Donafenib and GSK‐J4 Synergistically Induce Ferroptosis in Liver Cancer by Upregulating HMOX1 Expression

Abstract Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. Donafenib is a multi‐receptor tyrosine kinase inhibitor approved for the treatment of patients with advanced HCC, but its clinical effect is very limited. Here, through integrated screening of a small‐molecule inhibitor library and a druggable CRISPR library, that GSK‐J4 is synthetically lethal with donafenib in liver cancer is shown. This synergistic lethality is validated in multiple HCC models, including xenograft, orthotopically induced HCC, patient‐derived xenograft, and organoid models. Furthermore, co‐treatment with donafenib and GSK‐J4 resulted in cell death mainly via ferroptosis. Mechanistically, through integrated RNA sequencing (RNA‐seq) and assay for transposase‐accessible chromatin with high throughput sequencing (ATAC‐seq) analyses, that donafenib and GSK‐J4 synergistically promoted the expression of HMOX1 and increased the intracellular Fe2+ level is found, eventually leading to ferroptosis. Additionally, through cleavage under targets & tagmentation followed by sequencing (CUT&Tag‐seq), it is found that the enhancer regions upstream of HMOX1 promoter significantly increased under donafenib and GSK‐J4 co‐treatment. A chromosome conformation capture assay confirmed that the increased expression of HMOX1 is caused by the significantly enhanced interaction between the promoter and upstream enhancer under dual‐drug combination. Taken together, this study elucidates a new synergistic lethal interaction in liver cancer.