Addressing Clinical Limitations of Glutaminase Inhibitors: Novel Strategies for Osimertinib‐Resistant Lung Cancer by Exploiting Glutamine Metabolic Dependency

Abstract Overcoming acquired resistance to Osimertinib remains a critical challenge in treating NSCLC. This research indicates that Osimertinib‐resistant cells exhibit a strong dependence on glutamine metabolism. However, targeting GLS1 shows limited anticancer effects, probably because it cannot fully block the glutamine metabolic pathway. The investigation reveals that a more effective strategy involves simultaneously inhibiting both ASCT2 and GLS1. After confirming the efficacy of this dual‐targeting approach against Osimertinib‐resistant cells in preclinical models, the potential of utilizing a broad‐spectrum glutamine metabolism antagonist is further explored to achieve superior antitumor efficacy. DON, broad‐spectrum glutamine antagonist, presents toxicity issues. Herein, the high NQO1 expression in Osimertinib‐resistant NSCLC cells is leveraged to design an NQO1‐responsive DON prodrug, 10e (LBJ‐10e). This prodrug demonstrates superior safety compared to natural DON and greater antitumor activity against resistant tumors compared to the clinical phase II drug DRP104. These findings may address the clinical limitations of GLS1 allosteric inhibitors and underscore prodrug strategies in effectively treating Osimertinib‐resistant lung cancer, providing a foundation for future clinical trials.