Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM.Analyses revealed that therapy resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and forcemediated ECM remodeling through activation of actin-dependent mechanosensors Yesassociated protein (YAP) and Myocardin-related transcription factor (MRTF). Shortterm inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP and MRTFdependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacological targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCEThese findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing and therapy resistance.

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Advanced cutaneous melanoma is one of the most challenging cancers to treat because of its high plasticity, metastatic potential, and resistance to treatment. New targeted therapies and immunotherapies have shown remarkable clinical efficacy. However, such treatments are limited to a subset of patients and relapses often occur, warranting validation of novel targeted therapies. Posttranslational modification of proteins by ubiquitin coordinates essential cellular functions, including ubiquitin-proteasome system (UPS) function and protein homeostasis. Deubiquitinating enzymes (DUB) have been associated to multiple diseases, including cancer. However, their exact involvement in melanoma development and therapeutic resistance remains poorly understood. Using a DUB trap assay to label cellular active DUBs, we have observed an increased activity of the proteasome-associated DUB, USP14 (Ubiquitin-specific peptidase 14) in melanoma cells compared with melanocytes. Our survey of public gene expression databases indicates that high expression of correlates with melanoma progression and with a poorer survival rate in metastatic melanoma patients. Knockdown or pharmacologic inhibition of USP14 dramatically impairs viability of melanoma cells irrespective of the mutational status of, or and their transcriptional cell state, and overcomes resistance to MAPK-targeting therapies both and in human melanoma xenografted mice. At the molecular level, we find that inhibition of USP14 rapidly triggers accumulation of poly-ubiquitinated proteins and chaperones, mitochondrial dysfunction, ER stress, and a ROS production leading to a caspase-independent cell death. Our results provide a rationale for targeting the proteasome-associated DUB USP14 to treat and combat melanomas. .

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O-GlcNAcylation on serine/threonine is a post-translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O-GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O-GlcNAcylation in the context of pancreatic β-cell glucotoxicity. FoxO1 was O-GlcNAcylated in INS-1 832/13 β cells and isolated rat pancreatic islets. O-GlcNAcylation of FoxO1 resulted in a 2-fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3-fold increase in the expression of the insulin-like growth factor-binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin-like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O-GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS-1 832/13 cells from high-glucose- or O-glcNAcylation-induced cell death. In contrast, FoxO1 down-regulation by siRNA led to 30 to 60% protection of INS-1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O-GlcNAcylation inhibits Akt through an IGFBP1-mediated autocrine pathway, the deleterious effects of FoxO1 O-GlcNAcylation on cell survival appeared to be independent of this pathway.

<p>Morphology and focal adhesions complexes of parental and BRAFi-resistant cells on hydrogels of various stiffness</p>

<p>F-actin and fibronectin stainings in M238P and M238R cells</p>

<p>BRAF and MEK inhibition induces F-actin remodeling, YAP/MRTF activation and focal adhesion assembly in BRAF-mutant melanoma cells</p>