In cutaneous melanoma, 52% of cases harbour mutations in the BRAF gene. Although targeted therapy, which involves the combination of BRAF and MEK inhibitors (BRAFi+MEKi), has revolutionized the treatment of BRAF-mutated melanoma, most patients experience disease progression within 8-12 months due to acquired drug resistance. Additionally, there are poor responders to immunotherapy-based second-line treatments. Understanding resistance mechanisms is thus of significant clinical interest. A previous study, using an in vitro resistance model revealed that BRAFi+MEKi resistance acquisition involves the enrichment of clones overexpressing genes characteristic of invasive and mesenchymal-like phenotypes, such as SPOCK1. Hence, our aim is to elucidate the role of SPOCK1 as biomarker and potential therapeutic vulnerability in resistance acquisition scenario.

SPOCK1 RNA expression was analysed in silico in other melanoma BRAFi+MEKi resistant in vitro models (GSE134432 and GSE75299) and in melanoma tissue before treatment and at progression (GSE77940 and GSE61992). Additionally, an in vitro SPOCK1 knock-down was stablished using shRNA technique and functional studies of resistance reversion, proliferation, migration and invasion were performed by using MTT, wound-healing and transwell assays.

In silico analysis corroborated that SPOCK1 gene expression was increased in other melanoma cell lines resistant to targeted therapy. Furthermore, SPOCK1 expression was mainly higher in the resistant tumours compared to the basal ones. Functional in vitro analyses revealed that inhibiting the SPOCK1 gene did not reverse resistance to BRAF+MEK inhibitors, suggesting that SPOCK1 does not directly contribute to the molecular processes underlying resistance acquisition. However, SPOCK1 inhibition significantly reduced cell proliferation, migration, and invasion, suggesting its potential role in mesenchymal phenotype development and as a therapeutic vulnerability. Nevertheless, further studies in advanced preclinical models are guaranteed.

SPOCK1 is identified as a biomarker of acquired resistance to BRAFi+MEKi and it postulates as potential therapeutic vulnerability in resistance acquisition context.