Abstract 369: A novel class of multitarget small molecule PI3K-CDK4/6-CDK9-AURAKA/B inhibitor harnesses Warburg effect against non-small cell lung cancer

Abstract Introduction: In silico designed novel multitarget small molecule PI3K-CDK4/6-CDK9-AURKA/B inhibitor LCI139 is nanomolar potent against NSCLC cells of mixed histologic and mutational backgrounds. We previously established that higher NSCLC cells metabolic activity promotes LCI139 potency. Here, we describe how LCI139 harnesses the Warburg effect to induce synthetic lethality in NSCLC. Methods: LCI139-sensitive (NCI-H1703) and resistant (NCI-H1781) human NSCLC cells, identified from prior IC50 experiments, were treated with LCI139 (0.25 and 0.5μM) or single agent inhibitors PI3Ki (BKM120), CDK4/6i (Ribociclib), CDK9i (AZD4573), and AURKAi (Alisertib). Apoptosis and senescence were measured by flow cytometry (Annexin V/7-AAD or β-gal). Mitochondrial respiration and mitochondrial complexes activity was measured with Clark-type electrode. Target inhibition, glycolysis and OXPHOS enzymes, AMPK-FoxO3 signaling, Bcl2-family proteins, and caspase expression/activation were evaluated by Western blot and qRT-PCR on whole cellular extract or isolated mitochondria. Results: LCI139 induced caspase-dependent apoptosis in sensitive H1703 cells by promoting Cas8 activation, BIM expression, Bax-Bak complex formation, mitochondrial fission and apoptosome formation (Cas9). In resistant H1781 cells, LCI139 promoted cellular senescence by inducing mitochondrial respiration and oxidative stress (ROS-forming complexes I-III activity), while repressing caspase activation. Bioenergetic stress sensor AMP-activated protein kinase (AMPK) and downstream forkhead box O3 transcription factor (FOXO3) signaling played a key role in LCI139 response. Upon treatment of H1703 sensitive cells, pAMPK levels increased and FoxO3 was expressed/stabilized, leading to subsequent pro-apoptotic Cas8/BIM expression. Compared with H1781 resistant cells, H1703 cells had greater baseline glycolytic activity and mitochondrial respiration. Consequently, LCI139 profoundly disrupted both glycolytic-ATP (glucose uptake [Glut1] and catabolism [PFK, LDH] reduction), and OXPHOS-ATP production (mitochondrial complexes I-IV loss) in H1703 sensitive but not H1781 resistant cells. Single target inhibition experiments showed that the metabolic stress response to LCI139 was primarily driven by PI3Ki and CDK4/6i warheads and promoted both Cas8 and BIM. Inhibition of CDK9 alone was a potent inducer of subsequent Cas8 activation and, together with AURKAi, promoted cellular apoptosis in H1703 sensitive cells Conclusions: Multitarget inhibitor, LCI139 is uniquely designed to harness the Warburg effect to induce synthetic lethality in glycolysis-reliant NSCLC by 1) inducing metabolic stress (pAMPK), 2) lowering the threshold to metabolic stress response (FoxO3 stabilization), and 3) enhancing caspase-dependent cell death non-canonical Cas8 activation. Citation Format: Anna V. Ivanina Foureau, Cody C. McHale, Hailey L. Dryden, Krishnaiah Maddeboina, David M. Foureau, Fei Guo, Donald L. Durden, Kathryn F. Mileham. A novel class of multitarget small molecule PI3K-CDK4/6-CDK9-AURAKA/B inhibitor harnesses Warburg effect against non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 369.