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.

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.

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.

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.

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.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication¹. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer^2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood^4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.