Shikonin sensitises osimertinib-resistant lung adenocarcinoma to ferroptosis through HES1-mediated ATF3 upregulation.

Acquired resistance to osimertinib poses a major challenge in treating EGFR-mutant lung adenocarcinoma (LUAD). Here, we demonstrate that shikonin, a natural compound from Lithospermum erythrorhizon, synergizes with osimertinib to overcome this resistance by inducing ferroptosis. In osimertinib-resistant LUAD cells (PC9-OR/H1975-OR), the shikonin-osimertinib combination significantly enhances cytotoxicity and suppresses proliferation while triggering ferroptotic events, including reactive oxygen species accumulation, lipid peroxidation, and iron overload alongside glutathione depletion. RNA-sequencing identifies Hes family BHLH transcription factor 1 (HES1) downregulation as crucial to this process. HES1 knockdown replicates the combination's effects, confirming its key role. Mechanistically, ATF3 is identified as a direct transcriptional target of HES1, with HES1 binding to the ATF3 promoter (-786 to -779 nt) to repress its transcription. The shikonin-osimertinib combination relieves this repression, leading to ATF3-mediated suppression of the ferroptosis regulators SLC7A11 and GPX4. In vivo, this combination potently inhibits tumor growth in xenograft models without observable toxicity. Our study reveals a novel HES1/ATF3 axis governing ferroptosis and proposes shikonin as a promising therapeutic adjuvant to overcome osimertinib resistance in LUAD.