The AhR-SRC axis as a therapeutic vulnerability in BRAFi-resistant melanoma.

The nongenetic mechanisms required to control tumor phenotypic plasticity and shape drug-resistance remain unclear. We show here that the Aryl hydrocarbon Receptor (AhR) transcription factor directly regulates the gene expression program associated with the acquisition of resistance to BRAF inhibitor (BRAFi) in melanoma. In addition, we show in melanoma cells that canonical activation of AhR mediates the activation of the SRC pathway and promotes the acquisition of an invasive and aggressive resistant phenotype to front-line BRAFi treatment in melanoma. This nongenetic reprogramming identifies a clinically compatible approach to reverse BRAFi resistance in melanoma. Using a preclinical BRAFi-resistant PDX melanoma model, we demonstrate that SRC inhibition with dasatinib significantly re-sensitizes melanoma cells to BRAFi. Together we identify the AhR/SRC axis as a new therapeutic vulnerability to trigger resistance and warrant the introduction of SRC inhibitors during the course of the treatment in combination with front-line therapeutics to delay BRAFi resistance.

CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance.

Most genetic alterations that drive melanoma development and resistance to targeted therapy have been uncovered. In contrast, and despite their increasingly recognized contribution, little is known about the non-genetic mechanisms that drive these processes. Here, we performed in vivo gain-of-function CRISPR screens and identified SMAD3, BIRC3, and SLC9A5 as key actors of BRAFi resistance. We show that their expression levels increase during acquisition of BRAFi resistance and remain high in persister cells and during relapse. The upregulation of the SMAD3 transcriptional activity (SMAD3-signature) promotes a mesenchymal-like phenotype and BRAFi resistance by acting as an upstream transcriptional regulator of potent BRAFi-resistance genes such as EGFR and AXL. This SMAD3-signature predicts resistance to both current melanoma therapies in different cohorts. Critically, chemical inhibition of SMAD3 may constitute amenable target for melanoma since it efficiently abrogates persister cells survival. Interestingly, decrease of SMAD3 activity can also be reached by inhibiting the Aryl hydrocarbon Receptor (AhR), another druggable transcription factor governing SMAD3 expression level. Our work highlights novel drug vulnerabilities that can be exploited to develop long-lasting antimelanoma therapies.

Role of Flavonoids in the Prevention of AhR-Dependent Resistance During Treatment with BRAF Inhibitors.

BRAF and MEK inhibitors (BRAFi and MEKi) are the standard of care for the treatment of metastatic melanoma in patients with BRAFV600E mutations, greatly improving progression-free survival. However, the acquisition of resistance to BRAFi and MEKi remains a difficult clinical challenge, with limited therapeutic options available for these patients. Here, we investigated the therapeutic potential of natural flavonoids as specific AhR (Aryl hydrocarbon Receptor) transcription factor antagonists in combination with BRAFi. EXPERIMENTAL DESIGN: Experiments were performed in vitro and in vivo with various human melanoma cell lines (mutated for BRAFV600E) sensitive or resistant to BRAFi. We evaluated the role of various flavonoids on cell sensitivity to BRAFi and their ability to counteract resistance and the invasive phenotype of melanoma. RESULTS: Flavonoids were highly effective in potentiating BRAFi therapy in human melanoma cell lines by increasing sensitivity and delaying the pool of resistant cells that arise during treatment. As AhR antagonists, flavonoids counteracted a gene expression program associated with the acquisition of resistance and phenotype switching that leads to an invasive and EMT-like phenotype. CONCLUSIONS: The use of natural flavonoids opens new therapeutic opportunities for the treatment of patients with BRAF-resistant disease.

Sustained activation of the Aryl hydrocarbon Receptor transcription factor promotes resistance to BRAF-inhibitors in melanoma.

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

Pharmacological targeting of apelin impairs glioblastoma growth.

Glioblastoma are highly aggressive brain tumours that are associated with an extremely poor prognosis. Within these tumours exists a subpopulation of highly plastic self-renewing cancer cells that retain the ability to expand ex vivo as tumourspheres, induce tumour growth in mice, and have been implicated in radio- and chemo-resistance. Although their identity and fate are regulated by external cues emanating from endothelial cells, the nature of such signals remains unknown. Here, we used a mass spectrometry proteomic approach to characterize the factors released by brain endothelial cells. We report the identification of the vasoactive peptide apelin as a central regulator for endothelial-mediated maintenance of glioblastoma patient-derived cells with stem-like properties. Genetic and pharmacological targeting of apelin cognate receptor abrogates apelin- and endothelial-mediated expansion of glioblastoma patient-derived cells with stem-like properties in vitro and suppresses tumour growth in vivo. Functionally, selective competitive antagonists of apelin receptor were shown to be safe and effective in reducing tumour expansion and lengthening the survival of intracranially xenografted mice. Therefore, the apelin/apelin receptor signalling nexus may operate as a paracrine signal that sustains tumour cell expansion and progression, suggesting that apelin is a druggable factor in glioblastoma.

The E3 ubiquitin ligase MARCH3 controls the endothelial barrier.

Cell-cell contacts coordinate the endothelial barrier function in response to external cues. To identify new mediators involved in cytokine-promoted endothelial permeability, we screened a siRNA library targeting E3 ubiquitin ligases. Here, we report that silencing of the late endosome/lysosomal membrane-associated RING-CH-3 (MARCH3) enzyme protects the endothelial barrier. Furthermore, transcriptome analysis unmasked the upregulation of the tight junction-encoding gene occludin (OCLN) in MARCH3-depleted cells. Indeed, MARCH3 silencing results in the strengthening of cell-cell contacts, as evidenced by the accumulation of junctional proteins. From a molecular standpoint, the FoxO1 forkhead transcription repressor was inactivated in the absence of MARCH3. This provides a possible molecular link between MARCH3 and the signaling pathway involved in regulating the expression of junctional proteins and barrier integrity.

Desert Hedgehog/Patch2 Axis Contributes to Vascular Permeability and Angiogenesis in Glioblastoma.

Glioblastoma multiforme (GBM) constitutes the most common and the most aggressive type of human tumors affecting the central nervous system. Prognosis remains dark due to the inefficiency of current treatments and the rapid relapse. Paralleling other human tumors, GBM contains a fraction of tumor initiating cells with the capacity to self-renew, initiate and maintain the tumor mass. These cells were found in close proximity to brain vasculature, suggesting functional interactions between brain tumor-initiating cells (BTICs) and endothelial cells within the so-called vascular niche. However, the mechanisms by which these cells impact on the endothelium plasticity and function remain unclear. Using culture of BTICs isolated from a cohort of 14 GBM patients, we show that BTICs secretome promotes brain endothelial cell remodeling in a VEGF-independent manner. Gene array analysis unmasked that BTICs-released factors drove the expression of Ptch2 in endothelial cells. Interestingly, BTICs produce desert hedgehog (DHH) ligand, enabling a paracrine DHH/Ptch2 signaling cascade that conveys elevated permeability and angiogenesis. Finally, DHH silencing in BTICs dramatically reduced tumor growth, as well as vascularization and intra-tumor permeability. Collectively, our data unveil a role for DHH in exacerbated tumor angiogenesis and permeability, which may ultimately favor glioblastoma growth, and thus place the DHH/Ptch2 nexus as a molecular target for novel therapies.

The guanine exchange factor SWAP70 mediates vGPCR-induced endothelial plasticity.

BACKGROUND: The viral G protein-coupled receptor (vGPCR) is proposed to act as one of the predominant mediators of Kaposi's sarcoma (KS), a human herpes virus 8 (HHV8)-elicited disease. The actions of vGPCR manifest pathogenesis, in part, through increased permeability of endothelial cells. Endothelial cell-cell junctions have indeed emerged as an instrumental target involved in the vasculature defects observed within the tumor microenvironment. The pathway leading to adherens junction destabilization has been shown to involve the activation of the small GTPase Rac, in the context of either latent infection or the sole expression of vGPCR. However, the precise molecular mechanisms governed by vGPCR in vascular leakage require further elucidation. FINDINGS: Guanine exchange factors (GEFs) function as critical molecular switches that control the activation of small GTPases. We therefore screened the effects of 80 siRNAs targeting GEFs on vGPCR-driven endothelial permeability and identified switch-associated protein 70 (SWAP70) as necessary for its elevating effects. Pull-down experiments further showed that Rac activation by vGPCR was dependent on SWAP70. Examination of tissues and cells from HHV8-positive patients revealed that SWAP70 was ubiquitously expressed. Furthermore, SWAP70 was found to be crucial for vGPCR-driven endothelial tube formation and endothelial sprouting in vitro. CONCLUSIONS: SWAP70 appears to act as a molecular intermediate between vGPCR and endothelial activation. Because of the important role of vGPCR-mediated endothelial plasticity in KS pathogenesis, inhibition of SWAP70 function could be of interest for blocking vGPCR-driven activities in HHV8-defined diseases.

Control of CXCR2 activity through its ubiquitination on K327 residue.

BACKGROUND: The interleukin-8 chemokine (IL-8) G-protein coupled receptor CXCR2 governs pro-inflammatory and pro-angiogenic responses in leukocytes and endothelial cells. At a molecular standpoint, CXCR2 is widely reported to operate through calcium flux, phosphoinoisitide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK). While CXCR2 trafficking is suspected to be intertwined with its signaling, the exact mechanism is not fully elucidated. RESULTS: Here, we identified the lysine 327 within the CXCR2 C-terminal tail as a key residue for ubiquitination, internalization, and signaling. First, the substitution to an arginine of K327 mutation was associated with a reduction in CXCR2 poly-ubiquitination. While WT CXCR2 was rapidly internalized following IL-8 administration, K327R mutant remained at the plasma membrane. Finally, K327R mutant failed to promote the recruitment of ß-arrestin2, as estimated by imagery and bioluminescence resonance transfer. As a consequence, the activation of intracellular signaling, including both early events such as ERK phosphorylation and the increase in calcium flux, and the latter activation of the AP1 and NF-κB transcription factors, was blunted. CONCLUSIONS: Overall, our results demonstrate that CXCR2 ubiquitination on K327 residue modulates agonist-activated CXCR2 cell sorting and intracellular signaling. Thus, the inhibition of K327 ubiquitination might emerge as an effective mean to curb exacerbated CXCR2 signaling in several pathological conditions, such as inflammatory diseases and cancer.

A catalytic-independent role for the LUBAC in NF-κB activation upon antigen receptor engagement and in lymphoma cells.

Antigen receptor-mediated nuclear factor κB (NF-κB) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell-like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-κB activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-κB activation, dynamically integrates the CBM and marshals NF-κB optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-κB activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy.

The C-terminus region of ß-arrestin1 modulates VE-cadherin expression and endothelial cell permeability.

BACKGROUND: The endothelial specific cell-cell adhesion molecule, VE-cadherin, modulates barrier function and vascular homeostasis. In this context, we have previously characterized that VEGF (vascular endothelial growth factor) leads to VE-cadherin phosphorylation, ß-arrestin2 recruitment and VE-cadherin internalization in mouse endothelial cells. However, exactly how this VE-cadherin/ß-arrestin complex contributes to VEGF-mediated permeability in human endothelial cells remains unclear. In this study, we investigated in-depth the VE-cadherin/ß-arrestin interactions in human endothelial cells exposed to VEGF. FINDINGS: First, we demonstrated that VEGF induces VE-cadherin internalization in a clathrin-dependent manner in human umbilical vein endothelial cells (HUVEC). In addition to the classical components of endocytic vesicles, ß-arrestin1 was recruited and bound to phosphorylated VE-cadherin. Molecular mapping of this interaction uncovered that the C-terminus tail of ß-arrestin1, that comprises amino acids 375 to 418, was sufficient to directly interact with the phosphorylated form of VE-cadherin. Interestingly, the expression of the C-terminus tail of ß-arrestin1 induced loss of surface exposed-VE-cadherin, promoted monolayer disorganization and enhanced permeability. Finally, this effect relied on decreased VE-cadherin expression at the transcriptional level, through inhibition of its promoter activity. CONCLUSIONS: Altogether, our results demonstrate that ß-arrestin1 might play multiple functions collectively contributing to endothelial barrier properties. Indeed, in addition to a direct implication in VE-cadherin endocytosis, ß-arrestin1 could also control VE-cadherin transcription and expression. Ultimately, understanding the molecular mechanisms involved in VE-cadherin function might provide therapeutic tools for many human diseases where the vascular barrier is compromised.

Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34.

BACKGROUND: NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated. FINDINGS: Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor. CONCLUSIONS: Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.