Suppression of Lung Adenocarcinoma Progression by Nkx2-1

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras[superscript LSL-G12D/+];p53[superscript flox/flox] mice initiates lung adenocarcinoma development4. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limitsmetastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions ofNkx2-1 in the sametumourNational Institutes of Health (U.S.) (grant U01-CA84306 )National Institutes of Health (U.S.) (grant K99-CA151968)Howard Hughes Medical InstituteLudwig Center for Molecular OncologyNational Cancer Institute (U.S.) (Cancer Center Support (core) grant P30-CA14051

A versatile system to record cell-cell interactions.

Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of the cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes

An Arntl2-Driven Secretome Enables Lung Adenocarcinoma Metastatic Self-Sufficiency

The ability of cancer cells to establish lethal metastatic lesions requires the survival and expansion of single cancer cells at distant sites. The factors controlling the clonal growth ability of individual cancer cells remain poorly understood. Here, we show that high expression of the transcription factor ARNTL2 predicts poor lung adenocarcinoma patient outcome. Arntl2 is required for metastatic ability in vivo and clonal growth in cell culture. Arntl2 drives metastatic self-sufficiency by orchestrating the expression of a complex pro-metastatic secretome. We identify Clock as an Arntl2 partner and functionally validate the matricellular protein Smoc2 as a pro-metastatic secreted factor. These findings shed light on the molecular mechanisms that enable single cancer cells to form allochthonous tumors in foreign tissue environments

LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer.

LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the cancer state that stems from Lkb1 deficiency can be reverted remains unknown. To identify the processes governed by LKB1 in vivo, we generated an allele which enables Lkb1 inactivation at tumor initiation and subsequent Lkb1 restoration in established tumors. Restoration of Lkb1 in oncogenic KRAS-driven lung tumors suppressed proliferation and led to tumor stasis. Lkb1 restoration activated targets of C/EBP transcription factors and drove neoplastic cells from a progenitor-like state to a less proliferative alveolar type II cell-like state. We show that C/EBP transcription factors govern a subset of genes that are induced by LKB1 and depend upon NKX2-1. We also demonstrate that a defining factor of the alveolar type II lineage, C/EBPα, constrains oncogenic KRAS-driven lung tumor growth in vivo. Thus, this key tumor suppressor regulates lineage-specific transcription factors, thereby constraining lung tumor development through enforced differentiation

An Arntl2-Driven Secretome Enables Lung Adenocarcinoma Metastatic Self-Sufficiency.

The ability of cancer cells to establish lethal metastatic lesions requires the survival and expansion of single cancer cells at distant sites. The factors controlling the clonal growth ability of individual cancer cells remain poorly understood. Here, we show that high expression of the transcription factor ARNTL2 predicts poor lung adenocarcinoma patient outcome. Arntl2 is required for metastatic ability in vivo and clonal growth in cell culture. Arntl2 drives metastatic self-sufficiency by orchestrating the expression of a complex pro-metastatic secretome. We identify Clock as an Arntl2 partner and functionally validate the matricellular protein Smoc2 as a pro-metastatic secreted factor. These findings shed light on the molecular mechanisms that enable single cancer cells to form allochthonous tumors in foreign tissue environments

Abstract 827: A journey to deconvolute the multifaceted functions and context-dependency of cancer driver genes

Abstract Lung cancer is a lethal and genomically-complex disease. Structural genomics has largely advanced our knowledge of genomic alterations, yet the function of a majority of altered genes remains less clear. Previous in silico and in vitro functional genomics data often lead to contradictory conclusions on gene functions. Genetically-engineered mouse models are reliable approaches for in vivo functional analyses, but development of these models are lagging behind due to the throughput limit. To overcome this throughput limit, we developed tumor barcoding and ultradeep barcode sequencing (Tuba-seq) that precisely quantifies the growth metrics of hundreds of tumor genotypes, which is a huge leap forward. Through this approach, we have begun a journey to create a quantitative functional taxonomy of tumor suppression in oncogenic KRAS-driven lung cancer. For example, STAG2 and CDKN2C emerged as novel functional tumor suppressor genes in the lung, when they were often overlooked by computational analyses due to relatively low mutation prevalence. Interestingly, STK11 and PTEN, both playing an important role in tumor growth, exhibit distinct roles in tumor initiation. These findings suggest that structural genomics is not sufficient to predict cancer driver genes, and calls for closer investigation of tumor suppressor functions in specific tumorigenesis stages. Furthermore, the quantitative nature of our data has enabled systematic characterization of interactions between tumor suppressor genes. For instance, RNF43 exhibits different tumor suppression modes in the presence or absence of STK11 or TRP53, while TRP53 can play opposite roles in PTEN- and RB1-deficient tumors. In addition, Foggetti et al. (2021) reported that tumor suppressors can play opposite roles in the contexts of different oncogenes. Collectively, these findings suggest that cooccurring mutations shift the functional landscape of tumor suppressors even in the same pathological subtype of cancer. Given the genomic diversity of lung cancer patients, driver genes may change case by case. We are now investigating the molecular mechanisms underlying these tumor suppressors and their genetic interactions. Our findings underscore the necessity of determining the consequences of enormous combinations of genomic alterations in their natural environment, which is challenging but critical for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. Citation Format: Hongchen Cai, Su Kit Chew, Chuan Li, Christopher W. Murray, Laura Andrejka, Jess D. Hebert, Min K. Tsai, Rui Tang, Nicholas W. Hughes, Emily G. Shuldiner, Emily L. Ashkin, Shi Ya C. Lee, Maryam Yousefi, Dmitri A. Petrov, Charles Swanton, Monte W. Winslow. A journey to deconvolute the multifaceted functions and context-dependency of cancer driver genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 827

Zmat3 Is a Key Splicing Regulator in the p53 Tumor Suppression Program.

Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens in vivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression

A Functional Taxonomy of Tumor Suppression in Oncogenic KRAS-Driven Lung Cancer.

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. SIGNIFICANCE: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS-driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models.This article is highlighted in the In This Issue feature, p. 1601

A multiplexed in vivo approach to identify driver genes in small cell lung cancer

Summary: Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets