Edison T. Liu - US grants

Aberrant cellular oncogenes (c-oncs) have been associated with a variety of human tumors. The role of these c-oncs is yet unelucidated partly because of our relative inability to manipulate these resident genes. I propose to use a new technique of antisense mRNA inhibition to attenuate the action of one mutant c-onc, EJ ras. This technique involves the subcloning of a gene that transcribes for mRNA complementary to the native mRNA thus allowing for RNA:RNA interactions. This has been shown to attenuate the function of the herpes simplex TK gene. An antisense EJ ras construct has been made where the EJ ras gene is flipped and is transcriptionally driven in the opposite direction by an MSV LTR; furthermore, this vector has a neo gene for dominant selection, and the dhfr gene for amplification. This antisense construct will be transfected into transformed cells bearing a copy of the sense EJ ras and cells will be selected for resistance to G418. The resultant clones will be scored for the number of revertants as compared to controls. The converse experiment where cells bearing the antisense ras are transfected with the sense EJ ras gene and the number of transformants assessed will also be done. If during the mRNA analysis of these cells, we find the antisense message in suboptimal quantities, amplification of the antisense gene can be accomplished through thedhfr construct. In this well defined murine system, I hope to optimize the conditions for the inhibition of the oncogene function by antisense mRNA. once the system is established, I propose to introduce this antisense EJ ras plasmid into the EJ human bladder carcinoma cell line from which the EJ ras gene was isolated and attempt to inactivate the resident mutant ras. If successful, this work has broad applicability as a technique to test the function of a eukaryotic gene by inactivation.

Aberrant cellular oncogenes (c-oncs) have been associated with a variety of human tumors. The role of these c-oncs is yet unelucidated partly because of our relative inability to manipulate these resident genes. I propose to use a new technique of antisense mRNA inhibition to attenuate the action of one mutant c-onc, EJ ras. This technique involves the subcloning of a gene that transcribes for mRNA complementary to the native mRNA thus allowing for RNA:RNA interactions. This has been shown to attenuate the function of the herpes simplex TK gene. An antisense EJ ras construct has been made where the EJ ras gene is flipped and is transcriptionally driven in the opposite direction by an MSV LTR; furthermore, this vector has a neo gene for dominant selection, and the dhfr gene for amplification. This antisense construct will be transfected into transformed cells bearing a copy of the sense EJ ras and cells will be selected for resistance to G418. The resultant clones will be scored for the number of revertants as compared to controls. The converse experiment where cells bearing the antisense ras are transfected with the sense EJ ras gene and the number of transformants assessed will also be done. If during the mRNA analysis of these cells, we find the antisense message in suboptimal quantities, amplification of the antisense gene can be accomplished through thedhfr construct. In this well defined murine system, I hope to optimize the conditions for the inhibition of the oncogene function by antisense mRNA. once the system is established, I propose to introduce this antisense EJ ras plasmid into the EJ human bladder carcinoma cell line from which the EJ ras gene was isolated and attempt to inactivate the resident mutant ras. If successful, this work has broad applicability as a technique to test the function of a eukaryotic gene by inactivation.

The genetic basis of human tumor progression was studied by examining two hematopoietic disorders, chronic myelogenous leukemia (CML), and myelodysplasia (MDS). These diseases are characterized by a preleukemic or premalignant phase which progresses to acute leukemia. Using a sensitive tumorigenicity assay, point mutations in the ras protooncogenes were identified in the earliest preleukemic stage of MDS, but in the late or acute leukemic phase of CML. Furthermore, in MDS, the presence of aberrant ras alleles predicted for subsequent progression to acute leukemia. These data suggest that mutant ras genes are important factors in leukemogenesis, but that these mutant ras alleles have different progression functions depending on the disease context. Underscoring the complexity of the progression process, a novel transforming gene (gene X) was identified in the blood cells from two patients with CML. In light of these data, a study of ras mutations in preleukemic states is proposed using a sensitive method for detecting point mutations in small quantities of DNA: polymerase chain reaction. In this fashion, will extend our initial findings in a large number of patients and will determine the clinical significance of these findings. Blood cells will be separated into lineage specific fractions (i.e. granulocytes, monocytes, and lymphocytes) and the stem cell which sustained the mutation in ras can be identified. Lastly, gene X will be cloned from a secondary nude mouse transformant using cosmid vectors. Once cloned, the critical genetic lesion(s) which converted the normal cellular homologue of gene X into its transforming counterpart can be determined.

We propose to establish the UNC Interdisciplinary Program in Breast Cancer Research to serve as a national Specialized Program of Research Excellence (SPORE) in Breast Cancer for the National Cancer Institute. The UNC SPORE will have a unique set of goals, objectives, and programs that capitalize on and extend in novel ways the existing excellence in multidisciplinary breast cancer research at UNC and the UNC Lineberger Comprehensive Cancer Center. The goal of the UNC SPORE is to reduce breast cancer mortality and incidence in North Carolina through an interdisciplinary program of research and intervention that: 1. Integrates efforts in cancer prevention and control, molecular epidemiology, clinical research, and laboratory clinical research; and 2. Targets behavioral and biological issues relevant to Black women. The six primary objectives of the UNC SPORE are: 1. To identify the determinants of the Black/White gap in breast cancer mortality in North Carolina, and then to use novel community and provider interventions to close that gap; 2. To initiate a long-term, population-based study of breast cancer in a defined North Carolina population containing 1.6 million women, 27% of whom are minorities; 3. To combine molecular biology and epidemiology in the investigation of the environmental determinants of and genetic predispositions to breast cancer in blacks and whites in the defined population; 4. To improve the treatment, quality-of-life, and prognosis of late-stage breast cancer, a common presentation among Black women in North Carolina; 5. To develop new clinical markers for neoplastic proliferation of breast cancer cells; and 6. To complement the NCI and other SPOREs in the national effort to reduce breast cancer mortality and incidence with a focus on minority issues, development of a defined population, and research programs in cancer prevention and control, molecular epidemiology, clinical research, and the isolation and molecular analysis of novel regulatory genes.

Overexpression or amplification of the HER-2 oncogene has been associated with poor survival in node positive breast cancers. We, in CALGB, have discovered that despite this worse outcome, those with perturbations of HER-2 significantly benefit from dose intensive adjuvant chemotherapy resulting in a doubling of the 5 year actuarial survival rates. Our analysis suggests that the important agent in this dose interaction is Adriamycin. Equally important is that patients with no abnormalities of HER-2 do not appear to show the same benefit with high dose treatment. Thus the HER-2 status may define the specific agents and doses that are appropriate for this patient subset. We propose to verify and extend these initial findings by assessing the HER-2/dose interactions in a larger data set. We will study the mechanism of this interaction by determining the whether the expression of the neu differentiation factor (NDF), p53, and the expression and amplification of Topoisomerase II and PRAD-1 will contribute to this dose effect. We will also assess the impact of S phase fraction and the levels of circulating HER-2/c-erbB2 extracellular domain on this interaction. To further enhance this approach, we will obtain data sets from the International Breast Cancer Study Group node positive adjuvant trial, and other CALGB adjuvant trials where the HER-2 status is known, to determine whether specific chemotherapeutic agents, such as Adriamycin, are important in this oncogene/dose interaction.

The overall goal of our research has been to examine the molecular genetics of human myeloid leukemia. To this end we have studied two preleukemic syndromes chronic myelogenous leukemia (CML) and myelodysplasia (MDS). During our analysis of CML samples, we isolated a novel transforming gene in 2/8 patients. The molecular cloning and characterization of this gene, which we call axl, revealed that it is a novel receptor tyrosine kinase with subtle transforming activity and is localized to chromosome 19 where perturbations commonly occur in the progression to CML blast crisis. Preliminary data also suggests a role for axl in hematopoietic differentiation, especially towards the megakaryocytic lineage. We propose to expand our work on this novel oncogene by determining the genetic lesion that converts the normal axl to a transforming axl allele. We plan to raise antibodies to axl in order to better define its biochemical function, and to examine the role of this protein in human leukemias. Finally, we will pursue biological experiments to elucidate the function of axl in leukemogenesis and in hematopoietic differentiation. The results of our work will not only expand our understanding of a new class of receptor tyrosine kinases, but potentially will also provide clinicians with a new biochemical/genetic marker for leukemic progression and a new target for gene directed therapies.

breast neoplasms; health science research; cancer risk; neoplasm /cancer epidemiology; neoplasm /cancer genetics; cancer prevention;

The basis of the Molecular Epidemiology Program is the coupling of detailed epidemiologic analysis with state-of-the-art molecular biological approaches applied to studying important epidemiologic questions. To this end, a strong and well organic Molecular Analysis Facility is critical to the success of this mission. The Molecular Analysis Core Facility is responsible for the conduct of the genetic analyses proposed in the individual projects. All genetic tests for the molecular epidemiology projects will be performed in this Core Facility. The centralized nature of this facility has permitted stringent quality control (including control of PCR contamination) and economic utilization of laboratory resources. Unique aspects of this core have featured the technical versatility through cross training of laboratory personnel, the centralized database and tissue tracking procedures, a matrix leadership approach, and provisions for exploratory projects that require attention to techniques development. In addition, we have addressed the particular requirements for population based molecular studies: reproducibility, speed, and the development of screening algorithms that improve sensitivity and specificity.

breast neoplasms; training; neoplasm /cancer epidemiology; neoplasm /cancer genetics;

DESCRIPTION (provided by applicant): We have developed a new technology called Gene Identification Signature Pair-end-diTag (GIS-PET), which captures precisely and joins the 5'and 3'most 18 base pair of any clone insert in a library. The resultant "ditags" are signatures of the boundaries of any cDNA or genomic fragment within the library. When coupled with advanced sequencing technologies, GIS-PET can annotate an entire transcriptome 300-500 fold faster and less expensively than contemporary cloning and sequencing. We have captured the transcriptome snapshot of the MCF-7 breast cancer cell line to >500,000 full length cDNA equivalents and have captured the state of the transcriptome of this breast cancer cell line with unprecedented resolution. With this approach, we found that we can identify, again with precision, candidate fusion transcripts where the beginning of one gene is fused with the end of another. These fusion transcripts are cancer-specific and can function as biomarkers, as monitors of treatment effect, and as targets for new therapeutics. We therefore propose to expand and validate this technology on primary breast cancers and to confirm the significant list of candidate fusion transcripts already identified in the MCF-7 cell line. We will clone and sequence all the fusion transcripts accessible to these technologies and will assess if their presence in primary breast cancers is associated with cancer behavior and clinical outcome. We also propose to optimize the GIS-PET technology to genomic DNA so as to map the precise location of genomic rearrangements in cancer cells. When put together, the combined power of the technology can uncover the complete rearrangement map of any cancer genome. Because we have brought this technology beyond conceptualization, we are submitting this application as an R33. Relevance to public health: Cancer is a genetic disease and the understanding of the genetic abnormalities in cancer has been shown to uncover new biomarkers and new treatments in the management of the disease. We have developed a new technology that can map the consequences of all the important abnormal rearrangements in a cancer cell. We propose to perfect this technology and to apply it to human breast cancer tissues.

DESCRIPTION (provided by applicant): The Jackson Laboratory Cancer Center (JAXCC) provides a unique concentration of genetic and genomic expertise focusing on applications of murine model systems and systems genomics in addressing cancer questions. The JAXCC has 47 members in one Research Program, Genetic Models for Precision Cancer Medicine, comprising 3 related themes in cancer cell robustness, genetic and genomic complexity, and progenitor cell biology. Collectively, we seek to determine the molecular drivers of intrinsic cancer resistance, cancer evolution, adaptation, and self-renewal. Innovations In genomic technologies, computational analytics and mouse models for human cancers are integral to all themes, providing a robust interdisciplinary structure. The JAXCC now operates on 3 campuses: JAX Mammalian Genetics (Bar Harbor, ME) with a focus on mouse models for complex genetics; JAX Genomic Medicine (Farmington, CT), with a focus on human cancer genomics; and JAX-West (Sacramento, CA) with a focus on pre-clinical trials using patient-derived-xenograft (PDX) mouse models of human cancer. The scientific leadership of JAX has direct oversight of the Cancer Center: Dr. Edison Liu is President and CEO of JAX and Director of the JAXCC. Dr. Robert Braun Is Vice President for Research of JAX and Deputy Director of the Cancer Center. Drs. Carol Bult, Chengkai Dai and Frank McKeon are co-programming Leaders. Associate Directors for Regional Translational Partnerships (Dr. Kevin Mills) and Research Administration (Dr. Barbara Tennent) expand the leadership structure. A dedicated 7-member External Advisory Board (EAB) advises them. The new leadership, combined with recruitment of new senior and junior investigators, and a significant increase in institutional resources for cancer research heightens the impact ofthe JAXCC. This has been realized In the expanded JAXCC translational outreach through regional and national partnerships (e.g., SWOG). Support is requested for Shared Resources: a) Computational Sciences, for advanced computational and statistical analysis; b) Genome Technologies, for innovations in next generation sequencing, and in metabolomic and proteomic analyses; c) Cancer Model Development for consultation and project management in developing new models; d) Genetic Engineering Technologies for manipulating germline and somatic gene expression in mice; and e) Phenotyping Technologies for microscopy, cytogenetics, clinical assessment, histology and flow cytometry. Funds are also requested for support of annual EAB meetings and JAXCC member retreats. Developmental funds significantly supplemented by institutional funds are requested for a pilot project program to stimulate new cancer research opportunities and for support of new investigators to the JAXCC.

anticancer research; Applications Grants; Award; base; Cancer Center; Cancer Center Support Grant; Commit; cost; Data; Development; Feasibility Studies; Fee-for-Service Plans; Funding; Grant; Malignant Neoplasms; meetings; Mus; Pilot Projects; Preparation; programs; Request for Proposals; Research; Research Support; Students; Testing; The Jackson Laboratory; Time; Wages

The Jackson Laboratory (TJL) Cancer Center program planning and evaluation process is intrinsic to the continuous strategic planning effort for the parent institution. The institutional plan lays out five-year objectives for growth in research, education, and training, which guide budgetary decisions, faculty recruiting, facilities design and construction, resource and service expansion, equipment purchases, and other activities. The Cancer Center receives priority in all such decisions. Drs. Woychik and Knowles are members of the executive-level Senior Management Team, which advises Dr. Woychik on the administration of TJL and the Cancer Center. Drs. Woychik and Knowles are also advised by the Cancer Center Internal Advisory Committee composed of an interdisciplinary group of Cancer Center members and senior managers. The Scientific Advisory Council composed of six TJL faculty members, advises the Director on long-range planning, evaluation, and operation of TJL research mission, including the Cancer Center. An annual Scientific Staff retreat serves as the forum for planning that involves all Cancer Center members. External advice and evaluation are provided by the Board of Scientific Overseers. These planning and evaluation functions are supported through institutional funds. The Seminar Program serves the planning function by organizing a series of presentations by invited speakers. Speakers visit for up to three days, present seminars of Cancer Center-wide interest, and meet individually with Cancer Center members with related research interests. Drs. Woychik and Knowles use these opportunities to gain advice on long-range planning for the Center and to promote interdisciplinary collaborations. CCSG support is requested for sponsoring distinguished speakers of special relevance to cancer research. Priority will be given to speakers who can open doors for translational research partnerships and/or collaborations with other NCI-designated cancer centers or NCI-funded initiatives. Goals for the Cancer Center for the next grant cycle include: hiring one to two new Cancer Center members annually; adding research space for bioinformatics and computational biology; establishing interdisciplinary and transdisciplinary inter-institutional collaborations locally, nationally, and internationally to promote translational research; providing facilities and pioneering methods for continued improvements in efficiency of genetic resources; supporting research growth with requisite expansion and technological improvements in the Scientific Services; expanding the educational programs that serve the Center's outreach goals; and securing the necessary bandwidth and infrastructure to support anticipated data transmission needs.

Cancer Center; Grant;

The JAXCC developed a new strategic plan in 2012 that lays out initiatives for growth in genomic technologies, computational analytics, and mouse model systems that serve as robust research platforms for interrogating the genomic complexity of human cancers. These strategies converge on the common goal of establishing new preventative and therapeutic treatments based on the individual cancer genome. The plan sets objectives for growth in research, resources and education, which guide budgetary decisions, faculty recruiting, facilities design, shared resource expansion, equipment purchases, and other activities. The JAXCC External Advisory Board (EAB) meets annually with the JAXCC senior leadership to review progress toward meeting the strategic objectives; advise on strategic partnerships, especially those that advance the JAXCC's translational outreach; review JAXCC policies and governance practices; review the shared resources; and evaluate the usage of developmental funds. Chaired by Edward Benz, MD (Dana Farber Cancer Institute), the EAB members are senior leaders in NCI-designated Cancer Centers and include Michael Friedman, MD (City of Hope Cancer Center) Mark Israel, MD (Norris Cotton Cancer Center); Jill Mesirov, PhD (The Broad Institute and Koch Institute for Integrative Cancer Research); Stuart Orkin, MD (Dana Farber Cancer Institute); Pier Paolo Pandolfi, MD, PhD (Beth Israel Deaconess Cancer Center); Frank Torti, MD, PhD (University of Connecticut Health Center); and Barbara Vance, PhD, CRA (NYU Cancer Institute). CCSG funds are requested for support ofthe annual EAB meeting. Funds are requested to support the annual EAB meeting. The JAXCC Scientific Executive Committee (SEC) comprises the senior leadership planning and implementation group. The eight members represent all three JAXCC campuses and have overall responsibility for implementing the strategic plan and recommendations by the EAB. They direct the JAXCC pilot project program with emphasis on using the CCSG developmental funds to advance the strategic goals. The Seminar Program serves the planning function by bringing external cancer researchers to campus. 67 distinguished speakers presented seminars of cancer-relevance to the JAXCC members in the previous grant cycle. In the next grant cycle, the seminar program will be institutionally funded. Priority will be given to speakers who can open doors for translational research partnerships.

This renewal comes at a time of positive change for The Jackson Laboratory Cancer Center (JAXCC). The institutional strategic plan, adopted in August, 2012 (described below) sets the goals of JAX and the JAXCC for the next five years. In this plan, we enunciated the expansion of genomics, computational biology, and capabilities in mouse model creation to augment our fundamental cancer research and to move us into translational research in genomic medicine. To implement the plan, the JAXCC has established a new organizational framework that bridges the three-campus structure, strengthens the translational research capabilities, and establishes partnerships with clinical institutions. Entitled

The Jackson Laboratory Cancer Center (JAXCC) has undergone a major change in leadership with the recruitment of Edison Liu, MD in 2012 to serve as Director (4.8 months calendar effort). Robert Braun, PhD, serves as Deputy Director (2.4 calendar months). Dr. Liu, who is the President and CEO of JAX, was appointed by and reports to the JAX Board of Trustees. He sets the scientific direction and makes all appointments to the JAXCC. He has final authority on all budget decisions, and is responsible for the allocation of all central discretionary and philanthropic funds, space, and equipment for JAX and the JAXCC. Dr. Liu is a physician and researcher whose work on functional genomics of human cancers, particularly breast cancer spans the fields of molecular oncogenesis, epidemiology, genomics, and genetics. He has substantial leadership experience, most recently serving as Executive Director of the Genome Institute of Singapore, which he developed from its inception to an internationally recognized genome center. Dr. Braun, who is the Vice President for Research of JAX, reports to Dr. Liu and works closely with him to foster and implement strategic initiatives and appoint, develop and promote JAXCC members. He has primary responsibility for coordinating research across the 3 JAXCC campuses. Dr. Braun is an expert in male reproduction with a focus on RNA biology, adult stem cells and androgen action. Expansion of the Senior Leadership team was an immediate priority culminating in the appointment of additional senior leaders from the three JAXCC campuses to a Scientific Executive Committee (SEC) with Drs. Liu and Braun to guide the ongoing growth ofthe JAXCC, These include: Carol Bult, PhD, Chengkai Dai, MD, PhD, Neal Goodwin, PhD, Frank McKeon, PhD, Kevin Mills, PhD and Yijun Ruan, PhD. Drs, Bult, Dai and McKeon dedicate 1.2 calendar months to their roles as co-Program Leaders and serve the SEC in that capacity. Dr. Mills serves as Associate Director, Regional Translational Partnerships (1.2 calendar months), and leads the development of collaborative research with New England clinical centers. Dr. Goodwin, Senior Fellow, devotes 0.3 calendar months to integrating the PDX resource with the JAXCC research goals. Dr. Ruan (0.3 months effort) directs the genome technologies development initiative. The SEC meets monthly to plan large collaborative cancer research projects; coordinate and engage the JAXCC membership in the major technology and model development initiatives of the JAXCC; and direct the pilot project program in order to align research development with the strategic goals of the JAXCC. Strengthening translational outreach is a major goal. The senior leadership has formed collaborative relationships with regional cancer care centers in Maine and Connecticut, and has developed relationships with members of SWOG, culminating in membership as a Translational Medicine Member in 2013.

The JAXCC developed a new strategic plan in 2012 that lays out initiatives for growth in genomic technologies, computational analytics, and mouse model systems that serve as robust research platforms for interrogating the genomic complexity of human cancers. These strategies converge on the common goal of establishing new preventative and therapeutic treatments based on the individual cancer genome. The plan sets objectives for growth in research, resources and education, which guide budgetary decisions, faculty recruiting, facilities design, shared resource expansion, equipment purchases, and other activities. The JAXCC External Advisory Board (EAB) meets annually with the JAXCC senior leadership to review progress toward meeting the strategic objectives; advise on strategic partnerships, especially those that advance the JAXCC's translational outreach; review JAXCC policies and governance practices; review the shared resources; and evaluate the usage of developmental funds. Chaired by Edward Benz, MD (Dana Farber Cancer Institute), the EAB members are senior leaders in NCI-designated Cancer Centers and include Michael Friedman, MD (City of Hope Cancer Center) Mark Israel, MD (Norris Cotton Cancer Center); Jill Mesirov, PhD (The Broad Institute and Koch Institute for Integrative Cancer Research); Stuart Orkin, MD (Dana Farber Cancer Institute); Pier Paolo Pandolfi, MD, PhD (Beth Israel Deaconess Cancer Center); Frank Torti, MD, PhD (University of Connecticut Health Center); and Barbara Vance, PhD, CRA (NYU Cancer Institute). CCSG funds are requested for support of the annual EAB meeting. Funds are requested to support the annual EAB meeting. The JAXCC Scientific Executive Committee (SEC) comprises the senior leadership planning and implementation group. The eight members represent all three JAXCC campuses and have overall responsibility for implementing the strategic plan and recommendations by the EAB. They direct the JAXCC pilot project program with emphasis on using the CCSG developmental funds to advance the strategic goals. The Seminar Program serves the planning function by bringing external cancer researchers to campus. 67 distinguished speakers presented seminars of cancer-relevance to the JAXCC members in the previous grant cycle. In the next grant cycle, the seminar program will be institutionally funded. Priority will be given to speakers who can open doors for translational research partnerships.

Phenotyping Technologies (PT) incorporates the Histopathology and Imaging Sciences and Flow Cytometry Service functions to deliver unparalleled access to a complete phenotyping platform. Long-established Histopathology Sciences (HPS), led by Ms. Lesley Bechtold, combines scanning and transmission electron microscopy technologies, histology, and clinical assessment. The seven full-time ASCP-certified histotechnologists [HT (ASCP)] provide a comprehensive resource for preparation and morphological analysis of specimens. These Staff deliver tissue processing, paraffin and plastic embedding, cryoembedding and step, serial and cryo-sectioning; the service supports over 135 special stains and delivers immunohistochemistry services. Two full-time employees (FTE) deliver clinical chemistry and hematology analysis. Two full-time electron microscopists deliver specimen processing and preparation for examination by transmission and scanning electron microscopy; custom protocol development and consultative services are also available. Led by Dr. James Denegre, the Imaging Sciences component of the Phenotyping Technologies group delivers Light and Confocal Microscopy, Cytogenetics and Flow Cytometry Services, three Microscopy FTE provide extensive wide-field, confocal, laser-capture, image analysis and cytogenetics offerings. Instruments include two confocal microscopes, three wide field fluorescent microscopes, two stereo scopes (one of which is equipped for fluorescence), a digital pathology slide scanner, a spectral karyotyping system, and a 4Pi microscope. The Flow Cytometry function, staffed by two experienced cytometrists, is equipped with five analytical cytometers, one sorting cytometer, one imaging cytometer and a magnetic cell sorter as well as analytical workstations. Comprehensive training is provided for all microscopy and cytometry platforms, enabling authorized users independent operational access 24- hours-a-day. All Staff offer experimental design consultation, sample preparation, data acquisifion and analysis services. Advanced training is also provided through Service facilitated seminars. An extensive antibody reagent repository for flow and microscopy applications is maintained by the core. Users can work with facility staff to develop and execute analytical routines using workstations within the facility, and staff also develop scripts for automation of image analysis tasks. Collectively these highly interactive services offer JAX Cancer Center members a comprehensive platform for the characterization of phenotypes and assessment of experimentally manipulated animal models.

The long-term goal ofthe research program. Genetic Models for Precision Cancer Medicine (Drs. Bult, Dai, and McKeon co-Leaders), is to develop novel therapeutic and diagnostic concepts for cancer by modeling and interrogating the complex genomics of human cancers. Research is organized around three inter-related themes, all of which involve the systems genomics ofthe cancer cell and its host environment. The first theme. Cancer Cell Robustness, aims to define the dynamic genome alterations at a systems level that generate primary drug resistance in a cancer and its subclones. The second theme. Genetic and Genomic Complexity, aims to measure and quantify genomic instability in primary cancers; to describe the associated genomic configurations; and to use defective maintenance systems for genome integrity for therapeutic advantage. The third theme. Progenitor Cell Biology, aims to define the genomic parameters of certain cancer progenitors; to reconstruct cancer states in primary cells using systems approaches; and to use this knowledge to provide effective combinatorial therapeutics. All program goals rely on interactions with program members who are developing advanced cross cutting technological approaches. The first approach, precision model development, includes efficient methods for humanizing cancer-relevant genomic regions in the mouse; tool strains for directing and visualizing temporal and tissue-specific gene expression; optimized immunodeficient mice for sustained engraftment of primary human cancers; and populations of mice that model human genomic heterogeneity. The second approach, genomic technologies, develops new high throughput DNA interrogation methodologies for detailed assessment of structural and sequence mutations and their transcriptional consequences. The third key technology is computational, including algorithms for modeling gene networks; ontology development that supports data integration and enables in silico modeling of gene functional associations; and informatics and database systems are essential for integrative studies and comparative genomic analyses. The 47 program members include 35 at Bar Harbor, 8 at Farmington, and 1 at Sacramento, together with 3 adjunct members from UC Davis Comprehensive Cancer Center, Eastern Maine Healthcare Systems, and Trinity University (TX). Through its emphasis on basic research and unique technology development, the program leverages funding from multiple NIH ICs in support of cancer focused research. In total the program is supported by $40,150,131 total costs in NCI and other peer reviewed cancer-related support in the last budget year. Over the past grant cycle, program members produced 1022 publications, including 10% intra-programmatic collaborations and 62% with collaborators external to JAX

DESCRIPTION (provided by applicant): The Integrated Translational Science Center (ITSC) formed between SWOG, Cold Spring Harbor Laboratory (CSHL), and The Jackson Laboratory (JAX) provides a powerful combination of clinical and basic research expertise for application to cancer medicine. CSHL and JAX are both NCI-designated Cancer Centers and represent a significant collective experience in cancer gene discovery, the functional genomics of cancer, innovative tumor modeling, and the development of novel technologies to interrogate the disease. SWOG is a premier clinical trials organization that has established the standard of care for multiple cancer types over the past several decades. This alliance will bridge the gap between the laboratory and the clinic by elucidating the key clinical problems and challenges in oncology that can be addressed in the laboratory, discovering new diagnostic and therapeutic approaches that can be integrated into clinical trials, and providing a conduit for clinical trial results to be re-interpreted in the laboratory. This relationship will lead to a clearer understanding of the mechanisms underlying cancer and advance translational research aimed at developing effective targeted therapies. Both CSHL and JAX have used their extensive knowledge of the molecular alterations in human cancer to generate a diverse array of genetically modified or primary human- derived (PDX) xenograft mouse models of human cancer, and these models will be collectively utilized to identify new therapeutic vulnerabilities, determine mechanisms of primary or secondary drug resistance, and develop novel diagnostic strategies. Such information will be used to generate new hypotheses in regard to potential predictive biomarkers that can be studied in randomized clinical trials being conducted by SWOG. Specific examples of novel expertise include single cell sequencing, circulating DNA analysis, co-clinical trials in animal models, computational prediction of resistance from genomic data, and in silico and animal modeling of therapeutic combinations to optimize initial potency and avoid resistance. The scientific endeavors of CSHL and JAX will therefore inform ongoing clinical trials and establish a mechanism for fostering translational cancer research.

The Jackson Laboratory Cancer Center (JAXCC) is an organizational unit with dedicated administrative oversight that is strategically integrated with the parent organization's administration. Dr. Edison Liu, JAXCC Director, heads the Cancer Center administration, working with other members ofthe JAX executive committee, the Senior Management Team; Drs. Braun, (Center Deputy Director), Donahue (Director of Genetic Resource Science and co-Project Leader Cancer Models Development Resource) and Hewett (JAX Executive Vice President and Chief Operating Officer). Dr. Barbara Tennent, Associate Director for Research Administration provides dedicated administrative support to the JAXCC and reports to Dr. Liu. Two committees comprise the formal administrative structure ofthe JAXCC. The Operations Committee, chaired by Dr. Liu comprises Drs. Braun, Hewett, Nair and Tennent, and works to ensure that the overall direction ofthe JAXCC operations anticipates and supports the needs of JAXCC members. The Shared Resources Management Committee, which comprises Ms. Valerie Scott, Senior Director of Scientific Services, Ms. Yu-Hui Rogers, Site Director, JAX Genomic Medicine, and Drs. Donahue and Tennent coordinate the operations and development of CCSG-supported shared resources across campuses. Funds are requested for partial support of Dr. Tennent's administrative responsibilities. She oversees the necessary planning, policy development, budget development and research developmental activities for the ongoing growth ofthe JAXCC cancer research focus. Dr. Tennent is the primary coordinator for scientific and administrative management of the JAXCC. She is an ex officio member of the Scientific Executive Committee and serves on the Operations Executive Committee and the Shared Resources Committee. She works closely with all members ofthe administrative team to coordinate the activities ofthe JAXCC, including organization of the External Advisory Board meetings, management ofthe pilot project process, and membership records. Dr. Tennent is the primary administrative contact for NCI Cancer Center program officials and is responsible for management ofthe renewal, progress reports and requests from NCI as required. Institutional funds support dedicated effort for Ms. Scoff, who oversees the four institutional scientific services for which CCSG support is requested. She is responsible for advocacy, policy development and application, budget management, operations, quality assurance, technical staffing and development, planned technology acquisition and implementation as well as user communications. Institutional funds also support Ms. Roger's together in implementing processes, procedures and management structure that facilitate efficient resource sharing and access to CCSG-supported resources. The JAXCC is strongly supported by institutional administrative offices including the Office of Sponsored Programs for pre and post award administration, compliance, and financial management of the CCSG; Financial Services for budget development and account management; and Scientific Program Development, for securing external funding from foundations and federal agencies for research, education, and resource programs.

Introduction The Jackson Laboratory (J/\X) founded in 1929, is an independent, nonprofit institute dedicated to research and education in mammalian genetics and its application to precision medicine, as well as to empowering others in their scientific enterprise through access to our genetic resources. The JAX Cancer Center (JAXCC) comprises the cancer research and education activities at the Laboratory, as well as institutional shared resources that are used in cancer research. The large-scale distribution colonies that supply mice to the external research community are managed independently from the JAXCC by an administrative arm called JAX Mice & Services. The distribution colonies are financially self-supporting and, as a not-for-profit organization, generate surpluses that support fundamental research. The JAXCC occupies facilities on three JAX campuses, described below. The main JAX campus is an ~ 750,000 ft[2] facility in Bar Harbor Maine. We have launched a major expansion with the opening of a campus adjacent to the University of Connecticut Health Center (UCHC) in Farmington, Connecticut. This new campus, called The Jackson Laboratory for Genomic Medicine (JAX Genomic Medicine), will be a 189,000 ft[2] facility, to be completed in 2014. JAXCC members on this campus focus on human cancer genomics, computational biology and their application for precision medicine. The third campus, the JAX-West facility in Sacramento, California?has expanded to include a large, collaborative resource of patient-derived xenografted (PDX) primary human cancers, significantly enhancing the cancer resources ofthe JAXCC. The original campus in Bar Harbor has been renamed The Jackson Laboratory for Mammalian Genetics (JAX Mammalian Genetics) to reflect its role within the expanded three-campus framework. In total, we have an institution that has remarkable infrastructure to innovate and conduct science in mammalian genetics. The geographically distributed nature ofthe campuses is to our advantage in being able to capitalize on local (state) resources and talent, but unified by leadership, organization, and our focus on genetics and genomics.

Major Strengths Of The JAXCC The major focus of the JAXCC is to understand the complex genomics of cancer that leads to primary resistance to therapy, that enables cancer cell adaptation and evolution, and that pushes progenitor cell transformation. Our ability to address these topics comes from our capabilities in developing technologies and analytics for interrogating the cancer cell genome combined with the exceptional ability ofthe JAXCC to query the function of single genes and large-scale genomic changes using a diverse range of genetically defined mouse strains, reference populations, and unique heterogeneous stocks. Collaboration and programmatic integration are deeply embedded in the scientific culture of The Jackson Laboratory. The limited size of our faculty demands such an operating principle. The JAXCC provides a leadership and administrative structure, and is organized as one research program to further enhance transdisciplinary collaboration and integration within our three-campus structure. Throughout the previous grant cycle, this emphasis on collaboration has been shown in both the number and breadth of joint publications and grants from JAXCC members. Figure 2 depicts the network of interactions among current JAXCC members, based on the number of joint authored publications from the JAXCC as well as funded and pending joint grant submissions in which JAXCC members are co-PIs or named as key personnel. The external collaborative network is also robust, as evidenced by the 58% of publications in the last grant cycle co-authored with external investigators.

The Cancer Model Development Resource (CMDR) at JAX is a component of Genetic Resource Science (GRS), a group of over 100 people with broad expertise in mouse genetics and molecular biology necessary to manipulate the mouse genome. Dr. Leah Rae Donahue, Director of Genetic Resource Sciences, and Dr. Leonard Shultz, Professor, co-lead the CMDR. The CMDR provides JAX Cancer Center (JAXCC) investigators access to existing cancer models and supports development of new mouse models tailored to specific cancer research questions of importance to the Cancer Center. In turn, these models are made available to the JAXCC and wider scientific community through the GRS Repository. The CMDR provides JAXCC investigators access to mouse strains, the expertise of scientists in the Genetic Resource Science program, and project management. The GRS Repository is the largest single collection of genetically defined mouse strains anywhere in the world, and maintains an enormous variety of live strains (~1,600 at any given time), while other strains are maintained as cryopreserved stocks. To encourage use ofthe live resource, JAXCC members are supplied with mice free of charge, either as breeding pairs, small numbers of mutants and controls from mutant strains, or individual mice from strain panels. Additionally, the CMDR provides project management and genetic expertise to facilitate the development of cancer models by xenografts of human cancer samples into the NSG (NOD.Cg-Prkcdc[scid]//2rg[tm1Wji]/SzJ) mouse developed by Dr. Shultz. The CMDR advises JAXCC members who wish to develop specialized host strains tailored for particular scientific questions. Models are also developed for other research applications by combining mutant alleles to make compound mutant stocks. The CMDR and coordinates the model development process, working with the Genetic Engineering Technologies and Phenotyping Technologies resources as needed. The CMDR project manager coordinates animal care, management of biological materials, and surgical services to facilitate model development. The long-term objective is assist JAXCC members in their research by enabling the use ofthe full suite of genetic manipulation techniques and mouse model development.

PROJECT SUMMARY We have identified a group of genome instability configurations called the Tandem Duplicator Phenotypes (TDPs) that are found in ~50% of triple negative breast, ovarian and endometrial cancers and are characterized by the massive genome-wide distribution of somatic tandem duplications (TDs) of specific span sizes. We have identified the bona fide genetic drivers of these configurations, demonstrated that loss of Trp53 and Brca1 in the mouse mammary gland is sufficient to induce tumors with the short-span TDP configuration found in TP53- and BRCA1-deficient human cancers, and shown that upon loss of Brca1, TDs are formed through the aberrant repair of stalled replication forks. Here, we propose to deploy a combination of computational analyses, in vivo modelling and in vitro experimentation to achieve a deep mechanistic understanding of how the distinct TDP genomic configurations emerge and impact the course of breast tumorigenesis. Specifically, we will investigate the molecular mechanisms leading to de novo TD formation across the different TDP groups by exploring how local DNA features associated with DNA replication and fork stalling contribute to the generation of new TDs across a large pan-cancer dataset representing all TDP groups and all TDP genetic drivers (Aim 1A) and how loss of BRCA1 activity may modulate the spread and location of the de novo TDs formed in the context of the short-span TDP (Aim 1B). We will establish new genetically engineered mouse models (GEMMs) of breast cancer to validate that activation of the Ccne1 pathway or loss of Cdk12 activity, both in conjunction with Trp53 loss of function, induces medium- and long-span TDP configurations that mimic their human counterparts both in terms of TD span size and distribution (Aim 2A) and of the genomic features and genetic elements that are associated with and affected by TD formation (Aim 2B). We will also assess the tumor neo-antigen load of the TDP tumors emerging from the newly developed GEMMs and test whether immuno-oncology agents are effective against mammary tumors with the TDP configuration, as suggested by recently emerging clinical observations (Aim 2A). We will then use isogenic human cancer cell lines that are either proficient or deficient for BRCA1 activity, to determine the dynamics of de novo TD formation under different modes of cellular perturbation and as a function of BRCA1 status (Aim 3A). Finally, we will use the newly developed GEMMs to understand the evolutionary path to genome-wide TD distribution in the mammary gland, and to discern the dynamics of TDP emergence, both in terms of the rate of de novo TD formation and with respect to the timeline of breast tumorigenesis (Aim 3B). If successful, this proposal will uncover the root causes of a significant form of genomic instability in human cancer, the TDP, define the mutational dynamics leading to cancer formation in this condition, and generate model systems that can lead to the development of new and directed therapeutics against cancer growth.