New signaling pathways implicated in resistance to targeted therapy in tumors with alterations of the MAPK pathway

Abstract

Mutations in the oncogenic protein kinase BRAF are involved in the onset and progression of several tumours, including more than 50% of melanoma cases and around 8% of all cancers worldwide. Currently, BRAF/MEK inhibitors (BRAF/MEKi) are the standard therapy for BRAFmutated melanoma and have radically changed the treatment landscape of advanced BRAF mutation-positive tumours. However, limited efficacy and emergence of drug resistance are major handicaps for successful treatments. The channel protein connexin43 (Cx43), allows the exchange of small molecules such as ions, metabolites or small RNAs (sRNAs) via hemichannels, gap junctions and thought small extracellular vesicles (EVs), affecting of cell growth, metabolism, and differentiation. Cx43 has also been detected at the mitochondrial associated membranes (MAMs) and it has been identified as a potential tumour suppressor in primary melanoma. However, the molecular basis of its role in disease progression remains controversial. In this project, we aim to unravel de role of Cx43 in the context of BRAF mutated cancer and resistance to therapy in BRAF-mutated tumours. Our findings show that Cx43 enhances the efficacy of BRAF/MEKi in various BRAF-driven tumours in vitro and in vivo models, by attracting DNA repair complexes to the nuclear membrane by interacting with lamin-A/C and different DDR proteins such as HMGB1, PRPF19, XCRR6 or CDCL5. This nuclear compartmentalisation restrict HR pathways and delay DNA repair leading to persistent DNA damage, contribuing to genome instability and synthetic lethality. The results of this thesis demonstrated that targeting Cx43 to interfere with DNA repair could emerge as a potential therapeutic strategy to increase efficacy and to overcome drug resistance in tumours with a BRAF mutant background. In light of our findings, we designed an innovative drug combination by using EVs as drug delivery systems to deliver the full-Cx43 (mRNA and protein) in combination with the BRAF/MEKi. Our results have shown that Cx43 not only increases the internalisation capacity of EVs into receptor cells but also modifies their cargo through the recruitment of new proteins and different molecules of RNAs. The modification of the cargo of these EVs could have an impact on their physiological functions, as we have observed that both sensitive and resistant tumour cells are affected by treatment with these particles, especially when they are in combination with BRAF/MEKi. Furthermore, it has been observed that Cx43 impacts the metabolism of tumour cells by enhancing mitochondrial content and increasing their capacity for respiration through oxidative phosphorylation in BRAFV600E melanoma cells. In addition, Cx43 is present in the endoplasmic reticulum (ER) of melanoma cells, suggesting its role as a novel regulator of ERrelated stress pathways, potentially leading to cell death. Overall, our study highlights novel functions of Cx43, demonstrating for the first time that Cx43 is a novel factor contributing to DNA repair response and BRAF/MEKi efficacy, as well as a key player in the regulation of ER 15 stress, highlighting the potential of Cx43 as an antitumour agent and enhancer of therapy efficacy, as well as a bypass for drug resistance acquisition in BRAF-mutant tumours.