Abstract 5429: NRF2 activation sensitizes the Fe-S cluster synthesis pathway to iron restriction

Abstract Enhanced mitochondrial metabolism in human lung tumors relative to normal lung and lung tumorigenesis offers a potential therapeutic vulnerability in non-small cell lung cancer (NSCLC) due to the dependence on iron-sulfur (Fe-S) clusters. Recent data indicates that the incorporation of Fe-S clusters into recipient proteins is influenced by iron availability, with possible hierarchical loading when iron is limited. The labile iron pool which Fe-S clusters derive iron from is partly regulated by the transcription factor NRF2, which coordinates restriction of the labile iron pool through increased storage and efflux of free iron when activated. Given the aberrant activation of NRF2 in a significant proportion of NSCLC cases due to amplification or mutations in its negative regulator, KEAP1, we hypothesized that NRF2 regulation of intracellular iron availability may sensitize the Fe-S cluster synthesis pathway in NSCLC when iron is limiting. To investigate this potential vulnerability, we stratified 14 NSCLC lines based on the activation status of NRF2 and evaluated the sensitivity of Fe-S cluster biology to iron chelation with deferoxamine (DFO). Seahorse analyses of mitochondrial function showed that the decline in mitochondrial respiratory function under iron starvation is exacerbated in NRF2-high cells compared to NRF2-low counterparts. Furthermore, 13C-glucose tracing under DFO treatment revealed a significantly greater reduction in M+2 isotopologue enrichment of TCA cycle intermediates in NRF2-high cells relative to NRF2-low cells. In association with this enhanced mitochondrial metabolic dysfunction, we observe a more rapid loss of Fe-S protein activities such as aconitase and respiratory super complexes I-III and II-III in NRF2-high cells relative to NRF2-low cells following DFO treatment. Importantly, we find that NRF2 knockout protects against the loss of Fe-S protein function in response to iron restriction. Moreover, we find that the loss of mitochondrial iron in response to DFO treatment is dampened in NRF2 knockout cells, indicating that NRF2 coordinates mitochondrial iron availability in a manner relevant to Fe-S cluster biology. Overall, our data indicates that NRF2 activation augments the sensitivity of the Fe-S cluster synthesis pathway to iron restriction in NSCLC, presenting a vulnerability with potential therapeutic relevance. Citation Format: Maddison A. Olley, Nathan P. Ward. NRF2 activation sensitizes the Fe-S cluster synthesis pathway to iron restriction [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 5429.