Oncogenic context shapes the fitness landscape of tumor suppression.

Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged-the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context-and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.

Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAFV600E-Driven Lung Tumorigenesis.

Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty-mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers. Among the genes identified was Rbms3, which encodes an RNA-binding protein previously implicated as a putative tumor suppressor. Silencing of RBMS3 via CRISPR/Cas9 gene editing promoted growth of BRAFV600E lung organoids and promoted development of malignant lung cancers with a distinct micropapillary architecture in BRAFV600E and EGFRL858R GEM models. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of Ctnnb1, Ccnd1, Axin2, Lgr5, and c-Myc mRNAs, suggesting that RBMS3 silencing elevates signaling through the WNT/ß-catenin signaling axis. Although RBMS3 silencing rendered BRAFV600E-driven lung tumors resistant to the effects of dabrafenib plus trametinib, the tumors were sensitive to inhibition of porcupine, an acyltransferase of WNT ligands necessary for their secretion. Analysis of The Cancer Genome Atlas patient samples revealed that chromosome 3p24, which encompasses RBMS3, is frequently lost in non-small cell lung cancer and correlates with poor prognosis. Collectively, these data reveal the role of RBMS3 as a lung cancer suppressor and suggest that RBMS3 silencing may contribute to malignant NSCLC progression. SIGNIFICANCE: Loss of RBMS3 cooperates with BRAFV600E to induce lung tumorigenesis, providing a deeper understanding of the molecular mechanisms underlying mutant BRAF-driven lung cancer and potential strategies to more effectively target this disease.

KRAS-related proteins in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with a high mortality rate. Genetic and biochemical studies have shown that RAS signaling mediated by KRAS plays a pivotal role in disease initiation, progression and drug resistance. RAS signaling affects several cellular processes in PDAC, including cellular proliferation, migration, cellular metabolism and autophagy. 90% of pancreatic cancer patients harbor somatic oncogenic point mutations in KRAS, which lead to constitutive activation of the molecule. Pancreatic cancers lacking KRAS mutations show activation of RAS via upstream signaling through receptor mediated tyrosine kinases, like EGFR, and in a small fraction of patients, oncogenic activation of the downstream B-RAF molecule is detected. RAS-stimulated signaling of RAF/MEK/ERK, PI3K/AKT/mTOR and RalA/B is active in human pancreatic cancers, cancer cell lines and mouse models of PDAC, although activation levels of each signaling arm appear to be variable across different tumors and perhaps within different subclones of single tumors. Recently, several targeted therapies directed towards MEK, ERK, PI3K and mTOR have been assayed in pancreatic cancer cell lines and in mouse models of the disease with promising results for their ability to impede cellular growth or delay tumor formation, and several inhibitors are currently in clinical trials. However, therapy-induced cross activation of RAS effector molecules has elucidated the complexities of targeting RAS signaling. Combinatorial therapies are now being explored as an approach to overcome RAS-induced therapeutic resistance in pancreatic cancer.

TP53 Silencing Bypasses Growth Arrest of BRAFV600E-Induced Lung Tumor Cells in a Two-Switch Model of Lung Tumorigenesis.

Lung carcinogenesis is a multistep process in which normal lung epithelial cells are converted to cancer cells through the sequential acquisition of multiple genetic or epigenetic events. Despite the utility of current genetically engineered mouse (GEM) models of lung cancer, most do not allow temporal dissociation of the cardinal events involved in lung tumor initiation and cancer progression. Here we describe a novel two-switch GEM model for BRAF(V600E)-induced lung carcinogenesis allowing temporal dissociation of these processes. In mice carrying a Flp recombinase-activated allele of Braf (Braf(FA)) in conjunction with Cre-regulated alleles of Trp53, Cdkn2a, or c-MYC, we demonstrate that secondary genetic events can promote bypass of the senescence-like proliferative arrest displayed by BRAF(V600E)-induced lung adenomas, leading to malignant progression. Moreover, restoring or activating TP53 in cultured BRAF(V600E)/TP53(Null) or BRAF(V600E)/INK4A-ARF(Null) lung cancer cells triggered a G1 cell-cycle arrest regardless of p19(ARF) status. Perhaps surprisingly, neither senescence nor apoptosis was observed upon TP53 restoration. Our results establish a central function for the TP53 pathway in restricting lung cancer development, highlighting the mechanisms that limit malignant progression of BRAF(V600E)-initiated tumors.

Diminished WNT -> ß-catenin -> c-MYC signaling is a barrier for malignant progression of BRAFV600E-induced lung tumors.

Oncogene-induced senescence (OIS) is proposed as a cellular defense mechanism that restrains malignant progression of oncogene-expressing, initiated tumor cells. Consistent with this, expression of BRAF(V600E) in the mouse lung epithelium elicits benign tumors that fail to progress to cancer due to an apparent senescence-like proliferative arrest. Here we demonstrate that nuclear ß-catenin → c-MYC signaling is essential for early stage proliferation of BRAF(V600E)-induced lung tumors and is inactivated in the subsequent senescence-like state. Furthermore, either ß-catenin silencing or pharmacological blockade of Porcupine, an acyl-transferase essential for WNT ligand secretion and activity, significantly inhibited BRAF(V600E)-initiated lung tumorigenesis. Conversely, sustained activity of ß-catenin or c-MYC significantly enhanced BRAF(V600E)-induced lung tumorigenesis and rescued the anti-tumor effects of Porcupine blockade. These data indicate that early stage BRAF(V600E)-induced lung tumors are WNT-dependent and suggest that inactivation of WNT → ß-catenin → c-MYC signaling is a trigger for the senescence-like proliferative arrest that constrains the expansion and malignant progression of BRAF(V600E)-initiated lung tumors. Moreover, these data further suggest that the trigger for OIS in initiated BRAF(V600E)-expressing lung tumor cells is not simply a surfeit of signals from oncogenic BRAF but an insufficiency of WNT → ß-catenin → c-MYC signaling. These data have implications for understanding how genetic abnormalities cooperate to initiate and promote lung carcinogenesis.

MEK1/2 inhibition elicits regression of autochthonous lung tumors induced by KRASG12D or BRAFV600E.

Genetically engineered mouse (GEM) models of lung tumorigenesis allow careful evaluation of lung tumor initiation, progression, and response to therapy. Using GEM models of oncogene-induced lung cancer, we show the striking similarity of the earliest stages of tumorigenesis induced by KRAS(G12D) or BRAF(V600E). Cre-mediated expression of KRAS(G12D) or BRAF(V600E) in the lung epithelium of adult mice initially elicited benign lung tumors comprising cuboidal epithelial cells expressing markers of alveolar pneumocytes. Strikingly, in a head-to-head comparison, oncogenic BRAF(V600E) elicited many more such benign tumors and did so more rapidly than KRAS(G12D). However, despite differences in the efficiency of benign tumor induction, only mice with lung epithelium expression of KRAS(G12D) developed malignant non-small cell lung adenocarcinomas. Pharmacologic inhibition of mitogen-activated protein (MAP)-extracellular signal-regulated kinase (ERK) kinase (MEK)1/2 combined with in vivo imaging showed that initiation and maintenance of both BRAF(V600E)- or KRAS(G12D)-induced lung tumors was dependent on MEK→ERK signaling. Although the tumors dramatically regressed in response to MEK1/2 inhibition, they regrew following cessation of drug treatment. Together, our findings show that RAF→MEK→ERK signaling is both necessary and sufficient for KRAS(G12D)-induced benign lung tumorigenesis in GEM models. The data also emphasize the ability of KRAS(G12D) to promote malignant lung cancer progression compared with oncogenic BRAF(V600E).