RNA helicase DDX5 mediates adaptive response to multi-kinase inhibitors in liver cancer

Earlier studies linked sorafenib effectiveness to induction of ferroptosis. Herein we demonstrate sorafenib and mTKIs downregulate DDX5 in vitro and in vivo. To understand the effect of DDX5 downregulation, we compared TCGA-derived HCCs expressing low vs. high DDX5 focusing on ferroptosis-related genes. Glutathione Peroxidase 4 (GPX4), a key ferroptosis regulator, was significantly overexpressed in DDX5LOW HCCs. Importantly, DDX5-knockdown (DDX5KD) HCC cell lines lacked lipid peroxidation by GPX4 inhibition, indicating DDX5 downregulation suppresses ferroptosis. RNAseq analyses comparing wild type vs. DDX5KD cells in the presence or absence of sorafenib, identified a unique set of genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps genes from Wnt/ß-catenin and non-canonical NF-?B pathways, including NF-?B inducing Kinase required for non-canonical NF-?B activation. Pharmacologic inhibition of these pathways in combination with sorafenib reduced DDX5KD cell viability. Mechanistically, sorafenib-mediated NF-?B activation induced NRF2 transcription, while DDX5KD extended NRF2 half/life by stabilizing p62/SQSTM1, leading to enhanced expression of GPX4 and ferroptosis escape. Conclusion: Sorafenib/mTKI-mediated DDX5 downregulation results in adaptive mTKI resistance by enhancing NRF2 expression, leading to ferroptosis escape. We propose inhibition of the pathways leading to NRF2 expression will enhance the therapeutic effectiveness of sorafenib/mTKIs. Overall design: HepAD38WT and HepAD38-DDX5KD cells were treated with Sorafenib (7.5 µM) for 3 days prior to harvesting. Three independent biological replicates were prepared for RNA isolation and RNA sequencing. Total RNA submitted to Novogene for quality assessment and next-generation sequencing. Paired-end 2x50 bp sequencing performed using a HiSeq2500 system (Illumina).