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

<p indent="0mm">Acquired resistance to osimertinib poses a major challenge in treating EGFR-mutant lung adenocarcinoma (LUAD). Here, we demonstrate that shikonin, a natural compound from <italic>Lithospermum erythrorhizon</italic>, 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. <italic>HES1</italic> knockdown replicates the combination’s effects, confirming its key role. Mechanistically, <italic>ATF3</italic> is identified as a direct transcriptional target of <italic>HES1</italic>, with <italic>HES1</italic> binding to the <italic>ATF3</italic> promoter (-786 to <sc>-779 nt)</sc> to repress its transcription. The shikonin-osimertinib combination relieves this repression, leading to ATF3-mediated suppression of the ferroptosis regulators SLC7A11 and GPX4. <italic>In vivo</italic>, 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.