Exploiting mitochondrial dysfunction to overcome BRAF inhibitor resistance in advanced melanoma: the role of disulfiram as a copper ionophore

Abstract Resistance to targeted therapies poses a significant challenge in advanced melanoma with BRAF mutations. Even with a BRAF + MEK inhibitor combination, about 70% of patients experience disease progression within two years, highlighting the need for novel strategies beyond MAPK signaling inhibition. This study investigates whether mitochondrial dysfunction induced by the copper ionophore disulfiram (DSF) can effectively counteract resistance to BRAF inhibitors. We established two BRAF inhibitor (BRAFi)-resistant melanoma cell lines using BRAF mutant 451Lu and UACC62. In vivo experiments were conducted using subcutaneous implantation in nude mice. Cell viability and colony formation assays assessed treatment efficacy, while mitochondrial morphology was evaluated via transmission electron microscopy. Mitochondrial respiration was measured using a Seahorse metabolic analyzer, and oxidative stress was assessed through flow cytometry and confocal microscopy. RNA sequencing identified downstream factors regulated by intracellular copper levels, and the CRISPR-Cas9 system was used to knock out candidate genes in BRAFi-resistant cells for mechanistic validation. We provided evidence that DSF induced cell death in BRAFi-resistant melanoma in a copper-dependent manner, severely impairing mitochondrial structure and function through increased oxidative stress. RNA-seq and immunoblotting revealed that thioredoxin-interacting protein (TXNIP) expression significantly increased in response to DSF. TXNIP knockout reduced DSF-induced cytotoxicity by mitigating oxidative stress. These findings were supported by in vivo experiments. Furthermore, we demonstrated that the oxidative damage mediated by TXNIP involved its interaction with thioredoxin 2 (TRX2). In conclusion, targeting mitochondrial function with disulfiram effectively inhibits BRAFi-resistant melanoma cells, independent of MAPK signaling blockage. These results point to the potential of combining disulfiram with BRAF inhibitors as a promising strategy to overcome BRAFi resistance.