Second-generation BRAF inhibitor Encorafenib resistance is regulated by NCOA4-mediated iron trafficking in the drug-resistant malignant melanoma cells

The current study established the first in vitro Encorafenib resistance protocol in BRAF-mutated malignant melanoma (MM) cells and investigated the resistance-related mechanisms. RNA-Seq results exhibited altered epigenetic regulation of resistance; particularly ferritin family members, ion transport pathways. Then, increased NCOA4, FTH1, and iron levels detected in A375-R suggest that the iron metabolism-related mechanism, such as ferritinophagy, might be triggered, which was supported by TEM and oxidative stress analysis. Iron storage, transport, and ferritinophagy have the promising potential to be targeted for combining with BRAF-targeted therapy to reverse Encorafenib resistance in MM. Moreover, this is the first study evaluating in vitro Encorafenib resistance mechanisms, and we suggest that our findings contribute to improving new drug combinations targeting BRAF and iron metabolism in different MM cells. To determine the effect of Encorafenib resistance on transcriptional regulation, transcriptome sequencing was performed. Total RNA extraction was carried out using the RNeasy Mini Kit (Hilden, Germany) from resist and sensitive cells. To perform RNA sequencing, mRNA and small RNA libraries were generated using the NGS sequencing Illumina NextSeq 500 platform with the SMARTer smRNA-Seq Kit (Illumina, Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. The libraries were gel-purified and assessed using the D1000 ScreenTape System (Agilent Technologies, Waldbronn, Germany).