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Purpose: BRAFV600E-mutated colorectal cancer exhibits a strong correlation with DNA hypermethylation, suggesting that this subgroup of tumors presents unique epigenomic phenotypes. Nonetheless, 5-azacitidine, which inhibits DNA methyltransferase activity, is not efficacious in BRAFV600E colorectal cancer in vivo., Experimental Design: We randomized and treated mice implanted with patient-derived tumor xenografts harboring BRAFV600E mutation with control, 5-azacitidine, vemurafenib (BRAF inhibitor), or the combination. Comprehensive epigenomic profiling was conducted on control and 5-azacitidine-treated tumor samples, including DNA methylation, histone modifications, chromatin accessibility, and gene expression. Combinations of epigenetic agents were explored in preclinical BRAFV600E colorectal cancer models., Results: A profound reduction of DNA methylation levels upon 5-azacitidine treatment was confirmed, however, transcriptional repression was not relieved. This study unbiasedly explored the adaptive engagement of other epigenomic modifications upon 5-azacitidine treatment. A loss of histone acetylation and a gain of histone methylations, including H3K27 and H3K4 trimethylation, were observed around these hypomethylated regions, suggesting the involvement of polycomb repressive complex (PRC) activity around the genome with loss of DNA methylation, therefore maintaining the repression of key tumor-suppressor genes. Combined inhibition of PRC activity through EZH2 inhibition with 5-azacitidine treatment additively improved efficacies in BRAFV600E colorectal cancer cells., Conclusions: In conclusion, DNA hypomethylation by 5-azacitidine exhibits a close association with H3K27me3 and PRC activity in BRAFV600E colorectal cancer, and simultaneous blockade of DNA methyltransferase and EZH2 holds promise as a potential therapeutic strategy for patients with BRAFV600E-mutated colorectal cancer., (©2024 American Association for Cancer Research.)

Grover disease is an acquired epidermal blistering disorder in which keratinocytes lose intercellular connections. While its pathologic features are well defined, its etiology remains unclear, and there is no FDA-approved therapy. Interestingly, Grover disease was a common adverse event in clinical trials for cancer using B-RAF inhibitors, but it remained unknown how B-RAF blockade compromised skin integrity. Here, we identified ERK hyperactivation as a key driver of Grover disease pathology. We leveraged a fluorescent biosensor to confirm that the B-RAF inhibitors dabrafenib and vemurafenib paradoxically activated ERK in human keratinocytes and organotypic epidermis, disrupting cell-cell junctions and weakening epithelial integrity. Consistent with clinical data showing that concomitant MEK blockade prevents Grover disease in patients receiving B-RAF inhibitors, we found that MEK inhibition suppressed ERK and rescued cohesion of B-RAF-inhibited keratinocytes. Validating these results, we demonstrated ERK hyperactivation in patient biopsies from vemurafenib-induced Grover disease and from spontaneous Grover disease, revealing a common etiology for both. Finally, in line with our recent identification of ERK hyperactivation in Darier disease, a genetic disorder with identical pathology to Grover disease, our studies uncovered that the pathogenic mechanisms of these diseases converge on ERK signaling and support MEK inhibition as a therapeutic strategy.

With an increasing incidence of malignant melanoma, new prognostic biomarkers for clinical decision making have become more important. In this study, we evaluated the role of ecto-NOX disulfide-thiol exchanger 2 (ENOX2/tNOX), a cancer- and growth-associated protein, in the prognosis and therapy of primary malignant melanoma. We conducted a tissue microarray analysis of immunohistochemical ENOX2 protein expression and The Cancer Genome Atlas (TCGA) ENOX2 RNA expression analysis, as well as viability assays and Western blots of melanoma cell lines treated with the ENOX2 inhibitor phenoxodiol (PXD) and BRAF inhibitor (BRAFi) vemurafenib. We discovered that high ENOX2 expression is associated with decreased overall (OS), disease-specific (DSS) and metastasis-free survival (MFS) in primary melanoma (PM) and a reduction in electronic tumor-infiltrating lymphocytes (eTILs). A gradual rise in ENOX2 expression was found with an increase in malignant potential from benign nevi (BNs) via PMs to melanoma metastases (MMs), as well as with an increasing tumor thickness and stage. These results highlight the important role of ENOX2 in cancer growth, progression and metastasis. The ENOX2 expression was not limited to malignant cell lines but could also be found in keratinocytes, fibroblasts and melanocytes. The viability of melanoma cell lines could be inhibited by PXD. A reduced induction of phospho-AKT under PXD could prevent the development of acquired BRAFi resistance. In conclusion, ENOX2 may serve as a potential prognostic marker and therapeutic target in malignant melanoma.

Enteroviruses, which infect via the gut, have been implicated in type 1 diabetes (T1D) development. Prolonged faecal shedding of enterovirus has been associated with islet autoimmunity. Additionally, enteroviral proteins and viral RNA have been detected in the pancreatic islets of individuals with recent-onset T1D, implicating their possible role in beta cell destruction. Despite this, no approved antiviral drugs currently exist that specifically target enterovirus infections for utilisation in disease interventions. Drug repurposing allows for the discovery of new clinical uses for existing drugs and can expedite drug discovery. Previously, the cancer drug Vemurafenib demonstrated unprecedented antiviral activity against several enteroviruses. In the present study, we assessed the efficacy of Vemurafenib and an analogue thereof in preventing infection or reducing the replication of enteroviruses associated with T1D. We tested Vemurafenib in intestinal epithelial cells (IECs) and insulin-producing beta cells. Additionally, we established a protocol for infecting human stem cell-derived islets (SC-islets) and used Vemurafenib and its analogue in this model. Our studies revealed that Vemurafenib exhibited strong antiviral properties in IECs and a beta cell line. The antiviral effect was also seen with the Vemurafenib analogue. SC-islets expressed the viral receptors CAR and DAF, with their highest expression in insulin- and glucagon-positive cells, respectively. SC-islets were successfully infected by CVBs and the antiviral activity of Vemurafenib and its analogue was confirmed in most SC-islet batches. In summary, our observations suggest that Vemurafenib and its analogue warrant further exploration as potential antiviral agents for the treatment of enterovirus-induced diseases, including T1D., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Stromal interaction molecule 1 (STIM1) is the endoplasmic reticulum Ca 2+ sensor for store-operated calcium entry and is closely associated with carcinogenesis and tumor progression. Previously, we found that STIM1 is upregulated in melanoma cells resistant to the serine/threonine-protein kinase B-raf inhibitor vemurafenib, although the mechanism underlying this upregulation is unknown. Here, we show that vemurafenib resistance upregulates STIM1 through an epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR)-Yes-associated protein 1 (YAP1)/TEA domain transcription factor 2 (TEAD2) signaling axis. Vemurafenib resistance can lead to an increase in EGF and EGFR levels, causing activation of the EGFR signaling pathway, which promotes YAP1 nuclear localization to increase the expression of STIM1. Our findings not only reveal the mechanism by which vemurafenib resistance promotes STIM1 upregulation, but also provide a rationale for combined targeting of the EGF/EGFR-YAP1/TEAD2-STIM1 axis to improve the therapeutic efficacy of BRAF inhibitor in melanoma patients., (© 2024 Federation of European Biochemical Societies.)

Context: Melanoma is one of the cancers with the highest mortality rate for its ability to metastasize. Several targets have undergone investigation for the development of drugs against this pathology. One of the main targets is the kinase BRAF (RAF, rapidly accelerated fibrosarcoma). The most common mutation in melanoma is BRAFV600E and has been reported in 50-90% of patients with melanoma. Due to the relevance of the BRAFV600E mutation, inhibitors to this kinase have been developed, vemurafenib-OMe and dabrafenib. Ursolic acid (UA) is a pentacyclic triterpene with a privileged structure, the pentacycle scaffold, which allows to have a broad variety of biological activity; the most studied is its anticancer capacity. In this work, we reported the interaction profile of vemurafenib-OMe, dabrafenib, and UA, to define whether UA has binding capacity to BRAFWT, BRAFV600E, and BRAFV600K. Homology modeling of BRAFWT, V600E, and V600K; molecular docking; and molecular dynamics simulations were carried out and interactions and residues relevant to the binding of the inhibitors were obtained. We found that UA, like the inhibitors, presents hydrogen bond interactions, and hydrophobic interactions of van der Waals, and π-stacking with I463, Q530, C532, and F583. The ΔG of ursolic acid in complex with BRAFV600K (- 63.31 kcal/mol) is comparable to the ΔG of the selective inhibitor dabrafenib (- 63.32 kcal/mol) in complex to BRAFV600K and presents a ΔG like vemurafenib-OMe with BRAFWT and V600E. With this information, ursolic acid could be considered as a lead compound for design cycles and to optimize the binding profile and the selectivity towards mutations for the development of new selective inhibitors for BRAFV600E and V600K to new potential melanoma treatments., Methods: The homology modeling calculations were executed on the public servers I-TASSER and ROBETTA, followed by molecular docking calculations using AutoGrid 4.2.6, AutoDockGPU 1.5.3, and AutoDockTools 1.5.6. Molecular dynamics and metadynamics simulations were performed in the Desmond module of the academic version of the Schrödinger-Maestro 2020-4 program, utilizing the OPLS-2005 force field. Ligand-protein interactions were evaluated using Schrödinger-Maestro program, LigPlot + , and PLIP (protein-ligand interaction profiler). Finally, all of the protein figures presented in this article were made in the PyMOL program., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chemotherapy resistance is one of the main challenges in melanoma treatment. Violacein, a natural pigment produced by Chromobacterium violaceum , induces apoptosis in a variety of tumours, including melanoma. Here, we used BRAF-mutated melanoma spheroids to test the potential of violacein as a sensitizer of cellular viability and levels of the proteins p62 and fatty acid synthase (FASN). Importantly, violacein in combination with vemurafenib (ViVe) was able to interfere with spheroid survival at subtoxic concentrations. The results demonstrated that the ViVe protocol triggered cell death assessed by calcein and ethidium homodimer dyes. Accordingly, melanoma cells in 2D systems also showed a higher apoptosis rate when treated with ViVe. In the current study, we show evidence that ViVe downregulates crucial mediators like FASN, which partially explains how it acts as a sensitizer and ultimately improves the effectiveness of vemurafenib against melanoma cells.

Tetracaine, a local anesthetic, exhibits potent cytotoxic effects on multiple cancer; however, the precise underlying mechanisms of its anti-cancer activity remain uncertain. The anti-cancer activity of tetracaine was found to be the most effective among commonly used local anesthetics in this study. After tetracaine treatment, the differentially expressed genes in melanoma cells were identified by the RNAseq technique and enriched in the lysosome signaling pathway, cullin family protein binding, and proteasome signaling pathway through Kyoto Encyclopedia of Genes and Genomes. Additionally, the ubiquitin-like neddylation signaling pathway, which is hyperactivated in melanoma, could be abrogated due to decreased NAE2 expression after tetracaine treatment. The neddylation of the pro-oncogenic Survivin, which enhances its stability, was significantly reduced following treatment with tetracaine. The activation of neddylation signaling by NEDD8 overexpression could reduce the antitumor efficacy of tetracaine in vivo and in vitro. Furthermore, vemurafenib-resistant melanoma cells showed higher level of neddylation, and potential substrate proteins undergoing neddylation modification were identified through immunoprecipitation and mass spectrometry. The tetracaine treatment could reduce drug resistance via neddylation signaling pathway inactivation in melanoma cells. These findings demonstrate that tetracaine effectively inhibits cell proliferation and alleviates vemurafenib resistance in melanoma by suppressing the neddylation signaling pathway, providing a promising avenue for controlling cancer progression., (© 2024. The Author(s).)

Neoantigen-based immunotherapy is an attractive potential treatment for previously intractable tumors. To effectively broaden the application of this approach, stringent biomarkers are crucial to identify responsive patients. ARID1A, a frequently mutated subunit of SWI/SNF chromatin remodeling complex, has been reported to determine tumor immunogenicity in some cohorts; however, mutations and deletions of ARID1A are not always linked to clinical responses to immunotherapy. In this study, we investigated immunotherapeutic responses based on ARID1A status in targeted therapy-resistant cancers. Mouse and human BRAFV600E melanomas with or without ARID1A expression were transformed into resistant to vemurafenib, an FDA-approved specific BRAFV600E inhibitor. Anti-PD-1 antibody treatment enhanced antitumor immune responses in vemurafenib-resistant ARID1A-deficient tumors but not in ARID1A-intact tumors or vemurafenib-sensitive ARID1A-deficient tumors. Neoantigens derived from accumulated somatic mutations during vemurafenib resistance were highly expressed in ARID1A-deficient tumors and promoted tumor immunogenicity. Furthermore, the newly generated neoantigens could be utilized as immunotherapeutic targets by vaccines. Finally, targeted therapy resistance-specific neoantigen in experimental human melanoma cells lacking ARID1A were validated to elicit T-cell receptor responses. Collectively, the classification of ARID1A-mutated tumors based on vemurafenib resistance as an additional indicator of immunotherapy response will enable a more accurate prediction to guide cancer treatment. Furthermore, the neoantigens that emerge with therapy resistance can be promising therapeutic targets for refractory tumors. Significance: Chemotherapy resistance promotes the acquisition of immunogenic neoantigens in ARID1A-deficient tumors that confer sensitivity to immune checkpoint blockade and can be utilized for developing antitumor vaccines, providing strategies to improve immunotherapy efficacy., (©2024 American Association for Cancer Research.)

Programmed death 1 (PD-1)/programmed death-ligand 1 inhibitors are commonly used to treat various cancers, including melanoma. However, their efficacy as monotherapy is limited, and combination immunotherapies are being explored to improve outcomes. In this study, we investigated a combination immunotherapy involving an anti-PD-1 antibody that blocks the major adaptive immune-resistant mechanisms, a BRAF inhibitor that inhibits melanoma cell proliferation, and multiple primary immune-resistant mechanisms, such as cancer cell-derived immunosuppressive cytokines, and a Toll-like receptor 7 agonist that enhances innate immune responses that promote antitumor T-cell induction and functions. Using a xenogeneic nude mouse model implanted with human BRAF-mutated melanoma, a BRAF inhibitor vemurafenib was found to restore T-cell-stimulatory activity in conventional dendritic cells by reducing immunosuppressive cytokines, including interleukin 6, produced by human melanoma. Additionally, intravenous administration of the Toll-like receptor 7 agonist DSR6434 enhanced tumor growth inhibition by vemurafenib through stimulating the plasmacytoid dendritic cells/interferon-α/natural killer cell pathways and augmenting the T-cell-stimulatory activity of conventional dendritic cells. In a syngeneic mouse model implanted with murine BRAF-mutated melanoma, the vemurafenib and DSR6434 combination synergistically augmented the induction of melanoma antigen gp100-specific T cells and inhibited tumor growth. Notably, only triplet therapy with vemurafenib, DSR6434, and the anti-PD-1 antibody resulted in complete regression of SIY antigen-transduced BRAF-mutated melanoma in a CD8 T-cell-dependent manner. These findings indicate that a triple-combination strategy targeting adaptive and primary resistant mechanisms while enhancing innate immune responses that promote tumor-specific T cells may be crucial for effective tumor eradication., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Mutant BRAF is a critical oncogenic driver in melanoma, making it an attractive therapeutic target. However, the success of targeted therapy using BRAF inhibitors vemurafenib and dabrafenib has been limited due to development of resistance, restricting their clinical efficacy. A prior knowledge of resistance mechanisms to BRAFi or any cancer drug can lead to development of drugs that overcome resistance thus improving clinical outcomes. In vitro cellular models are powerful systems that can be utilized to mimic and study resistance mechanisms. In this study, we employed a multi-omics approach to characterize a panel of BRAF mutant melanoma cell lines to develop and systematically characterize BRAFi persister and resistant cells using exome sequencing, proteomics and phosphoproteomics. Our datasets revealed frequently observed intrinsic and acquired, genetic and non-genetic mechanisms of BRAFi resistance that have been studied in patients who developed resistance. In addition, we identified proteins that can be potentially targeted to overcome BRAFi resistance. Overall, we demonstrate that in vitro systems can be utilized not only to predict resistance mechanisms but also to identify putative therapeutic targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pseudopodium-enriched atypical kinase 1 (PEAK1) has been demonstrated to be upregulated in human malignancies and cells. Enhanced PEAK1 expression facilitates tumor cell survival and chemoresistance. However, the role of PEAK1 inhibition to anaplastic thyroid carcinoma cell (ATC) and vemurafenib resistance is still unknown. Here, we observed that targeting PEAK1 inhibited cell viability and colony formation, but not cell apoptosis in both of the 8505C and Hth74 cells in vitro . Targeting PEAK1 sensitized 8505C and Hth74 cells to vemurafenib by inducing cell apoptosis, and thereby decreasing cell viability. Mechanistically, vemurafenib treatment upregulated PEAK1 expression. Combined PEAK1 depletion and Vemurafenib treatment upregulated Bim expression. Targeting PEAK1 sensitized vemurafenib-induced apoptosis by upregulating Bim. In conclusion, vemurafenib resistance in ATC cells harboring BRAF V600E is associated with PEAK1 activation, resulting in the inhibition of pro-apoptotic Bim protein. Therefore, targeting PEAK1 may be an effective strategy to sensitize ATC harboring BRAF V600E to vemurafenib., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Despite the targeted- and immunotherapies used in the past decade, survival rate among patients with metastatic melanoma remains low, therefore, melanoma is responsible for the majority of skin cancer-related deaths. The ongoing investigation of natural antitumor agents, the nonpsychoactive cannabinoid, cannabigerol (CBG) found in Cannabis sativa is emerging as a promising candidate. CBG offers a potential therapeutic role in the treatment of melanoma demonstrating cell growth inhibition in some tumors. Its low water solubility and bioavailability hinder the potential effectiveness. To address these challenges, a modified CBG, namely LE-127/2 was synthesized by Mannich-type reaction. The aim was to investigate the effect of this novel compound on cell proliferation as well as the mechanism of cell death with a particular focus on autophagy and apoptosis. Human cutan melanoma cell lines, WM35, A2058 and WM3000 were utilized for the present study. Cell proliferation of the cells after the treatment with LE-127/2, parent CBG or vemurafenib was assessed by Cell Titer Blue Assay. Cells were treated with a 1.25-80 µM of the above-mentioned compounds, and it was found that at 20 μM of all drugs showed a comparable effective inhibition of cell proliferation, however, vemurafenib and CBG proved to be more effective than LE-127/2. In addition, clonogenic cell survival assays were performed to examine the inhibitory effect of LE-127/2 on the colony formation ability of melanoma cell lines. Cells treated with 20 µM of LE-127/2 for 14 days showed about a 50% suppression of clonogenic cell survival. LE-127/2 exerted the most intensive inhibition on A2058 cell colonies. Furthermore, notably, LDH cytotoxicity assay performed on HaCaT cell line, proved LE-127/2 to be cytotoxic only at higher concentration, such as 80 μM, while the parent CBG was cytotoxic at concentration as low as 5 μM, suggesting that the new CBG derivative as a drug candidate may be applied in human pharmacotherapy without causing a substantial damage in intact epidermal cells. Analysis of protein expression revealed the impact of LE-127/2 on the expression of basic proteins (LC-3, Beclin-1 and p62) involved in the process of autophagy in the three different melanoma cell lines studied. Elevated expression of these proteins was detected as a result of LE-127/2 (20 µM) treatment. LE-127/2 also induced the expression of some proteins involved in apoptosis, and it is particularly noteworthy the increased level of cleaved PARP. Based on the results obtained, it can be concluded that LE-127/2 induced autophagy could lead to the inhibition of cell proliferation and death in melanoma cells., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: BRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors., Methods: Cell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array., Results: The resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC 50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRβ receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion., Conclusions: In summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies., (© 2024. The Author(s).)

Raf kinases play key roles in signal transduction in cells for regulating proliferation, differentiation, and survival. Despite decades of research into functions and dynamics of Raf kinases with respect to other cytosolic proteins, understanding Raf kinases is limited by the lack of their full-length structures at the atomic resolution. Here, we present the first model of the full-length CRaf kinase obtained from artificial intelligence/machine learning algorithms with a converging ensemble of structures simulated by large-scale temperature replica exchange simulations. Our model is validated by comparing simulated structures with the latest cryo-EM structure detailing close contacts among three key domains and regions of the CRaf. Our simulations identify potentially new epitopes of intramolecule interactions within the CRaf and reveal a dynamical nature of CRaf kinases, in which the three domains can move back and forth relative to each other for regulatory dynamics. The dynamic conformations are then used in a docking algorithm to shed insight into the paradoxical effect caused by vemurafenib in comparison with a paradox breaker PLX7904. We propose a model of Raf-heterodimer/KRas-dimer as a signalosome based on the dynamics of the full-length CRaf., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. All rights reserved.)

Malignant melanoma, an aggressive skin cancer with a poor prognosis, frequently features BRAFV600E mutation resulting in activation of the MAPK pathway and melanocyte proliferation and survival. BRAFV600E inhibitors like vemurafenib and dabrafenib have enhanced patient survival, yet drug resistance remains a significant challenge. We investigated the role of the ERK5 pathway in BRAFV600E melanoma cells and cells with acquired resistance to PLX4720 (vemurafenib) and dabrafenib. In BRAFV600E melanoma, ERK5 inhibition minimally affected viability compared to ERK1/2 inhibition. In vemurafenib-resistant cells, ERK5 inhibition alone didn't impact viability or restore drug sensitivity to vemurafenib. However, in dabrafenib-resistant cells, ERK5 inhibition reduced viability and enhanced the anti-proliferative effect of MEK1/2 inhibition. Targeting the ERK5 pathway may represent a therapeutic opportunity in dabrafenib-resistant melanoma., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Exosomes derived from neighboring v-raf murine sarcoma viral oncogene homolog B1 inhibitor (BRAFi)-resistant melanoma cells mediate the formation of resistance in melanoma cells sensitive to BRAFi. The function and molecular mechanisms of exosomal miRNA in BRAFi resistance of melanoma have not been studied. We found that the expression of miR-19a in BRAFi resistant melanoma cells was significantly higher than that in sensitive cells, and miR-19a contributes to the resistance of melanoma cells to BRAFi by targeting immunoglobulin-like domains protein 1 (LRIG1). miR-19a was highly enriched in exosomes secreted from BRAFi resistant melanoma cells, and these exosomal miR-19a promote the spread of BRAFi resistant. The reactivation of Protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways is the main reason for the BRAFi resistant of melanoma cells. We demonstrated that exosomal miR-19a derived from melanoma cell promotes the formation and spread of BRAFi resistant in melanoma through targeting LRIG1 to reactivate AKT and MAPK pathway. Therefore, miR-19a may serve as a potential therapeutic target in melanoma patients with acquired drug resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Melanoma cells express high levels of CD73 that produce extracellular immunosuppressive adenosine. Changes in the CD73 expression occur in response to tumor environmental factors, contributing to tumor phenotype plasticity and therapeutic resistance. Previously, we have observed that CD73 expression can be up-regulated on the surface of melanoma cells in response to nutritional stress. Here, we explore the mechanism by which melanoma cells release soluble CD73 under low nutrient availability and whether this might be affected by agents targeting the proto-oncogene B-Raf (BRAF). We found that starved melanoma cells can release high levels of CD73, able to convert AMP into adenosine, and this activity is abrogated by selective CD73 inhibitors, APCP or PSB-12489. The release of CD73 from melanoma cells is mediated by the matrix metalloproteinase MMP-9. Indeed, MMP-9 inhibitors significantly reduce the levels of CD73 released from the cells, while its surface levels increase. Of relevance, melanoma cells, harboring an activating BRAF mutation, upon treatment with dabrafenib or vemurafenib, show a strong reduction of CD73 cell expression and reduced levels of CD73 released into the extracellular space. Conversely, melanoma cells resistant to dabrafenib show high expression of membrane-bound CD73 and soluble CD73 released into the culture medium. In summary, our data indicate that CD73 is released from melanoma cells. The expression of CD73 is associated with response to BRAF inhibitors. Melanoma cells developing resistance to dabrafenib show increased expression of CD73, including soluble CD73 released from cells, suggesting that CD73 is involved in acquiring resistance to treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-β signalling. In this study, the role of integrins and TGF-β signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-β pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-β signalling, specifically ITGA5 , ITGB3 , PAI1 , and p21 , may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.

Background: BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms., Methods: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts., Results: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings., Conclusions: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors., (© 2024. The Author(s).)

Targeted therapies against mutant BRAF are effectively used in combination with MEK inhibitors (MEKi) to treat advanced melanoma. However, treatment success is affected by resistance and adverse events (AEs). Approved BRAF inhibitors (BRAFi) show high levels of target promiscuity, which can contribute to these effects. The blood vessel lining is in direct contact with high plasma concentrations of BRAFi, but effects of the inhibitors in this cell type are unknown. Hence, we aimed to characterize responses to approved BRAFi for melanoma in the vascular endothelium. We showed that clinically approved BRAFi induced a paradoxical activation of endothelial MAPK signaling. Moreover, phosphoproteomics revealed distinct sets of off-targets per inhibitor. Endothelial barrier function and junction integrity were impaired upon treatment with vemurafenib and the next-generation dimerization inhibitor PLX8394, but not with dabrafenib or encorafenib. Together, these findings provide insights into the surprisingly distinct side effects of BRAFi on endothelial signaling and functionality. Better understanding of off-target effects could help to identify molecular mechanisms behind AEs and guide the continued development of therapies for BRAF-mutant melanoma., (© 2024 Bromberger et al.)

The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

BRAF V600E is the most prevalent mutation in thyroid cancer and correlates with poor prognosis and therapy resistance. Although selective inhibitors of BRAF V600E have been developed, more advanced tumors such as anaplastic thyroid carcinomas show a poor response in clinical trials. Therefore, the study of alternative survival mechanisms is needed. Since metabolic changes have been related to malignant progression, in this work we explore metabolic dependencies of thyroid tumor cells to exploit them therapeutically. Our results show that respiration of thyroid carcinoma cells is highly dependent on fatty acid oxidation and, in turn, fatty acid mitochondrial availability is regulated through macroautophagy/autophagy. Furthermore, we show that both lysosomal inhibition and the knockout of the essential autophagy gene, ATG7 , lead to enhanced lipolysis; although this effect is not essential for survival of thyroid carcinoma cells. We also demonstrate that following inhibition of either autophagy or fatty acid oxidation, thyroid tumor cells compensate oxidative phosphorylation deficiency with an increase in glycolysis. In contrast to lipolysis induction, upon autophagy inhibition, glycolytic boost in autophagy-deficient cells is essential for survival and, importantly, correlates with a higher sensitivity to the BRAF V600E selective inhibitor, vemurafenib. In agreement, downregulation of the glycolytic pathway results in enhanced mitochondrial respiration and vemurafenib resistance. Our work provides new insights into the role of autophagy in thyroid cancer metabolism and supports mitochondrial targeting in combination with vemurafenib to eliminate BRAF V600E -positive thyroid carcinoma cells. Abbreviations : AMP: adenosine monophosphate; ATC: anaplastic thyroid carcinoma; ATG: autophagy related; ATP: adenosine triphosphate; BRAF: B-Raf proto-oncogene, serine/threonine kinase; Cas9: CRISPR-associated protein; CREB: cAMP responsive element binding protein; CRISPR: clustered regularly interspaced short palindromic repeats; 2DG: 2-deoxyglucose; FA: fatty acid; FAO: fatty acid oxidation; FASN: fatty acid synthase; FCCP: trifluoromethoxy carbonyl cyanide phenylhydrazone; LAMP1: lysosomal associated membrane protein 1; LIPE/HSL: lipase E, hormone sensitive type; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation; PRKA/PKA: protein kinase cAMP-activated; PTC: papillary thyroid carcinoma; SREBF1/SREBP1: sterol regulatory element binding transcription factor 1.

Extracellular vesicles (EVs) constitute a vital component of intercellular communication, exerting significant influence on metastasis formation and drug resistance mechanisms. Malignant melanoma (MM) is one of the deadliest forms of skin cancers, because of its high metastatic potential and often acquired resistance to oncotherapies. The prevalence of BRAF mutations in MM underscores the importance of BRAF-targeted therapies, such as vemurafenib and dabrafenib, alone or in combination with the MEK inhibitor, trametinib. This study aimed to elucidate the involvement of EVs in MM progression and ascertain whether EV-mediated metastasis promotion persists during single agent BRAF (vemurafenib, dabrafenib), or MEK (trametinib) and combined BRAF/MEK (dabrafenib/trametinib) inhibition.Using five pairs of syngeneic melanoma cell lines, we assessed the impact of EVs - isolated from their respective supernatants - on melanoma cell proliferation and migration. Cell viability and spheroid growth assays were employed to evaluate proliferation, while migration was analyzed through mean squared displacement (MSD) and total traveled distance (TTD) measurements derived from video microscopy and single-cell tracking.Our results indicate that while EV treatments had remarkable promoting effect on cell migration, they exerted only a modest effect on cell proliferation and spheroid growth. Notably, EVs demonstrated the ability to mitigate the inhibitory effects of BRAF inhibitors, albeit they were ineffective against a MEK inhibitor and the combination of BRAF/MEK inhibitors. In summary, our findings contribute to the understanding of the intricate role played by EVs in tumor progression, metastasis, and drug resistance in MM., (© 2024. The Author(s).)

Vemurafenib has been used as first-line therapy for unresectable or metastatic melanoma with BRAFV600E mutation. However, overall survival is still limited due to treatment resistance after about one year. Therefore, identifying new therapeutic targets for melanoma is crucial for improving clinical outcomes. In the present study, we found that lowering intracellular cholesterol by knocking down DHCR24, the limiting synthetase, impaired tumor cell proliferation and migration and abrogated the ability to xenotransplant tumors. More importantly, administration of DHCR24 or cholesterol mediated resistance to vemurafenib and promoted the growth of melanoma spheroids. Mechanistically, we identified that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol synthesized by the enzyme cytochrome P450 27A1 (CYP27A1), reproduces the phenotypes induced by DHCR24 or cholesterol administration and activates Rap1-PI3K/AKT signaling. Accordingly, CYP27A1 is highly expressed in melanoma patients and upregulated by DHCR24 induction. Dafadine-A, a CYP27A1 inhibitor, attenuates cholesterol-induced growth of melanoma spheroids and abrogates the resistance property of vemurafenib-resistant melanoma cells. Finally, we confirmed that the effects of cholesterol on melanoma resistance require its metabolite 27HC through CYP27A1 catalysis, and that 27HC further upregulates Rap1A/Rap1B expression and increases AKT phosphorylation. Thus, our results suggest that targeting 27HC may be a useful strategy to overcome treatment resistance in metastatic melanoma., (© 2024. The Author(s).)

Regardless of the clinical response and improved patient survival observed following treatment with BRAFi like Vemurafenib (Vem), rapid development of resistance still remains as a major obstacle in melanoma therapy. In this context, we developed and characterized two acquired Vem-resistant melanoma cell lines, A375V and SK-MEL-28V, and an intrinsically Vem-resistant cell line, RPMI-7951. Altered morphology and growth rate of the resistant cell lines displayed spindle-shaped cells with filopodia formation and enhanced proliferation rate as compared to parental cells. Further in vitro characterization in 2D models confirmed the emergence of a resistant phenotype in melanoma cells. To mimic the in vivo tumor microenvironment, spheroids were developed for both parental and resistant cell lines to recognize materialization of invadopodia structures demonstrating elevated invasiveness and proliferation of resistant cells-based spheroids, especially A375V. Importantly, we validated A375V cell line in vivo to prove its tumorigenicity and drug resistance in tumor xenograft model. Taken together, our established clinically relevant Vem-resistant tumor model could be beneficial to elucidate drug resistance mechanisms, screen and identify novel anticancer therapies to overcome BRAFi resistance in melanoma., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Evodiamine, a novel alkaloid, was isolated from the fruit of tetradium. It exerts a diversity of pharmacological effects and has been used to treat gastropathy, hypertension, and eczema. Several studies reported that evodiamine has various biological effects, including anti-nociceptive, anti-bacterial, anti-obesity, and anti-cancer activities. However, there is no research regarding its effects on drug-resistant cancer. This study aimed to investigate the effect of evodiamine on human vemurafenib-resistant melanoma cells (A375/R cells) proliferation ability and its mechanism. Cell activity was assessed using the cell counting kit-8 (CCK-8) method. Flow cytometry assay was used to assess cell apoptosis and cell cycle. A xenograft model was used to analyze the inhibitory effects of evodiamine on tumor growth. Bioinformatics analyses, network pharmacology, and molecular docking were used to explore the potential mechanism of evodiamine in vemurafenib-resistant melanoma. RT-qPCR and Western blotting were performed to reveal the molecular mechanism. The alkaloid extract of the fruit of tetradium, evodiamine showed the strongest tumor inhibitory effect on vemurafenib-resistant melanoma cells compared to treatment with vemurafenib alone. Evodiamine inhibited vemurafenib-resistant melanoma cell growth, proliferation, and induced apoptosis, conforming to a dose-effect relationship and time-effect relationship. Results from network pharmacology and molecular docking suggested that evodiamine might interact with IRS4 to suppress growth of human vemurafenib-resistant melanoma cells. Interestingly, evodiamine suppressed IRS4 expression and then inhibited PI3K/AKT signaling pathway, and thus had the therapeutic action on vemurafenib-resistant melanoma., (© 2024. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Melanoma is an aggressive malignant tumor with a poor prognosis. Vemurafenib (PLX4032, vem) is applied to specifically treat BRAF V600E-mutated melanoma patients. However, prolonged usage of vem makes patients resistant to the drug and finally leads to clinical failure. We previously tested the combination regimen of tubulin inhibitor VERU-111 with vem, as well as USP14 selective inhibitor b-AP15 in combination with vem, both of which have showed profound therapeutic effects in overcoming vem resistance in vitro and in vivo. Most importantly, we discovered that vem-resistant melanoma cell lines highly expressed E3 ligase SKP2 and DUB enzyme USP14, and we have demonstrated that USP14 directly interacts and stabilizes SKP2, which contributes to vem resistance. These works give us a clue that USP14 might be a promising target to overcome vem resistance in melanoma. MitoCur-1 is a curcumin derivative, which was originally designed to specifically target tumor mitochondria inducing redox imbalance, thereby promoting tumor cell death. In this study, we have demonstrated that it can work as a novel USP14 inhibitor, and thus bears great potential in providing an anti-tumor effect and sensitizing vem-resistant cells by inducing ferroptosis in melanoma. Application of MitoCur-1 dramatically induces USP14 inhibition and inactivation of GPX4 enzyme, meanwhile, increases the depletion of GSH and decreases SLC7A11 expression level. As a result, ferrous iron-dependent lipid ROS accumulated in the cell, inducing ferroptosis, thus sensitizes the vem-resistant melanoma cell. Interestingly, overexpression of USP14 antagonized all the ferroptosis cascade events induced by MitoCur-1, therefore, we conclude that MitoCur-1 induces ferroptosis through inhibition of USP14. We believe that by inhibition of USP14, vem resistance can be reversed and will finally benefit melanoma patients in future., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cobimetinib/vemurafenib combination therapy is approved for treatment of adults with unresectable or metastatic BRAF V600 mutated malignant melanoma (mM). The non-interventional post-authorisation safety study coveNIS collected real-world data on cobimetinib/vemurafenib treatment focussing on overall survival (OS), safety and utilization. MM patients with brain metastases are usually excluded from clinical studies. coveNIS observed 2 cohorts: mM patients without (Cohort A) and with cerebral metastases (Cohort B), aiming to close the data gap for the latter population. A direct comparison of the 2 cohorts was not intended. The primary effectiveness objective was OS; the safety objective was the incidence of all and of serious adverse events (AEs). Secondary objectives included progression-free survival (PFS), time to development of cerebral metastasis (Cohort A) and time to central nervous system relapse (Cohort B). All statistical analyses were descriptive. Between 2017 and 2021, 95 patients were included (Cohort A: 54, Cohort B: 41 patients) at 32 sites in Germany. Median OS was 21.6 months in Cohort A, 7.4 months in Cohort B. Median PFS was 6.9 months in Cohort A, 5.2 months in Cohort B. The proportion of patients experiencing any AEs was 83.3% (Cohort A) and 87.8% (Cohort B). The two most common AEs in Cohort A were 'diarrhoea' (37%), 'vomiting' (20.4%) and 'pyrexia' (20.4%); in Cohort B 'diarrhoea' (36.6%) and 'fatigue' (22%). In conclusion, the OS rates in Cohort A and Cohort B of coveNIS are in line with the OS data from other trials with BRAF/MEK inhibitors for mM. No new safety signals were observed., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The outcome of Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remain dismal despite the development of treatment. Targeted therapy is gaining more and more attention in improving prognosis., Methods: Expression of BRAF was analyzed by RT-qPCR in AML and MDS patients. Cells viability treated by drugs was measured by CCK-8 assay. Network pharmacology and RNA-sequence were used to analyze the mechanism of drugs and verified in vitro and xenograft tumor model., Results: Here we showed that BRAF was overexpressed in AML and MDS patients, and correlated with poor prognosis. The BRAF inhibitor-Vemurafenib (VEM) could significantly induce senescence, proliferation inhibition and apoptosis in AML cells, which can be enhanced by Bortezomib (BOR). This inhibitory effect was also verified in CD34 + cells derived from AML patients. Mechanistically, we showed that VEM combined with BOR could turn on HIPPO signaling pathway, thereby inducing cellular senescence in AML cells and xenograft mouse., Conclusions: Taken together, our findings demonstrate a significant upregulation of BRAF expression in AML and MDS patients, which is associated with unfavorable clinical outcomes. We also discovered that the BRAF inhibitor Vemurafenib induces cellular senescence through activation of the HIPPO signaling pathway. Analysis of BRAF expression holds promise as a prognostic indicator and potential therapeutic target for individuals with AML and MDS., (© 2024. The Author(s).)

BRAF V600 -mutated melanoma brain metastases (MBMs) are responsive to BRAF inhibitors, but responses are generally less durable than those of extracranial metastases. We tested the hypothesis that the drug efflux transporters P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) expressed at the blood-brain barrier (BBB) offer MBMs protection from therapy. We intracranially implanted A375 melanoma cells in wild-type (WT) and Abcb1a/b;Abcg2 -/- mice, characterized the tumor BBB, analyzed drug levels in plasma and brain lesions after oral vemurafenib administration, and determined the efficacy against brain metastases and subcutaneous lesions. Although contrast-enhanced MRI demonstrated that the integrity of the BBB is disrupted in A375 MBMs, vemurafenib achieved greater antitumor efficacy against MBMs in Abcb1a/b;Abcg2 -/- mice compared with WT mice. Concordantly, P-gp and BCRP are expressed in MBM-associated brain endothelium both in patients and in A375 xenografts and expression of these transporters limited vemurafenib penetration into A375 MBMs. Although initially responsive, A375 MBMs rapidly developed therapy resistance, even in Abcb1a/b;Abcg2 -/- mice, and this was unrelated to pharmacokinetic or target inhibition issues. Taken together, we demonstrate that both intrinsic and acquired resistance can play a role in MBMs., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1 nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Melanoma, a frequently encountered cutaneous malignancy characterized by a poor prognosis, persists in presenting formidable challenges despite the advancement in molecularly targeted drugs designed to improve survival rates significantly. Unfortunately, as more therapeutic choices have developed over time, the gradual emergence of drug resistance has become a notable impediment to the effectiveness of these therapeutic interventions. The hepatocyte growth factor (HGF)/c-met signaling pathway has attracted considerable attention, associated with drug resistance stemming from multiple potential mutations within the c-met gene. The activation of the HGF/c-met pathway operates in an autocrine manner in melanoma. Notably, a key player in the regulatory orchestration of HGF/c-met activation is the long non-coding RNA MEG3., Methods: Melanoma tissues were collected to measure MEG3 expression. In vitro validation was performed on MEG3 to prove its oncogenic roles. Bioinformatic analyses were conducted on the TCGA database to build the MEG3-related score. The immune characteristics and mutation features of the MEG3-related score were explored., Results: We revealed a negative correlation between HGF and MEG3. In melanoma cells, HGF inhibited MEG3 expression by augmenting the methylation of the MEG3 promoter. Significantly, MEG3 exhibits a suppressive impact on the proliferation and migration of melanoma cells, concurrently inhibiting c-met expression. Moreover, a predictive model centered around MEG3 demonstrates notable efficacy in forecasting critical prognostic indicators, immunological profiles, and mutation statuses among melanoma patients., Conclusions: The present study highlights the potential of MEG3 as a pivotal regulator of c-met, establishing it as a promising candidate for targeted drug development in the ongoing pursuit of effective therapeutic interventions., (© 2023 John Wiley & Sons, Ltd.)

Background: BRAF inhibitors are approved in BRAF V600 -mutated metastatic melanoma, non-small-cell lung cancer (NSCLC), Erdheim-Chester disease (ECD), and thyroid cancer. We report here the efficacy, safety, and long-term results of single-agent vemurafenib given in the AcSé vemurafenib basket study to patients with various BRAF-mutated advanced tumours other than BRAF V600 -mutated melanoma and NSCLC., Patients and Methods: Patients with advanced tumours other than BRAF V600E melanoma and progressing after standard treatment were eligible for inclusion in nine cohorts (including a miscellaneous cohort) and received oral vemurafenib 960 mg two times daily. The primary endpoint was the objective response rate (ORR) estimated with a Bayesian design. The secondary outcomes were disease control rate, duration of response, progression-free survival (PFS), overall survival (OS), and vemurafenib safety., Results: A total of 98 advanced patients with various solid or haematological cancers, 88 with BRAF V600 mutations and 10 with BRAF nonV600 mutations, were included. The median follow-up duration was 47.7 months. The Bayesian estimate of ORR was 89.7% in hairy cell leukaemias (HCLs), 33.3% in the glioblastomas cohort, 18.2% in cholangiocarcinomas, 80.0% in ECD, 50.0% in ovarian cancers, 50.0% in xanthoastrocytomas, 66.7% in gangliogliomas, and 60.0% in sarcomas. The median PFS of the whole series was 8.8 months. The 12-, 24-, and 36-month PFS rates were 42.2%, 23.8%, and 17.9%, respectively. Overall, 54 patients died with a median OS of 25.9 months, with a projected 4-year OS of 40%. Adverse events were similar to those previously reported with vemurafenib., Conclusion: Responses and prolonged PFS were observed in many tumours with BRAF mutations, including HCL, ECD, ovarian carcinoma, gliomas, ganglioglioma, and sarcomas. Although not all cancer types responded, vemurafenib is an agnostic oncogene therapy of cancers., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The mutation of BRAF V600E often occurred in melanoma and results in tumorigenesis. BRAF mutation drives hyperactivation of the RAF-MAPK-ERK pathway. The acquired drug resistance upon prolonged use of BRAF inhibitors (such as vemurafenib) still remains the main obstacle. Previously, we have found that E3 ligase Skp2 over-expresses vemurafenib-resistant melanoma cells, and knockdown of Skp2 enhances the anti-tumor effect of vemurafenib. Interestingly, the literature has reported that the selective USP14/UCHL5 inhibitor b-AP15 displays great potential in melanoma therapy; however, the molecular mechanism still remains unknown., Methods: In vitro, the effect of the combination regimen of vemurafenib (Vem, PLX4032) and b-AP15 on vem-sensitive and vem-resistant melanoma has been investigated by wound healing, colony formation, transwell invasion assay, flow cytometry, lysosome staining, and ROS detection. In vivo, the combination effect on vem-resistant melanoma has been evaluated with a nude mice xenograft tumor model. GST-pulldown and co-immunoprecipitation (co-IP) assays have been applied to investigate the interactions between USP14, UCHL5, and Skp2. Cycloheximide (CHX) assay and ubiquitination assays have been used to explore the effect of USP14 on Skp2 protein half-life and ubiquitination status., Results: In the present study, we have revealed that repression of USP14 sensitizes vemurafenib resistance in melanoma through a previously unappreciated mechanism that USP14 but not UCHL5 stabilizes Skp2, blocking its ubiquitination. K119 on Skp2 is required for USP14-mediated deubiquitination and stabilization of Skp2. Furthermore, the mutated catalytic activity amino acid cysteine (C) 114 on USP14 abrogates stabilization of Skp2. Stabilization of Skp2 is required for USP14 to negatively regulate autophagy. The combination regimen of Skp2 inhibitor vemurafenib and USP14/UCHL5 inhibitor b-AP15 dramatically inhibits cell viability, migration, invasion, and colony formation in vemurafenib-sensitive and vemurafenib-resistant melanoma. Vemurafenib and b-AP15 hold cells in the S phase thus leading to apoptosis as well as the formation of the autophagic vacuole in vemurafenib-resistant SKMEL28 cells. The enhanced proliferation effect of USP14 and Skp2 is mainly due to a more effective reduction of cell apoptosis and autophagy. Further evaluation of various protein alterations has revealed that the increased expression of cleaved-PARP, LC3, and decreased Ki67 are more obvious in the combination of vemurafenib and b-AP15 treatment than those in single-drug treatment. Moreover, the co-treatment of vemurafenib and b-AP15 dramatically inhibits the growth of vemurafenib-resistant melanoma xenograft in vivo. Collectively, our findings have demonstrated that the combination of Skp2 inhibitor and USP14 inhibitor provides a new solution for the treatment of BRAF inhibitor resistance melanoma., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Over the last years, the incidence of melanoma, the deadliest form of skin cancer, has risen significantly. Nearly half of the melanoma patients exhibit the BRAF V600E mutation. Although the use of BRAF and MEK inhibitors (BRAFi and MEKi) showed an impressive success rate in melanoma patients, durability of response remains an issue because tumor quickly becomes resistant. Here, we generated and characterized Lu1205 and A375 melanoma cells resistant to vemurafenib (BRAFi). Resistant cells (Lu1205R and A375R) exhibit higher IC 50 (5-6 fold increase) and phospho-ERK levels and 2-3 times reduced apoptosis than their sensitive parents (Lu1205S and A375S). Moreover, resistant cells are 2-3 times bigger, display a more elongated morphology and have a modulation of migration capacity. Interestingly, pharmacological inhibition of sphingosine kinases, that prevents sphingosine-1-phosphate production, reduces migration of Lu1205R cells by 50 %. In addition, although Lu1205R cells showed increased basal levels of the autophagy markers LC3II and p62, they have decreased autophagosome degradation and autophagy flux. Remarkably, expression of Rab27A and Rab27B, which are involved in the release of extracellular vesicles are dramatically augmented in resistant cells (i.e. 5-7 fold increase). Indeed, conditioned media obtained from Lu1205R cells increased the resistance to vemurafenib of sensitive cells. Hence, these results support that resistance to vemurafenib modulates migration and the autophagic flux and may be transferred to nearby sensitive melanoma cells by factors that are released to the extracellular milieu by resistant cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

B-Raf kinase inhibitors such as vemurafenib (PLX4032) and dabrafenib have limited therapeutic efficacy on BRAF-mutated thyroid cancer. Cancer stem cells (CSCs) play important roles in tumor recurrence, drug resistance, and metastasis. Whether CSCs play a role in dampening the antitumor activity of B-Raf kinase inhibitors remains unknown. Here, we report that vemurafenib (PLX4032) induced the expression of several stemness-related genes including Gli1, Snail, BMI1, and SOX2 in two anaplastic thyroid cancer cell lines, SW1736 and 8505C, but decreased the expression of these genes in A375 cells, a human melanoma cell line. PLX4032 promoted thyroid cancer stem cell self-renewal, as evidenced by increased numbers of aldehyde dehydrogenase-positive cells and thyrospheres. Mechanistically, PLX4032 activates the PI-3 and mitogen-activated protein kinase pathways through HER3 to cross-activate Gli1, a transcription factor of the sonic hedgehog (Shh) pathway. GANT61, a specific inhibitor of Gli1, blocked the expression of the stemness-related genes in PLX4032-treated thyroid cancer cells in vitro and in vivo in two thyroid cancer xenograft models. GANT61 treatment alone weakly inhibited SW1736 tumor growth but enhanced the antitumor activity of PLX4032 when used in combination. Our study provides mechanistic insights into how thyroid cancer poorly responds to B-Raf kinase inhibitors and suggests that targeting B-Raf and the Shh pathway in combination may overcome thyroid cancer drug resistance.

Cutaneous melanoma is the deadliest skin cancer. Most have Ras-MAPK pathway (BRAF V600E or NRAS) mutations and highly effective targeted therapies exist; however, they and immune therapies are limited by resistance, in part driven by small GTPase (Rho and Rac) activation. To facilitate preclinical studies of combination therapies to provide durable responses, we describe the first mouse melanoma lines resistant to BRAF inhibitors. Treatment of mouse lines, YUMM1.7 and YUMMER, with vemurafenib (Vem), the BRAF V600E -selective inhibitor, resulted in high-level resistance (IC 50 shifts 20-30-fold). Resistant cells showed enhanced activation of Rho and the downstream transcriptional coactivator, myocardin-related transcription factor (MRTF). Resistant cells exhibited increased stress fibers, nuclear translocation of MRTF-A, and an increased MRTF-A gene signature. Pharmacological inhibition of the Rho/MRTF pathway using CCG-257081 reduced viability of resistant lines and enhanced sensitivity to Vem. Remarkably, co-treatment of parental lines with Vem and CCG-257081 eliminated resistant colony development. Resistant cells grew more slowly in vitro, but they developed highly aggressive tumors with a shortened survival of tumor-bearing mice. Increased expression of immune checkpoint inhibitor proteins (ICIs) in resistant lines may contribute to aggressive in vivo behavior. Here, we introduce the first drug-resistant mouse melanoma models for assessing combinations of targeted and immune therapies.

BRAF V600E mutation is one of the most therapeutic targets in thyroid cancers. However, its specific inhibitors have shown little clinical benefit because they can reactivate the MAPK/ERK and PI3K/AKT pathways by feedback upregulating the transcription of HER3. Peptidyl-prolyl cis/trans isomerase Pin1 has been proven to be closely associated with tumor progression. Here, we aimed to determine antitumor activity of Pin1 inhibitor API-1 in thyroid cancer and its effect on cellular response to BRAF inhibitors. The results showed that API-1 exhibited strong antitumor activity against thyroid cancer. Meanwhile, it improved the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4032 and there was a synergistic effect between them. Specially, a combination therapy of API-1 and PLX4032 significantly inhibited cell proliferation, colony formation, and the growth of xenograft tumors as well as induced cell apoptosis in BRAF-mutant thyroid cancer cells compared with API-1 or PLX4032 monotherapy. Similar results were also observed in transgenic mice with Braf V600E -driven thyroid cancer. Mechanistically, API-1 enhanced XPO5 ability to export pre-microRNA 20a (pre-miR-20a) from the nucleus to cytoplasm, thereby promoting the maturation of miR-20a-5p. Further studies showed that miR-20a-5p specifically targeted and down-regulated HER3, thereby blocking the reactivation of MAPK/ERK and PI3K/AKT signaling pathways caused by PLX4032. These results, taken together, demonstrate that Pin1 inhibitor API-1 significantly improves the sensitivity of BRAF-mutant thyroid cancer cells to PLX4032. Thus, this study not only determines the potential antitumor activity of Pin1 inhibitor API-1 in thyroid cancer but also offers an alternative therapeutic strategy for BRAF-mutant thyroid cancers by a combination of Pin1 inhibitor and BRAF kinase inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study aims to discern the possible molecular mechanism of the effect of ubiquitin-specific peptidase 18 (USP18) on the resistance to BRAF inhibitor vemurafenib in BRAF V600E mutant melanoma by regulating cyclic GMP-AMP synthase (cGAS). The cancer tissues of BRAF V600E mutant melanoma patients before and after vemurafenib treatment were collected, in which the protein expression of USP18 and cGAS was determined. A BRAF V600E mutant human melanoma cell line (A2058R) resistant to vemurafenib was constructed with its viability, apoptosis, and autophagy detected following overexpression and depletion assays of USP18 and cGAS. Xenografted tumors were transplanted into nude mice for in vivo validation. Bioinformatics analysis showed that the expression of cGAS was positively correlated with USP18 in melanoma, and USP18 was highly expressed in melanoma. The expression of cGAS and USP18 was up-regulated in cancer tissues of vemurafenib-resistant patients with BRAF V600E mutant melanoma. Knockdown of cGAS inhibited the resistance to vemurafenib in A2058R cells and the protective autophagy induced by vemurafenib in vitro. USP18 could deubiquitinate cGAS to promote its protein stability. In vivo experimentations confirmed that USP18 promoted vemurafenib-induced protective autophagy by stabilizing cGAS protein, which promoted resistance to vemurafenib in BRAF V600E mutant melanoma cells. Collectively, USP18 stabilizes cGAS protein expression through deubiquitination and induces autophagy of melanoma cells, thereby promoting the resistance to vemurafenib in BRAF V600E mutant melanoma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Introduction: Acquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation., Objectives: We identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study., Methods: DIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co‑immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays., Results: We found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently., Conclusion: This study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

BRAF V600 mutations are the most common oncogenic alterations in melanoma cells, supporting proliferation, invasion, metastasis and immune evasion. In patients, these aberrantly activated cellular pathways are inhibited by BRAFi whose potent antitumor effect and therapeutic potential are dampened by the development of resistance. Here, by using primary melanoma cell lines, generated from lymph node lesions of metastatic patients, we show that the combination of two FDA-approved drugs, the histone deacetylate inhibitor (HDCAi) romidepsin and the immunomodulatory agent IFN-α2b, reduces melanoma proliferation, long-term survival and invasiveness and overcomes acquired resistance to the BRAFi vemurafenib (VEM). Targeted resequencing revealed that each VEM-resistant melanoma cell line and the parental counterpart are characterized by a distinctive and similar genetic fingerprint, shaping the differential and specific antitumor modulation of MAPK/AKT pathways by combined drug treatment. By using RNA-sequencing and functional in vitro assays, we further report that romidepsin-IFN-α2b treatment restores epigenetically silenced immune signals, modulates MITF and AXL expression and induces both apoptosis and necroptosis in sensitive and VEM-resistant primary melanoma cells. Moreover, the immunogenic potential of drug-treated VEM-resistant melanoma cells results significantly enhanced, given the increased phagocytosis rate of these cells by dendritic cells, which in turn exhibit also a selective down-modulation of the immune checkpoint TIM-3. Overall, our results provide evidence that combined epigenetic-immune drugs can overcome VEM resistance of primary melanoma cells by oncogenic and immune pathways reprogramming, and pave the way for rapidly exploiting this combination to improve BRAFi-resistant metastatic melanoma treatment, also via reinforcement of immune checkpoint inhibitor therapy., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Necroptosis, a programmed cell death with necrotic-like morphology, has been recognized as an important driver in various inflammatory diseases. Inhibition of necroptosis has shown potential promise in the therapy of multiple human diseases. However, very few necroptosis inhibitors are available for clinical use as yet. Here, we identified an FDA-approved anti-cancer drug, Vemurafenib, as a potent inhibitor of necroptosis. Through direct binding, Vemurafenib blocked the kinase activity of receptor-interacting protein kinases 1 (RIPK1), impeded the downstream signaling and necrosome complex assembly, and inhibited necroptosis. Compared with Necrostain-1, Vemurafenib stabilized RIPK1 in an inactive DLG-out conformation by occupying a distinct allosteric hydrophobic pocket. Furthermore, pretreatment with Vemurafenib provided strong protection against necroptosis-associated diseases in vivo. Altogether, our results demonstrate that Vemurafenib is an effective RIPK1 antagonist and provide rationale and preclinical evidence for the potential application of approved drug in necroptosis-related diseases., (© 2023. The Author(s).)

Despite the successes of immunotherapy, melanoma remains one of the deadliest cancers, therefore, the need for innovation remains high. We previously reported anti-melanoma compounds that work by downregulating spliceosomal proteins hnRNPH1 and H2. In a separate study, we reported that these compounds were non-toxic to Balb/C mice at 50 mg/kg suggesting their utility in in vivo studies. In the present study, we aimed to assess the efficacy of these compounds by testing them in A375 cell-line xenograft in nude athymic mice. Animals were randomized into four groups (n = 12/group): 10 mg/kg vemurafenib, and 25 mg/kg 2155-14 and 2155-18 thrice a week for 15 days along with a control group. The results revealed that both 2155-14 and 2155-18 significantly decreased the growth of A375 tumors, which was comparable to vemurafenib. These results were confirmed by tumor volume, weight, and histopathological examination. In conclusion, these results demonstrate the therapeutic potential of targeting spliceosomal proteins hnRNPH1 and H2.

Despite the advancements in targeted therapy for BRAFV600E-mutated metastatic colorectal cancer (mCRC), the development of resistance to BRAFV600E inhibition limits the response rate and durability of the treatment. Better understanding of the resistance mechanisms to BRAF inhibitors will facilitate the design of novel pharmacological strategies for BRAF-mutated mCRC. The aim of this study was to identify novel protein candidates involved in acquired resistance to BRAFV600E inhibitor vemurafenib in BRAFV600E-mutated colon cancer cells using an integrated proteomics approach. Bioinformatic analysis of obtained proteomics data indicated actin-cytoskeleton linker protein ezrin as a highly ranked protein significantly associated with vemurafenib resistance whose overexpression in the resistant cells was additionally confirmed at the gene and protein level. Ezrin inhibition by NSC305787 increased anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant cells in an additive manner, which was accompanied by downregulation of CD44 expression and inhibition of AKT/c-Myc activities. We also detected an increased ezrin expression in vemurafenib-resistant melanoma cells harbouring the BRAFV600E mutation. Importantly, ezrin inhibition potentiated anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant melanoma cells in a synergistic manner. Altogether, our study suggests a role of ezrin in acquired resistance to vemurafenib in colon cancer and melanoma cells carrying the BRAFV600E mutation and supports further pre-clinical and clinical studies to explore the benefits of combined BRAF inhibitors and actin-targeting drugs as a potential therapeutic approach for BRAFV600E-mutated cancers.

Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAF V600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect was related to a cellular phosphatidylinositol 4-kinase type IIIβ (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevented infection efficiently in acute cell models, eradicated infection in a chronic cell model, and lowered virus amounts in pancreas and heart in an acute mouse model. Altogether, instead of acting through the RAF/MEK/ERK pathway, vemurafenib affects the cellular PI4KB and, hence, enterovirus replication, opening new possibilities to evaluate further the potential of vemurafenib as a repurposed drug in clinical care. IMPORTANCE Despite the prevalence and medical threat of enteroviruses, presently, there are no antivirals against them. Here, we show that vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAF V600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIβ (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses., Competing Interests: The authors declare a conflict of interest. H.Hy is a minor shareholder and member of the board of Vactech Ltd, which develops vaccines against picornaviruses. No other potential conflicts of interest relevant to this article were reported.

BRAF represents one of the most frequently mutated protein kinase genes and BRAF V600E mutation may be found in many types of cancer, including hairy cell leukemia (HCL), anaplastic thyroid cancer (ATC), colorectal cancer and melanoma. Herein, a fluorescent probe, based on the structure of the highly specific BRAF V600E inhibitor Vemurafenib (Vem, 1) and featuring the NIR fluorophore cyanine-5 (Cy5), was straightforwardly synthesized and characterized (Vem-L-Cy5, 3), showing promising spectroscopic properties. Biological validation in BRAF V600E -mutated cancer cells evidenced the ability of 3 to penetrate inside the cells, specifically binding to its elective target BRAF V600E with high affinity, and inhibiting MEK phosphorylation and cell growth with a potency comparable to that of native Vem 1. Taken together, these data highlight Vem-L-Cy5 3 as a useful tool to probe BRAF V600E mutation in cancer cells, and suitable to acquire precious insights for future developments of more informed BRAF inhibitors-centered therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Objectives: More and more evidences show that circular RNAs (circRNAs) can be used as miRNA sponge to regulate the drug resistance of malignancies, including melanoma. However, how exosomal circRNAs participate in the therapeutic resistance of melanoma remains ambiguous., Methods: Vemurafenib-resistant A375 cells were cultured and then the circRNA profile of exosomes from the parental A375 and A375-resistant cells were sequenced. Transmission electron microscopy (TEM), exogenous nanoparticle tracking analysis (NTA) and Western Blot assays were leveraged to confirm the successful collection of exosomes from A375 and A375R cells. Another five published RNA-seq data and microRNA-seq data, and seven miRNA databases were collected to construct a competing endogenous RNA (ceRNA) network. Comprehensive bioinformatic analysis was adopted to identify key molecules related to the drug resistance, including multiscale embedded gene co-expression network analysis (MEGENA). Then, qRT-PCR, cell viability and colony formation were used to estimate the function of hub circRNAs. The role of has_circ_0001005 in vivo was verified via xenograft assay. The Tumor online Prognostic analyses Platform (ToPP) was leveraged to develop the has_circ_0001005-related prognostic models for melanoma patients based on TCGA data., Results: Compared with parental cells, hsa_circ_0001005 expression was significantly increased in resistant cells and their exosomes. The elevated level of hsa_circ_0001005 was related to the poor clinical prognosis of melanoma patients. Hsa_circ_0001005 found in melanoma was mainly secreted by drug-resistant cells as exosomes. Exosomal hsa_circ_0001005 activated multiple canonical pathways related to drug resistance through sponging four miRNAs, thus suppressing the drug sensitivity of melanoma. Knocking down hsa_circ_0001005 in vitro, we found that the inhibition of hsa_circ_0001005 could hinder the clone formation of melanoma. Further in vivo animal experiments suggested that suppression of hsa_circ_0001005 can increase the sensitivity to Vemurafenib of melanoma cells. Finally, we also constructed the functional regulatory ceRNA network and prognostic risk models for hsa_circ_0001005, and further survival analysis reveals that the regulatory network and prognostic risk models obviously affected the prognosis of melanoma patients., Conclusions: This study confirmed that the level of hsa_circ_0001005 in exosomes is the key factor affecting drug resistance of melanoma, which provides a new potential therapeutic target for melanoma patients., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Malignant melanoma is challenging to treat, and metastatic cases need chemotherapy strategies. Targeted inhibition of commonly mutant BRAF V600E by inhibitors is efficient but eventually leads to resistance and progression in the vast majority of cases. Numerous studies investigated the mechanisms of resistance in melanoma cell lines, and an increasing number of in vivo or clinical data are accumulating. In most cases, bypassing BRAF and resulting reactivation of the MAPK signaling, as well as alternative PI3K-AKT signaling activation are reported. However, several unique changes were also shown. We developed and used a patient-derived tumor xenograft (PDTX) model to screen resistance evolution in mice in vivo, maintaining tumor heterogeneity. Our results showed no substantial activation of the canonical pathways; however, RNAseq and qPCR data revealed several altered genes, such as GPR39 , CD27 , SLC15A3 , IFI27 , PDGFA , and ABCB1 . Surprisingly, p53 activity, leading to apoptotic cell death, was unchanged. The found biomarkers can confer resistance in a subset of melanoma patients via immune modulation, microenvironment changes, or drug elimination. Our resistance model can be further used in testing specific inhibitors that could be used in future drug development, and combination therapy testing that can overcome inhibitor resistance in melanoma.

BRAF inhibitors have improved the treatment of advanced or metastatic melanoma in patients that harbor a BRAF T1799A mutation. Because of new insights into the role of aberrant glycosylation in drug resistance, we designed and studied three novel vemurafenib derivatives possessing pentose-associated aliphatic ligands-methyl-, ethyl-, and isopropyl-ketopentose moieties-as potent BRAF V600E kinase inhibitors. The geometries of these derivatives were optimized using the density functional theory method. Molecular dynamic simulations were performed to find interactions between the ligands and BRAF V600E kinase. Virtual screening was performed to assess the fate of derivatives and their systemic toxicity, genotoxicity, and carcinogenicity. The computational mapping of the studied ligand-BRAF V600E complexes indicated that the central pyrrole and pyridine rings of derivatives were located within the hydrophobic ATP-binding site of the BRAF V600E protein kinase, while the pentose ring and alkyl chains were mainly included in hydrogen bonding interactions. The isopropyl-ketopentose derivative was found to bind the BRAF V600E oncoprotein with more favorable energy interaction than vemurafenib. ADME-TOX in silico studies showed that the derivatives possessed some desirable pharmacokinetic and toxicologic properties. The present results open a new avenue to study the carbohydrate derivatives of vemurafenib as potent BRAF V600E kinase inhibitors to treat melanoma.