Abstract 5752: Enhancer amplification defines lineage addiction in human lung adenocarcinoma
Authors: Pulice John Louis , Meyerson Matthew 
Published: April 2023

Abstract
Lung cancer is the most common cause of death from cancer worldwide, and lung adenocarcinoma (LUAD) is the most common type of lung cancer. NKX2-1 (also known as TTF-1, TITF-1) is a lineage defining transcription factor for normal lung development. In LUAD, NKX2-1 is a highly specific and sensitive marker for both primary and metastatic LUADs, with 85-90% expressing NKX2-1. We and others identified that NKX2-1 as the most significantly amplified gene in LUAD, with 22.5% of primary LUADs exhibiting copy number gains. 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 tumors, we identify this region as a alveolar lineage super-enhancer (NKX2-1 SE) that is specifically active in NKX2-1(+) LUAD cell lines and primary tumors. Notably, this region is co-amplified with NKX2-1 in 96-100% of NKX2-1-amplified samples, suggesting this region a critical component of the NKX2-1 amplicon. 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 demonstrate that the key enhancer elements can activate transcription of the NKX2-1 promoter. 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 RNA-seq, ChIP-seq, and ATAC-seq, we find that NKX2-1 controls the transcriptional and epigenomic landscape of lung adenocarcinoma. 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, SFTPA1/2, and HOPX, at which NKX2-1 mediates lineage enhancer accessibility to regulate target gene expression. Global clustering of LUAD cell lines by RNA-seq or H3K27ac ChIP-seq identifies a distinct NKX2-1(+) cluster, and we find that NKX2-1 directly regulates the genes (57/97) and enhancers that distinguish this subpopulation. Using genome-wide shRNA and CRISPR screens, we identify a NKX2-1 dependency in NKX2-1(+) LUAD cell lines, and find that the majority of NKX2-1(+) LUAD cell lines (11/14) assayed are dependent on NKX2-1.
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 Louis Pulice, Matthew Meyerson. Enhancer amplification defines lineage addiction in human lung adenocarcinoma. [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 5752.