From drug resistance mechanisms to microRNA function in melanoma

Cutaneous melanoma is an aggressive skin cancer that emerges from the unrestrained proliferation of melanocytes, which are the pigment producing cells in the basal layer of the epidermis. Despite the fact that it only accounts for approximately 5% of all skin cancers, melanoma is responsible for the vast majority of skin cancer-related deaths. As more than half of the patients with sporadic melanoma harbour activating mutations in the protein kinase BRAF, the development of small kinase inhibitors targeting mutated BRAF led to an increased overall survival of patients with metastatic melanoma. Despite the initially promising results, the rapidly emerging resistance to these targeted therapies remains a serious clinical issue.
To investigate the mechanisms underlying resistance to targeted therapies, we used in vitro BRAF-mutant drug-sensitive and drug-resistant melanoma cell models that were generated in our laboratory. First, we performed a kinase inhibitor library screening with the aim to identify novel kinase inhibitor combinations to circumvent or delay BRAF inhibitor-induced resistance. We have characterised synergistic kinase inhibitors targeting the MAPK pathway and the cell cycle showing promising effects in BRAF-mutant drug-sensitive and -resistant cells, which could be used as an effective sequential or alternative treatment option for late-stage melanoma patients. Additionally, we investigated the impact of BRAF inhibitors at the transcriptional level by comparing miRNome and transcriptome changes in drug-sensitive and -resistant melanoma cells. We identified miRNAs (e.g. miR-509, miR-708) and genes (e.g. PCSK2, AXL) that were distinctly differentially expressed in resistant compared to sensitive cells. Subsequent co-expression analyses revealed a low MITF/AXL ratio in a subset of resistant cell lines, suggesting that miRNAs might be involved in the switch from one molecular phenotype to another, thus conferring tolerance to targeted therapies.
Finally, we applied a method based on cross-linking ligation and sequencing of hybrids (qCLASH) to provide a comprehensive snapshot of the miRNA targetome in our BRAF-mutant melanoma cells. To our knowledge, we implemented for the first time a CLASH-based method to cancer cells, and identified over 8k direct and distinct miRNA-target interactions in melanoma cells, including many with non-predicted and non-canonical binding characteristics, thus expanding the pool of miRNA-target interactions. Taken together, these results provide new insights into complex and heterogeneous responses to BRAF inhibition, adding an additional level of complexity to drug-induced (post-) transcriptional network rewiring in melanoma.