The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has been shown to be an essential regulator of a broad spectrum of biological activities required for maintaining the body’s vital functions. AhR also plays a critical role in tumorigenesis. Its role in cancer is complex, encompassing both pro- and anti-tumorigenic activities. Its level of expression and activity are specific to each tumor and patient, increasing the difficulty of understanding the activating or inhibiting roles of AhR ligands. We explored the role of AhR in tumor cell lines and patients using genomic data sets and discuss the extent to which AhR can be considered as a therapeutic target.

BRAF and MEK inhibitors (BRAFi and MEKi) are the standard of care for the treatment of metastatic melanoma in patients with BRAF^V600E 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 BRAF^V600E) 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.

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 and these genes promote the tumor growth capability of persister cells. 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 expands our understanding of the biology of persister cells and highlight novel drug vulnerabilities that can be exploited to develop long-lasting antimelanoma therapies.