Inactivation of kindlin-3 increases human melanoma aggressiveness through the collagen-activated tyrosine kinase receptor DDR1.

The role of the focal adhesion protein kindlin-3 as a tumor suppressor and its interaction mechanisms with extracellular matrix constitute a major field of investigation to better decipher tumor progression. Besides the well-described role of kindlin-3 in integrin activation, evidence regarding modulatory functions between melanoma cells and tumor microenvironment are lacking and data are needed to understand mechanisms driven by kindlin-3 inactivation. Here, we show that kindlin-3 inactivation through knockdown or somatic mutations increases BRAFV600mut melanoma cells oncogenic properties via collagen-related signaling by decreasing cell adhesion and enhancing proliferation and migration in vitro, and by promoting tumor growth in mice. Mechanistic analysis reveals that kindlin-3 interacts with the collagen-activated tyrosine kinase receptor DDR1 (Discoidin domain receptor 1) modulating its expression and its interaction with ß1-integrin. Kindlin-3 knockdown or mutational inactivation disrupt DDR1/ß1-integrin complex in vitro and in vivo and its loss improves the anti-proliferative effect of DDR1 inhibition. In agreement, kindlin-3 downregulation is associated with DDR1 over-expression in situ and linked to worse melanoma prognosis. Our study reveals a unique mechanism of action of kindlin-3 in the regulation of tumorigenesis mediated by the collagen-activated tyrosine kinase receptor DDR1 thus paving the way for innovative therapeutic targeting approaches in melanoma.

Combined PDE4+MEK inhibition shows antiproliferative effects in NRASQ61 mutated melanoma preclinical models.

Upregulation of phosphodiesterase type 4 (PDE4) has been associated with worse prognosis in several cancers. In melanomas harboring NRAS mutations, PDE4 upregulation has been shown to trigger a switch in signaling from BRAF to RAF1 which leads to mitogen-activated protein kinase pathway activation. Previous in vitro evidence showed that PDE4 inhibition induced death in NRASQ61mut melanoma cells and such a strategy may thus be a relevant therapeutic option in those cases with no molecular targeted therapies approved to date. In this study, we generated patient-derived xenografts (PDX) from two NRASQ61mut melanoma lesions. We performed ex vivo histoculture drug response assays and in vivo experiments. A significant ex vivo inhibition of proliferation with the combination of roflumilast+cobimetinib was observed compared to dimethyl sulfoxide control in both models (51 and 67%). This antiproliferative effect was confirmed in vivo for PDX-1 with a 56% inhibition of tumor growth. To decipher molecular mechanisms underlying this effect, we performed transcriptomic analyses and revealed a decrease in MKI67, RAF1 and CCND1 expression under bitherapy. Our findings strengthen the therapeutic interest of PDE4 inhibitors and support further experiments to evaluate this approach in metastatic melanoma.

TERT Expression Induces Resistance to BRAF and MEK Inhibitors in BRAF-Mutated Melanoma In Vitro.

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.

Novel treatment strategy for NRAS-mutated melanoma through a selective inhibitor of CD147/VEGFR-2 interaction.

More than 70% of human NRASmut 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 NRASmut melanoma cells. It significantly inhibited the malignant properties of NRASmut melanomas ex vivo and in vivo. Importantly, NRASmut 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 NRASmut melanoma when combined with MEKi.

CD147 Promotes Tumor Lymphangiogenesis in Melanoma via PROX-1.

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.

Phase I-II Open-Label Multicenter Study of Palbociclib + Vemurafenib in BRAF V600MUT Metastatic Melanoma Patients: Uncovering CHEK2 as a Major Response Mechanism.

PURPOSE: In BRAF V600MUT 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 BRAF V600E/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.

Mitogen-activated protein kinase blockade in melanoma: intermittent versus continuous therapy, from preclinical to clinical data.

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.

Discoidin Domain Receptors in Melanoma: Potential Therapeutic Targets to Overcome MAPK Inhibitor Resistance.

Melanoma is a highly malignant skin cancer with high propensity to metastasize and develop drug resistance, making it a difficult cancer to treat. Current therapies targeting BRAF (V600) mutations are initially effective, but eventually tumors overcome drug sensitivity and reoccur. This process is accomplished in part by reactivating alternate signaling networks that reinstate melanoma proliferative and survival capacity, mostly through reprogramming of receptor tyrosine kinase (RTK) signaling. Evidence indicates that the discoidin domain receptors (DDRs), a set of RTKs that signal in response to collagen, are part of the kinome network that confer drug resistance. We previously reported that DDR1 is expressed in melanomas, where it can promote tumor malignancy in mouse models of melanoma, and thus, DDR1 could be a promising target to overcome drug resistance. In this review, we summarize the current knowledge on DDRs in melanoma and their implication for therapy, with emphasis in resistance to MAPK inhibitors.

FGF2 Induces Resistance to Nilotinib through MAPK Pathway Activation in KIT Mutated Melanoma.

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.

Intermittent Versus Continuous Dosing of MAPK Inhibitors in the Treatment of BRAF-Mutated Melanoma.

The development of BRAF and MEK inhibitors (BRAFi/MEKi) has led to major advances in melanoma treatment. However, the emergence of resistance mechanisms limits the benefit duration and a complete response occurs in less than 20% of patients receiving BRAFi ± MEKi. In this study, we evaluated the impact of an intermittent versus continuous dosing schedule of BRAF/MEK inhibition in a melanoma model mildly sensitive to a BRAF inhibitor. The combination of a BRAFi with three different MEKi was studied with a continuous or intermittent dosing schedule in vivo, in a xenografted melanoma model and ex vivo using histoculture drug response assays (HDRAs) of patient-derived xenografts (PDX). To further understand the underlying molecular mechanisms of therapeutic efficacy, a biomarker pharmacodynamic readout was evaluated. An equal impact on tumor growth was observed in monotherapy or bitherapy regimens whether we used continuous and intermittent dosing schedules, with no significant differences in biomarkers expression between the treatments. The antitumoral effect was mostly due to modulations of expression of cell cycle and apoptotic mediators. Moreover, ex vivo studies did not show significant differences between the dosing schedules. In this context, our preclinical and pharmacodynamic results converged to show the similarity between intermittent and continuous treatments with either BRAFi or MEKi alone or with the combination of both.

CD147 Is a Promising Target of Tumor Progression and a Prognostic Biomarker.

Microenvironment plays a crucial role in tumor development and progression. Cancer cells modulate the tumor microenvironment, which also contribute to resistance to therapy. Identifying biomarkers involved in tumorigenesis and cancer progression represents a great challenge for cancer diagnosis and therapeutic strategy development. CD147 is a glycoprotein involved in the regulation of the tumor microenvironment and cancer progression by several mechanisms-in particular, by the control of glycolysis and also by its well-known ability to induce proteinases leading to matrix degradation, tumor cell invasion, metastasis and angiogenesis. Accumulating evidence has demonstrated the role of CD147 expression in tumor progression and prognosis, suggesting it as a relevant tumor biomarker for cancer diagnosis and prognosis, as well as validating its potential as a promising therapeutic target in cancers.

Baseline Genomic Features in BRAFV600-Mutated Metastatic Melanoma Patients Treated with BRAF Inhibitor + MEK Inhibitor in Routine Care.

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' 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.

Discoidin domain receptors: A promising target in melanoma.

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.