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

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 find that disruption of dNTP homeostasis plays a critical role in determining sensitivity to the EGFR inhibitor osimertinib (Osi) and in shaping mechanisms of acquired resistance. Transcriptomic and biochemical analyses revealed that Osi suppresses RRM2 expression, a key regulator of dNTP synthesis, through downregulation of the transcription factor MYBL2. In response to Osi-mediated replication stress and dNTP depletion, cells activated a compensatory pathway involving the stress-inducible ribonucleotide reductase subunit RRM2B via a novel transcriptional regulator, TNNT3. We further identified CHK2 signaling as 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 Osi resistance both in vitro and in xenograft models. Collectively, these findings reveal that EGFR-mutant NSCLC cells rely on a dynamic EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory axis to maintain dNTP pool balance under therapeutic pressure. Disruption of this axis sensitizes tumors to Osi and impairs the acquisition of resistance, highlighting dNTP metabolism as a critical vulnerability and actionable target in EGFR-driven lung cancer. SIGNIFICANCE: Targeting dNTP metabolism sensitizes EGFR-mutant NSCLC to osimertinib treatment and delays drug resistance, revealing a novel and actionable therapeutic vulnerability in EGFR-driven lung cancer.