Regulating the response to targeted MEK inhibition in melanoma: Enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/β-catenin activation

The limitations of revolutionary new mutation-specific inhibitors of BRAF^V600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF^V600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAF^V600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.     

Activities of multiple cancer-related pathways are associated with BRAF mutation and predict the resistance to BRAF/MEK inhibitors in melanoma cells

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.     

Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS‐mutant melanoma

The MEK inhibitor MEK162 is the first targeted therapy agent with clinical activity in patients whose melanomas harbor NRAS mutations; however, median PFS is 3.7 months, suggesting the rapid onset of resistance in the majority of patients. Here, we show that treatment of NRAS‐mutant melanoma cell lines with the MEK inhibitors AZD6244 or trametinib resulted in a rebound activation of phospho‐ERK (pERK). Functionally, the recovery of signaling was associated with the maintenance of cyclin‐D1 expression and therapeutic escape. The combination of a MEK inhibitor with an ERK inhibitor suppressed the recovery of cyclin‐D1 expression and was associated with a significant enhancement of apoptosis and the abrogation of clonal outgrowth. The MEK/ERK combination strategy induced greater levels of apoptosis compared with dual MEK/CDK4 or MEK/PI3K inhibition across a panel of cell lines. These data provide the rationale for further investigation of vertically co‐targeting the MAPK pathway as a potential treatment option for NRAS‐mutant melanoma patients.     

SHOC2 and CRAF Mediate ERK1/2 Reactivation in Mutant NRAS-mediated Resistance to RAF Inhibitor

ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAF^V600E melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAF^V600E/NRAS^Q61K co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAF^V600 cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAF^V600E/NRAS^Q61K cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAF^V600E/NRAS^Q61K cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.     

Anti-leukemic effects of simvastatin on NRASG12D mutant acute myeloid leukemia cells

The statins are a group of therapeutic drugs widely used for lowering plasma cholesterol level, while it has also been reported to induce cell death in human acute myeloid leukemia (AML) cells. To determine antitumor activity triggered by simvastatin, four AML cell lines—U937, KG1, THP1 (NRAS^G12D mutant) and HL60 (NRAS^Q61L mutant)—were cultured with simvastatin and cell viability was assessed using the CellTiter-Glo reagent. For understanding mechanism of antitumor activity, immunoblot analysis for pAkt (Ser473), Akt, pMEK, MEK, pERK (Thr202/Tyr204) and ERK (Thr202/Tyr204) was performed. Apoptotic cell population was calculated using the Annexin V-FITC assay, and cell cycle state was assessed by flow cytometry. Simvastatin showed different cytotoxic effect among AML cells, of which NRAS^G12D mutant THP1 was the most statin sensitive cell line (IC₅₀ values: 1.96 uM in HL60, 7.87 uM in KG1, 0.83 uM in THP1 and 1.37 uM in U937). Western blot analysis revealed that Ras downstream signaling molecules including Akt, MEK, and ERK1/2 were markedly inhibited in THP1 cells compared to other AML cells when exposed to simvastatin. In addition, only in THP1 cells, increased apoptosis and cell cycle arrest by simvastatin was observed. The combination of simvastatin and MEK inhibitor AZD6244 synergistically reduced THP1 cell proliferation compared to simvastatin alone and AZD6244 alone (IC₅₀ values: 0.88 uM in simvastatin, 0.32 uM in AZD6244, and 0.23 uM in combination of simvastatin and AZD6244). Simvastatin exhibited anti-leukemic effect in human AML cells in vitro, especially at NRAS^G12D mutant AML cell line.

     

Activation of Wnt/β-Catenin Signaling Increases Apoptosis in Melanoma Cells Treated with Trail

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation.     

Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway

Background

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF^V600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.

Methodology/Principal Findings

The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.

Conclusions/Significance

Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.     

NVP-LDE225, a Potent and Selective SMOOTHENED Antagonist Reduces Melanoma Growth In Vitro and In Vivo

Melanoma is one of the most aggressive cancers and its incidence is increasing worldwide. So far there are no curable therapies especially after metastasis. Due to frequent mutations in members of the mitogen-activated protein kinase (MAPK) signaling pathway, this pathway is constitutively active in melanoma. It has been shown that the SONIC HEDGEHOG (SHH)-GLI and MAPK signaling pathway regulate cell growth in many tumors including melanoma and interact with each other in the regulation of cell proliferation and survival.Here we show that the SHH-GLI pathway is active in human melanoma cell lines as they express downstream target of this pathway GLI1. Expression of GLI1 was significantly higher in human primary melanoma tissues harboring BRAF^V600E mutation than those with wild type BRAF. Pharmacologic inhibition of BRAF^V600E in human melanoma cell lines resulted in decreased expression of GLI1 thus demonstrating interaction of SHH-GLI and MAPK pathways. Inhibition of SHH-GLI pathway by the novel small molecule inhibitor of smoothened NVP-LDE225 was followed by inhibition of cell growth and induction of apoptosis in human melanoma cell lines, interestingly with both BRAF^V600E and BRAF^{Wild Type} status. NVP-LDE225 was potent in reducing cell proliferation and inducing tumor growth arrest in vitro and in vivo, respectively and these effects were superior to the natural compound cyclopamine.Finally, we conclude that inhibition of SHH-GLI signaling pathway in human melanoma by the specific smoothened inhibitor NVP-LDE225 could have potential therapeutic application in human melanoma even in the absence of BRAF^V600E mutation and warrants further investigations.     

The BRAFV600E causes widespread alterations in gene methylation in the genome of melanoma cells

Although BRAF^V600E is well known to play an important role in the tumorigenesis of melanoma, its molecular mechanism, particularly the epigenetic aspect, has been incompletely understood. Here, we investigated the role of BRAF^V600E signaling in altering gene methylation in the genome of melanoma cells using a methylated CpG island amplification/CpG island microarray system and searched for genes coupled to the BRAF^V600E signaling through methylation aberrations. The results indicated that a wide range of genes with broad functions were linked to BRAF^V600E signaling through their hyper- or hypomethylation. Expression of 59 genes hypermethylated upon BRAF knockdown was selectively tested and found to be largely correspondingly underexpressed, suggesting that these genes were naturally hypomethylated and overexpressed with BRAF^V600E in melanoma. This BRAF^V600E-promoted hypomethylation was confirmed on genes selectively examined in primary melanoma tumors. Some of these genes were functionally tested and demonstrated to play a role in melanoma cell proliferation and invasion. As a mechanism of aberrant gene methylation driven by BRAF^V600E, expression of the DNA methyltransferase 1 and histone methyltransferase EZH2 was profoundly affected by BRAF^V600E. We have thus uncovered a previously unrecognized prominent epigenetic mechanism in the tumorigenesis of melanoma driven by BRAF^V600E. Many of the functionally important genes controlled by the BRAF^V600E signaling through aberrant methylation may prove to be novel therapeutic targets for melanoma.Key words: BRAF mutation, DNA methylation, melanoma, MAP kinase pathway, gene hypomethylation, gene hypermethylation     

The BRAFV600E inhibitor,PLX4032, increases type I collagen synthesis in melanoma cells

Vertical growth phase (VGP) melanoma is frequently metastatic, a process mediated by changes in gene expression, which are directed by signal transduction pathways in the tumor cells. A prominent signaling pathway is the Ras-Raf-Mek-Erk MAPK pathway, which increases expression of genes that promote melanoma progression. Many melanomas harbor a mutation in this pathway, BRAF^V600E, which constitutively activates MAPK signaling and expression of downstream target genes that facilitate tumor progression. In BRAF^V600E melanoma, the small molecule inhibitor, vemurafenib (PLX4032), has revolutionized therapy for melanoma by inducing rapid tumor regression. This compound down-regulates the expression of many genes. However, in this study, we document that blocking the Ras-Raf-Mek-Erk MAPK pathway, either with an ERK (PLX4032) or a MEK (U1026) signaling inhibitor, in BRAF^V600E human and murine melanoma cell lines increases collagen synthesis in vitro and collagen deposition in vivo. Since TGFß signaling is a major mediator of collagen synthesis, we examined whether blocking TGFß signaling with a small molecule inhibitor would block this increase in collagen. However, there was minimal reduction in collagen synthesis in response to blocking TGFß signaling, suggesting additional mechanism(s), which may include activation of the p38 MAPK pathway. Presently, it is unclear whether this increased collagen synthesis and deposition in melanomas represent a therapeutic benefit or an unwanted “off target” effect of inhibiting the Ras-Raf-Erk-Mek pathway.     

The BRAFV600E causes widespread alterations in gene methylation in the genome of melanoma cells

Although BRAF^V600E is well known to play an important role in the tumorigenesis of melanoma, its molecular mechanism, particularly the epigenetic aspect, has been incompletely understood. Here, we investigated the role of BRAF^V600E signaling in altering gene methylation in the genome of melanoma cells using a methylated CpG island amplification/CpG island microarray system and searched for genes coupled to the BRAF^V600Esignaling through methylation aberrations. The results indicated that a wide range of genes with broad functions were linked to BRAF^V600E signaling through their hyper- or hypomethylation. Expression of 59 genes hypermethylated upon BRAF knockdown was selectively tested and found to be largely correspondingly underexpressed, suggesting that these genes were naturally hypomethylated, and overexpressed with BRAF^V600E in melanoma. This BRAF^V600E-promoted hypomethylation was confirmed on genes selectively examined in primary melanoma tumors. Some of these genes were functionally tested and demonstrated to play a role in melanoma cell proliferation and invasion. As a mechanism of aberrant gene methylation driven by BRAF^V600E, expression of the DNA methyltransferase 1 and histone methyltransferase EZH2 was profoundly affected by BRAF^V600E. We have thus uncovered a previously unrecognized prominent epigenetic mechanism in the tumorigenesis of melanoma driven by BRAF^V600E. Many of the functionally important genes controlled by the BRAF^V600E signaling through aberrant methylation may prove to be novel therapeutic targets for melanoma.     

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.     

PIK3CA‐mutated melanoma cells rely on cooperative signaling through mTORC1/2 for sustained proliferation

Malignant conversion of BRAF‐ or NRAS‐mutated melanocytes into melanoma cells can be promoted by PI3′‐lipid signaling. However, the mechanism by which PI3′‐lipid signaling cooperates with mutationally activated BRAF or NRAS has not been adequately explored. Using human NRAS‐ or BRAF‐mutated melanoma cells that co‐express mutationally activated PIK3CA, we explored the contribution of PI3′‐lipid signaling to cell proliferation. Despite mutational activation of PIK3CA, melanoma cells were more sensitive to the biochemical and antiproliferative effects of broader spectrum PI3K inhibitors than to an α‐selective PI3K inhibitor. Combined pharmacological inhibition of MEK1/2 and PI3K signaling elicited more potent antiproliferative effects and greater inhibition of the cell division cycle compared to single‐agent inhibition of either pathway alone. Analysis of signaling downstream of MEK1/2 or PI3K revealed that these pathways cooperate to regulate cell proliferation through mTORC1‐mediated effects on ribosomal protein S6 and 4E‐BP1 phosphorylation in an AKT‐dependent manner. Although PI3K inhibition resulted in cytostatic effects on xenografted NRAS^Q61H/PIK3CA^H1047R melanoma, combined inhibition of MEK1/2 plus PI3K elicited significant melanoma regression. This study provides insights as to how mutationally activated PIK3CA acts in concert with MEK1/2 signaling to cooperatively regulate mTORC1/2 to sustain PIK3CA‐mutated melanoma proliferation.     

Inhibition of MEK signaling enhances the ability of cytarabine to induce growth arrest and apoptosis of acute myelogenous leukemia cells

The mitogen-activated protein kinase/ERK kinase (MEK)/ERK pathway was shown to be constitutively activated in a large number of acute myelogenous leukemia (AML) cells, suggesting the important roles of this pro-survival signaling in leukemogenesis and proliferation of AML cells. This study explored the impact of the MEK inhibitor AZD6244 on the effect of cytarabien (AraC), one of the most commonly used anti-leukemia agents, to induce growth arrest and apoptosis of AML cells. AZD6244 effectively blocked AraC-induced MEK/ERK activation and enhanced its ability to induce growth arrest and apoptosis of NB4 and HL60 cells in parallel with induction of DNA damage as measured by detection of γ-H2AX by Western Blot analysis, resulting in enhanced expression of p21 ^waf1 and downregulation of c-Myc and Bcl-xl in these cells. Enhanced induction of apoptosis mediated by combination of AZD6244 and AraC was also shown in freshly isolated AML cells (n = 3). Taken together, concomitant administration of AraC and the inhibitor of MEK/ERK signaling may be useful for treatment of individuals with AML.     

Inhibition of mutant BRAF splice variant signaling by next‐generation,selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAF^V600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF^V600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAF^V600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF^V600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAF^V600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.     

Combined tazemetostat and MAPKi enhances differentiation of papillary thyroid cancer cells harbouring BRAFV600E by synergistically decreasing global trimethylation of H3K27

Clinical efficacy of differentiation therapy with mitogen-activated protein kinase inhibitors (MAPKi) for lethal radioiodine-refractory papillary thyroid cancer (RR-PTC) urgently needs to be improved and the aberrant trimethylation of histone H3 lysine 27 (H3K27) plays a vital role in BRAF^V600E-MAPK-induced cancer dedifferentiation and drug resistance. Therefore, dual inhibition of MAPK and histone methyltransferase (EZH2) may produce more favourable treatment effects. In this study, BRAF^V600E-mutant (BCPAP and K1) and BRAF-wild-type (TPC-1) PTC cells were treated with MAPKi (dabrafenib or selumetinib) or EZH2 inhibitor (tazemetostat), or in combination, and the expression of iodine-metabolizing genes, radioiodine uptake, and toxicity were tested. We found that tazemetostat alone slightly increased iodine-metabolizing gene expression and promoted radioiodine uptake and toxicity, irrespective of the BRAF status. However, MAPKi induced these effects preferentially in BRAF^V600E mutant cells, which was robustly strengthened by tazemetostat incorporation. Mechanically, MAPKi-induced decrease of trimethylation of H3K27 was evidently intensified by tazemetostat in BRAF^V600E-mutant cells. In conclusion, tazemetostat combined with MAPKi enhances differentiation of PTC cells harbouring BRAF^V600E through synergistically decreasing global trimethylation of H3K27, representing a novel differentiation strategy.     

Loss of PI(4,5)P2 5-Phosphatase A Contributes to Resistance of Human Melanoma Cells to RAF/MEK Inhibitors

Past studies have shown that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase (PIB5PA), is commonly downregulated or lost in melanomas, which contributes to elevated activation of phosphatidylinositol 3-kinase (PI3K)/Akt in melanoma cells. In this report, we provide evidence that PIB5PA deficiency plays a role in resistance of melanoma cells to RAF/mitogen-activated protein kinase kinase (MEK) inhibitors. Ectopic expression of PIB5PA enhanced apoptosis induced by the RAF inhibitor PLX4720 in BRAF^V600E and by the MEK inhibitor U0126 in both BRAF^V600E and wild-type BRAF melanoma cells. This was due to inhibition of PI3K/Akt, as co-introduction of an active form of Akt (myr-Akt) abolished the effect of overexpression of PIB5PA on apoptosis induced by PLX4720 or U0126. While overexpression of PIB5PA triggered activation of Bad and down-regulation of Mcl-1, knockdown of Bad or overexpression of Mcl-1 recapitulated, at least in part, the effect of myr-Akt, suggesting that regulation of Bad and Mcl-1 is involved in PIB5PA-mediated sensitization of melanoma cells to the inhibitors. The role of PIB5PA deficiency in BRAF inhibitor resistance was confirmed by knockdown of PIB5PA, which led to increased growth of BRAF^V600E melanoma cells selected for resistance to PLX4720. Consistent with its role in vitro, overexpression of PIB5PA and the MEK inhibitor selumetinib cooperatively inhibited melanoma tumor growth in a xenograft model. Taken together, these results identify loss of PIB5PA as a novel resistance mechanism of melanoma to RAF/MEK inhibitors and suggest that restoration of PIB5PA may be a useful strategy to improve the therapeutic efficacy of the inhibitors in the treatment of melanoma.     

Inhibition of both BRAF and MEK in BRAFV600E mutant melanoma restores compromised dendritic cell (DC) function while having differential direct effects on DC properties

Purpose

Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF^V600 mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF^V600 mutant melanoma.

Methods

We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib.

Results

Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF^V600E mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations.

Conclusions

BRAF^V600E mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.     

Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS^G12C/G13D or BRAF^V600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.     

Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the NRAS or BRAF oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS‐mutant cells, and with oncogenic BRAF in BRAF^V600E‐mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS‐mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF^V600E‐mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.