Abstract A003: Enhancer amplification defines lineage addiction in human lung adenocarcinoma

Abstract Lung cancer is the most common cause of death from cancer worldwide, and lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. NKX2-1 (also known as TTF-1, TITF-1) is a lineage defining transcription factor throughout normal lung development. In LUAD, NKX2-1 is a highly specific and sensitive marker for 85-90% of both primary and metastatic LUADs. We and others identified NKX2-1 as the most significantly amplified gene in LUAD. NKX2-1 is amplified as the earliest stages of LUAD development, and NKX2-1 amplification is a truncal event in multi-region LUAD evolution. Despite strong evidence for an oncogenic role for NKX2-1, little is known about the mechanisms of NKX2-1 activation, or how its oncogenic regulation drives LUAD. Here, we identify recurrent focal amplification targeting a super-enhancer (SE) of NKX2-1 as a driving event in LUAD. Using epigenomic data from LUAD cell lines and primary samples, we identify this region as a lineage super-enhancer that is specifically active in NKX2-1(+) cell lines and patients. Notably, this region is co-amplified with NKX2-1 in 95.9% (143/149) of NKX2-1-amplified samples, suggesting this region is a hallmark of NKX2-1 activation, through focal or co-amplification with NKX2-1. Using endogenous ChIP-seq and exogenous luciferase assays, we show the enhancer activity of the NKX2-1 SE is comprised of three constituent enhancers, and map the transcriptional activity of the strongest enhancer to individual binding motifs for AP-1 and ETS. Using CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) in LUAD cell lines expressing high or low levels of NKX2-1, we show that activity of the NKX2-1 SE defines endogenous NKX2-1 expression. Using genome-wide shRNA and CRISPR screens, we identify a NKX2-1 dependency in NKX2-1 positive LUAD cell lines, and validate this dependency in clonogenic and proliferation assays. Using RNA sequencing (RNA-seq), we find that NKX2-1 knockdown activates an epithelial-mesenchymal transition (EMT) gene signature and downregulates an alveolar differentiation signature, including critical markers of LUAD differentiation, such as NAPSA, LMO3, and SLC34A2. Using a global clustering approach to CCLE RNA-seq of LUAD cell lines, we find that NKX2-1(+) LUAD cell lines cluster apart from NKX2-1(–), and that NKX2-1 knockdown regulates the majority (57/97) of genes that distinguish NKX2-1(+) LUADs. Similarly, we find that global clustering of H3K27ac ChIP-seq in LUAD cell lines separates NKX2-1(+) cells, and identify enhancers that are uniquely active NKX2-1(+) LUAD cell lines. We find these enhancers are bound and activated by NKX2-1, regulating genes we identified above such as NAPSA and LMO3. Our data demonstrates that enhancer amplification is a hallmark of oncogenic NKX2-1 activation in LUAD, through which NKX2-1 drives a lineage addicted state and oncogenic cell proliferation. This suggests that NKX2-1 is a critical defining oncogene for LUAD, and that targeting of NKX2-1 or its enhancer may suppress LUAD. Citation Format: John L. Pulice, Matthew Meyerson. Enhancer amplification defines lineage addiction in human lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A003.