Genome-Wide CRISPR/Cas9 Screening Reveals Lipid Metabolism and Inflammatory Signalling as Modulators of Ganoderic Acid DM Cytotoxicity
This study investigates the molecular mechanisms underlying the cytotoxic activity of Ganoderic Acid DM (GA DM), a lanostane-type triterpenoid derived from the medicinal mushroom Ganoderma lucidum. Despite its demonstrated anticancer activity in breast, melanoma and prostate cancer models, the precise genetic dependencies governing cellular sensitivity and resistance to GA-DM remain poorly characterised. To address this, we performed a genome-wide CRISPR/Cas9 knockout screen in the human melanoma cell line A375 using the Brunello pooled lentiviral library, comprising 76,441 single guide RNAs (sgRNAs) targeting 19,114 protein-coding genes. Cells were treated with GA-DM at the half-maximal inhibitory concentration (50 uM) or with DMSO in biological duplicates and sgRNA representation was profiled by next-generation sequencing at two timepoints (Day 7 and Day 14 post-treatment). Differential sgRNA abundance was analysed using the MAGeCK algorithm to identify genes whose loss conferred resistance (enriched sgRNAs) or sensitivity (depleted sgRNAs) to GA-DM. The screen identified key modulators of GA-DM cytotoxicity, including components of the SREBP-mediated lipid biosynthesis pathway, the ubiquitin-proteasome system, the Hippo signalling pathway and, at later timepoints, inflammatory NF-kB and Toll-like receptor signalling pathways. These findings implicate cholesterol homeostasis and inflammatory signalling as central determinants of GA-DM-induced cytotoxicity and provide a functional genomic framework for understanding the mechanism of action of this natural compound. This dataset comprises the raw FASTQ files from all sequenced samples, enabling independent reanalysis of sgRNA abundance and screen hit identification.