Abstract LB322: Investigating the role of <i>Nfe2l2</i> in early <i>Alk</i>-dependent lung adenocarcinoma tumorigenesis

Abstract ALK fusions are the third most-prevalent mutation in lung adenocarcinoma (LUAD). Afflicting never-smokers, ALK-positive lung cancer presents unique diagnostic challenges and treatment resistance compared to other LUAD subtypes. To better understand the time course of tumorigenesis of Alk-mutant LUAD, to make inferences regarding cell of origin, and to understand the earliest cellular changes that occur in ALK-dependent transformation, we studied a previously published mouse tumor model whereby the Eml4-Alk translocation is created using an adenovirus carrying the CRISPR/Cas9 machinery (Ad5-EA-Cas9) administered via intratracheal instillation. Lung tissues were collected from these mice at intervals ranging from 2 to 12 weeks post-infection and were either fixed for histological staining or flash frozen for nuclear extraction and single-nucleus sequencing analysis. Detection of Eml4-Alk fusion gene expression was confirmed through fluorescent in situ hybridization (FISH) and RT-qPCR. We conducted single-nucleus RNA sequencing (snRNA-seq) and ATAC-sequencing (snATAC-seq), to characterize cellular states, gene expression, and chromatin status. Our analysis revealed an increase in Alk-expressing cells over time and identified two distinct tumorigenic pathways: one originating from alveolar type 2 cells (AT2) and another from club cells which gain plasticity upon ALK activation. Beyond identifying putative cells of origin and tumorigenesis trajectories, we examined changes in transcription factor activity. Of particular interest, the motif of nuclear factor erythroid 2-like 2 (Nfe2l2, also known as Nrf2), a key regulator of oxidative stress, was enriched in our snATAC-seq data, and its target genes were significantly up-regulated. To validate these findings, we induced Eml4-Alk translocation in Nfe2l2-knockout mice and observed significant differences in tumor formation. 8 weeks after Eml4-Alk induction in the lung, Nfe2l2 -/- mice showed significantly larger, but fewer tumor lesions compared to controls with differential gene expression of various lung epithelial cell markers. Data in human ALK-positive patients also suggests upregulation of NRF2 activity in early-stage tumors. Altogether, our data points to Nfe2l2 and its downstream pathways playing critical roles in promoting early tumorigenesis and possibly plasticity in ALK-mutant LUAD. Citation Format: Yusoo Lee, Ling Mengkun Yang, Xu Cheng, Xiangning Dong, Indrani Talukder, Micheal S. Downey, Lila Tudrick, Zhiwei Xiao, Tristan Frum, Varun Ponnusammy, Mark D. Slayton, Yatrik M. Shah, Sofia D. Merajver, Jason R. Spence, Peggy P. Hsu. Investigating the role of Nfe2l2 in early Alk-dependent lung adenocarcinoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB322.