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Simple Summary: Approximately 60% of all melanoma patients have cancer with an activating BRAF mutation that can be effectively treated with a BRAF inhibitor. Unfortunately, such treatment often leads to rapid development of BRAF inhibitor-resistance. These BRAF inhibitor-resistant melanomas are also characterized by increased progression and metastasis. The present study was undertaken to identify an effective antimetastatic therapy for these patients. We found increased activity of the cytoskeletal-associated MARCKS protein in BRAF inhibitor-resistant melanoma cells compared to BRAF inhibitor-sensitive melanoma cells and explored the mechanism behind this increased activity. We also showed that inhibition of MARCKS activity not only reduced the invasion and migration of BRAF inhibitor-resistance melanoma cells, but also their metastatic capacity. Our study reveals that MARCKS is a critical mediator of enhanced metastasis in BRAF inhibitor-resistant melanoma cells and thus a reasonable, novel antimetastatic therapeutic target in patients with BRAF inhibitor-resistant melanoma. Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells. [ABSTRACT FROM AUTHOR]

Single-cell RNA-sequencing (scRNA-seq) has become a powerful tool for biomedical research by providing a variety of valuable information with the advancement of computational tools. Lineage analysis based on scRNA-seq provides key insights into the fate of individual cells in various systems. However, such analysis is limited by several technical challenges. On top of the considerable computational expertise and resources, these analyses also require specific types of matching data such as exogenous barcode information or bulk assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) data. To overcome these technical challenges, we developed a user-friendly computational algorithm called "LINEAGE" (label-free identification of endogenous informative single-cell mitochondrial RNA mutation for lineage analysis). Aiming to screen out endogenous markers of lineage located on mitochondrial reads from label-free scRNA-seq data to conduct lineage inference, LINEAGE integrates a marker selection strategy by feature subspace separation and de novo "low cross-entropy subspaces" identification. In this process, the mutation type and subspace-subspace "cross-entropy" of features were both taken into consideration. LINEAGE outperformed three other methods, which were designed for similar tasks as testified with two standard datasets in terms of biological accuracy and computational efficiency. Applied on a label-free scRNA-seq dataset of BRAF-mutated cancer cells, LINEAGE also revealed genes that contribute to BRAF inhibitor resistance. LINEAGE removes most of the technical hurdles of lineage analysis, which will remarkably accelerate the discovery of the important genes or cell-lineage clusters from scRNA-seq data. [ABSTRACT FROM AUTHOR]

BRAF inhibitors are some of the most effective drugs against melanoma; however, their clinical application is largely limited by drug resistance. Juglone, isolated from walnut trees, has demonstrated anti-tumour activity. In the present study, it was investigated whether juglone could enhance the responses to a BRAF inhibitor in melanoma cells (A375R and SK-MEL-5R) with an acquired resistance. These cells were treated with juglone alone, BRAF inhibitor (PLX4032) alone, or juglone combined with PLX4032. It was demonstrated that the combination of juglone and PLX4032 had synergistic effects on BRAF inhibitor-resistant melanoma cells. Juglone potentiated PLX4032-induced cytotoxicity and mitochondrial apoptosis in both A375R and SK-MEL-5R cells, which was accompanied by a decline in mitochondrial membrane potential and a decrease in Bcl-2/Bax ratio. Moreover, juglone combined with PLX4032 markedly increased the intracellular level of reactive oxygen species (ROS) and activated p38 and p53, as compared with juglone alone or PLX4032 alone. Pre-treatment with N-acetyl-L-cysteine, a ROS scavenger, completely reversed the cytotoxicity induced by juglone combined with PLX4032. In conclusion, juglone potentiated BRAF inhibitor-induced apoptosis in resistant melanoma cells, and these effects occurred partially through ROS and the p38-p53 pathway, suggesting the potential of juglone as a sensitizer to BRAF inhibitors in the treatment of melanoma. [ABSTRACT FROM AUTHOR]

Therapeutic success of targeted therapy with BRAF inhibitors (BRAFi) for melanoma is limited by resistance development. Observations from preclinical mouse models and recent insights into the immunological effects caused by BRAFi give promise for future development of combination therapy for human melanoma. In our study, we used the transplantable D4M melanoma mouse model with the BRAFV600E mutation and concomitant PTEN loss in order to characterize alterations in tumor‐infiltrating effector immune cells when tumors become resistant to BRAFi. We found that BRAFi‐sensitive tumors displayed a pronounced inflammatory milieu characterized by high levels of cytokines and chemokines accompanied by an infiltration of T and NK cells. The tumor‐infiltrating effector cells were activated and produced high levels of IFN‐γ, TNF‐α and granzyme B. When tumors became resistant and progressively grew, they reverted to a low immunogenic state similar to untreated tumors as reflected by low mRNA levels of proinflammatory cytokines and chemokines and fewer tumor‐infiltrating T and NK cells. Moreover, these T and NK cells were functionally impaired in comparison to their counterparts in BRAFi‐sensitive tumors. Their effector cell function could be restored by additional peritumoral treatment with the TLR7 agonist imiquimod, a clinically approved agent for nonmelanoma skin cancer. Indeed, resistance to BRAFi therapy was delayed and accompanied by high numbers of activated T and NK cells in tumors. Thus, combining BRAFi with an immune stimulating agent such as a TLR ligand could be a promising alternative approach for the treatment of melanoma. What's new? While inhibitors targeting mutant BRAF proteins can induce melanoma regression, many tumors become resistant to these agents, possibly owing to immunological effects of BRAF inhibitor therapy. Here, using a preclinical mouse model, the authors show that during the early treatment phase with BRAF inhibitors, melanomas are highly immunogenic, with infiltrating T cells and natural killer cells. When resistance develops, however, tumors regress toward low immunogenicity, similar to untreated tumors. Experiments show that in the BRAF‐sensitive phase, peritumoral injection of the TLR7 ligand imiquimod preserves immunogenicity and delays resistance, thus representing a potentially effective novel therapeutic strategy for melanoma. [ABSTRACT FROM AUTHOR]

One of the most frequently found mutations in human melanomas is in the B-raf gene, making its protein BRAF a key target for therapy. However, in patients treated with BRAF inhibitor (BRAFi), although the response is very good at first, relapse occurs within 6 months, on the average. In order to overcome this drug resistance to BRAFi, various combinations of BRAFi with other drugs have been explored, and some are being applied clinically, such as a combination of BRAF and MEK inhibitors. Experimental data for melanoma in mice show that under continuous treatment with BRAFi, the pro-cancer MDSCs and chemokine CCL2 initially decrease but eventually increase to above their original level, while the anticancer T cells continuously decrease. In this paper, we develop a mathematical model that explains these experimental results. The model is used to explore the efficacy of combinations of BRAFi with anti-CCL2, anti-PD-1 and anti-CTLA-4, with the aim of eliminating or reducing drug resistance to BRAFi. [ABSTRACT FROM AUTHOR]

Colorectal cancers (CRCs) harboring the BRAF(V600E) mutation are associated with aggressive disease and resistance to BRAF inhibitors by feedback activation of the receptor tyrosine kinase (RTK)→RAS→MAPK pathway. The oncogenic MUC1-C protein promotes progression of colitis to CRC; whereas there is no known involvement of MUC1-C in BRAF(V600E) CRCs. The present work demonstrates that MUC1 expression is significantly upregulated in BRAF(V600E) vs wild-type CRCs. We show that BRAF(V600E) CRC cells are dependent on MUC1-C for proliferation and BRAF inhibitor (BRAFi) resistance. Mechanistically, MUC1-C integrates induction of MYC in driving cell cycle progression with activation of the SHP2 phosphotyrosine phosphatase, which enhances RTK-mediated RAS→ERK signaling. We demonstrate that targeting MUC1-C genetically and pharmacologically suppresses (i) activation of MYC, (ii) induction of the NOTCH1 stemness factor, and (iii) the capacity for self-renewal. We also show that MUC1-C associates with SHP2 and is required for SHP2 activation in driving BRAFi-induced feedback of ERK signaling. In this way, targeting MUC1-C in BRAFi-resistant BRAF(V600E) CRC tumors inhibits growth and sensitizes to BRAF inhibition. These findings demonstrate that MUC1-C is a target for the treatment of BRAF(V600E) CRCs and for reversing their resistance to BRAF inhibitors by suppressing the feedback MAPK pathway. • MUC1 gene expression is upregulated in BRAF(V600E), as compared to wild-type (WT) BRAF, CRCs. • The MUC1-C subunit is necessary for proliferation and BRAFi resistance of BRAF(V600E) CRC cells. • MUC1-C is necessary for activation of SHP2 and induction of RTK→RAS→MAPK pathway in response to BRAF inhibition. • Targeting MUC1-C inhibits BRAF(V600E) tumorigenicity and abrogates BRAFi resistance. • MUC1-C is a target for the treatment of BRAF(V600E) CRCs and for reversing their resistance to BRAF inhibitors. [ABSTRACT FROM AUTHOR]

The BRAFV600E mutation is the most common oncogenic mutation in thyroid cancer, suggesting an aggressive subtype of thyroid cancer and poor prognosis. Vemurafenib, a selective inhibitor of BRAFV600E, may provide therapeutic benefit in various cancers including thyroid cancer. However, the prevalence of drug resistance remains a challenge because of the feedback activation of the MAPK/ERK and PI3K/AKT pathways. In treating thyroid cancer cells with vemurafenib, we have detected reactivation of the MAPK/ERK signaling pathway as a result of the release of multiple receptor tyrosine kinases (RTKs) from the negative feedback of ERK phosphorylation. SHP2 is an important target protein downstream of the RTK signaling pathway. Decreasing it through SHP2 knockdown or the use of an inhibitor of SHP2 (SHP099) was found to significantly increase the early sensitivity and reverse the late resistance to vemurafenib in BRAFV600E mutant thyroid cancer cells. Overall, our findings suggest that blocking SHP2 reverses the reactivation of the MAPK/ERK signaling pathway caused by the activation of RTKs and improves the sensitivity of thyroid cancer to vemurafenib, which has potential implications for mechanism-based early combination strategies to treat thyroid cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The activation of transcriptional coactivators YAP and its paralog TAZ has been shown to promote resistance to anti-cancer therapies. YAP/ TAZ activity is tightly coupled to actin cytoskeleton architecture. However, the influence of actin remodeling on cancer drug resistance remains largely unexplored. Here, we report a pivotal role of actin remodeling in YAP/ TAZ-dependent BRAF inhibitor resistance in BRAF V600E mutant melanoma cells. Melanoma cells resistant to the BRAF inhibitor PLX4032 exhibit an increase in actin stress fiber formation, which appears to promote the nuclear accumulation of YAP/ TAZ. Knockdown of YAP/ TAZ reduces the viability of resistant melanoma cells, whereas overexpression of constitutively active YAP induces resistance. Moreover, inhibition of actin polymerization and actomyosin tension in melanoma cells suppresses both YAP/ TAZ activation and PLX4032 resistance. Our si RNA library screening identifies actin dynamics regulator TESK1 as a novel vulnerable point of the YAP/ TAZ-dependent resistance pathway. These results suggest that inhibition of actin remodeling is a potential strategy to suppress resistance in BRAF inhibitor therapies. [ABSTRACT FROM AUTHOR]

Abstract: BRAF mutations are identified in 40–50% of patients with melanoma. Treatment of these patients with either of two BRAF inhibitors (vemurafenib, dabrafenib) or the MEK inhibitor trametinib is associated with improved clinical benefit (response rate, progression free survival, and overall survival) compared with treatment with chemotherapy in three phase III trials. Unfortunately, most patients, including those who experience initial, profound tumour regression, have evidence of disease progression within 6–8months after commencing therapy with one of these agents. The mechanisms of resistance are varied and include activation of alternative signalling pathways as well as reactivating the MAP kinase pathway through alternative means. This review describes relevant aspects of MAP kinase pathway signalling, summarises the clinical data with BRAF and MEK inhibitors, presents the known resistance mechanisms to BRAF inhibitor therapy, and provides some strategies for how resistance may be overcome. [Copyright &y& Elsevier]

PLX4032 is a BRAF-selective inhibitor shown to be efficacious in the treatment of melanomas presenting with the BRAF mutation. However, favorable responses to treatment are short-lived, and complete remission is rarely observed. Therefore, it is important to identify novel therapies designed to enhance treatment responses and to increase the longevity of initial response to BRAF inhibitors. To this end, we characterized the effects of the 225.28 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibody (mAb) capable of blocking multiple signaling pathways important to cell growth, migration, and survival. Addition of 225.28 to the treatment regimen enhanced the in vitro response magnitude and the duration efficacy of PLX4032 in treating CSPG4, BRAF melanoma cells (melanoma cells). Data presented in this report demonstrated that (1) treatments comprised of PLX4032 and mAb 225.28 were more effective at inhibiting melanoma cell growth than either agent alone, (2) mAb 225.28 prevented/delayed the development of resistance in melanoma cells to PLX4032, and (3) the mechanism of action of the combination therapy caused a down-regulation in multiple signaling pathways. This study provides a foundation for future investigations designed to improve BRAF inhibitor effectiveness in vitro and in vivo for treating melanoma cells in combination with a CSPG4-specific mAb. [ABSTRACT FROM AUTHOR]

Single-cell RNA-sequencing (scRNA-seq) has become a powerful tool for biomedical research by providing a variety of valuable information with the advancement of computational tools. Lineage analysis based on scRNA-seq provides key insights into the fate of individual cells in various systems. However, such analysis is limited by several technical challenges. On top of the considerable computational expertise and resources, these analyses also require specific types of matching data such as exogenous barcode information or bulk assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) data. To overcome these technical challenges, we developed a user-friendly computational algorithm called "LINEAGE" (label-free identification of endogenous informative single-cell mitochondrial RNA mutation for lineage analysis). Aiming to screen out endogenous markers of lineage located on mitochondrial reads from label-free scRNA-seq data to conduct lineage inference, LINEAGE integrates a marker selection strategy by feature subspace separation and de novo "low cross-entropy subspaces" identification. In this process, the mutation type and subspace-subspace "cross-entropy" of features were both taken into consideration. LINEAGE outperformed three other methods, which were designed for similar tasks as testified with two standard datasets in terms of biological accuracy and computational efficiency. Applied on a label-free scRNA-seq dataset of BRAF-mutated cancer cells, LINEAGE also revealed genes that contribute to BRAF inhibitor resistance. LINEAGE removes most of the technical hurdles of lineage analysis, which will remarkably accelerate the discovery of the important genes or cell-lineage clusters from scRNA-seq data., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Transcriptional regulators (TRs) participate in essential processes in cancer pathogenesis and are critical therapeutic targets. Identification of drug response-related TRs from cell line-based compound screening data is often challenging due to low mRNA abundance of TRs, protein modifications, and other confounders (CFs). In this study, we developed a regression-based pharmacogenomic and ChIP-seq data integration method (RePhine) to infer the impact of TRs on drug response through integrative analyses of pharmacogenomic and ChIP-seq data. RePhine was evaluated in simulation and pharmacogenomic data and was applied to pan-cancer datasets with the goal of biological discovery. In simulation data with added noises or CFs and in pharmacogenomic data, RePhine demonstrated an improved performance in comparison with three commonly used methods (including Pearson correlation analysis, logistic regression model, and gene set enrichment analysis). Utilizing RePhine and Cancer Cell Line Encyclopedia data, we observed that RePhine-derived TR signatures could effectively cluster drugs with different mechanisms of action. RePhine predicted that loss-of-function of EZH2/PRC2 reduces cancer cell sensitivity toward the BRAF inhibitor PLX4720. Experimental validation confirmed that pharmacological EZH2 inhibition increases the resistance of cancer cells to PLX4720 treatment. Our results support that RePhine is a useful tool for inferring drug response-related TRs and for potential therapeutic applications. The source code for RePhine is freely available at https://github.com/coexps/RePhine., (Copyright © 2022. Published by Elsevier B.V.)

We describe here the evaluation of the cytotoxic efficacy of two platinum (II) complexes bearing an N-heterocyclic carbene (NHC) ligand, a pyridine ligand and bromide or iodide ligands on a panel of human metastatic cutaneous melanoma cell lines representing different genetic subsets including BRAF-inhibitor-resistant cell lines, namely A375, SK-MEL-28, MeWo, HMCB, A375-R, SK-MEL-5-R and 501MEL-R. Cisplatin and dacarbazine were also studied for comparison purposes. Remarkably, the iodine-labelled Pt-NHC complex strongly inhibited proliferation of all tested melanoma cells after 1-h exposure, likely due to its rapid uptake by melanoma cells. The mechanism of this inhibitory activity involves the formation of DNA double-strand breaks and apoptosis. Considering the intrinsic chemoresistance of metastatic melanoma cells of current systemic treatments, these findings are promising and could give research opportunities in the future to improve the prognosis of patients suffering from unresectable metastatic melanoma that are not eligible or that do not respond to the most effective drugs available to date, namely BRAF inhibitors and the anti-PD-1 monoclonal antibody (mAb). [ABSTRACT FROM AUTHOR]

Metastatic melanoma is the most aggressive skin cancer. Although BRAF inhibitor treatment has achieved great success in melanoma, resistance develops within 12 months. Icariside II (IS), a natural compound extracted from Herba Epimedii, exerts anticancer properties. In the present study, we determined by MTT, flow cytometry and western blotting, respectively that IS potentiated the PLX4032‑induced downregulation of cell viability and increase in apoptosis and autophagy in BRAF inhibitor‑resistant melanoma. In addition, we also revealed by flow cytometry and western blotting, respectively, that IS combined with PLX4032 increased mitochondrial and intracellular reactive oxygen species (ROS) generation and subsequently promoted depolarization of mitochondria and release of apoptotic proteins. N‑acetyl cysteine (NAC) and glutathione (GSH), ROS scavengers, reversed the IS‑induced enhancement of the response to PLX4032. Microphthalmia‑associated transcription factor (MITF) and tyrosine‑protein kinase Met (c‑Met) are well‑known factors that contribute to BRAF inhibitor resistance. Furthermore, c‑Met is a direct transcriptional target of MITF in melanocytes and melanoma cells. It was also revealed that IS markedly inhibited MITF and c‑Met expression partially by increasing ROS production in BRAF inhibitor‑resistant melanoma cells.

Limited therapeutic interventions and development of resistance to targeted therapy within few months of therapy pose a great challenge in the treatment of melanoma. Current work was aimed to investigate; (a) Anticancer activity of a novel class of compound - Bromodomain and Extra-Terminal motif (BET) protein degrader in sensitive and vemurafenib-resistant melanoma (b) Preformulation studies and formulation development. ARV-825 (ARV), a molecule designed using PROteolysis-TArgeting Chimeric (PROTAC) technology, degrades BRD4 protein instead of merely inhibiting it. Based on extensive preformulation studies, ARV loaded self-nanoemulsifying preconcentrate (ARV-SNEP) was developed and optimized. ARV showed extremely poor aqueous solubility (<7 μg/mL) and pH dependent hydrolytic degradation. CaCO-2 cell uptake assay and human liver microsome studies proved that ARV is a substrate of CYP3A4 but not of P-gp efflux pump. Optimized ARV-SNEP spontaneously formed nanoglobules of 45.02 nm with zeta potential of −3.78 mV and significantly enhanced solubility of ARV in various aqueous and bio-relevant media. Most importantly, ARV showed promising cytotoxicity, anti-migration and apoptotic activity against vemurafenib-resistant melanoma cells. ARV-SNEP could be potentially novel therapeutic approach for the treatment of drug-resistant melanoma. This is the very first paper investigating a PROTAC class of molecule for the treatment of drug resistant cancer, preformulation and formulation studies. Unlabelled Image [ABSTRACT FROM AUTHOR]

Melanoma is an aggressive form of skin cancer characterized by poor prognosis and high mortality. The development of targeted agents based on the discovery of driver mutations as well as the implementation of checkpoint inhibitor-based immunotherapy represents a major breakthrough in the treatment of metastatic melanoma. However, in both cases the development of drug resistance and immune escape mechanisms as well as the lack of predictive biomarkers limits their extraordinary clinical efficacy. In this article, we summarize the available therapeutic options for patients with metastatic melanoma, outline the mechanisms implicated in the resistance to both targeted agents and immunotherapy, discuss potential predictive biomarkers and outline future therapeutic approaches under investigation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

BRAF inhibitor (BRAFi) has been used for treatment of melanomas harboring V600E mutation. Despite a high initial response rate, resistance to BRAFi is inevitable. Here, we demonstrate that BRAFi-resistant (BR) melanomas are susceptible to arginine deprivation due to inability to initiate re-expression of argininosuccinate synthetase (ASS1, a key enzyme for arginine synthesis) as well as ineffective autophagy. Autophagy and ASS1 re-expression are known to protect melanoma cells from cell death upon arginine deprivation. When melanoma cells become BR cells by long-term in vitro incubation with BRAFi, c-Myc-mediated ASS1 re-expression and the levels of autophagy-associated proteins (AMPK-α1 and Atg5) are attenuated. Furthermore, our study uncovers that downregulation of deubiquitinase USP28 which results in more active c-Myc degradation via ubiquitin-proteasome machinery is the primary mechanism for inability to re-express ASS1 upon arginine deprivation in BR cells. Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival. On the other hand, overexpression of Atg5 or AMPK-α1 in BR cells can redirect arginine deprivation-induced apoptosis toward autophagy. The xenograft models also confirm that BR tumors possess lower expression of ASS1 and are hypersensitive to arginine deprivation. These biochemical changes in BRAFi resistance which make them vulnerable to arginine deprivation can be exploited for the future treatment of BR melanoma patients.

The field of melanoma oncology has recently awakened with groundbreaking scientific advances and innovative therapeutic strategies. New groups of small-molecule kinase inhibitors targeting the aberrant mitogen-activated protein kinase (MAPK) pathway activation mediating tumor growth and survival have revolutionized the therapeutic approach to advanced melanoma. BRAF and MEK inhibitors are the first groups of agents that improved all clinical efficacy endpoints, including response rate, progression-free survival (PFS) and overall survival (OS), in patients with BRAF-mutated advanced melanoma when compared with standard chemotherapy in randomized phase III studies. However, despite the impressive clinical responses in patients with BRAF mutant advanced melanoma, duration of response to MAPK pathway-targeted therapy remains limited, implicating rapid emergence of drug resistance. Diverse strategies to overcome tumor resistance to MAPK inhibitors, the focus of today's translational and clinical research, will further improve the clinical outcome for patients with BRAF-mutated advanced melanoma in the near future.

Control of BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and acquired resistance. Growth factor receptor up-regulation is among the mechanisms underlying BRAF-I resistance of melanoma cells. Here we demonstrate for the first time that PDGFRα up-regulation causes BRAF-I resistance. PDGFRα inhibition by PDGFRα-specific short hairpin (sh)RNA and by PDGFRα inhibitors restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. This effect reflects the inhibition of ERK and AKT activation which is associated with BRAF-I resistance of melanoma cells. PDGFRα up-regulation is mediated by Sonic Hedgehog Homolog (Shh) pathway activation which is induced by BRAF-I treatment. Similarly to PDGFRα inhibition, Shh inhibition by LDE225 restores and increases melanoma cells' sensitivity to BRAF-I. These effects are mediated by PDGFRα down-regulation and by ERK and AKT inhibition. The clinical relevance of these data is indicated by the association of PDGFRα up-regulation in melanoma matched biopsies of BRAF-I +/- MEK inhibitor treated patients with shorter time to disease progression and less tumor regression. These findings suggest that monitoring patients for early PDGFRα up-regulation will facilitate the identification of those who may benefit from the treatment with BRAF-I in combination with clinically approved PDGFRα or Shh inhibitors.