Abstract A003: Enhancer amplification defines lineage addiction in human lung adenocarcinoma
Authors: Pulice John L. , Meyerson Matthew 
Published: December 2022

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.