NUAK1-Mediated Phosphorylation of NADK Mitigates ROS Accumulation to Promote Osimertinib Resistance in Non-Small Cell Lung Carcinoma

Abstract Osimertinib, a third generation epidermal growth factor receptor tyrosine kinase inhibitor, is approved as a first-line therapy in patients with advanced non–small cell lung carcinoma (NSCLC) with EGFR-activating mutations or the T790M resistance mutation. However, the efficacy of osimertinib is limited due to acquired resistance, highlighting the need to elucidate resistance mechanisms to facilitate the development of improved treatment strategies. Here, we screened for significantly upregulated genes encoding protein kinases in osimertinib-resistant NSCLC cells and identified NUAK1 as a pivotal regulator of osimertinib resistance. NUAK1 was highly expressed in osimertinib-resistant NSCLC and promoted the emergence of osimertinib resistance. Genetic or pharmacological blockade of NUAK1 restored the sensitivity of resistant NSCLC cells to osimertinib in vitro and in vivo. Mechanistically, NUAK1 directly interacted with and phosphorylated nicotinamide adenine dinucleotide kinase (NADK) at serine 64 (S64), which mitigated osimertinib-induced accumulation of reactive oxygen species (ROS) and contributed to the acquisition of osimertinib resistance in NSCLC. Furthermore, virtual drug screening identified T21195 as an inhibitor of NADK-S64 phosphorylation, and T21195 synergized with osimertinib to reverse acquired resistance by inducing ROS accumulation. Collectively, these findings highlight the role of the NUAK1-NADK axis in governing osimertinib resistance in NSCLC and indicate the potential of targeting this axis as a strategy for circumventing resistance. Significance: Phosphorylation of NADK by NUAK1 diminishes ROS accumulation and confers resistance to osimertinib, identifying NUAK1-NADK signaling as a potential therapeutic target for improving the response to EGFR inhibition in lung cancer.