Transcriptome sequencing analysis of BRAF-mutant melanoma metastases.

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. In this study, we used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was β-sitosterol, a well-tolerated and brain-penetrable phytosterol. We found that β-sitosterol attenuated melanoma cell growth in vitro and significantly inhibited brain metastasis in vivo. Large-scale phosphoproteomic and in silico analyses indicated that the therapeutic potential of β-sitosterol was linked to mitochondrial interference. Mechanistically, β-sitosterol effectively reduced mitochondrial respiration and respiratory capacity in melanoma cells, mediated by a selective inhibition of mitochondrial complex I. This led to increased oxidative stress and apoptosis. Notably, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either β-sitosterol or a functional knockdown of mitochondrial complex I. Based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, β-sitosterol represents a promising candidate for drug repurposing in patients with, or at risk for, melanoma brain metastases. A total of 12 samples were analyzed by transcriptome sequencing (RNA-seq) in this study. The study included metastaic xenograft tumors derived from intracardiac injection of H1_DL2 melanoma cells into 5 mice. Metastases to brain, brain, adrenals, ovaries, and femurs in each mouse were identified by bioluminescence imaging, and metastic tumor cells were collected and counted by cell sorting for GFP fluorescence. Replicate tumor cell samples (triplicates) with the highest number of cells from each organ metastasis were selected for RNA-seq analysis.