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Because BRAF-mutated melanomas are addicted to the Mitogen Activated Protein Kinase (MAPK) pathway they show a high response rate to BRAF and MEK inhibitors. However, the clinical responses to these inhibitors are often short-lived with the rapid onset of resistance to treatment. Deciphering the molecular mechanisms driving resistance has been the subject of intense research. Recent in vitro and clinical data have suggested a link between expression of telomerase and resistance to targeted therapy in melanoma. TERT promoter mutations are the main mechanism for the continuous upregulation of telomerase in melanoma and co-occur frequently with BRAF alterations. To understand how TERT promoter mutations could be associated with resistance to targeted therapy in melanoma, we conducted translational and in vitro studies. In a cohort of V600E-BRAF-mutated melanoma patients, we showed that the TERT promoter mutation status and TERT expression tended to be associated with response to BRAF and MEK inhibitors. We demonstrated that TERT overexpression in BRAF-mutated melanoma cells reduced sensitivity to BRAF and MEK independently of TERT’s telomer maintenance activity. Interestingly, inhibition of TERT reduced growth of BRAF-mutated melanoma including resistant cells. TERT expression in melanoma can therefore be a new biomarker for resistance to MAPK inhibitors as well as a novel therapeutic target.

Tables S1, S2, S3, S4

Figure S2. (A) Change in the tumor burden from baseline over time according to RECIST for all the included patients. The tumor burden was measured as the sum of the longest diameters of target lesions. Each line represents a patient. (B) Kaplan-Meier plot of the overall survival for all the included patients (N =18). (C) Kaplan-Meier plot of the progression-free survival for all the included patients (N =18).

Purpose:In BRAFV600MUT metastatic melanoma, cyclin D–CDK4/6–INK4–Rb pathway alterations are involved in resistance to MAPK inhibitors, suggesting a clinical benefit of cyclin-dependent kinase 4 (CDK4) inhibitors. In this phase I–II study, we aimed to establish the MTD of palbociclib when added to vemurafenib.Patients and Methods:Patients with BRAFV600E/KMUT metastatic melanoma harboring CDKN2A loss and RB1 expression were included and stratified into two groups according to previous BRAF inhibitor treatment (no:strata 1; yes:strata 2). Treatment comprised palbociclib once daily for 14 days followed by a 7-day break + continuous dosing of vemurafenib. The primary endpoint was the occurrence of dose-limiting toxicity (DLT), and the secondary endpoints included the best response, survival, pharmacokinetics, and tumor molecular profiling.Results:Eighteen patients were enrolled, with 15 in strata 2. Characteristics at inclusion were American Joint Committee on Cancer stage IVM1c (N = 16; 88.9%), high lactate dehydrogenase (N = 9; 50.0%), and median number of previous treatments of 2. One and 5 patients experienced DLT in strata 1 and 2, respectively, defining the MTD at palbociclib 25 mg and vemurafenib 960 mg in strata 2. No significant evidence for drug–drug interactions was highlighted. The median progression-free survival was 2.8 months, and 5 (27.8%) patients showed a clinical response. The baseline differential mRNA expression analysis and in vitro data revealed the role of CHEK2 in the response to palbociclib.Conclusions:Although the combination of palbociclib + fixed-dose vemurafenib did not allow an increased palbociclib dosage above 25 mg, a significant clinical benefit was achieved in pretreated patients with melanoma. An association between the transcriptomic data and clinical response was highlighted.

Supplementary Materials and Methods

Purpose: In BRAFV600MUT metastatic melanoma, cyclin D–CDK4/6–INK4–Rb pathway alterations are involved in resistance to MAPK inhibitors, suggesting a clinical benefit of cyclin-dependent kinase 4 (CDK4) inhibitors. In this phase I–II study, we aimed to establish the MTD of palbociclib when added to vemurafenib. Patients and Methods: Patients with BRAFV600E/KMUT metastatic melanoma harboring CDKN2A loss and RB1 expression were included and stratified into two groups according to previous BRAF inhibitor treatment (no:strata 1; yes:strata 2). Treatment comprised palbociclib once daily for 14 days followed by a 7-day break + continuous dosing of vemurafenib. The primary endpoint was the occurrence of dose-limiting toxicity (DLT), and the secondary endpoints included the best response, survival, pharmacokinetics, and tumor molecular profiling. Results: Eighteen patients were enrolled, with 15 in strata 2. Characteristics at inclusion were American Joint Committee on Cancer stage IVM1c (N = 16; 88.9%), high lactate dehydrogenase (N = 9; 50.0%), and median number of previous treatments of 2. One and 5 patients experienced DLT in strata 1 and 2, respectively, defining the MTD at palbociclib 25 mg and vemurafenib 960 mg in strata 2. No significant evidence for drug–drug interactions was highlighted. The median progression-free survival was 2.8 months, and 5 (27.8%) patients showed a clinical response. The baseline differential mRNA expression analysis and in vitro data revealed the role of CHEK2 in the response to palbociclib. Conclusions: Although the combination of palbociclib + fixed-dose vemurafenib did not allow an increased palbociclib dosage above 25 mg, a significant clinical benefit was achieved in pretreated patients with melanoma. An association between the transcriptomic data and clinical response was highlighted.

Purpose of review Although targeted therapy provides a high response rate and rapid disease control in advanced melanoma, most patients experience disease progression due to acquired resistance mechanisms leading to reactivation of mitogen-activated protein kinase pathway. The purpose of this article is to review the recently published data on the impact of an intermittent versus continuous dosing schedule of BRAF and MEK inhibition in advanced melanoma to determine the best approach in clinical practice. Recent findings Some preclinical studies have highlighted the concept that drug-resistant cells may also display drug dependency, such that intermittent dosing of targeted therapy may prevent the emergence of lethal drug resistance. Moreover, clinical observations have suggested that repeated treatment after a break or an intervening therapy may provide clinical benefit. However, recent preclinical and clinical studies have also failed to demonstrate an advantage of intermittent dosing and showed a similar efficacy of the intermittent versus continuous regimens of BRAF and MEK inhibitors in mice models and phase 2 clinical trial. Summary Owing to these discordant results, continuous dosing of BRAF and MEK inhibitors remains the optimal therapeutic approach until additional clinical data demonstrate the superiority of another combination or dosing regimen.

More than 70% of human NRAS melanomas are resistant to MEK inhibitors highlighting the crucial need for efficient therapeutic strategies for these tumors. CD147, a membrane receptor, is overexpressed in most cancers including melanoma and is associated with poor prognosis. We show here that CD147i, a specific inhibitor of CD147/VEGFR-2 interaction represents a potential therapeutic strategy for NRAS melanoma cells. It significantly inhibited the malignant properties of NRAS melanomas ex vivo and in vivo. Importantly, NRAS patient’s-derived xenografts, which were resistant to MEKi, became sensitive when combined with CD147i leading to decreased proliferation ex vivo and tumor regression in vivo. Mechanistic studies revealed that CD147i effects were mediated through STAT3 pathway. These data bring a proof of concept on the impact of the inhibition of CD147/VEGFR-2 interaction on melanoma progression and represents a new therapeutic opportunity for NRAS melanoma when combined with MEKi., This work was supported by La Ligue Nationale contre le Cancer, the Institut National de la Santé et de la Recherche Médicale (INSERM) and La Société Française de Dermatologie. AL was supported by a PhD funding from La Ligue Nationale contre le Cancer and the Spanish Ministry of Science and Innovation (PID2019-110167RB-I00). We thank technological platform of the Institut Recherche Saint-Louis (IRSL) for confocal microscopy analyses and Dr Benoit Souquet for technical support.

Simple Summary Melanoma is one of the most aggressive skin cancers, characterized by metastasis to the lymph nodes and a high capacity to develop drug resistance. There is a lack of knowledge on the mechanisms contributing to lymphatic vessel formation and metastasis regulation in malignant melanoma. We previously reported the involvement of CD147, a transmembrane glycoprotein overexpressed in melanoma, in the regulation of the tumor microenvironment and angiogenesis. The aim of our study was to further determine how CD147 is involved in lymphangiogenesis regulation. Our results revealed that high CD147 expression is correlated with the number of lymphatic vessels in the human melanoma lymph nodes and that paracrine CD147 upregulates lymphangiogenesis through lymphangiogenic mediators in vitro and in vivo, suggesting that CD147 could be a promising target for melanoma-associated lymphangiogenesis inhibition. Abstract Malignant melanoma is one of the most aggressive skin cancers and is characterized by early lymph node metastasis and the capacity to develop resistance to therapies. Hence, understanding the regulation of lymphangiogenesis through mechanisms contributing to lymphatic vessel formation represents a treatment strategy for metastatic cancer. We have previously shown that CD147, a transmembrane glycoprotein overexpressed in melanoma, regulates the angiogenic process in endothelial cells. In this study, we show a correlation between high CD147 expression levels and the number of lymphatic vessels expressing LYVE-1, Podoplanin, and VEGFR-3 in human melanoma lymph nodes. CD147 upregulates in vitro lymphangiogenesis and its related mediators through the PROX-1 transcription factor. In vivo studies in a melanoma model confirmed that CD147 is involved in metastasis through a similar mechanism as in vitro. This study, demonstrating the paracrine role of CD147 in the lymphangiogenesis process, suggests that CD147 could be a promising target for the inhibition of melanoma-associated lymphangiogenesis.

// Baptiste Louveau 1, 2, 3 , Julie Delyon 1, 2, 4 , Coralie Reger De Moura 2, 3 , Maxime Battistella 1, 5, 6 , Fanelie Jouenne 1, 2, 3 , Lisa Golmard 7 , Aurelie Sadoux 1, 2, 3 , Marie-Pierre Podgorniak 1, 2, 3 , Ichrak Chami 4 , Oren Marco 8 , Julie Caramel 9 , Stephane Dalle 9, 10 , Jean-Paul Feugeas 11 , Nicolas Dumaz 1, 2 , Celeste Lebbe 1, 2, 4, * and Samia Mourah 1, 2, 3, * 1 Paris-Diderot University, Sorbonne Paris Cite, Paris, France 2 Paris-Diderot University, Inserm, UMR_S976, Paris, France 3 Department of Pharmacogenomics, Saint-Louis Hospital, AP-HP, Paris, France 4 Department of Dermatology, Saint-Louis Hospital, AP-HP, Paris, France 5 Department of Pathology, Saint-Louis Hospital, AP-HP, Paris, France 6 Paris Diderot University, Inserm, UMR_S1165, Paris, France 7 Department of Genetics, Pole de Medecine Diagnostique et Theranostique, Institut Curie, Paris, France 8 Department of Plastic, Reconstructive and Esthetic Surgery, Saint-Louis Hospital, AP-HP, Paris, France 9 Universite de Lyon, Universite Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Leon Berard, Cancer Research Center of Lyon, Equipe Labellisee Ligue contre le Cancer, Lyon, France 10 Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Pierre Benite, France 11 Universite de Franche-Comte, Inserm, UMR_1137, Paris, France * co-senior authors Correspondence to: Samia Mourah, email: samia.mourah@aphp.fr Keywords: melanoma; BRAF inhibitors; targeted genomic alteration; predictive analysis; targeted therapy resistance Received: March 22, 2018 Accepted: January 31, 2019 Published: March 01, 2019 ABSTRACT Several mechanisms have been described to elucidate the emergence of resistance to MAPK inhibitors in melanoma and there is a crucial need for biomarkers to identify patients who are likely to achieve a better and long-lasting response to BRAF inhibitors therapy. In this study, we developed a targeted approach combining both mRNA and DNA alterations analysis focusing on relevant gene alterations involved in acquired BRAF inhibitor resistance. We collected baseline tumor samples from 64 melanoma patients at BRAF inhibitor treatment initiation and showed that the presence, prior to treatment, of mRNA over-expression of genes’ subset was significantly associated with improved progression free survival and overall survival. The presence of DNA alterations was in favor of better overall survival. The genomic analysis of relapsed-matched tumor samples from 20 patients allowed us to uncover the largest landscape of resistance mechanisms reported to date as at least one resistance mechanism was identified for each patient studied. Alterations in RB1 have been most frequent and hence represent an important additional acquired resistance mechanism. Our targeted genomic analysis emerges as a relevant tool in clinical practice to identify those patients who are more likely to achieve durable response to targeted therapies and to exhaustively describe the spectrum of resistance mechanisms. Our approach can be adapted to new targeted therapies by including newly identified genetic alterations.

InPatients withEighteen patients were enrolled, with 15 in strata 2. Characteristics at inclusion were American Joint Committee on Cancer stage IVM1c (Although the combination of palbociclib + fixed-dose vemurafenib did not allow an increased palbociclib dosage above 25 mg, a significant clinical benefit was achieved in pretreated patients with melanoma. An association between the transcriptomic data and clinical response was highlighted.

KIT is a bona fide oncogene in a subset of melanoma and, ex vivo, KIT inhibitors are very efficient at killing KIT-mutant melanoma cell lines. However, KIT-mutant melanoma tumors tend to show a de novo resistance in most cases and a limited duration of response when response is achieved. We performed pharmacodynamic studies on patients with KIT-mutated melanoma treated with nilotinib, which suggested that the FGF2 axis may be a mechanism of resistance in this subset of melanoma. Using several melanoma cell lines, which are dependent on oncogenic KIT, we showed that although KIT inhibition markedly decreased cell viability in melanoma cell lines with distinct KIT mutations, this effect was lessened in the presence of FGF2 due to inhibition of BIM expression by MAPK pathway activation. Addition of a MEK inhibitor reversed the FGF2-driven resistance for all KIT mutants. We confirmed the expression of FGF2 and activation of MEK-ERK in melanoma patients using in situ data from a clinical trial. Therefore, the combined inhibition of KIT with FGFR or MEK may be a next-step effective clinical strategy in KIT-mutant melanoma., SCOPUS: ar.j, info:eu-repo/semantics/published

The discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family that signals in response to collagen and that has been implicated in cancer progression. In the present study, we investigated the expression and role of DDR1 in human melanoma progression. Immunohistochemical staining of human melanoma specimens (n = 52) shows high DDR1 expression in melanoma lesions that correlates with poor prognosis. DDR1 expression was associated with the clinical characteristics of Clark level and ulceration and with BRAF mutations. Downregulation of DDR1 by small interfering RNA (siRNA) in vitro inhibited melanoma cells malignant properties, migration, invasion, and survival in several human melanoma cell lines. A DDR tyrosine kinase inhibitor (DDR1-IN-1) significantly inhibited melanoma cell proliferation in vitro, and ex vivo and in tumor xenografts, underlining the promising potential of DDR1 inhibition in melanoma.

In BRAFV600mut metastatic melanoma, the combination of BRAF and MEK inhibitors (BRAFi, MEKi) has undergone multiple resistance mechanisms, limiting its clinical benefit and resulting in the need for response predicting biomarkers. Based on phase III clinical trial data, several studies have previously explored baseline genomic features associated with response to BRAFi + MEKi. Using a targeted approach that combines the examination of mRNA expression and DNA alterations in a subset of genes, we performed an analysis of baseline genomic alterations involved in MAPK inhibitors&rsquo, resistance in a real-life cohort of BRAFV600mut metastatic melanoma patients. Twenty-seven patients were included in this retrospective study, and tumor samples were analyzed when the BRAFi + MEKi therapy was initiated. The clinical characteristics of our cohort were consistent with previously published studies. The BRAFi + MEKi treatment was initiated in seven patients as a following-line treatment, and had a specific transcriptomic profile exhibiting 14 genes with lower mRNA expression. However, DNA alterations in CCND1, RB1, and MET were only observed in patients who received BRAFi + MEKi as the first-line treatment. Furthermore, KIT mRNA expression was significantly higher in patients showing clinical benefit from the combined therapy, emphasizing the tumor-suppressor role of KIT already described within the context of BRAF-mutant melanoma.

Kindlin-3 (FERMT-3) is a key integrin activating protein belonging to the Kindlin family which includes three members Kindlin-1, 2 and 3. Kindlins can directly bind to various classes of integrins and participate in their activation, thus playing a key role in the regulation of cell-matrix adhesions. Kindlin-3 has been known to be central in the control of hemostasis and thrombosis as its deficiency disrupts platelet aggregation and causes Leukocyte Adhesion Deficiency disease. Its expression has so far been only described in hematopoietic cells, and more recently in endothelial cells. The current manuscript highlights key findings from our recent research describing a novel role of Kindlin-3 as a tumor suppressor in cancer. We present data showing that Kindlin-3 is expressed more ubiquitously than previously thought, and that through gene inactivation mechanisms, its expression is significantly reduced in several human solid cancer lesions. The increase in the malignant properties of tumor cells when Kindlin-3 expression is silenced and the inverse expression of Kindlin-3 with the tumor promoter EMMPRIN/CD147 corroborate the tumor suppressor role of Kindlin-3. A schematic model based on our research data describing Kindlin-3 mode of action in tumor cells is presented.