Adaptive Regulation of dNTP Homeostasis Confers Osimertinib Resistance in EGFR-Mutant Non-small Cell Lung Carcinoma

Abstract Maintaining sustained deoxyribonucleotide triphosphate (dNTP) pools is essential for DNA replication fidelity and genome stability. In EGFR-mutant non-small cell lung carcinoma (NSCLC), we found that disruption of dNTP homeostasis plays a critical role in determining sensitivity to the EGFR inhibitor osimertinib and in shaping mechanisms of acquired resistance. Transcriptomic and biochemical analyses revealed that osimertinib suppresses RRM2 expression, a key regulator of dNTP synthesis, through downregulation of the transcription factor MYBL2. In response to osimertinib-mediated replication stress and dNTP depletion, cells activated a compensatory pathway involving the stress-inducible ribonucleotide reductase subunit RRM2B via a transcriptional regulator, TNNT3. CHK2 signaling was essential for TNNT3 nuclear translocation and RRM2B transcriptional activation. Inhibition of CHK2 or combined CHK1/2 blockade impaired RRM2B induction, exacerbated replication stress, and delayed the development of osimertinib resistance both in cell lines and in xenograft models. Collectively, these findings reveal that EGFR-mutant NSCLC cells rely on dynamic signaling through EGFR–MYBL2–RRM2 and CHK2–TNNT3–RRM2B regulatory pathways to maintain dNTP pool balance under therapeutic pressure. Disruption of this signaling network sensitizes tumors to osimertinib and impairs the acquisition of resistance, linking metabolic regulation to therapeutic resistance and disease progression.