Abstract 12: Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression
Authors: DeBlasi Janine M. , Falzone Aimee , Caldwell Samantha , Kang Yun Pyo , Prieto-Farigua Nicolas , Prigge Justin R. , Schmidt Edward E. , Chio Iok In Christine , Karreth Florian A. , DeNicola Gina M. 
Published: June 2023

Abstract
KEAP1 negatively regulates the transcription factor NRF2, which regulates the response to oxidative stress. Mutations in the KEAP1-NRF2 pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases, leading to constitutive activation of NRF2 and resistance to therapy in patients. To study the role of NRF2 activation in lung cancer, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and performed a thorough interrogation of how these mutations affect tumor initiation and progression. Following targeted activation of these alleles in the lung with adenoviral-Cre, immunohistochemical analysis of Nrf2 and its target Nqo1 revealed that the Keap1R554Q/R554Q mutant was the most activating towards Nrf2, followed by the Nrf2D29H/+ and Keap1R554Q/+ mutants. We found that chronic Nrf2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors p53 or Lkb1. We also found that constitutive Nrf2 activation promotes lung tumor initiation and early progression of KrasG12D cells to low-grade tumors.
To analyze the influence of Keap1/Nrf2 mutation on later progression, we crossed these mutations into the KrasG12D; Trp53fl/fl model of lung adenocarcinoma. Surprisingly, we found that Keap1R554Q/R554Q mutation blocked progression to advanced-grade tumors. We also found that advanced-grade tumors downregulated Nrf2 expression and activity, suggesting that excess Nrf2 activation is detrimental to tumor progression. Next, to test whether the progression block in the Keap1R554Q/R554Q model was dependent on Nrf2, we performed single copy deletion of Nrf2. We found that lowering Nrf2 levels in this model rescued the impairment in tumor progression. Finally, using a NSCLC cell culture system of NRF2 overexpression, we found that high NRF2 levels impaired cell proliferation, viability, and anchorage-independent growth. In our in vitro studies, we observed that NRF2 expression was rapidly downregulated to basal levels to restore proliferation.
Our findings indicate that NRF2 promotes lung tumor initiation and early progression but impairs late progression in a dosage-dependent manner. Our preliminary data also suggests that NRF2 hyperactivation is detrimental to tumor cell proliferation. Collectively, our results establish the context-dependence and threshold for NRF2 during the tumorigenic process. Future work will include interrogating the mechanism of NRF2 downregulation in vivo and use of additional cell lines and systems to investigate how NRF2 overexpression can impair tumor cell proliferation.
Citation Format: Janine M. DeBlasi, Aimee Falzone, Samantha Caldwell, Yun Pyo Kang, Nicolas Prieto-Farigua, Justin R. Prigge, Edward E. Schmidt, Iok In Christine Chio, Florian A. Karreth, Gina M. DeNicola. Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 12.