to an induction in p-EGFR and p-AKT levels, concomitant with the expected suppression of p-MEK and p-ERK. MEK inhibition, by AZD6244 treatment, similarly led to the rebound phosphorylation of EGFR. Based on earlier literature showing that the ERK kinase phosphorylates Cdc25c, activating its phosphatase activity, and that Cdc25c can dephosphorylate EGFR, Prahallad et al. went on to show that Cdc25c knockdown mimicked vemurafenib treatment in inducing p-EGFR levels. As predicted, vemurafenib treatment of CRC cell line inhibited Cdc25c phosphorylation at a key threonine (Thr 48), which was previously demonstrated to be a key event for its phosphatase activity. Addition of an EGFRi (cetuximab or gefitnib) to the BRAFi vemurafenib treatment downregulated the baseline level of p-ERK and the BRAFi-induced p-AKT level (but not the baseline p-AKT level). Moreover, addition of an EGFRi sensitized CRC cell lines to growth inhibition by vemurafenib in vitro but did not induce tumor regression in vivo, again suggesting incomplete survival signaling blockade. Accordingly, it has been shown that the effect of vemurafenib in shrinking CRC tumor xenografts was enhanced by combining with an AKT inhibitor (MK-2206)¹⁴. Moreover, in this study, the addition of vemurafenib to erlotinib treatment also resulted in increased anti-tumor activity and improved survival in xenograft models. It should be pointed out that Prahallad et al. did not formally assess BRAFi and EGFRi synergy, nor did they examine the diversity of RTK overexpression/activity and its contribution to downstream survival signaling (e.g., AKT).These works, along with prior studies^4,10, highlight the importance of expression and activity level of RTKs as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines (Figure 1). An important question remains as to whether the diversity of RTK overexpression and/or upregulation participates in and contributes to the full BRAFi resistance phenotype. A recent study afforded us a systems-wide view of the RTKinome reprogramming in response to MEK inhibition in the so-called triple-negative breast cancer cell lines¹⁵. The balance of the MAPK vs. RTK network signaling may be dynamically influenced by kinase inhibitors targeting RAF or MEK. This daunting diversity of RTK expression/activity may corner us into abandoning a combination of RTK inhibitors (already approved for clinical usage) with a BRAF inhibitor. Instead, we might need to resort to downstream pathway inhibitors not yet approved for clinical usage (e.g., an inhibitor of MEK with an inhibitor of the PI3K-AKT-mTORC1/2 axis) before we have a chance to corner BRAF mutant cancers into death.Open in a separate windowFigure 1Upregulation of receptor tyrosine kinase(s) (RTKs) as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines. (A) In BRAF mutant melanoma cell lines, RTKs are generally expressed at very low levels and contribute minimally to survival signaling, resulting in a strong addiction to mutant BRAF signaling and sensitivity to BRAFi. When BRAF mutant melanoma cell lines acquire BRAFi resistance, they upregulate the expression and activity of PDGFRb and other RTKs, resulting in reactivation of MEK-ERK as well as MAPK-redundant PI3K-AKT survival signaling. (B) In BRAF mutant colorectal carcinoma (CRC) cell lines, EGFR and other RTKs are upregulated by overexpression and some level of activation, resulting in MAPK-redundant survival signaling and conferring innate or primary BRAFi resistance. Treatment of CRC cell lines wth a BRAF or a MEK inhibitor can further activate EGFR and potentially other RTKs and stimulate GTP-RAS levels, consolidating innate BRAFi resistance. Red denotes mutated protein (e.g., BRAF); gray symbols denote weak signaling or interactions; multiplicity of protein symbols denotes overexpression; P in blue denotes activation-associated phosphorylation.     

Modification,Biological Evaluation and SAR Studies of Novel 1H‐Pyrazol Derivatives Containing N,N′‐Disubstituted Urea Moiety as Potential Anti‐melanoma Agents

Malignant melanomas are amongst the most aggressive cancers. BRAF Inhibitors have exhibited therapeutic effects against BRAF‐mutant melanoma. In continuation of our earlier studies on anti‐melanoma agents based on 1H‐pyrazole skeleton, two sets of novel compounds that include 1H‐pyrazole‐4‐amines FA 1 – FA13 and corresponding urea derivatives FN 1 – FN13 have been synthesized and evaluated for their BRAF^V600E inhibitory and antiproliferation activities. Compound FN 10 displayed the most potent biological activity against BRAF^V600E (IC₅₀ = 0.066 μm ) and the A375 human melanoma cell line (GI₅₀ = 0.81 μm ), which was comparable to the positive control vemurafenib, and more potent than our previously reported 1H‐pyrazole‐3‐amines and their urea derivatives. The results of SAR studies and molecular docking can guide further optimization and may help to improve potency of these pyrazole‐based anti‐melanoma agents.     

Intermittent Versus Continuous Dosing of MAPK Inhibitors in the Treatment of BRAF-Mutated Melanoma

The development of BRAF and MEK inhibitors (BRAFi/MEKi) has led to major advances in melanoma treatment. However, the emergence of resistance mechanisms limits the benefit duration and a complete response occurs in less than 20% of patients receiving BRAFi ± MEKi. In this study, we evaluated the impact of an intermittent versus continuous dosing schedule of BRAF/MEK inhibition in a melanoma model mildly sensitive to a BRAF inhibitor. The combination of a BRAFi with three different MEKi was studied with a continuous or intermittent dosing schedule in vivo, in a xenografted melanoma model and ex vivo using histoculture drug response assays (HDRAs) of patient-derived xenografts (PDX). To further understand the underlying molecular mechanisms of therapeutic efficacy, a biomarker pharmacodynamic readout was evaluated.An equal impact on tumor growth was observed in monotherapy or bitherapy regimens whether we used continuous and intermittent dosing schedules, with no significant differences in biomarkers expression between the treatments. The antitumoral effect was mostly due to modulations of expression of cell cycle and apoptotic mediators. Moreover, ex vivo studies did not show significant differences between the dosing schedules.In this context, our preclinical and pharmacodynamic results converged to show the similarity between intermittent and continuous treatments with either BRAFi or MEKi alone or with the combination of both.     

Inhibition of melanoma development in the Nras(Q61K)::Ink4a−/− mouse model by the small molecule BI‐69A11

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15–20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI‐69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon‐ and cell death‐related genes that were associated with responsiveness of melanoma cell lines to BI‐69A11. Strikingly, the administration of BI‐69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras^(Q61K)::Ink4a^?/?). Biweekly administration of BI‐69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI‐69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI‐69A11‐resistant Nras^(Q61K)::Ink4a^?/? tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI‐69A11 for clinical assessment.     

Wnt5A promotes an adaptive,senescent‐like stress response,while continuing to drive invasion in melanoma cells

We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAF^V600E mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence‐associated β‐galactosidase (SA‐β‐gal), senescence‐associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA‐β‐gal and SAHF, these Wnt5A‐high cells are able to colonize the lungs in in vivo tail vein colony‐forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.     

Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab

Metastatic melanoma has a poor prognosis with high resistance to chemotherapy and radiation. Recently, the anti-CTLA-4 antibody ipilimumab has demonstrated clinical efficacy, being the first agent to significantly prolong the overall survival of inoperable stage III/IV melanoma patients. A major aim of patient immune monitoring is the identification of biomarkers that predict clinical outcome. We studied circulating myeloid-derived suppressor cells (MDSC) in ipilimumab-treated patients to detect alterations in the myeloid cell compartment and possible correlations with clinical outcome. Lin^? CD14⁺ HLA-DR^? monocytic MDSC were enriched in peripheral blood of melanoma patients compared to healthy donors (HD). Tumor resection did not significantly alter MDSC frequencies. During ipilimumab treatment, MDSC frequencies did not change significantly compared to baseline levels. We observed high inter-patient differences. MDSC frequencies in ipilimumab-treated patients were independent of baseline serum lactate dehydrogenase levels but tended to increase in patients with severe metastatic disease (M1c) compared to patients with metastases in skin or lymph nodes only (M1a), who had frequencies comparable to HD. Interestingly, clinical responders to ipilimumab therapy showed significantly less lin^? CD14⁺ HLA-DR^? cells as compared to non-responders. The data suggest that the frequency of monocytic MDSC may be used as predictive marker of response, as low frequencies identify patients more likely benefitting from ipilimumab treatment. Prospective clinical trials assessing MDSC frequencies as potential biomarkers are warranted to validate these observations.     

Seasonal variation in effect of anti-PD-1 initiation on overall survival among patients with advanced melanoma

Melanoma is a highly immunogenic cancer, and circannual rhythms influence the activity of the immune system. We retrospectively collected information on all cases with metastatic melanoma (ocular melanoma excluded) that initiated treatment with BRAF-inhibitor-based therapy (BRAFi) or anti-PD-1 monotherapy (PD-1). Cases were divided in two groups based on treatment initiation in the summer half-year (April to September) or winter half-year (October to March). We collected a total of 1054 (BRAF-i) and 1205 (PD-1) patient cases. Median follow-up was 39.7 (BRAFi) and 47.5 (PD-1) months. We did not observe differences in outcomes across patients who were treated in summer versus winter in the BRAFi cohort. Furthermore, we did not observe significant differences in ORR, CRR, and PFS in the PD-1 cohort. However, in patients with BRAF wild-type disease of the PD-1 cohort, treatment initiation in summer was associated with an improved OS (mOS 39.7 months [summer] versus 21.3 months [winter]; HR 0.70, 95% CI 0.57–0.86, p = .0007). This result remained robust to multivariable proportional hazards adjustment (HR 0.70, 95% CI 0.57–0.87, p = .001). Initiation of immunotherapy in summer is associated with prolonged survival in patients with BRAF wild-type melanoma living in Denmark.     

Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5′ partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in‐frame to six N‐terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAF^V600E mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.     

Near‐genomewide RNAi screening for regulators of BRAFV600E‐induced senescence identifies RASEF,a gene epigenetically silenced in melanoma

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene‐induced senescence (OIS), a highly potent in vivo tumor‐suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near‐genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF^V600E‐induced arrest. With genome‐wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence‐associated (SA)‐β‐galactosidase activity, interleukins, and tumor suppressor p15^INK4B. Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.     

MicroRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis

The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh‐frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumours and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation. Associations with prognosis were replicated for miR‐150‐5p, miR‐142‐3p and miR‐142‐5p. Co‐analysis of miRNA and mRNA uncovered a network associated with poor prognosis (PP) that paradoxically favoured expression of miRNAs opposing tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves. Robust association of miR‐150‐5p and the miR‐142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with PP in an autoregulatory fashion will not be suitable therapeutic targets.     

Characterization of stress response in human retinal epithelial cells

The pathogenesis of age‐related macular degeneration (AMD) involves demise of the retinal pigment epithelium and death of photoreceptors. In this article, we investigated the response of human adult retinal pigmented epithelial (ARPE‐19) cells to 5‐(N,N‐hexamethylene)amiloride (HMA), an inhibitor of Na⁺/H⁺ exchangers. We observed that ARPE‐19 cells treated with HMA are unable to activate ‘classical’ apoptosis but they succeed to activate autophagy. In the first 2 hrs of HMA exposure, autophagy is efficient in protecting cells from death. Thereafter, autophagy is impaired, as indicated by p62 accumulation, and this protective mechanism becomes the executioner of cell death. This switch in autophagy property as a function of time for a single stimulus is here shown for the first time. The activation of autophagy was observed, at a lesser extent, with etoposide, suggesting that this event might be a general response of ARPE cells to stress and the most important pathway involved in cell resistance to adverse conditions and toxic stimuli.     

T‐type calcium channel blockers inhibit autophagy and promote apoptosis of malignant melanoma cells

We have recently reported that human melanoma cells express a variety of voltage‐gated calcium (Ca²⁺) channel types, including low‐voltage‐activated T‐type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T‐type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G₁‐S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in‐depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca²⁺ homeostasis, autophagy, and cell death were mimicked by T‐type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.     

Targeted BRAF Inhibition Impacts Survival in Melanoma Patients with High Levels of Wnt/β-Catenin Signaling

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of β-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.