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Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery., (© 2022. The Author(s).)

More than half of metastatic melanoma patients receiving standard therapy fail to achieve a long-term survival due to primary and/or acquired resistance. Tumor cell ability to switch from epithelial to a more aggressive mesenchymal phenotype, attributed with AXL high molecular profile in melanoma, has been recently linked to such event, limiting treatment efficacy. In the current study, we investigated the therapeutic potential of the AXL inhibitor (AXLi) BGB324 alone or in combination with the clinically relevant BRAF inhibitor (BRAFi) vemurafenib. Firstly, AXL was shown to be expressed in majority of melanoma lymph node metastases. When treated ex vivo, the largest reduction in cell viability was observed when the two drugs were combined. In addition, a therapeutic benefit of adding AXLi to the BRAF-targeted therapy was observed in pre-clinical AXL high melanoma models in vitro and in vivo. When searching for mechanistic insights, AXLi was found to potentiate BRAFi-induced apoptosis, stimulate ferroptosis and inhibit autophagy. Altogether, our findings propose AXLi as a promising treatment in combination with standard therapy to improve therapeutic outcome in metastatic melanoma., (© 2022. The Author(s).)

We identified fermitin family member 2 (FERMT2, also known as kindlin-2) as a potential target in A375 cell line by siRNA library screening. Drugs that target mutant BRAF kinase lack durable efficacy in the treatment of melanoma because of acquired resistance, thus the identification of novel therapeutic targets is needed. Immunohistochemistry was used to identify kindlin-2 expression in melanoma samples. The interaction between kindlin-2 and Rac1 or p-Rac/Cdc42 guanine nucleotide exchange factor 6 (α-Pix) was investigated. Finally, the tumor suppressive role of kindlin-2 was validated in vitro and in vivo. Analysis of clinical samples and Oncomine data showed that higher levels of kindlin-2 predicted a more advanced T stage and M stage and facilitated metastasis and recurrence. Kindlin-2 knockdown significantly inhibited melanoma growth and migration, whereas kindlin-2 overexpression had the inverse effects. Further study showed that kindlin-2 could specifically bind to p-α-Pix(S13) and Rac1 to induce a switch from the inactive Rac1-GDP conformation to the active Rac1-GTP conformation and then stimulate the downstream MAPK pathway. Moreover, we revealed that a Rac1 inhibitor suppressed melanoma growth and metastasis and the combination of the Rac1 inhibitor and vemurafenib resulted in a better therapeutic outcome than monotherapy in melanoma with high kindlin-2 expression and BRAF mutation. Our results demonstrated that kindlin-2 promoted melanoma progression, which was attributed to specific binding to p-α-Pix(S13) and Rac1 to stimulate the downstream MAPK pathway. Thus, kindlin-2 could be a potential therapeutic target for treating melanoma., (© 2021. The Author(s).)

Cancer cells within tumors display a high degree of phenotypic variability. This variability is thought to allow some of the cells to survive and persist after seemingly effective drug treatments. Studies on vemurafenib, a signaling inhibitor that targets an oncogenic BRAF mutation common in melanoma, suggested that cell-to-cell variation in drug resistance, measured by long-term proliferation, originates from epigenetic differences in gene expression that pre-exist treatment. However, it is still unknown whether reactivation of signaling downstream to the inhibited BRAF, thought to be a key step for resistance, is heterogeneous across cells. While previous studies established that signaling reactivation takes place many hours to days after treatment, they monitored reactivation with bulk-population assays unsuitable for detecting cell-to-cell heterogeneity. We hypothesized that signaling reactivation is heterogeneous and is almost instantaneous for a small subpopulation of resistant cells. We tested this hypothesis by monitoring signaling dynamics at a single-cell resolution and observed that despite highly uniform initial inhibition, roughly 15% of cells reactivated signaling within an hour of treatment. Moreover, by tracking cell lineages over multiple days, we established that these cells indeed proliferated more than neighboring cells, thus establishing that rapid signaling reactivation predicts long-term vemurafenib resistance., (© 2021. The Author(s).)

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a member of the TIPE/TNFAIP8 family which regulates tumor growth and survival. Our goal is to delineate the detailed oncogenic role of TNFAIP8 in skin cancer development and progression. Here we demonstrated that higher expression of TNFAIP8 is associated with basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma development in patient tissues. Induction of TNFAIP8 expression by TNFα or by ectopic expression of TNFAIP8 in SCC or melanoma cell lines resulted in increased cell growth/proliferation. Conversely, silencing of TNFAIP8 decreased cell survival/cell migration in skin cancer cells. We also showed that miR-205-5p targets the 3'UTR of TNFAIP8 and inhibits TNFAIP8 expression. Moreover, miR-205-5p downregulates TNFAIP8 mediated cellular autophagy, increased sensitivity towards the B-RAF V600E mutant kinase inhibitor vemurafenib, and induced cell apoptosis in melanoma cells. Collectively our data indicate that miR-205-5p acts as a tumor suppressor in skin cancer by targeting TNFAIP8.

Identification of mechanisms underlying sensitivity and response to targeted therapies, such as the BRAF inhibitor vemurafenib, is critical in order to improve efficacy of these therapies in the clinic and delay onset of resistance. Glycolysis has emerged as a key feature of the BRAF inhibitor response in melanoma cells, and importantly, the metabolic response to vemurafenib in melanoma patients can predict patient outcome. Here, we present a multiparameter genome-wide siRNA screening dataset of genes that when depleted improve the viability and glycolytic response to vemurafenib in BRAF V600 mutated melanoma cells. These datasets are suitable for analysis of genes involved in cell viability and glycolysis in steady state conditions and following treatment with vemurafenib, as well as computational approaches to identify gene regulatory networks that mediate response to BRAF inhibition in melanoma.

Cutaneous malignant melanoma (CMM) is the deadliest form of skin cancer and clinically challenging due to its propensity to develop therapy resistance. Reactive oxygen species (ROS) can induce DNA damage and play a significant role in CMM. MTH1 protein protects from ROS damage and is often overexpressed in different cancer types including CMM. Herein, we report that MTH1 inhibitor TH1579 induced ROS levels, increased DNA damage responses, caused mitotic arrest and suppressed CMM proliferation leading to cell death both in vitro and in an in vivo xenograft CMM zebrafish disease model. TH1579 was more potent in abrogating cell proliferation and inducing cell death in a heterogeneous co-culture setting when compared with CMM standard treatments, vemurafenib or trametinib, showing its broad anticancer activity. Silencing MTH1 alone exhibited similar cytotoxic effects with concomitant induction of mitotic arrest and ROS induction culminating in cell death in most CMM cell lines tested, further emphasizing the importance of MTH1 in CMM cells. Furthermore, overexpression of receptor tyrosine kinase AXL, previously demonstrated to contribute to BRAF inhibitor resistance, sensitized BRAF mutant and BRAF/NRAS wildtype CMM cells to TH1579. AXL overexpression culminated in increased ROS levels in CMM cells. Moreover, silencing of a protein that has shown opposing effects on cell proliferation, CAV-1, decreased sensitivity to TH1579 in a BRAF inhibitor resistant cell line. AXL-MTH1 and CAV-1-MTH1 mRNA expressions were correlated as seen in CMM clinical samples. Finally, TH1579 in combination with BRAF inhibitor exhibited a more potent cell killing effect in BRAF mutant cells both in vitro and in vivo. In summary, we show that TH1579-mediated efficacy is independent of BRAF/NRAS mutational status but dependent on the expression of AXL and CAV-1.

Emerging research suggests that multiple tumor compartments can influence treatment responsiveness and relapse, yet the search for therapeutic resistance mechanisms remains largely focused on acquired genomic alterations in cancer cells. Here we show how treatment-induced changes occur in multiple tumor compartments during tumor relapse and can reduce benefit of follow-on therapies. By using serial biopsies, next-generation sequencing, and single-cell transcriptomics, we tracked the evolution of multiple cellular compartments within individual lesions during first-line treatment response, relapse, and second-line therapeutic interventions in an autochthonous model of melanoma. We discovered that although treatment-relapsed tumors remained genetically stable, they converged on a shared resistance phenotype characterized by dramatic changes in tumor cell differentiation state, immune infiltration, and extracellular matrix (ECM) composition. Similar alterations in tumor cell differentiation were also observed in more than half of our treatment-relapsed patient tumors. Tumor cell-state changes were coincident with ECM remodeling and increased tumor stiffness, which alone was sufficient to alter tumor cell fate and reduce treatment responses in melanoma cell lines in vitro. Despite the absence of acquired mutations in the targeted pathway, resistant tumors showed significantly decreased responsiveness to second-line therapy intervention within the same pathway. The ability to preclinically model relapse and refractory settings-while capturing dynamics within and crosstalk between all relevant tumor compartments-provides a unique opportunity to better design and sequence appropriate clinical interventions.

Although the utilization of selective BRAF V600E inhibitors is associated with improved overall survival in patients with metastatic melanoma, a growing challenge of drug resistance has  emerged. CDC7 has been shown to be overexpressed and associated with poor prognosis in various cancers including melanoma. Thus, we aimed to elucidate the biological role of CDC7 in promoting Vemurafenib resistance and the anticipated benefits of dual targeting of BRAF V600E and CDC7 in melanoma cells. We performed exosomes-associated microRNA profiling and functional assays to determine the role of CDC7 in drug resistance using Vemurafenib-sensitive and resistant melanoma cells. Our results demonstrated that Vemurafenib-resistant cells exhibited a persistent expression of CDC7 in addition to prolonged activity of MCM2 compared to drug-sensitive cells. Reconstitution of miR-3613-3p in resistant cells downregulated CDC7 expression and reduced the number of colonies. Treatment of cells with low concentrations of CDC7 inhibitor TAK-931 sensitized resistant cells to Vemurafenib and reduced the number of cell colonies. Taken together, CDC7 overexpression and downregulation of miR-3613-3p were associated with Vemurafenib resistance in BRAF V600E - bearing melanoma cells. Dual targeting of CDC7 and BRAF V600E reduced the development of resistance against Vemurafenib. Further studies are warranted to investigate the clinical effect of targeting CDC7 in metastatic melanoma.

de novo fatty acid biosynthesis (DNFA) is a hallmark adaptation of many cancers that supports survival, proliferation, and metastasis. Here we elucidate previously unexplored aspects of transcription regulation and clinical relevance of DNFA in cancers. We show that elevated expression of DNFA genes is characteristic of many tumor types and correlates with poor prognosis, especially in melanomas. Elevated DNFA gene expression depends on the SREBP1 transcription factor in multiple melanoma cell lines. SREBP1 predominantly binds to the transcription start sites of DNFA genes, regulating their expression by recruiting RNA polymerase II to promoters for productive transcription elongation. We find that SREBP1-regulated DNFA represents a survival trait in melanoma cells, regardless of proliferative state and oncogenic mutation status. Indeed, malignant melanoma cells exhibit elevated DNFA gene expression after the BRAF/MEK signaling pathway is blocked (e.g. by BRAF inhibitors), and DNFA expression remains higher in melanoma cells resistant to vemurafenib treatment than in untreated cells. Accordingly, DNFA pathway inhibition, whether by direct targeting of SREBP1 with antisense oligonucleotides, or through combinatorial effects of multiple DNFA enzyme inhibitors, exerts potent cytotoxic effects on both BRAFi-sensitive and -resistant melanoma cells. Altogether, these results implicate SREBP1 and DNFA enzymes as enticing therapeutic targets in melanomas.

Drug resistance imposes severe limitations to the efficacy of targeted therapy in BRAF-mutated metastatic melanoma. Although this issue has been mitigated by the development of combination therapies with BRAF plus MEK inhibitors, drug resistance inevitably occurs with time and results in clinical recurrences and untreatable disease. Hence, there is strong need of developing new combination therapies and non-invasive diagnostics for the early identification of drug-resistant patients. We report here that the development of drug resistance to BRAFi is dominated by a dynamic deregulation of a large population of miRNAs, leading to the alteration of cell intrinsic proliferation and survival pathways, as well as of proinflammatory and proangiogenic cues, where a prominent role is played by the miR-199b-5p/VEGF axis. Significant alterations of miRNA expression levels are detectable in tumor biopsies and plasma from patients after disease recurrence. Targeting these alterations blunts the development of drug resistance.

The BRAF inhibitors dabrafenib and vemurafenib induce remarkable clinical responses in patients with BRAF-mutated melanomas. However, adverse events, including the emergence of secondary tumors and drug resistance, have been reported. Studies have revealed that undesirable RAF dimerization induced by inhibitors promotes these adverse effects. Here, we developed highly sensitive biosensors of RAF dimerization in cells utilizing the split enhanced click beetle luciferase (Emerald Luc, ELuc) complementation technique. We demonstrated that our biosensor system works effectively for high-throughput screens in the microplate format. A comprehensive analysis of commercially available RAF inhibitors performed using this assay system revealed that the inhibitors exhibit various potencies in inducing the dimerization of RAF isoforms, and their dimerization potencies do not always correlate with the RAF enzyme inhibition. This sensitive assay system will become a powerful tool to discover next-generation BRAF inhibitors with safer profiles.

The maximum response and 10-year survival rate for metastatic melanoma patients treated with standardised chemotherapy is still less than 15% and 10%, respectively. In contrast, oncogene targeting was found a promising tool for killing of BRAF V600 mutated melanoma cells. Nevertheless, despite improved response and survival rates, resistance acquisition remains an ongoing problem. In this context, the impact of chronic BRAF inhibition on the efficacy of commonly applied cytostatics is still unknown. In our study, human melanoma cells with BRAF V600E mutation were treated with chemotherapeutics and a BRAF inhibitor. Resistance patterns were analysed by microelectrode array-based impedance spectroscopy, XTT and flow cytometric apoptosis/proliferation assay. BRAF V600E melanoma cells acquired a time- and concentration-dependent desensitisation up to 100-fold towards oncogene-specific PLX4032 and chemotherapeutic dacarbazine after twelve months treatment. The impact of multiple drug insensitivity on molecular melanoma characteristics was elaborated via mRNA and protein quantification. Following BRAF V600E targeting, melanoma cells developed an increasingly aggressive, dacarbazine-insensitive phenotype. Thereby, hyperactivated canonical alternative MAPK and bypass PI3K/AKT signalling caused cross-resistance of differently acting drugs. With these results, we are the first to show that long-term melanoma therapy with BRAF inhibitors can prevent further therapeutic success with dacarbazine due to acquisition of cross-resistance.

While being the rarest skin cancer, melanoma is also the deadliest. To further drug discovery and improve clinical translation, new human cell-based in vitro models are needed. Our work strives to mimic the melanoma microenvironment in vitro as an alternative to animal testing. We used the self-assembly method to produce a 3D human melanoma model exempt of exogenous biomaterial. This model is based on primary human skin cells and melanoma cell lines while including a key feature for tumor progression: blood and lymphatic capillaries. Major components of the tumor microenvironment such as capillaries, human extracellular matrix, a stratified epidermis (involucrin, filaggrin) and basement membrane (laminin 332) are recapitulated in vitro. We demonstrate the persistence of CD31 + blood and podoplanin + /LYVE-1 + lymphatic capillaries in the engineered tissue. Chronic treatment with vemurafenib was applied to the model and elicited a dose-dependent response on proliferation and apoptosis, making it a promising tool to test new compounds in a human-like environment.

While advances in laboratory automation has dramatically increased throughout of compound screening efforts, development of robust cell-based assays in relevant disease models remain resource-intensive and time-consuming, presenting a bottleneck to drug discovery campaigns. To address this issue, we present a modified gene trap approach to efficiently generate pathway-specific reporters that result in a robust "on" signal when the pathway of interest is inhibited. In this proof-of-concept study, we used vemurafenib and trametinib to identify traps that specifically detect inhibition of the mitogen-activated protein kinase (MAPK) pathway in a model of BRAFV600E driven human malignant melanoma. We demonstrate that insertion of our trap into particular loci results in remarkably specific detection of MAPK pathway inhibitors over compounds targeting any other pathway or cellular function. The accuracy of our approach was highlighted in a pilot screen of ~6000 compounds where 40 actives were detected, including 18 MEK, 10 RAF, and 3 ERK inhibitors along with a few compounds representing previously under-characterized inhibitors of the MAPK pathway. One such compound, bafetinib, a second generation BCR/ABL inhibitor, reduced phosphorylation of ERK and when combined with trametinib, both in vitro and in vivo, reduced growth of vemurafenib resistant melanoma cells. While piloted in a model of BRAF-driven melanoma, our results set the stage for using this approach to rapidly generate reporters against any transcriptionally active pathway across a wide variety of disease-relevant cell-based models to expedite drug discovery efforts.

Multiple genetic mutations within melanoma not only cause lesion-specific responses to targeted therapy but also alter the molecular route of resistance to that therapy. Inactivation of PTEN occurs in up to 30% of melanomas, frequently with a concurrent activating BRAF mutation. PTEN loss regulates both acquired and intrinsic drug resistance. Here we show that AXL/AKT axis mediated-resistance to BRAF inhibitor (BRAFi) depends upon PTEN status in melanoma. Hyperactivation of both ERK and AKT pathways was associated with BRAFi resistance in melanoma with wildtype PTEN. The PTEN-impaired melanoma cells required only the ERK resistance mechanism. Moreover, we identified AXL as a key upstream effector of AKT pathway-associated resistance to BRAFi in melanoma with wildtype PTEN, but not in melanoma with impaired PTEN. Notably, we confirmed that blocking AXL by shRNA and a small molecular inhibitor could rescue the sensitivity of resistant melanoma cells with wildtype PTEN to BRAFi and inhibit their growth in vitro and in vivo. Our study has uncovered a mechanism by which PTEN status contributes to acquired resistance to BRAFi and offers a rational strategy to guide clinical testing in pre-identified subsets of patients who relapse during treatment with BRAFi. The identified protein AXL represents a promising therapeutic target for BRAF mutant melanoma patients with wildtype PTEN.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation 2,3 . Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U 34 ) tRNA (U 34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U 34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U 34 enzymes. Mechanistically, U 34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U 34 enzymes and HIF1α. Together, these results demonstrate that U 34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.

BRAF mutations occur in ~10% of colorectal cancer (CRC) and are associated with poor prognosis. Inhibitors selective for the BRAF V600E oncoprotein, the most common BRAF mutant, elicit only poor response rates in BRAF-mutant CRC as single agents. This unresponsiveness was mechanistically attributed to the loss of negative feedbacks on the epidermal growth factor receptor (EGFR) and initiated clinical trials that combine BRAF (and MEK) inhibitors, either singly or in combination, with the anti-EGFR antibodies cetuximab or panitumumab. First results of these combinatorial studies demonstrated improved efficacy, however, the response rates still were heterogeneous. Here, we show that BRAF inhibition leads to the upregulation of a variety of receptor tyrosine kinases (RTKs) in CRC cell lines, including not only the EGFR, but also human epidermal growth factor receptor (HER) 2 and HER3. Importantly, combination of the BRAF inhibitors (BRAFi) vemurafenib (PLX4032), dabrafenib, or encorafenib with inhibitors dually targeting the EGFR and HER2 (such as lapatinib, canertinib, and afatinib) significantly reduced the metabolic activity and proliferative potential of CRC cells. This re-sensitization was also observed after genetic depletion of HER2 or HER3. Interestingly, BRAF inhibitors did not only upregulate RTKs, but also increased the abundance of the GRB2-associated binders (Gab) 1 and Gab2, two important amplifiers of RTK signaling. An allele-specific shRNA-mediated knockdown of BRAF V600E revealed that Gab2 upregulation was directly dependent on the loss of the oncoprotein and was not caused by an "off-target" effect of these kinase inhibitors. Furthermore, Gab2 and Gab2-mediated Shp2 signaling were shown to be functionally important in BRAFi resistance. These findings highlight potential new escape mechanisms to these targeted therapies and indicate that a broad suppression of RTK signaling might be beneficial and should be taken into account in future research addressing targeted therapy in BRAF-mutant CRC.

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.