抑制谷氨酰胺代谢联合放射破坏线粒体功能并损害同源重组

A mainstay of lung cancer treatment is radiotherapy. With our ever-increasing ability to genomically stratify patients, it is now possible to identify biomarkers that contribute to radioresistance or radiosensitivity. Mutations in the gene Kelch-like ECH-associated protein 1 (KEAP1) are associated with resistance to radiotherapy resulting in greater local recurrence and poorer patient outcomes. Mutations in KEAP1 lead to Nuclear Factor (erythroid-derived) 2-like Factor (Nrf2) nuclear translocation where it acts as a transcription factor promoting the formation of antioxidant proteins. However, while tumors harboring KEAP1 mutations are more resistant to therapy, they also rely heavily on the amino acid glutamine, which is processed by the enzyme Glutaminase-1 (GLS1) to meet the metabolic demands associated with the constitutive activation of Nrf2. Disrupting glutamine metabolism in KEAP1 mutant cells and tumors using small molecule inhibitors has shown promise as a radiosensitizing strategy. At a basic mechanistic level this has been attributed to a reduced glutamate level, leaving cells unable to produce the antioxidant glutathione, therefore increasing oxidative stress following radiotherapy. This in turn contributes to greater levels of DNA damage and more cell death. Our data support the radiosensitizing effect of GLS1 inhibition in KEAP1 mutant cells, for both photons and protons, with associated increases in oxidative stress, specifically mitochondrial superoxide. In addition to this basic concept, we hypothesize that the profound metabolic disruption induced by inhibiting glutamine metabolism has implications for not only DNA damage induction but also DNA repair. To investigate this, we first explored DNA damage response (ATM) and pathway specific DNA repair (Rad51) proteins, alongside the comet assay to measure total DNA damage. Our results identified that the inhibitor IACS-6274, which inhibits GLS1, increased total DNA damage measured by the alkaline comet assay but abrogated foci formation for both phosphorylated-ATM and Rad51 (homologous recombination [HR]). We also observed significant synergy when IACS-6274 was combined with PARP or ATR inhibitors in the absence of radiation. In parallel, we determined that mitochondrial membrane potential measured using the dye, TMRM, is significantly reduced along with reduced ATP production in GLS1 inhibited cells. In summary, our results show that KEAP1 mutant lung cancer cells treated with a GLS1 inhibitor can be significantly radiosensitized to radiotherapy, and this in part maybe attributable to reduced HR proficiency. We propose that either bioenergetic crisis prevents fueling of HR processes or that impaired TCA cycle anaplerosis reduces alpha-ketogluterate levels, which is an important co-factor for chromatin remodeling preventing DNA end resection and resulting Rad51 foci formation.