AZ-628 sensitizes donafenib in hepatocellular carcinoma by targeting tyrosine kinase pathway and ferroptosis.

[OBJECTIVE] Hepatocellular carcinoma (HCC) represents a primary liver tumor characterized by rapid disease progression and unfavorable clinical outcomes. Most patients with HCC are identified in advanced stage, where targeted therapies are considered an effective treatment method for advanced disease. The tyrosine kinase inhibitor (TKI) donafenib has shown efficacy in managing HCC. However, drug resistance often occurs after treatment with donafenib, which limits its widespread clinical application. Thus, this study aims to identify small-molecule TKIs that can enhance the sensitivity of HCC to donafenib.

[MATERIAL AND METHODS] The HCC cells HepG2 and SNU449 were treated with five drugs, namely, dimethyl sulfoxide, AZ-628, SU-5402, TG-101209, and SPP-86, combined with donafenib to determine half-maximal inhibitory concentration values. RNA sequencing data obtained from The Cancer Genome Atlas (TCGA) were analyzed using Differential Expression analysis for Sequence data 2 (DESeq2)/limma and Gene Set Enrichment Analysis (GSEA). The effects of AZ-628 on proliferation, viability, apoptosis, and migration were assessed. The expression level of early growth response gene 1 (EGR1) was measured through Western blotting/quantitative polymerase chain reaction and silenced by cell transfection. Donafenib-resistant HepG2 cells negative control shRNA (shNC)/shRNA targeting EGR1 (shEGR1) were treated with AZ-628 combined with donafenib. Ferrous ion (Fe) and reactive oxygen species levels were measured after Erastin/RSL3 induction. The synergy between AZ-628 and donafenib was analyzed using Combenefit2. , tumor growth, and Ki67 expression were evaluated in nude mice treated with DMSO, AZ-628, donafenib, or their combination.

[RESULTS] This study showed that AZ-628 reduced donafenib resistance in HCC by targeting the tyrosine kinase (TK) pathway. Cell counting kit-8 and colony formation assay validated that AZ-628 significantly improved the sensitivity of HCC cells to donafenib ( < 0.0001). Rescue experiments showed that AZ-628 regulated HCC cell proliferation and drug resistance through EGR1 ( < 0.001). In addition, AZ-628 was found to affect donafenib resistance in HCC by regulating epithelial-mesenchymal transition, apoptosis, and ferroptosis ( < 0.0001). experiments demonstrated a combined anti-tumor efficacy of AZ-628 and donafenib in HCC models ( < 0.0001).

[CONCLUSION] The findings of this study reveal a new combination therapy targeting the TK pathway for the treatment of HCC and provide a theoretical foundation for addressing donafenib resistance.