Alison L. Dooley

Requirement for NF-kappaB signalling in a mouse model of lung adenocarcinoma

NF-κB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival. They have also been implicated in cellular transformation and tumorigenesis. However, despite extensive biochemical characterization of NF-κB signalling during the past twenty years, the requirement for NF-κB in tumour development in vivo, particularly in solid tumours, is not completely understood. Here we show that the NF-κB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-κB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-κB inhibition. Furthermore, the inhibition of NF-κB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo, from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-κB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-κB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.

Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase

The development of animal models of lung cancer is critical to our understanding and treatment of the human disease. Conditional mouse models provide new opportunities for testing novel chemopreventatives, therapeutics and screening methods that are not possible with cultured cell lines or xenograft models. This protocol describes how to initiate tumors in two conditional genetic models of human non-small cell lung cancer (NSCLC) using the activation of oncogenic K-ras alone or in combination with the loss of function of p53. We discuss methods for sporadic expression of Cre in the lungs through engineered adenovirus or lentivirus, and provide a detailed protocol for the administration of the virus by intranasal inhalation or intratracheal intubation. The protocol requires 1–5 min per mouse with an additional 30–45 min to set-up and allow for the recovery of mice from anesthesia. Mice may be analyzed for tumor formation and progression starting 2–3 weeks after infection.

Genetic and Clonal Dissection of Murine Small Cell Lung Carcinoma Progression by Genome Sequencing

Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs.

Nuclear factor I/B is an oncogene in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.

Requirement for NF-κB signalling in a mouse model of lung adenocarcinoma

NF-κB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival. They have also been implicated in cellular transformation and tumorigenesis. However, despite extensive biochemical characterization of NF-κB signalling during the past twenty years, the requirement for NF-κB in tumour development in vivo, particularly in solid tumours, is not completely understood. Here we show that the NF-κB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-κB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-κB inhibition. Furthermore, the inhibition of NF-κB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo, from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-κB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-κB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.

Requirement for NF-|[kgr]|B signalling in a mouse model of lung adenocarcinoma

NF-κB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival. They have also been implicated in cellular transformation and tumorigenesis. However, despite extensive biochemical characterization of NF-κB signalling during the past twenty years, the requirement for NF-κB in tumour development in vivo, particularly in solid tumours, is not completely understood. Here we show that the NF-κB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-κB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-κB inhibition. Furthermore, the inhibition of NF-κB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo, from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-κB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-κB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.

Requirement for NF-κB signaling in a mouse model of lung adenocarcinoma

NF-κB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival. They have also been implicated in cellular transformation and tumorigenesis. However, despite extensive biochemical characterization of NF-κB signalling during the past twenty years, the requirement for NF-κB in tumour development in vivo, particularly in solid tumours, is not completely understood. Here we show that the NF-κB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-κB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-κB inhibition. Furthermore, the inhibition of NF-κB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo, from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-κB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-κB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.

Abstract 1084: Comprehensive genomic analysis of murine small cell lung carcinoma uncovers recurrent Pten alterations that drive tumor progression and alter tumor genome evolution.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Small cell lung carcinoma (SCLC) is a highly lethal tumor for which few targetable genetic alterations have been identified. SCLC is tightly associated with an extended history of tobacco use, and recent DNA sequencing studies have revealed highly mutated SCLC cancer genomes. Interestingly, two studies analyzing the human SCLC genome report distinct sets of putative driver mutations, highlighting the challenge of identifying functional events in highly mutated cancers. In the absence of environmental mutagens, a previously established genetically engineered mouse model of SCLC initiated by combined loss of Trp53 and Rb1 recapitulates the salient clinical features of human SCLC including tumor histologic progression and frequent distant metastases. This model has been shown to acquire DNA copy number alterations shared with human SCLC, suggesting that cross-species cancer genomics may identify a subset of driver events in human cancers. In order to characterize the spectrum of acquired events in murine SCLC and identify evolutionarily conserved drivers of SCLC progression, we define the somatic genome and transcriptome of a large panel of murine SCLC at single nucleotide resolution using exome, genome and RNA sequencing. We uncover complex subclonality in primary murine SCLC tumors and detect evidence for clonal selection during metastatic spread. As expected in the absence of environmental mutagens, we demonstrate a low somatic mutation frequency in murine SCLC. Using integrative genomic analyses we uncover alterations in chromatin remodeling enzymes and identify Pten as a critical tumor suppressor in this model. Engineered Pten deletion in murine SCLC dramatically accelerates tumorigenesis and fundamentally alters the genomic evolution of these tumors. This work represents the first large-scale comprehensive genomic characterization of a genetically engineered mouse cancer model at single nucleotide resolution, has implications for the nature of tumor evolution in mouse cancer models and identifies a potential therapeutic target in a subset of human SCLC. Citation Format: David G. McFadden, Thales Papagiannakopoulos, Kristian Cibulskis, Chip Stewart, Scott Carter, Amaro Taylor-Weiner, Arjun Bhutkar, Carrie Sougnez, Alison Dooley, Erica Shefler, Eric Lander, Stacey Gabriel, Gad Getz, Tyler Jacks. Comprehensive genomic analysis of murine small cell lung carcinoma uncovers recurrent Pten alterations that drive tumor progression and alter tumor genome evolution. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1084. doi:10.1158/1538-7445.AM2013-1084

Abstract LB-146: Identification of copy number alterations in small cell lung cancer

Small cell lung cancer (SCLC) comprises 20% of all lung cancer cases and is a particularly malignant disease. The identification of genomic alterations in small cell tumors and metastases and characterization of the genes within those regions will aid in our understanding of SCLC development and progression. DNA copy number gains and losses in human and mouse tumors frequently highlight genes of functional importance. Mouse tumors are often less genomically complex than their human counterparts and several recent studies have highlighted the power of comparative genomic studies of mouse models to identify genes relevant to human tumor progression. In a mouse model, inactivation of p53 and Rb following Cre-mediated recombination results in the development of lung tumors that resemble human SCLC histopathologically. Using this model of malignant SCLC, we have identified DNA copy number alterations in tumors and metastases using Illumina9s Solexa massively parallel DNA sequencing technology. While the majority of the genome is unaltered, we observed several focal deletions and high-level amplifications. We detected an amplification of a region including the gene L-myc, a gene previously identified to be amplified in SCLC, and a region including Nuclear Factor 1/B (Nfib). Additionally, several cell lines derived from these lung tumors and metastases have high-level amplifications of Nfib. The DNA copy number data will be probed for conserved changes in tumors and metastases isolated from different animals and functionally validated using in vitro and in vivo assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-146.