Radiotherapy combined with anti-PD-1 immunotherapy promotes ferroptosis-driven control of hepatocellular carcinoma

Abstract Combination of radiotherapy (RT) and anti-PD-1 immunotherapy (IO) has shown significant efficacy in treating hepatocellular carcinoma (HCC). Nevertheless, yet the underlying mechanisms remain incompletely understood. A Hepa1-6 mouse HCC model was established to explore the anti-tumor mechanism of combination therapy in HCC. Notably, combination therapy effectively inhibited tumor growth in mice bearing Hepa1-6 tumors. Through MeRIP-sequencing, we indicated that combination therapy increased m6A modification and reduced mRNA expression of Hspb1, a negative regulator of ferroptosis, in tumors from mice. Both combination therapy and Hspb1 downregulation significantly induced Hepa1-6 cell ferroptosis. Metabolomics analysis revealed that Hspb1 downregulation further promoted abnormal lipid metabolism in Hepa1-6 tumor-bearing mice, enhancing pro-ferroptosis effects of combination therapy. Meanwhile, Hspb1 downregulation further enhanced RT and IO-induced anti-tumor immune response in tumor-bearing mice, as evidenced by significantly elevated numbers of cytotoxic CD8 + T cells. Additionally, combination therapy also significantly downregulated RNA demethylase Alkbh5 in tumor-bearing mice. Overexpression of Alkbh5 increased Hspb1 expression and inhibited ferroptosis, indicating that Alkbh5 regulates ferroptosis through targeting Hspb1. Targeting Alkbh5/Hspb1/ferroptosis axis may enhance anti-tumor effects in combination therapy, highlighting a potential therapeutic approach for HCC.