Allan Balmain - US grants

The purpose of this research is to develop the mouse skin carcinogenesis model as a paradigm for the study of tumor modifiers: their numbers and locations within the genome, their genetic interactions, biological functions and effects on the somatic genetic events of tumor development. Our ultimate goal is to clone tumor modifiers in the mouse for testing in human populations, with a view to improving the prospects for prediction of risk, prevention and therapy of human cancers. To achieve to broad aims set out in this proposal, we have set up an international consortium of groups and consultants with complementary expertise in the study of mouse tumor modifiers (P.Demant, Holland and T.Dragani, Italy), generation of novel models through germline manipulation, (T.Jacks, Boston;D.Hanahan and E.Epstein, San Francisco), physical induction of germline deletions for functional studies of modifier genes (T.Sato and M.Kusakabe, Japan) and the search for tumor modifiers in human populations (B.Ponder, UK). Our initial aim is to determine the number and chromosomal locations of skin tumor modifier loci in a variety of mouse strains, using a combination of genetic approaches including interspecific backcrosses between mus spretus and mus musculus. Since the detection of human modifiers of complex traits directly using human material involves approaches such as linkage disequilibrium with single nucleotide polymorphisms, we will pursue a parallel strategy in the mouse by investigating animals selected from a mixture of genetic backgrounds by virtue of sensitivity of resistance to skin carcinogenesis. This will be carried out using chemical carcinogenesis in skin as a method of tumor induction, and will provide us with information on the degree of overlap of these different approaches to the detection and analysis of tumor modifiers. A number of transgenic/knock out models for skin tumor induction will also be investigated. These include keratin promoter-driven ras or HPV transgenic mice, which develop multiple squamous carcinomas, and patched (Ptc) knock-out mice, which provide a model for the development of basal cell carcinomas after UV treatment. Modifiers of these transgene/knockout induced phenotypes will be analyzed using genetic mapping approaches in mus musculus and mus spretus crosses. In the same crosses, DNA repair capacity will be assessed (with J.Cleaver, San Francisco) in individual mice and the relationship between loci that control DNA repair and the tumor predisposition loci will be investigated. These studies may identify subsets of tumor modifiers which operate in skin independent of the mode of tumor induction, or which may be specific for a particular genetic insult or target cell within the skin. Together with Drs. Kusakabe and Sato in Japan, we will induce germline deletions in regions of the mouse genome that harbor potential tumor modifier or tumor suppressor genes as a prelude to functional studies in vivo. The relationship between these germline tumor modifiers and the somatic genetic alterations which take place during tumorigenesis will be investigated using microsatellite-based LOH, CGH and genomic arrays (with J.Gray, D.Pinkel and D.Albertson, San Francisco) to study patterns of gene loss or gain in tumors representing different stages of carcinogenesis. Finally, candidate modifiers identified using mouse approaches will be studied in humans to determine their relevance to the development of human skin or other tumor types.

DESCRIPTION (provided by applicant): Most human tumors arise from the self-renewing cells of the epithelia - the linings of the major tissues such as the skin, lung and intestine. Our hypothesis is that while some polymorphisms that influence cancer risk may act specifically in certain tissues, for example hormonal influences on the breast or prostate, many will control the basic underlying properties of growth control and genetic stability that are almost always deregulated during cancer development. We will look for these common genes and polymorphisms using mouse models of susceptibility to skin, lung, colon and prostate tumors. A combination of linkage analysis and haplotyping will be used to refine the regions containing genes that confer increased risk of developing epithelial tumors. Candidate genes will be selected by analysis of allele-specific genetic alterations in tumors using genome wide high density BAC arrays, together with gene expression microarrays to profile both normal tissues and tumors from backcross animals. This analysis will be facilitated by the availability of an extensive database and tissue/tumor bank derived from almost 2000 mice from a series of overlapping interspecific Mus spretus X Mus musculus crosses. In parallel with these studies on mouse models, we have set up an extensive network of collaborations involving multiple groups worldwide with expertise in human population genetics and, most importantly, collections of normal DNA samples from large human population-based case-control or cohort studies. These collaborators have access to DNA samples from patients with cancers from each of the tissues for which we have developed mouse models (skin, lung, colon, prostate), as well as from patients with breast and other cancers. Additional collaborators have focused on collections of human tumor DNA and/or RNA from patients with the same tumor types. Many biological and epidemiological studies have demonstrated relationships between cancer and other disease phenotypes such as inflammation or obesity. We will adopt a Systems Biology approach to genotype-phenotype relationships by setting up a large interspecific backcross designed to collect data on skin tumor incidence, pathology, progression, and metastasis. Serum and blood samples will be stored for subsequent proteomic and functional studies. A series of additional parameters of each mouse in the backcross will be measured including immune function, body weight/obesity, bone density, and inflammatory response. This is an ambitious attempt to collect and analyze large data sets containing information on cancer from both mouse and human perspectives. The results will be important for the prediction of human cancer risk, as well as for development of prevention or therapeutic strategies based on genotype-phenotype networks rather than single genetic targets.

DESCRIPTION (provided by applicant): Most human tumors arise from the self-renewing cells of the epithelia - the linings of the major tissues such as the skin, lung and intestine. Our hypothesis is that while some polymorphisms that influence cancer risk may act specifically in certain tissues, for example hormonal influences on the breast or prostate, many will control the basic underlying properties of growth control and genetic stability that are almost always deregulated during cancer development. We will look for these common genes and polymorphisms using mouse models of susceptibility to skin, lung, colon and prostate tumors. A combination of linkage analysis and haplotyping will be used to refine the regions containing genes that confer increased risk of developing epithelial tumors. Candidate genes will be selected by analysis of allele-specific genetic alterations in tumors using genome wide high density BAC arrays, together with gene expression microarrays to profile both normal tissues and tumors from backcross animals. This analysis will be facilitated by the availability of an extensive database and tissue/tumor bank derived from almost 2000 mice from a series of overlapping interspecific Mus spretus X Mus musculus crosses. In parallel with these studies on mouse models, we have set up an extensive network of collaborations involving multiple groups worldwide with expertise in human population genetics and, most importantly, collections of normal DNA samples from large human population-based case-control or cohort studies. These collaborators have access to DNA samples from patients with cancers from each of the tissues for which we have developed mouse models (skin, lung, colon, prostate), as well as from patients with breast and other cancers. Additional collaborators have focused on collections of human tumor DNA and/or RNA from patients with the same tumor types. Many biological and epidemiological studies have demonstrated relationships between cancer and other disease phenotypes such as inflammation or obesity. We will adopt a Systems Biology approach to genotype-phenotype relationships by setting up a large interspecific backcross designed to collect data on skin tumor incidence, pathology, progression, and metastasis. Serum and blood samples will be stored for subsequent proteomic and functional studies. A series of additional parameters of each mouse in the backcross will be measured including immune function, body weight/obesity, bone density, and inflammatory response. This is an ambitious attempt to collect and analyze large data sets containing information on cancer from both mouse and human perspectives. The results will be important for the prediction of human cancer risk, as well as for development of prevention or therapeutic strategies based on genotype-phenotype networks rather than single genetic targets.

Most human tumors arise from self-renewing cells of epithelial tissues. Tumors in the lung are the most deadly, with a projected 152,000 deaths in 2003 in the US alone. A strong genetic component contributes to lung cancer risk, as shown by evidence of familial clustering, and the fact that only 10-20% of smokers develop lung cancer. Identification of genetic variants that confer susceptibility to lung cancer will enable prediction of individual risk, and facilitate the development of novel therapeutic or preventive agents. A particular species of mouse (Musspretus) is genetically resistant to development of lung cancer, due to the presence of germline polymorphisms that confer resistance. Crosses between sensitive and resistant mouse strains will be used in a genetic approach to identify lung tumor resistance genes. This analysis will be facilitated by the availability of an extensive database and tissue/tumor bank derived from a large backcross between the lung tumor-susceptible KrasM2 mice on the FVB background, with the resistant Mus spretus species. Data are already available both on the genomic changes associated with lung cancer initiation and progression, and on the genetic basis of lung cancer susceptibility. These data have implicated the Kras2 gene as a major determinant of lung tumor development and susceptibility. This particular ras gene family member is frequently mutated in both human and mouse lung tumors, and is one of the candidates for the major mouse lung tumor susceptibility locus Past. Two engineered mouse models will be used to test the role of Kras2 in lung tumor development and susceptibility. One involves a knock-in of a mutant Kras2 allele into the endogenous locus (/

The Genome Analysis Core provides services and advice for DNA sequencing, genotyping, and gene expression analysis. The DNA sequencing services are instrumental in helping scientists determine causative mutations in the germline or somatic genes which lead to certain cancers or human diseases. DNA sequencing services are also used to interrogate the methylation status of genes. Loss of heterozygosity (LOH) and genetic linkage analyses are used for identifying regions of the genome likely to be involved in the occurrence and progression of malignancies. The Core has extensive experience performing micrbsatellite and single base extension SNP genotyping on the 3700 platform, and TaqMan allelic discrimination SNP typing on the 7900HT platform for these purposes. The sequencing capability is also used to determine whether there are exon-sized deletions in one allele of a cancer susceptibility gene once it has been determined that there are no point mutations. For these studies, the sequencer is used to analyze fluorescent fragments generated by the Multiplex Amplifiable Probe Hybridization (MAPH) or Multiplex Ligation-dependent Probe Amplification (MLPA), and Spectratyping techniques. A major focus of the Core's services is the use of quantitative real-time PCR (Q-PCR) on the 7900HT platform. Using an arsenal of TaqMan assays, the Core provides services for DNA copy number measurements and mRNA expression analyses, plus training courses for members of the Cancer Center to learn Q-PCR techniques. The Cancer Center Genome Analysis Core works to improve access of Cancer Center member to genetic and genomic technologies. The services from we offer, or propose to develop, are actively coordinated with the other UCSF genomics core facilities to avoid detrimental duplication of capacity. For example, Cancer Center members have access to extensive high throughput gene expression and genotyping technology (Affymetrix and Illumina) through core facilities at the Parnassus and Mission Bay campuses of UCSF.

Patients with organ transplants have an increased risk of cancer, and organ transplant recipients of European descent are at particularly high risk for developing non-melanoma skin cancers. Although there has been much published on clinical aspects of this risk, there have been relatively few studies of the mechanism underlying its increase. We propose herein an extensive, in depth such study using three individual Projects and two Cores. These Projects will investigate three aspects of the problem: 1. We will investigate the role of aberrant TGFI3 signaling in these tumors, the role of anti-rejection drugs in activating this signaling system, and the effects polymorphisms in the genes encoding members of this pathway have on susceptibility to this post-transplant complication. 2. We will compare genetic abnormalities in squamous cell carcinomas in organ transplant recipients with those present in these tumors in normal patients, expecting that such comparison will help elucidate pathways that are crucial to the increased risk in transplant recipients. Furthermore we will use the overlap of abnormalities in multiple tumors from the same patient to help identify genes with alleles that confer susceptibility vs. resistance to tumorigenesis. 3. We will develop a robust mouse model in which anti-rejection drug treatment enhances skin carcinogenesis and will use this model to test the contribution to enhancement by anti-rejection drug impairment of DNA repair and of immune function. These three will be supported by an Administrative Core and by a Tissue Core that will establish a collection of tumors and blood from very large numbers of organ transplant recipients with tumors and from appropriate controls, a collection that will be essential for the prosecution of the Projects and for future studies of this important clinical problem.

[unreadable] DESCRIPTION (provided by applicant): There are unprecedented opportunities to exploit the information emerging from the Human and Mouse Genome Projects, as well as the HapMap project, for assessment of individual susceptibility to common diseases such as cancer, diabetes, obesity, or cardiovascular disorders. Identification of individuals at risk will have major implications for prevention as well as treatment of these conditions, and will have a fundamental impact on approaches to health care. At present, in spite of the general agreement that "personalized medicine" is a major goal of the NIH, there is as yet no consensus regarding the methods and approaches that should be used to achieve this goal. UCSF is one of the foremost medical centers in the US, with an extremely large patient population and vast experience in the treatment of human disease. UCSF also has in depth experience in the development of mouse models of human disease, in particular cancer, cardiovascular and immunodeficiency. We therefore have a unique opportunity to exploit the patient and tissue resources, as well as the mouse models available across the campus by carrying out large scale genome-wide and targeted genotyping studies of DNA samples from appropriate patients and controls. This proposal is for the purchase of the Sequenom MassArray genotyping System for the analysis of DNA samples from patients and controls. Although much attention has recently been focused on approaches to high throughput whole genome scanning, there is a major requirement for a targeted approach that allows low-medium throughput analysis of specific candidate genes and SNPs. While the Affymetrix and Illumina systems are well suited to simultaneous analysis of large numbers of SNPs, the Sequenom system is most appropriate for low-intermediate SNP numbers that can be typed rapidly on large numbers of patient samples. At the moment, there is no cost -effective SNP genotyping service of this kind available on the campus. The projects that are planned or already in progress at UCSF include efforts to determine the genetic basis of a range of common diseases: diabetes, autoimmunity, multiple sclerosis, obesity, and cancer. The main projects outlined in this proposal are cancer related, and it is proposed to house the equipment within the Cancer Center. However, the Sequenom MassArray will be part of the newly formed Consolidated Genome Core and will be available to all participating institutions and departments at UCSF and its affiliated institutions. Relevance The Federal Government, through NIH/NCI funded initiatives, has made a major investment in the genetics of human disease. The human and mouse genome projects have cost billions of dollars, but the translation of this information into tangible benefits for patients is still in its infancy. These projects will exploit the vast patient and tissue resources at UCSF in an attempt to relate disease susceptibility to the underlying genetics of the host, and is thus fits with the overall strategic aims of the NIH. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cancer treatment is in the process of transitioning from the era of empirical treatments for all patients with a particular form of cancer, to a more individualized and consequently mechanism-based approach to patient care. The genetic background of individual patients affects susceptibility to cancer development, the probability of malignant progression, and the likelihood of responding to particular targeted therapies. The objective of this application is to develop an integrated systems genetics approach that exploits genetic diversity between mouse strains, and new network analysis tools to identify the genes and pathways involved in each of these stages of cancer development and treatment. Systems genetics, unlike traditional approaches to the analysis of disease that focus on single genes or proteins in isolation, attempts to integrate the complex interaction of many kinds of genetic and biological information - genomic DNA sequence, mRNA, microRNA and protein expression, and link these to cancer phenotypes. This project will use systems genetics to integrate genetic, gene expression and phenotypic data from a mouse model of skin cancer that has been used for over 40 years to dissect the biological and molecular events that are essential for initiation, promotion and progression to malignant, metastatic disease. The first Specific Aim is based on the premise that a prerequisite for understanding cancer susceptibility is an appreciation of how the normal host gene expression in the target tissue is controlled. This will be analyzed by gene expression network analysis of normal skin from interspecific backcross mice as well as from a series of recombinant inbred lines, to exploit both linkage analysis and haplotyping for expression quantitative trait locus (eQTL) mapping. These network approaches are capable of revealing expression motifs associated with tissue structure, as well as pathways linked to stem cells, the cell cycle, and inflammatory responses. The second Specific Aim will identify features of the normal genetic architecture of expression that are associated with susceptibility to malignant progression, and will also look for perturbations in networks within carcinomas to identify signaling pathways and genes that may provide novel therapeutic targets. Specific Aim 3 will take steps towards individualized cancer treatment by investigating the role of a specific gene, FBXW7, in determining response of primary tumors to inhibitors of mTOR - one of the main players in a well characterized oncogenic pathway. Specific Aim 4 involves a close collaboration with groups working on systems genetics of human cancer to identify molecular signatures of the response of metastatic tumors to inhibitors of the mTOR pathway.

DESCRIPTION (provided by applicant): This project will exploit mouse genetics to test the concepts and approaches that will be required to implement personalized medicine for human cancer. This will require joint studies, both within the mouse models of human cancer consortium (MMHCC) and together with other NCI-funded organizations, of systems genetics analysis of genetic background and gene expression networks in cancer susceptibility, progression and therapeutic responses. The value of systems-based approaches has been demonstrated from mouse studies of susceptibility to obesity and diabetes, in which the gene expression networks in normal tissues have revealed signaling pathways that reflect underlying susceptibility, as well as signaling hubs that are viable therapeutic targets. Similar studies of mouse models of skin cancer have identified hierarchical networks that control tissue structure and function, and provided novel insights into mechanisms that lead to skin tumor susceptibility. The application of these integrative systems approaches to cancer has major implications for all of the areas of research being pursued by the MMHCC. This project will include a Core Project, the purpose of which is to construct a browsable database of combined single nucleotide polymorphisms (SNPs) and gene expression data from tissues from interspecific backcross mice and from the Rl lines of the Collaborative Cross. Such a data base would be invaluable both for groups in the Cancer Susceptibility and Resistance cluster, but also more generally within the MMHCC and in other NCI-funded bodies. A tissue bank from the same animals will be available to members of the MMHCC for investigations of the genetic control of gene expression at the protein level. Pilot projects will be set up within the cancer susceptibility and resistance theme to use the same genetic approaches to investigate tumor susceptibility using different mouse models that represent the major forms of human cancer. A further goal is to investigate the interplay between germline polymorphisms and somatic events in cancer detection, progression and treatment responses. These aims overlap substantially with the Roadmap initiative, and in particular with the goals of the Integrated Cancer Biology Program (ICBP), the Specialized Programs of Research Excellence (SPORE) program, the Early Detection Research Network (EDRN) and the Cancer Family Registry (CFR). Representatives of each of these programs have provided supporting materials for this comparative mouse-human genetic approach to analysis of multiple aspects of cancer susceptibility.

DESCRIPTION (provided by applicant): Genes of the RAS family were found to be mutated in human cancers around 30 years ago, and the long term goal of this project is to develop a deeper understanding of the mechanisms by which these genes cause malignant transformation. The KRAS gene is the most commonly mutated family member, particularly in carcinomas of the pancreas, colon and lung. Together, these tumors account for hundreds of thousands of deaths worldwide each year. Using genetically engineered mouse models, this laboratory showed that a minor isoform of Kras (Kras4A) is essential for development of Kras mutant tumors. Mice deficient in this isoform are highly resistant to development of lung or skin tumors with Kras mutations, suggesting that inhibition of the function of Kras4A could result in therapeutic benefit for patients. This project involves a comprehensive analysis of the signaling pathway that is activated by this specific form of the Kras protein. A number of novel approaches will be used to understand how Kras4A signals within cells and tissues. A recently developed new imaging approach (Photo-activated localization microscopy, or PALM) will image the location of single molecules of Kras4A in cells, and find out whether there is an interaction between mutant and wild type Kras4A proteins. Signaling through canonical RAS pathway components in cells lacking Kras4A or both Kras4A and 4B, will be investigated in order to identify any specific signaling pathways that are deficient in these cells. Finally, an unbiased view of Kras signaling in vivo in mouse lungs will be developed, using novel gene expression network analysis tools. Comparison of gene expression networks from mice that are susceptible to lung cancer with those that are resistant due to deletion of Kras4A may identify pathways not previously associated with Ras that may account for the critical role of Kras4A in transformation. Using mouse models in vivo, the possible function of Kras4A in regeneration of lung epithelium from stem cells after damage will be studied, and new models in which Kras4A can be inhibited using inducible shRNA will be developed to investigate the role of this protein in the propagation and maintenance of tumors with mutant Kras genes.

DESCRIPTION (provided by applicant): Activating mutations in genes of the RAS family are the most common dominant acting oncogenic changes in human cancers. These tumors are particularly difficult to treat, and have a very poor prognosis. Mutations in RAS family members exhibit tissue specificity, but we do not know how tissue damage, inflammation and the resultant remodeling process results in early selection of cells carrying specific mutations in individual RAS isoforms. Mutations in the HRAS gene are mainly found in squamous carcinomas (SCCs) of the lung, skin, and head and neck, which share many histological and molecular characteristics and together constitute a major human cancer burden worldwide. In contrast, KRAS is the most commonly mutated oncogene in adenocarcinomas of the lung, pancreas, and colon. The same tissue specificity is observed in mouse cancer models: carcinogen-induced squamous tumors of the skin have almost 100% Hras mutations, whereas lung adenocarcinomas induced by the same carcinogen have almost 100% Kras mutations. The identification of the factors that underlie this specificity would provide important information tht may lead to tissue-specific or mutation-specific routes to cancer prevention or treatment. We generated novel mouse models in which the specificity for particular Ras isoforms in skin and lung has been reversed or eliminated, resulting in mice that develop Kras mutant skin carcinomas, Hras mutant lung carcinomas, or are completely resistant to chemical carcinogen treatment. This project will exploit these novel mouse models as well as computational network approaches to the identification of functions of Ras genes in normal tissue and carcinomas driven by either Hras or Kras. Mouse gene expression network data will be validated in human samples by integration with human gene expression datasets from squamous carcinomas of the lung and head and neck (generated by TCGA) or in primary cutaneous SCCs, in particular with human SCCs from BRAF-inhibitor treated patients which have an elevated frequency of HRAS (Q61L) gene mutations - exactly the same point mutation that is found in Hras mutant mouse skin tumors. We will use a Systems Biology approach to visualize the architecture of Ras signaling in whole tissues in vivo and in epithelial cells derived from mutant mice, to examine the relationships between inflammation and susceptibility to development of Hras- or Kras-driven malignancies. This comprehensive systems-based approach will reveal conserved features of squamous carcinoma formation that will first help us to understand the genesis of these neoplasms, and begin to formulate strategies for prevention or treatment.

DESCRIPTION (provided by applicant): Activating mutations in KRAS occur frequently in some of the most common and deadliest of human cancers but efforts to target KRAS for therapeutic purposes have not been successful. KRAS generates two highly homologous isoforms (KRAS4A and KRAS4B) as a result of alternative splicing. While the complete Kras gene knockout in mice causes embryonic lethality, knock-in of either Kras4A (Kras4AKI) or Kras4B (Kras4BKI) at the expense of the alternate isoform results in viable mice. Kras4AKI mice express only Kras4A and Kras4BKI mice express only Kras4B, enabling for the first time a comparison of the individual roles of these Kras isoforms in transformation in vivo. We have found that Kras4BKI mice are highly resistant to the development Kras mutant lung and skin tumors, suggesting that Kras4A is essential for transformation in 2 independent tissues. Our studies further showed that Kras4A is also responsible for the previously reported tumor suppressor activity of wild type Kras. The overall goal of this proposal is to exploit these novel mouse models to study the functions of both Kras4A and Kras4B in lung cancer development, both as oncogenes (mutant form) and as tumor suppressors (wild type form). A major strength of this proposal is the availability of viable strains uniquely expressing each isoform, representing powerful and complementary in vivo systems for studies into the isoform specific functions of this major human oncogene. Aim 1 of this proposal will investigate the oncogenic function of individual Kras isoforms in vivo using models of carcinogen induced lung carcinogenesis. Aim 2 will investigate the tumor suppressor activity of wild type Kras4A and Kras4B in vivo in models of conditionally activatable mutant Kras (i.e. KrasLA2 and KrasLSL-G12D). Aim 3 will take advantage of lung epithelial cell lines derived from the various mouse models for detailed in vitro analyses of Kras4A and Kras4B signaling pathways in order to gain mechanistic insights into their oncogenic activities. Aim 4 will evaluate the roles of these individual KRAS isoforms in the growth/maintenance of human KRAS mutant lung cancer cells. Insights into the isoform specific functions of KRAS will not only be important to the understanding of cancer development but could potentially have major implications for therapeutic design.

Identification of cancer drug targets using high throughput screens of tumor cell lines has led to a number of agents presently in clinical trials. In addition, recent advances in drugs that attack immune cells within tumors, such as ?CTLA4 and ?PD-1, have highlighted the importance of immune modulation as a strategy for cancer therapy. The next phase of cancer drug target discovery will seek to integrate these strategies to identify combinations of drugs that most efficiently target both tumor cells and the immune components in advanced cancers. The goal of this proposal is to identify and validate these combinations using large-scale data mining and mouse pre-clinical cancer models that mimic the major genetic features of human cancer. This proposal addresses both mechanisms of immune escape by a) finding genetic targets that may enhance tumor mutation load, and b) carrying out high throughout screens in T cells or myeloid cells for targets that promote immune cell infiltration. We will exploit unique mouse models that mirror major genetic categories of human cancer ? high vs low mutation load, and strong vs weak immune infiltrate. Applying single-cell RNAseq and mass cytometric proteomic analyses, cutting edge immune composition databases and novel computational network approaches to cancer target discovery using existing large databases, we propose to identify vulnerabilities addressed by combining small molecule drugs with immunotherapy. We will make immunologically ?cold? tumors, that do not engage the immune system, into ?hot? tumors that present more or stronger antigens, or that encourage infiltration by immune effector cells. To achieve this goal, we propose three highly innovative aims centered on perturbation of specific targets: first by a CRISP/Cas9 screen in immune cells of the tumor microenvironment, second through increasing antigen load in tumors to optimize immune recognition and finally through a network-based identification of tumor-expressed targets that may confer susceptibility to existing immune-oncology therapies. This represents a true `network' of our collective expertise as well as a measured collection of candidate and screening approaches. AIM 1 ?We will perform CRISPR screens in monocytes and T-cells to identify genes associated with tumor entry and function in two distinct tumor types. AIM 2? We will use genetic or pharmacological perturbation of newly generated candidate genes involved in metabolic stress and ROS-induced DNA damage to increase mutation load and antigen abundance in a tumor- specific manner, leading to improved responses to immunotherapy. AIM 3 ? We will exploit gene expression networks to identify druggable targets and pathways that augment immune responses. This proposal identifies pathways and perturbants for accelerating immunotherapies.

Abstract Systems Genetics Analysis of Tumor Evolution in the Mouse. Metastasis is the major cause of death due to cancer in humans, but many factors play a role in determining the route taken by initiated cells to reach this end stage. We know that tumors arise due to the combined effects of inherited genetic factors and environmental exposures, and have taken the long term view that successful modeling of human cancer in the mouse requires us to take account of interactions between genetics and environment. The over-arching goal of this research is to develop an integrated systems genetics view of the cells, signaling pathways and mutations in specific genes involved in evolution of metastasis from single initiated cells, using one of the best characterized models of multistage carcinogenesis of the skin. Based on major funding from the NCI MMHCC over the past 15 years, we have built up a unique tissue and tumor bank comprising thousands of samples of Ras-mutant squamous tumors from a genetically heterogeneous mouse population. These samples encompass all stages from benign lesions to metastases, and in particular include hundreds of matched primary carcinomas and distant metastases from the same animals. All tumors were induced by exposure to mutagens and tumor promoting agents, resulting in complex genomic landscapes that resemble, in mutation frequency and type, the genomic profiles of human squamous carcinomas of the skin, head and neck, and lung. All mice in the cohort have been genotyped genome-wide to facilitate linkage and eQTL analysis to identify genetic determinants of initiation, benign tumor formation, progression and metastatic dissemination. This project will exploit this unique tissue bank and database by using genomic technologies, together with novel mouse strains, to identify the cells of origin of benign and malignant tumors, biomarkers of risk of malignant progression, and genetic drivers of metastasis. Computational network analysis tools will be used to study the evolution of signaling pathways, for example the Ras pathway, through multiple stages of carcinogenesis, to identify changes in these networks that will identify potential novel cancer target genes. Finally we will initiate a new series of translational studies of immunotherapy, based on our observation that these chemically induced tumors harbor novel neo-antigens and show promising responses to inhibitors of immune checkpoint proteins.