Ronald A. DePinho, MD - US grants

Cataract disease represents an important health problem that accounts for 22% of all cases of blindness. Over 2 million cataract operations are performed in the US annually. The etiology of cataract disease remains unclear but is clearly multifactorial. Ther heritable predisposition to cataract disease suggest that genetic factors can contribute to the pathogenesis of cataracts. Congenital cataracts whcih manifest as a central (nuclear) opacifications are believed to represent perturbations in normal lens cell differentiation; however, molecular mechanisms governing cataract formation and normal lens differentiation are poorly understood. Myc-family genes (c- N- and L-myc) are believes to play key regulatory roles in normal cell growth and differentiation. Each gene is differentially expressed throughout mammalian development with dramatic changes in the expression of specific myc members coinciding with key developmental transitions in many cell lineages. We ahve demonstrated that as lens fiber cells progress through their developmental program myc-family genes are differentially regulated with respect to developmental stage. For instance, proliferating undifferentiated lens cells express c-myc and L-myc. As these immature cells undergo proliferative arrest and initiate differentiation, both c-myc and L-myc are down-regulated. In contrast, N-myc transcripts are not detectable in immature cells but become abundant at the onset of differentiation. The goal of these studies is to determine the physiological significance of myc in lens differentiation and in the development of cataract disease. To achieve these objectives, we will produce transgenic mice that aberrantly express myc-family genes in the developing lens. With the alphaA-crystallin promoter-enhancer element, we have demonstrated that forced expression of the L-myc gene in differentiating lens cells induces large nuclear congenital cataracts in all cases. Nuclear congenital cataracts may be related to defected lens development and suggests that myc serevs an important role in normal lens cell differentiation. The focus of the proposed studies are (1) to characterize further the structural and molecular features of these alphaA crystallin promoter-driven L-myc transgenic mice, (2) to generate and characterize similar transgenic animals with the c-myc gene, and (3) to utilize dominant interference to abrogate N-myc activity in actively differentiating lens fiber cells, (4) to explore the xpression and function of myc family homologues in the lens of Xenopus laevis.

The focus of this work is to define the regulatory mechanisms that govern the differential expression of myc-family genes. Myc-family oncoproteins (c-, N- and L-myc) are believed to function as regulators of normal development. Distinctive patterns and changes in expression of myc-family genes are correlated with critical developmental transitions in different cell lineages, suggesting that their differential expression may influence processes leading to cellular differentiation. The molecular mechanisms that govern the expression of myc-family genes are poorly understood. Nuclear run-on experiments indicate that transcriptional attenuation is an important mechanism in the regulation of myc-family gene expression with respect to tissue and developmental stage. The experiments outlined in this proposal are designed to (1) determine the mechanisms responsible for the tissue-specific and developmental stage-specific expression of the N-myc gene and (2) understand the physiological role of these regulatory mechanisms in normal development processes and in malignant transformation. The regulation of myc-family gene expression results from a complex regulatory strategy involving transcriptional initiation, attenuation and mRNA stability. In this proposal, we will focus primarily on the N-myc attenuator element because transcriptional attenuation appears to be the dominant mechanism regulating the tissue-specific and developmental stage-specific expression of the N-myc gene during mouse development. Regulatory sequences and factors that mediate attenuation will be identified by both biochemical and molecular genetic analysis. Candidate attenuator sequences identified by biochemical methods will be verified functionally in a transforming assay and gene-fusion experiments. Since myc gene expression studies using gene transfer into cultured cells and transgenic mice are subject to a number of artifacts, including chromosomal position effects and poor tissue-specific regulation, we will utilize gene targeting methods to mutate and analyze the attenuator sequence element in the endogenous N-myc gene. In situ sequence disruption will be performed first in a cell culture-based differentiation system and ultimately in the mouse germline. The biological impact of altered N-myc regulation will be examined. These studies will assess (1) the role of attenuation in the deregulation of myc expression in malignant transformation, (2) the significance of attenuation in the developmental expression of N-myc and (3) the significance of N-myc downregulation during cellular differentiation,

The purpose of this proposal is to obtain partial funding for an intensive training course on the design, execution, and analysis of transgenic mouse and gene targeting experiments. The Mouse Developmental Genetics course, offered at the Albert Einstein College of Medicine (AECOM) annually since 1992, teaches the basic techniques and concepts involved in the introduction of foreign DNA into the mouse germline (pronuclear microinjection), the alteration of endogenous genetiC information in embryonal stem (ES) cells and their introduction into the germline via chimera formation by blastocyst microinjection and co-culture, as well as related microsurgical skills required to harvest and return manipulated embryos to the female reproductive tract. In addition, the course will also cover methods of RNA in situ hybridization for the visualization of specific transcripts in tissue sections and whole mount preparations, and various immunohistochemiCal methods (e.g., TUNEL and BrdU assays) for the study of cell death and cellular proliferation in the developing embryo. In conjunction with the actual experimental experience the course provides daily research seminar discussions related to the development and utilization of transgenic mice as model systems for the study of human diseases. In addition to these formal presentations, informal lunchtime and dinnertime chalk talks will address more practical aspects of experimental design, specific protocols, as well as students' programs and future plans.

Elucidation of the molecular mechanisms governing cellular growth is central to our understanding of normal development and cancer. The goal of this proposal is to explore the regulation of the principal cell cycle checkpoint, the G1/S transition, in the context of the developing mouse lens. Our main focus will be on the regulation of a G1 cyclin-dependent kinase (cdk), cdk4, and its functional relationship to the retinoblastoma (Rb) protein. The cdk4 complex has captured center stage in the cell cycle field because of its documented importance in the proliferative response of cells to growth factors and of the involvement of its associated proteins in cancer pathogenesis. Activity of the cdk4 complex is regulated in large part through direct interactions with positive (cyclin D) and negative (inhibitors of cdk4, e.g. p15/INK4 and p16INK4) modulatory proteins. Owing to a foundation laid over the past 6 years, my laboratory is now uniquely positioned to address in a comprehensive manner the function of cdk4 and its associated regulatory proteins in normal mammalian development. This opportunity arises from (i) establishment of the lens as an ideal system for examining growth control in vivo (EMBO J, 1995) and demonstration that Rb, the substrate of cdk4, plays a central and essential role in lens cell growth control (Nature, 1994), (ii) characterization of the functional interrelationships among p16INK4, cdk4, Myc and Rb proteins in several cell culture systems (Science, 1995), (iii) generation of 4 different transgenic mouse lines (various cyclin D and cdk4 constructs) and a germline null mutation of the pl6INK4 gene, (iv) availability of cancer-prone transgenic mouse models that we and others have generated, and most importantly, (v) extensive experience in employing the mouse as an experimental system for the analysis of gene function. Characterization of this full complement of gain-of function and loss-of-function mouse models will allow us to understand the function of cyclin D, cdk4, and p15INK4, p16INK4 genes in the regulation of the G1/S transition in normal tissues in vivo and in the genesis of cancer. With respect to the cancer studies, we will examine and compare the onset and distribution of tumors arising in each mouse model separately, in combination with each other, or in conjunction with cancer- prone mice (such as p53-/- or some of our Myc transgenics). Finally, the connection of p16INK4 to familial melanoma will prompt an assessment of whether UV exposure of null p16INK mice accelerates the development of skin cancers.

biomedical facility; genetically modified animals; animal colony; microinjections; laboratory mouse;

gene targeting; biomedical facility; genetically modified animals; laboratory mouse;

DESCRIPTION (adapted from investigator's abstract): The goal of these studies is to test the telomere hypothesis: Cellular senescence is triggered by the shortening of telomere repeats to a critical length due to loss of telomerase function while cellular immortalization involves telomerase reactivation. The objectives of this proposal are : i) to assess the role of telomerase in the growth and development of normal cells during embryogenesis, ii) in the homeostasis of established organ systems of the adult and iii) to determine the requirement for telomerase activity in neoplasia. To achieve these objectives, Dr. De Pinho will make use of a knockout mouse model which is homozygous null for the gene encoding the essential RNA primer component of telomerase. The developmental and physiological consequences of telomerase deficiency will be monitored in organ systems that posses significant regenerative capacity. The cancer aspect of telomerase deficiency will be determined by breeding these mice with several well-established cancer-prone models.

The significance and impact of melanoma as a disease entity can not be understated. Despite the long history of clinical and molecular efforts directed towards this disease, surprisingly little is known about the precise genetic lesions leading to melanoma and even less is known with regard to how these few genetic lesions relate to disease classification or progression. Significant progress on both the basic and clinical fronts could be achieved through the production of an accurate mouse model of malignant melanoma that faithfully reproduces disease progression on the pathological and molecular levels. This proposal attempts to refine and validate further an established mouse model of cutaneous melanoma. To achieve this goal, mice will be engineered to possess several genetic lesions commonly observed in human melanomas, including activated MET, EGF receptors as well as disruption of the p16INK4a, PTEN and possibly Mxi1 genes. Evolving gene expression patterns and genomic changes at various tumor stages will be extensively cataloged as a means of validation. This refined model of melanoma should serve to advance our understanding of melanoma biology as well as to provide a system for melanoma gene discovery. The latter will include a combination of CGH, genome wide LOH, genetic mapping of susceptibility loci and candidate gene mutational analyses. The use of these melanoma mice in preclinical testing are outlined as well.

[unreadable] DESCRIPTION (provided by applicant): The principal focus of this renewal application is to explore the complex roles of telomeres, telomerase and p53 in tumor progression and metastases. Work from the current grant has utilized telomerase knockout mice to explore the complex roles of telomeres and telomerase in normal tissue regeneration, the aging process, acquired and inherited degenerative disorders, and cancer. Analysis of this model on the molecular, cellular and organismal levels has established that intact telomeres are essential for the long-term preservation of tissue stem cells and organ function, that telomere dysfunction is a key pathogenetic element in age-related and degenerative disorders, and that p53 is a nodal point in sensing telomere dysfunction and in executing a complex array of telomere checkpoint responses across different tissue compartments. Most relevant to the renewal application, this model also established that telomere dysfunction drives cancer initiation (particularly epithelial cancers) by provoking cancer-associated chromosomal structural aberrations, particularly amplifications and deletions. While cancer initiation is increased, the current work also suggests that ongoing telomere dysfunction and associated p53-dependent telomere checkpoint operate to constrain progression of these initiated neoplasms into advanced highly malignant disease. Upon this foundation, we now propose to explore how the telomere and p53 pathway interact to govern the survival or depletion of tissue stem cells and ultimately their transformation and evolution into fully malignant disease. Specifically, we propose the development of a novel inducible alleles for telomerase reverse transcriptase and p53 to assess the impact of somatic restoration of telomere function on stem cell depletion brought about by telomere dysfunction, to apply genetic screens to define the molecular circuitry of the telomere checkpoint response, to explore the relative contributions of telomerase reactivation and p53 inactivation in tumor progression, and to ascertain the molecular and biological response of established tumors to extinction of telomere activity and re-entry into telomere-based crisis. [unreadable] [unreadable]

The mouse core has been established to assist PPG member laboratories in the design, production and characterization of transgenic and knockout mouse strains. The mouse core will be responsible for all aspects of mouse engineering and will provide scientific advice on issues relating to mouse genetics and biology and to cancer modeling. Details on the construction of other mouse lines can be found in each of the accompanying proposals. We present the engineering of such mutant mouse strains as illustrative examples. These strains are the conditional PTEN knockout and the probascin-driven transgenic line. Specifically, for the production of transgenic mice, the facility will (i) maintain the core colony needed for production of the embryos and foster females, (ii) purify transgene inserts for microinjection, (iii) perform pronuclear microinjections, and (iv) conduct oviduct transfers, monitor births, and confirm transgenic founders. For the gene targeting experiments, the core responsibilities will be to (i) assist investigators in the design and construction of targeting vectors, (ii) conduct the ES transfections, selections, and clone picking and freezing, (iii) perform blastocyst microinjections of mutant ES cells, and (iv) perform uterine transfers and monitor chimera formation.

[unreadable] DESCRIPTION (provided by applicant): The aim of this proposal is to organize a scientific meeting entitled "Mouse Models for Cancer" at Disney's Contemporary Resort, Lake Buena Vista, Florida, from February 19-23, 2003. This American Association for Cancer Research (AACR) Special Conference is the third in a series of highly successful meeting on this topic. The major objectives of the meeting are: 1) to provide an overview of the field of mouse models for cancer by national and international experts; 2) To focus on new technical developments in the generation and utilization of mouse models; and 3) To provide a forum for interaction between scientists from different disciplines interested in developing and utilizing mouse models for cancer research. [unreadable] [unreadable] Animal models for human diseases are of crucial importance not only to understand the genetic factors that influence the phenotypic characteristics of the disease but also to utilize as a basis for developing rational intervention strategies. The last decade has witnessed an explosive increase in the development and characterization of mouse models for cancer. The first-generation mono-allelic cancer-prone models have been replaced by complex compound conditional TS gene knockouts/ transgenics that permit a more precise recapitulation of the temporal and spatial events in human cancer genesis, progression and maintenance. These advanced models are currently being coupled with novel noninvasive imaging modalities that enhance their suitability for preclinical testing of anti-oncologic lead compounds. Such systems hold significant promise in accelerating and making more accurate the evaluation of these compounds prior to entry into the clinic. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We have assembled a complementary team of basic cancer geneticists, mouse biologists, neuropathologists, neuro-oncologists, developmental neurobiologists, neurologists, and genomics-bioinformatics specialists to delineate the critical events in the genesis, progression and maintenance of malignant glioma. Malignant gliomas are aggressive, highly invasive and neurologically destructive tumors considered to be among the deadliest of human cancers. In its most aggressive manifestation, glioblastoma, median survival ranges from 9 to 12 months -- a fact that has changed little over several decades. It is indeed notable that, despite detailed knowledge of glioma-associated gene mutations, we know precious little about how such mutations contribute to the unique biology of this tumor type, whether such lesions play roles in both tumor genesis and maintenance, which cellular compartments serve as target for or origin of the transformation process, and why malignant gliomas remain refractory to existing therapy. It is our belief that the proposed studies will lead to meaningful insights that promise to validate specific mutations as essential or non-essential therapeutic targets as well as to identify biomarkers that will aide in glioma classification and ultimately clinical management. This new P01 application rests upon the hypotheses that: (1) genetic mutations involved in tumor genesis remain relevant to tumor maintenance; (2) tumor-associated genetic lesions play specific and discernable roles which relate to the unique biological features of glioma; and (3) genes/pathways controlling normal glia cell proliferation, survival and differentiation also contribute the pathogenesis of glioma. Drs. DePinho and Maher will exploit the experimental merits of the mouse to dissect how cellular differentiation and specific RTK and tumor suppressor gene mutations contribute to glioma progression and maintenance. Drs. Cavenee and Furnari will evaluate the genetic interactions of EGFR and PTEN through the com-bined use of expression profiling, genetic screens and cross-species model comparisons. Using neural stem cell technology and functional genomics, Drs. Rowitch and Bachoo will evaluate the role of key glioma-relevant mutations in the growth, survival and differentiation processes of normal glia. These projects will be aided by cores for Transgenic Mice and Neural Stem Cells (DePinho), Neuropathology (Louis), Genomics-Bioinformatics (Chin/Wong), and Administration (DePinho). The goals of this P01 mirror precisely the priorities articulated by the recent NCI/NINDS Brain Tumor PRG.

DESCRIPTION (provided by applicant): This consortium represents a multi-disciplinary team effort to launch an integrated program focused on model development/refinement, cancer gene discovery, preclinical experimental therapeutics and technology development. The model development initiative will seek to develop and characterize models that recapitulate two fundamental aspects of cancer in humans - epithelial cancer predisposition and episodic chromosomal instability - by engineering mice that experience transient telomere dysfunction and subsequent telomerase activation in the evolution of colorectal cancer and squamous cell carcinoma of the skin. In addition, a model of pancreatic ductal adenocarcinoma will be developed in which we attempt to ascertain whether telomere-linked chromosomal instability and compromised telomere checkpoints serve as a general mechanisms driving age-dependent epithelial carcinogenesis. In the cancer gene discovery area, we will conduct a comprehensive comparative oncogenomic analysis of human and mouse epithelial cancers (specifically colon, skin and pancreas) by taking advantage of powerful gene-specific array-CGH and informatics platforms. These comparative oncogenomic studies will not only serve to validate these models but also carry the potential to identify common and distinct molecular themes in human and murine carcinoma development. In therapeutics program, inducible oncogene melanoma models will be used to identify genetic resistance elements capable of replacing activated RAS, BRAF or AKT function in established tumors. Both genomic and forward genetic screens are proposed. Finally, an important mission will be technology development in the areas of genomics, informatics, imaging and nanoteclmology. Specifically, the team will develop the oligo-based CGH platform and develop of informatics tools for crossspecies genomic comparisons. The imaging component will feature several experiments such as the application of novel reagents to visualize the dynamic vascular changes occurring in regressing tumors as well as hypoxia-induced reporters capable to monitoring oxygen status in growing and regressing tumors. Finally, nanosensors will be developed that are capable to detecting telomerase activity and quantifying telomere reserves.

model; neoplasm /cancer

This project seeks to exploit established mouse PDAC models/alleles, a strong oncogenomics and[unreadable] validation infrastructure, and a comprehensive PDAC genomics database in order to elucidate how signature[unreadable] genetic mutations contribute to the initiation and progression of PDAC and to discover and validate new[unreadable] PDAC oncogenes residing in regions of recurrent focal amplications. This project is driven by the hypothesis[unreadable] that the development of effective targeted therapies demands a comprehensive understanding of how known[unreadable] and novel lesions interact and influence tumor phenotypes. In the context of the established K-Ras-driven[unreadable] PDAC model, Project 1 will examine systematically the role of Ink4a/Arf, p53 and/or Smad4 mutations. All of[unreadable] the projects will work as a cohesive unit to determine the impact of each of these mutations on tumor[unreadable] histopathologic progression, invasive and metastatic potential (this project), angiogenic and stromal[unreadable] response (project 3), signaling pathway activation (project 2), and status of the cancer stem cell[unreadable] compartment (4). The efforts of Project 1 to reconstruct the key genetic events in the mouse will not only,[unreadable] build a PDAC progression model in which genotype-phenotype correlations can be established, but will also[unreadable] establish a modeling foundation upon which roles of yet-to-be discovered PDAC oncogenes can be validated[unreadable] in vivo. Indeed, it has become evident that many PDAC-relevant genes are emerging from ongoing genome[unreadable] scanning and transcriptome profiling efforts. Project 1 will make use of a large high-resolution human PDAC[unreadable] genomic database (including genomic profiles of our mouse tumors) and a battery of established functional[unreadable] and biochemical assays to begin to identify and validate novel PDAC oncogenes. As an illustrative example,[unreadable] Project 1 will pursue a systematic discovery and validation approach to the genes residing within a highly[unreadable] recurrent and focal amplicon with potential relevance to the PI3K pathway. The assays utilized emphasize[unreadable] the target's prevalence on human PDAC samples (in collaboration with the Experimental Pathology and[unreadable] Biobank Cores), its linkage to critical cancer cell signaling pathways (particularly Ras-PI3K in collaboration[unreadable] with Project 2), its expression/activity state in the cancer stem cell compartment (in collaboration with Project[unreadable] 4), and its role in PDAC tumor biology (particularly in shaping/maintaining the tumor microenvironment in[unreadable] collaboration with Project 3). Finally, we develop an inducible system for the P01 in which genotypephenotype[unreadable] of known and yet-to-be-discovered PDAC oncogenes can be elucidated.

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer death, with nearly all cases following a rapid and merciless course of intractable pain, cachexia, hopelessness and death. Repeated cycles of clinical trial failures have underscored the need for an improved understanding of PDAC pathogenesis, motivating the formation of this multi-disciplinary team of basic, translational and clinical scientists, with knowledge in cancer cell signaling, developmental and stem cell biology, tumor biology and angiogenesis, mouse models of cancer, genomics and informatics, molecular imaging, experimental pathology, and small molecule chemistry and biotherapeutics. The goal of this P01 is to further elucidate the genetics and biology of the disease to a level that will guide the rational development of effective targeted agents, alone and in combination. Specifically, we seek to (i) refine mouse models of human PDAC in order to understand the tumor biological role of PDAC signature mutations, (ii) identify, validate and characterize new human PDAC oncogenes in order to set the stage for drug discovery, (iii) illuminate and validate the role of the PI3K pathway in PDAC using mouse and human systems with the goal of identifying key therapeutic targets and guiding clinical trials targeting this pathway, (iv) utilize genetically engineered mice and human tumors to define signaling events governing the evolution and maintenance of the PDAC microenvironment, and to use this information to design and conduct innovative preclinical trials in refined mouse models of human PDAC, (v) determine the PDAC cell of origin and its genetic events in the mouse so as to illuminate a possible path to chemoprevention, and (vi) identify and characterize PDAC cancer stem cells in murine and human tumors. State-of-the-art imaging and antibody technologies, as well as strong experimental pathology and biospecimens repositories, will enable in-depth and dynamic analyses of PDAC tumor biology and signaling in unprecedented detail. The long-term goal of these basic and preclinical efforts, employing and integrating both mouse and human systems, is to identify and guide opportunities for targeted drug discovery and for the development of diagnostic agents and biomarkers.

This project seeks to exploit established mouse PDAC models/alleles, a strong oncogenomics and validation infrastructure, and a comprehensive PDAC genomics database in order to elucidate how signature genetic mutations contribute to the initiation and progression of PDAC and to discover and validate new PDAC oncogenes residing in regions of recurrent focal amplications. This project is driven by the hypothesis that the development of effective targeted therapies demands a comprehensive understanding of how known and novel lesions interact and influence tumor phenotypes. In the context of the established K-Ras-driven PDAC model, Project 1 will examine systematically the role of Ink4a/Arf, p53 and/or Smad4 mutations. All of the projects will work as a cohesive unit to determine the impact of each of these mutations on tumor histopathologic progression, invasive and metastatic potential (this project), angiogenic and stromal response (project 3), signaling pathway activation (project 2), and status of the cancer stem cell compartment (4). The efforts of Project 1 to reconstruct the key genetic events in the mouse will not only, build a PDAC progression model in which genotype-phenotype correlations can be established, but will also establish a modeling foundation upon which roles of yet-to-be discovered PDAC oncogenes can be validated in vivo. Indeed, it has become evident that many PDAC-relevant genes are emerging from ongoing genome scanning and transcriptome profiling efforts. Project 1 will make use of a large high-resolution human PDAC genomic database (including genomic profiles of our mouse tumors) and a battery of established functional and biochemical assays to begin to identify and validate novel PDAC oncogenes. As an illustrative example, Project 1 will pursue a systematic discovery and validation approach to the genes residing within a highly recurrent and focal amplicon with potential relevance to the PI3K pathway. The assays utilized emphasize the target's prevalence on human PDAC samples (in collaboration with the Experimental Pathology and Biobank Cores), its linkage to critical cancer cell signaling pathways (particularly Ras-PI3K in collaboration with Project 2), its expression/activity state in the cancer stem cell compartment (in collaboration with Project 4), and its role in PDAC tumor biology (particularly in shaping/maintaining the tumor microenvironment in collaboration with Project 3). Finally, we develop an inducible system for the P01 in which genotypephenotype of known and yet-to-be-discovered PDAC oncogenes can be elucidated.

21+ years old; Abscission; Address; Adult; Alleles; Allelomorphs; Anti-Oncogenes; Antibodies; Antioncogenes; Apopain; Apoptosis; Apoptosis Pathway; Astrocytes; Astrocytic Neoplasm; Astrocytic Tumor; Astrocytoma; Astrocytoma, Grade IV; Astrocytus; Astroglia; Astroglioma; Automobile Driving; BZS; Belief; Biochemical; Biological; Biological Models; Brain; Brain Neoplasia; Brain Neoplasms; Brain Tumors; Bypass; CASP-3; CASP3; CD140B; CPP-32; CPP32; CPP32 protein; CPP32B; CPP32beta; Cancer Genes; Cancer-Promoting Gene; Cancers; Candidate Disease Gene; Candidate Gene; Caspase 3, Apoptosis-Related Cysteine Protease; Cell Communication and Signaling; Cell Cycle; Cell Cycle Arrest; Cell Cycle Control; Cell Cycle Regulation; Cell Cycle Regulation, Including Apoptosis; Cell Death, Programmed; Cell Division Cycle; Cell Growth in Number; Cell Line; Cell Lines, Strains; Cell Multiplication; Cell Proliferation; Cell Signaling; Cell-Death Protease; Cell/Tissue, Immunohistochemistry; CellLine; Cells; Cellular Proliferation; Cellular Transformation; Cessation of life; Classification; Clinical; Color; Combined Modality Therapy; Complement; Complement Proteins; Condition; Construction; Cysteine Protease CPP32; DNA Alteration; DNA mutation; Data; Death; Deep; Depth; Development; Diffuse; Disease; Disease Progression; Disorder; Drivings, Automobile; EGFR; EGFR Blocker; EGFR Inhibitor; EGFR Tyrosine Kinase Inhibitor; EGFR inhibition; EGFR-TK Inhibitor; ENPT; ERBB Protein; ERBB1; Embryo; Embryonic; Emerogenes; Encephalon; Encephalons; End Point; EndPointCode; Endpoints; Engineering; Engineerings; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Inhibitor; Epidermal Growth Factor Receptor Kinase; Epidermal Growth Factor Receptor Protein-Tyrosine Kinase; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Erlotinib; Evaluation; Event; Excision; Exons; Exposure to; Extinction; Extinction (Psychology); Extirpation; FLR; Facility Construction Funding Category; Failure (biologic function); Feedback; Foundations; Funding; Gene Alteration; Gene Mutation; Genes; Genes, Cancer Suppressor; Genes, Onco-Suppressor; Genetic; Genetic Alteration; Genetic Change; Genetic Markers; Genetic Models; Genetic defect; Genetic mutation; Genomics; Genotype; Glial Cell Tumors; Glial Neoplasm; Glial Tumor; Glioblastoma; Glioma; Glioma, Astrocytic; Gliomagenesis; Goals; Grade IV Astrocytic Neoplasm; Grade IV Astrocytic Tumor; Grant; HER1; Histopathology; Human; Human, Adult; Human, General; ICE-like protease; IHC; Immunohistochemistry; Immunohistochemistry Staining Method; In Vitro; Incidence; Injection of therapeutic agent; Injections; Intracellular Communication and Signaling; Intracranial Central Nervous System Neoplasms; Intracranial Central Nervous System Tumors; Intracranial Neoplasms; Intracranial Tumor; Investments; JTK12; Knock-in; Knock-in Mouse; Knock-out; Knockout; Lesion; MHAM; MMAC1; Maintenance; Maintenances; Malignant Glial Neoplasm; Malignant Glial Tumor; Malignant Glioma; Malignant Neoplasms; Malignant Neuroglial Neoplasm; Malignant Neuroglial Tumor; Malignant Tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Mice; Mitochondria; Model System; Modeling; Models, Biologic; Models, Genetic; Molecular; Multimodal Therapy; Multimodal Treatment; Multimodality Treatment; Murine; Mus; Mutation; Necrosis; Necrotic; Neoplasms; Neoplasms of Neuroglia; Nervous System, Brain; Neuroglial Neoplasm; Neuroglial Tumor; Oncogenes; Oncogenes, Recessive; Oncogenes-Tumor Suppressors; Oncogenic; Overexpression; PARP Cleavage Protease; PDGF-R-Beta; PDGFR; PDGFR1; PDGFRB; PDGFRB gene; PTEN; PTEN gene; PTEN1; PTK Receptors; Pathogenesis; Pattern; Phenocopy; Phenotype; Phosphatase and Tensin Homolog; Play; Position; Positioning Attribute; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Pre-Clinical Model; Preclinical Models; Primary Neoplasm; Primary Tumor; Principal Investigator; Programs (PT); Programs [Publication Type]; 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Treatment Protocols; Treatment Regimen; Treatment Schedule; Tumor Cell; Tumor Suppressing Genes; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor-Derived; Tumors; Tumors of Neuroglia; Tyrosine Kinase Growth Factor Receptor; Tyrosine Kinase Linked Receptors; Tyrosine Kinase Receptors; Validation; Work; Yama; Yama protein; adult human (21+); angiogenesis; base; behavioral extinction; biological signal transduction; c-erbB-1; c-erbB-1 Protein; caspase; caspase-3; combination therapy; combined modality treatment; combined treatment; comparative; cultured cell line; cystein protease; cystein proteinase; cysteine endopeptidase; cysteine protease P32; disease/disorder; driving; drug development; drug discovery; erbB-1; erbB-1 Proto-Oncogene Protein; erbBl; experiment; experimental research; experimental study; failure; gene product; genetic element; genome mutation; glioblastoma multiforme; glioma genesis; human disease; in vivo; inhibitor; inhibitor/antagonist; malignancy; metaplastic cell transformation; mitochondrial; mouse model; multimodality therapy; neoplasia; neoplasm/cancer; neoplastic cell; neoplastic growth; nestin; nestin protein; new therapeutics; next generation therapeutics; novel; novel therapeutics; oncosuppressor gene; overexpress; postnatal; pre-clinical; preclinical; programs; proto-oncogene protein c-erbB-1; receptor expression; research study; resection; resistant; response; social role; spongioblastoma multiforme; transcriptomics; transgenic; tumor; tumor suppressor; tumor xenograft; tumors in the brain

Abscission; Alleles; Allelomorphs; Animals; Area; Arm; Assay; Astrocytes; Astrocytoma, Grade IV; Astrocytus; Astroglia; BZS; Banking, Tissue; Bears; Bio-Informatics; Bioassay; Bioinformatics; Biologic Assays; Biologic Marker; Biological; Biological Assay; Biological Function; Biological Markers; Biological Models; Biological Process; Biology; Biopsy; Blastocyst; Blastocyst structure; Blastosphere; Blood Cells; Body Tissues; Brain; Brain Neoplasia; Brain Neoplasms; Brain Tumors; CDK6 Inhibitor p18; CDK6-associated protein p18; CDKN2C; CDKN2C Protein; CDKN6; CNS Tumor; CNS neoplasm; Cancer Genes; Cancer Model; Cancer-Promoting Gene; CancerModel; Cancers; Cell Line; Cell Lines, Strains; Cell/Tissue, Immunohistochemistry; CellLine; Cells; Central Nervous System Neoplasms; Chimera; Chimera organism; Chimerism; Chin; Clinical; Cloning; Collaborations; Collection; Colony-Forming Units, Neoplastic; Communities; Complementary DNA; Complex; Consent; Construction; Core Facility; Cultured Cells; Cultured Tumor Cells; Cyclin-Dependent Inhibitor; Cyclin-Dependent Kinase 4 Inhibitor C; Cyclin-Dependent Kinase 6 Inhibitor; Cyclin-Dependent Kinase 6 Inhibitor p18; Cyclin-Dependent Kinase Inhibitor 2C; Cyclin-Dependent Kinase Inhibitor 2C (p18, Inhibits CDK4); DNA, Complementary; Data; Detection; Development; ES cell; Embryo; Embryo, Preimplantation; Embryonic; Encephalon; Encephalons; Engineering; Engineerings; Environment; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Ethics Committees, Research; Evaluation; Evolution; Excision; Expertise, Technical; Extirpation; Facility Construction Funding Category; Female; Fostering; Freezing; Future; Gene Products, RNA; Gene Targeting; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genetically Engineered Mouse; Genetics, in situ Hybridization; Genome; Genome Instability; Genomic Instability; Genomics; Genotype; Germ-Line Mutation; Germline Mutation; Glial Cell Tumors; Glial Neoplasm; 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Monitor; Mothers; Mouse Strains; Murine; Mus; Mutation; Neoplasms of Neuroglia; Neoplastic Cells, Cultured; Nervous System, Brain; Neural Neoplasm; Neural Stem Cell; Neural Tumor; Neuroepithelial, Perineurial, and Schwann Cell Neoplasm; Neuroglial Neoplasm; Neuroglial Tumor; Numbers; Oncogenes; Operation; Operative Procedures; Operative Surgical Procedures; PTEN; PTEN gene; PTEN1; Partner in relationship; Pathogenesis; Pathology; Pathway interactions; Patient currently pregnant; Patient pregnant NOS; Patients; Pb element; Performance; Peripheral Blood Cell; Phosphatase and Tensin Homolog; Position; Positioning Attribute; Pregnancy not delivered; Pregnancy, gravid; Primary Neoplasm; Primary Tumor; Principal Investigator; Production; Productivity; Programs (PT); Programs [Publication Type]; Proteins; Protocol; Protocols documentation; Purpose; RNA; RNA, Non-Polyadenylated; Reagent; Recording of previous events; Removal; Research; Research Ethics Committees; Research Infrastructure; Research Personnel; 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cDNA; cell engineering; cell transformation; cellular engineering; concept; cost; cultured cell line; cyclin-dependent kinase inhibitor p18; design; designing; doubt; embryonic stem cell; established cell line; experience; gain of function; gene discovery; gene function; gene product; genome mutation; genome, mouse; glioblastoma multiforme; heavy metal Pb; heavy metal lead; host neoplasm interaction; human tissue; improved; in situ Hybridization Staining Method; in vivo; innovate; innovation; innovative; instrumentation; malignancy; mate; member; mimetics; molecular imaging; mouse genome; mouse model; neoplasm/cancer; nerve stem cell; neural progenitor cells; neuronal progenitor; neuronal progenitor cells; neuropathology; new technology; new therapeutics; next generation therapeutics; novel; novel therapeutics; p18; p18 protein; p18-INK4C; p18-INK6; p18INK4c protein; pathway; personnel; pregnant; programs; pup; quality assurance; reagent testing; repository; reproductive; resection; screening; screenings; severe combined immune deficiency; social role; spongioblastoma multiforme; stem; stem cell of embryonic origin; surgery; therapeutic target; tool; transcriptomics; transformed cells; transgenic; transmission process; transplant; tumor; tumor suppressor; tumors in the brain; tumors in the central nervous system; validation studies; vector

[unreadable] DESCRIPTION (provided by applicant): Tuberous Sclerosis (TSC) is caused by mutation in either TSC1 or TSC2 gene and is characterized by the formation of hamartomas in a variety of organs. However, the development of malignancy is very rare in TSC. TSC1-TSC2 complex is a critical negative regulator of the mammalian target of rapamycin (mTOR). It has been showed that an mTOR-mediated negative feedback loop leads to attenuation of Akt signaling in TSC-related tumors and compromised cell survival in TSC deficient cells, which might explain the rare progression to malignancy in TSC. However, uncertainties remain as to (i) the underlying mechanisms and key downstream effector(s) of Akt that mediate the pro-survival function of TSC and (ii) the basis for the attenuated cancer phenotype of the TSC-related tumors. Recent genetic studies from both mouse models and human cancers strongly suggest that the FoxO transcription factors represent key downstream effector arms of PI3K-AKT signaling network and play critical roles in cancer development. Indeed, neoplastic conversion of endothelial cells is a prominent phenotype upon loss of either TSC or FoxO, suggesting an epistatic relationship. Furthermore, FoxOs were shown to localize in the nucleus and active in TSC deficient cells and tumors, suggesting that FoxOs might play a critical role in TSC pathophysiology. Thus, a clear understanding the genetic and functional interactions between TSC and FoxO appears to be critical to understand TSC pathogenesis and ultimate management. This proposal aims to elucidate the underlying mechanisms of rare malignancy in TSC-related tumors. We hypothesize that the anti-apoptotic function of TSC is mainly mediated by suppression of FoxOs transcriptional activity through promoting Akt-mediated FoxO phosphorylation. Reactivation of FoxOs upon loss of TSC might contribute to the less severity of TSC-related tumors and thus compound deletion of both TSC and FoxOs would lead more malignant phenotypes. The specific aims are (1) to examine whether compound inactivation of FoxOs and TSC in the mouse would exacerbate the tumor phenotypes observed in TSC single mutants, (2) to study the impact of FoxOs on TSC-mediated cell biological activity in primary cells, and (3) to identify direct FoxO transcriptional targets which might mediate the cell biological functions of TSC. This proposal will provide important biochemical and biological insights into the role and essentiality of FoxO transcriptional factors in TSC development. The negative feedback regulation of Akt signaling by TSC-mTOR signaling suggests combined treatment of rapamycin analogues with inhibitors of PI3K-Akt signaling should be used in TSC clinical trials. Given the critical role of FoxO in PI3K-Akt signaling, our efforts to identify direct FoxO transcriptional targets involved in the regulation of TSC might expand drug development opportunities for TSC or, minimally, identify a FoxO biomarker which may provide a pharmacodynamic marker for monitoring the impact of anti-PI3K drugs in clinical trials. PUBLIC HEALTH RELEVANCE: This proposal aims to examine the role of FoxO transcriptional factors, a key downstream surrogate of PI3K-AKT signaling in cancer development, in TSC-mediated negative feedback regulation through combined genetic, genomics and cell biological approaches. The identification of validated FoxO targets with oncogenic activities playing essential roles in the regulation of TSC may expand drug target opportunities for this disease. 1 [unreadable] [unreadable] [unreadable]

This Administrative Core will ensure that effective communication and the flow of materials are maintained among the various projects and cores. The Core provide financial oversight and report scientific progress of the entire program to assure continued success. The Core director will work with program associates in ensuring that meetings between the PO1 investigators are scheduled regularly via face to face meeting as well as phone, emails, teleconferences, videoconferences, and WebEx presentations. These communications will ensure that the goals of the overall program projects are met and that all project and core budgets are administered properly. In addition, this core will act as a liaison between various performance sites to ensure effective utilization of institutional resources and as a catalyst to translate the discoveries made by the program project into early clinical testing in close collaborations with GI SOPRE of DFCI and other clinical departments. Finally, the Core will manage and ensure large datasets are properly deposited and useful reagents and animal models are distributed to research communities rapidly following the execution of material transfer agreement between institutions.

DESCRIPTION (provided by applicant): The University of Texas MD Anderson Cancer Center is a free-standing comprehensive cancer center within the University of Texas system. In 2011, the institution marked its 70th anniversary and it welcomed Ronald DePinho, M.D., as its fourth full-time president and PI of the CCSG. The mission of MD Anderson is to eliminate cancer in Texas, the nation and the world through outstanding integrated programs of patient care, research, education and prevention. MD Anderson is dedicated wholly to the study of cancer involving a continuum of basic, clinical and population-based investigation, with an emphasis on multidisciplinary translational research. During the last 5 years, the number of cancer center members has increased 14%, facilities including those under construction have increased 56% and new patients have increased to 34,000 annually. Annual citations in Web of Science have increased to 2507 in 2011 (16.5%), including 283 articles in journals with an impact factor >10, reflecting substantial contributions to cancer research. The total research budget increased 40% from $445 million to $624 million. NCI grant support has increased from $115M to $120M (3%) with the largest number of NCI grants for any center (>220), including 11 SPOREs and 13 P01s. Research Programs remain at 19 and support is requested for 16 shared resources. Since the last CCSG renewal, basic science has been strengthened substantially with recruitment of world class leaders in immunology, genomics, proteomics, drug discovery and cancer biology. Translational research has been enhanced and strategic planning implemented to accelerate reductions in cancer deaths in this decade, through MD Anderson's 'moon shot' efforts across the cancer care continuum focused on several major cancers. These planning efforts were made possible by the CCSG disease programs and their well-established multi-disciplinary interactions. Since the last renewal 21 new agents developed at MD Anderson have entered clinical trials, 8 additional agents are expected to enter clinical trials in the next year and 12 INDs were prepared by the cancer center. Clinical research has been strengthened with new leadership, further development of infrastructure and data bases, and emphasis on hypothesis driven, investigator initiated trials. Cancer is a global problem, and MD Anderson has built a network of 26 Sister Institutions in 19 countries to facilitate research and educational activities. Cancer prevention and survivorship is a priority for MD Anderson with an emphasis on molecular epidemiology, behavioral science, clinical cancer prevention and early detection research to reduce the burden of cancer within Texas and worldwide.

DESCRIPTION (provided by applicant): The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology, Preclinical Therapeutics, Computation, and an Administrative Core will enable these Projects.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death and is driven mostly by mutated Kras gene. Despite advances in our understanding of the pathogenesis of PDAC, the disease remains highly refractory to treatment. In this proposal, we will employ genome scale approaches to identify potential signaling nodes and druggable targets that will be used for therapeutics development for PDAC patients. Based on our preliminary data and technical capabilities, we proposed the following specific aims. Aim #1) To identify co-extinction targets for combination therapeutics against KRAS* PDAC. The goal of this aim is to identify candidate targets that when co-extinguished can lead to suppression or death of KRAS* PDAC tumor cells. Both genetic and pharmacological approaches will be used, taking advantage of strengths of both mouse and human systems, as well as leveraging the emerging comprehensive genomic data on human PDAC. Co-extinction target candidates identified in more than one system will be prioritized for in-depth biological, functional as well as clinical pathological validation. Aim #2) To identify resistance mechanism to KRas inhibition. The goal of this aim is to proactively anticipate and elucidate possible molecular basis for resistance in setting of Kras* inhibition. We will apply a novel in vivo context-specific genetic screen approach to identify and functionally validate genes that promote survival of PDAC cells following KRas inactivation, initially focusing on the kinases followed by genetic elements of interests defined by comprehensive genomics by ICGC PDAC project. Furthermore, we will engineer GEM models with the most promising resistant hits to validate resistance mechanism in vivo and to test new therapeutic strategy against such resistance mechanisms.

The construction and phenotypic analysis of genetically engineered mouse (GEM) strains are fundamental and integral for the study of pancreatic cancer, including the analysis of signaling molecules in this pathology, the discovery and analysis of novel genes and their linked networks, and the validation and assessment of novel therapeutic targets and associated molecular biomarkers. The Genetic Engineering Mouse Core (GEMC) of this PDAC POI will provide all the necessary expertise, reagents and services to generate four genetically engineered mouse (GEM) modeling projects per year of grant funding. The GEMC will work closely with POI project leaders, investigators to produce the most advanced cancer relevant GEM strains. Specifically: (A) Transgenics; the GEMC will support all aspects of the construction of transgenic mouse models, including advice, service, technologies and reagents for the optimal design and construction of each specific transgene. (B) Gene Targeting; We will support all aspects of the construction of knockout and knock-in mouse alleles, including provide services, support, advice, technologies and reagents for the optimal design, construction and production of each specific targeting vector and resulting mice. (C) Evaluate and implement new technologies for the construction of genetically engineered mice and derivative cells.

PROJECT SUMMARY (See instructions): The Translational and Analytical Chemistry Core (TACC) consists of a Chemistry Core Facility and a Nuclear Magnetic Resonance (NMR) Facility. The Chemistry Core began operations in July 2006 and was designated a CCSG developmental shared resource in July 2008. In 2012, the NMR Facility was moved from the Pharmacology & Analytical Facility to the TACC. The mission of the combined core is to assist researchers with the design, synthesis, and analysis of compounds for use in cancer research. The facility is a state-of-the-art organic chemistry lab, offering services ranging from drug design to pre-clinical drug development. The TACC offers two innovative services that other synthesis cores do not. The molecular modeling service allows design of ab inito new chemical agents. Additionally, the TACC offers small molecule X-ray crystallography. The Chemistry Core has experienced average yearly growth of 36% for design and synthesis services (Development, Modeling, and Custom Synthesis) and a decline of 7% for the analytical services (Mass Spec and X-ray). Over the last 5 years, the Chemistry Core has completed 69 projects and synthesized 144 compounds for 35 cancer center members, representing more than 11,200 hours of service. Major equipment for the Chemistry core includes Varian LC-940 HPLC systems, an Agilent Accurate-Mass TOF LC/MS and a Bruker SMART X2S Automated Bench Top X-ray system. The NMR facility is used primarily for structure determination of compounds developed in drug discovery, pharmacology, and diagnostic imaging laboratories. Other uses include macromolecular structure determination and metabolism studies. Usage of the NMR facility increased 147% over the past five years. Instrumentation consists of Bruker 300 MHz DPX, 500 MHz DRX and 600 MHz Avance NMR spectrometers. In the past five years researchers from thirty-two research groups have utilized the laboratory. Peer reviewed investigators account for 87% of utilization of chemistry services and 46% of utilization of the NMR Facility. For the coming grant cycle, TACC requests $232,209 (24%) support from the CCSG. The institution has provided $1,598,359 for purchase of TACC equipment. Publications cited using the TACC have appeared in J Clin Invest and J Natl Cancer Inst. Future plans include the proposed expansion of the facility to incorporate compound screening to provide for early stage hits and access to GMP capabilities to support clinical research studies. The NMR Facility will continue to support the drug discovery and imaging agent research at MD Anderson, and plans to expand into support of metabolomics research.

PROJECT SUMMARY (See instructions): The Patient-Reported Outcomes, Survey, and Population Research (PROSPR) shared resource is a centralized source of expertise in the science of collecting and managing participant-reported outcome (PRO) and behavioral data. These data are important in many treatment trials (e.g., to evaluate the impact of treatment on quality of life and symptoms), survivorship studies, and in cancer prevention research. The PROSPR offers services in 3 areas: research support services, which have seen a 100% increase in utilization over the past 5 years, data support services (12% increase), and fitness and body composition testing (new service in 2011). PROSPR has access to a SQL server, as well as equipment for testing fitness and body composition (Hologic bone densitometer, Biodex for measuring strength, 3 metabolic carts, 3 ECGs, 2 cycle ergometers, 1 treadmill, a perometer, and a bank of 150 actigraphs). In the past 5 years, the PROSPR facility has served 112 investigators in 17 programs. PROSPR has facilitated 47 publications with articles appearing in JAMA and J Clin Oncol. Center members with peer-reviewed funding account for 80% of users, and 32% of total costs are requested from the CCSG. The institution has provided $431,072 in financial support for the shared resource since 2008 for operational costs and for equipment, including an HP server, 3 Dell precision workstations, and 150GT3XE-plus triaxial activity monitors. In addition, the fitness and body composition equipment was purchased by the institution. Over the next 5 years, PROSPR will improve its expertise and expand services in design and data management for computer-assisted data collection (e.g., ecological momentary assessment, computer adaptive testing, and internet-based surveys) and expand its expertise in body composition testing.

PROJECT SUMMARY (See instructions): The Small Animal Imaging Facility (SAIF) provides Cancer Center Members with powerful imaging technologies, staff and expertise necessary to integrate imaging into preclinical cancer research. The facility is led by Drs. John D. Hazle and James A. Bankson and is supported by a network of faculty with research expertise in each imaging modality. Faculty and staff consult with users prior to planning of imaging experiments, and assist in all aspects of imaging services, including preparation of grants, experimental design, protocol optimization, data acquisition and analysis, interpretation, presentation, and publication of results. Instrumentation and full-service support are available for preclinical MRI (4.7T/40cm, 7.0T/30cm) including hyperpolarized

MD Anderson uses several external and internal review mechanisms for planning and evaluation to develop and improve the cancer center. External Reviews: For the past 27 years, MD Anderson has used funding from the CCSG to support an External Advisory Board (EAB), consisting of a group of 30 distinguished scientific advisors that meets annually at MD Anderson to provide advice to Dr. DePinho, CCSG senior leadership, CCSG program leaders, and CCSG shared resource directors. The objectives of the EAB are to review research programs and shared resources, and evaluate their productivity, excellence, and alignment with institutional goals; to identify gaps in scientific expertise or capabilities and to suggest options for closing these gaps; to consider new programs or restructuring of existing programs; to assist in recruiting outstanding faculty to the institution; to evaluate the extent of collaboration between basic scientists, translational/clinical scientists, and population-based investigators, recommending actions to enhance interactivity among these three groups: External reviews of shared resources consist of visits by an ad hoc EAB of 3 outside experts specific to each core and have been conducted for 7 of the 16 shared resources presented in this application. Plans are to extend these reviews to additional shared resources. Internal Reviews: Mechanisms for internal review of research programs, shared resources, and research planning include formal Research Council presentations of programs and shared resources, the Institutional Clinical Executive Committee (ICEC) and the Institutional Research Executive Committee (IREC), which advise Dr. DePinho on strategic issues related to clinical and research activities, and the CCSG Executive Committee which oversees CCSG program and shared resource development in the context of the broader institutional research strategy. Surveys, focus groups, and user committees have also reviewed each shared resource during the current grant period. Strategic Planning: A comprehensive planning exercise across a broad, multidisciplinary perspective, the Moon Shot developed proposals for 11 cancers that integrate all aspects of the cancer continuum - prevention, early detection, diagnosis, treatment, and survivorship to impact survival over the next several years. Five of these areas will be selected for intensive organization and support. All CCSG programs are intimately involved with the development of these plans, which will chart the course for future programmatic research.

PROJECT SUMMARY (See instructions): The primary function of the Protocol Review and Monitoring System is to ensure that all human subjects research is of the highest scientific merit and proceeds at an optimal pace. Over the past 5 years, 934 faculty members participating in human subjects research utilized the PRMS, and 273 new interventional protocols were reviewed by the PRMS in the last year. The PRMS is supported by 32 staff members under the direction of Dr. Aman Buzdar, Vice President for Clinical Research Administration. The core function of the PRMS is to provide a mechanism to ensure adequate internal oversight of the scientific and research aspects of all institutional clinical trials through a rigorous review of the scientific merit, progress, and priorities of the clinical research protocols conducted by the faculty. This function is coordinated by the PRMS as a single source of service, support and oversight The PRMS is made up of several subcommittees (described below) that are designated to provide scientific review and approval for new research protocols, as well as to monitor the progress of the ongoing trials. Importantly, the increasingly complex regulatory requirements and oversight activities have resulted in increased institutional staffing commitments and prompted provision of several new or expanded services, as described in greater detail in this application. During the last grant year, the funds used to support the PRMS function were $240,152 (8%) from the Cancer Center Support Grant (CCSG), $110,000 (4%) from IRB fees, and $2,540,868 (88%) from the institution. It is projected that in the next award cycle, the funds used to support the PRMS function will be $249,758 provided by the CCSG (9%), $110,000 from IRB fees (4%), and $2,406,755 (87%) from the institution. The PRMS consists of four Clinical Research Committees (CRCs) and one Psychosocial Behavioral Health Services Research Committee (PBHSRC) that review protocols for scientific merit. The Chairs of these committees are also members of the Electronic Protocol Accrual Auditing Committee (ePAAC) that selects and monitors all institutional protocols that are identified as slow-accruing. Eight hundred and one protocols were reviewed by ePAAC in the last fiscal year, and 115 (14%) were closed or withdrawn. During the last 6 months, substantive changes have been made 1) to make the initial review and prioritization of protocols by the CRC and the disease site programs and departments more rigorous, 2) to reduce the time required for review, and 3) to scrutinize accrual more carefully.

PROJECT SUMMARY (See instructions): The Tissue Biospecimen and Pathology Resource (TBPR) is comprised of mature, highly functional CCSG supported shared facilities and provides all basic science, translational, clinical, and population science investigators at MD Anderson with access to human tissues that have been removed by therapeutic resection or biopsy with research consent, and advanced histologic services for characterization of human and animal research tissues. The TBPR supports hypothesis-generating, -developing, and -testing studies, including both correlative and integrated marker studies in clinical trials. The TBPR biorepository has six -80° freezers and three isothermal liquid nitrogen freezers in its centralized and monitored storage facility, and a - 80° freezer in each of the two Surgical Pathology suites. The TBPR research histology core includes specialized equipment for immunohistochemistry, fluorescent in situ hybridization (FISH), laser capture microdissection, and tissue microarray construction. 190,235 tissue specimens from 37,296 cases were collected during the current reporting period for Yrs 33-38, and 63,288 specimens were distributed to 152 investigators. Research histology usage grew by 5.2% from 174,015 units in 2007-2008 to 183,134 in 2011- 2012. Of the 152 total individual users of the TBPR biorepository, 86% of users had peer-reviewed funding, and 81.2% of 422 TBPR research histology users had peer-reviewed funding. 42% of the total operating budget for the TBPR is requested from the CCSG. Publications cited using the CCLC have appeared in Nature, Nature Med and J Clin Oncol. Institutional administrative support was provided by the Research Support Office in the Division of Pathology & Laboratory Medicine. Future plans include: establishing collaborative efforts with Lyndon B. Johnson Hospital and Banner MD Anderson to increase specimen collections from underserved and community patient populations; increasing sterile tissue collection and processing for vaccine clinical trials; increasing specimen collection in clinic procedure rooms and Interventional Radiology; collaborating with the Office of Biorepository Regulatory Support to standardize operations in all federated biorepositories at MD Anderson; enhancing Tissue Station integration with clinical and research protocol management databases; increasing the roster of available tissue biomarkers; and implementing a more efficient and multifunctional Laboratory Information Management System for charge backs, inventory monitoring, and workload reporting.

The Research Animal Support Facility (RASF) exists to support ongoing research involving laboratory animals at MD Anderson. All animal facilities are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International, have Animal Welfare Assurance approval, and are registered as research animal facilities with the US Department of Agriculture. The RASF has two locations: RASF-Houston (RASF-H -112,990 ft^) and RASF-Smithville (RASF-S -30,000 ft^). Both RASF facilities provide housing, procedure space, veterinary care, and quality assurance programs for animals used in cancer research. Clinical, surgical, imaging, radiation therapy, and pathology laboratory facilities and services such as Genetic Services and Mutant Mouse Pathology Service are also provided. RASF-H veterinarians provide consultation services and participate in all relevant compliance committees. Major equipment includes individually ventilated rodent caging, automated bedding dispensing and waste collection, patient monitoring in vivo imaging systems, automated pathology specimen processing, and vehicles for materials animal transport. The RASF has 156 personnel, including 17 veterinarians and 97 animal care personnel. In the past 5 years, the RASF has been used by 378 investigators supporting all 19 CCSG programs. Publications cited using the RASF have appeared in several high impact journals such as Nature, Nature Med, Cell Stem Cell and PNAS. Peer-reviewed investigators represent 92% of the RASF-H and 95% of RASF-S user utilization. The RASF-H requests 5% of CCSG support for the next grant cycle, and 26.5% for RASF-S. User fees account for 52% (RASF-H) and 44% (RASF-S), institutional support is 43% (RASF-H) and 23% (RASF-S), and 2% other sources support RASF-S. FIASF-H. Future plans include renovation and expansion of the CRB basement animal facility, build-out of the animal imaging support holding facility on South Campus, and upgrading and implementing computer applications for business operations and preclinical drug development. RASF-S future plans include enhancing/expanding barrier facilities, continued customization of breeding colony management software, development of new research genetic services and new ante-mortem bioluminescence and fluorescence imaging services.

PROJECT SUMMARY (See instructions): The CCSG senior leadership includes the MD Anderson president and principal investigator. Dr. Ronald A. DePinho, the provost and executive vice president ad interim. Dr. Thomas Buchholz, the vice president for translational research and co-principal investigator. Dr. Robert Bast, the vice president for basic science and associate director for basic science. Dr. Mien-Chie Hung, the vice president for clinical research administration and associate director for clinical research. Dr. Aman Buzdar, the Vice President for Cancer Prevention and associate director for cancer prevention. Dr. Ernest Hawk, and the chair of the Department of Health Disparities Research and associate director for health disparities Dr. David Wetter. This leadership team comprises the CCSG Executive Committee. Dr. DePinho as president of MD Anderson has authority over all aspects of the institution, providing vision and ultimate oversight for all clinical, research, education, and prevention activities. Dr. DePinho also chairs the MD Anderson institutional executive committee providing leadership and strategic direction in support of MD Anderson's mission and vision. Dr. Buchholz provides executive leadership for the development of all research and academic activities. Dr. Bast meets with program leaders, reviews program membership, encourages program activities, promotes multidisciplinary grants, coordinates the physician-scientist and clinician-investigator programs, meets with shared resource directors, reviews capital equipment requests, oversees finances of the core facilities, helps to develop new shared resources, and recommends use of development funds. Dr. Hung is responsible for facilitating all aspects of basic research and strengthening the intraprogrammatic- and interprogrammatic collaborations of the basic science programs. Dr. Buzdar is responsible for clinical research infrastructure, facilitates all aspects of clinical research and ensures that MD Anderson adheres to the highest standards of clinical research compliance with federal regulations. Dr. Hawk provides strategic and tactical direction to the center's work in cancer prevention research, education, and clinical services, and helped to establish the institution's cancer control program to guide policy, educational, and service initiatives oriented toward community needs. Dr. Wetter coordinates efforts to assure minority participation in clinical trials working with other members of the administration to increase the number of minority patients cared for at MD Anderson.

PROJECT SUMMARY (See instructions): In January 2012, the CCSG-supported Genomics and DNA Analysis facilities consolidated their activities to form a comprehensive institutional genomics shared resource: the Sequencing and Microarray Facility (SMF). The mission of the SMF is to support genomics research at MD Anderson by providing investigators with access to state-of-the-art instrumentation and a high level of technical expertise in a centralized facility, thereby minimizing the duplication of expensive equipment while maintaining technical excellence. The SMF, located in a newly renovated 1600-square-foot laboratory space, provides a complete range of genomic services that includes project and technology consultation, RNA and DNA quality assessment and quantification, Sanger DNA sequencing, comprehensive lllumina next-generation sequencing services, gene resequencing, fluorescent fragment analysis, and TaqMan-based gene expression analysis. The SMF also provides complete DNA and RNA services for several microarray applications, including gene expression, single-nucleotide polymorphism, comparative genomic hybridization, chromatin immunoprecipitation microarrays, and microRNA profiling. MD Anderson has demonstrated its support and commitment to the SMF by providing more than $1.2 million in funding for the purchase of state-of-the-art instrumentation. Major equipment includes AB 3730 sequencers, lllumina Hiseq2000 sequencers, an AB 7900 Sequence Detection System, Affymetrix GeneChip systems, an Axon Genepix 4000B Scanner, a MAUI Hybridization System, and an lllumina BeadXpress and iScan. Since 2008, the SMF component facilities processed 785,638 samples for 399 Pis in 19 CCSG programs supporting 14 P01, 227 R01, and 14 SPORE grants. The total number of samples processed increased 39%, and the number of Pis using the SMF increased 32% over the previous grant period (3/1/02-2/28/07). Publications cited using the SMF have appeared in several high impact journals including Nat Genet, Lancet Oncol, Cell and Cancer Cell. Peer-reviewed Pis accounted for 96% of the samples processed. Funding for the new grant cycle is proposed to come from the CCSG (22%), user fees (77.8%) and institutional support (0.2%). Future plans include the expansion of the NGS service to include platforms for midscale and single-molecule sequencing. The SMF will continue to expand the range of NGS and validation services offered and develop methods that can successfully use MD Anderson's unique resource of archival tumor samples.

Since the last CCSG renewal, Development Funds have supported 1) the recruitment of nine outstanding young investigators, 2) development of three new shared resources and a new service in an existing shared resource, and 3) partial funding for eight MD Anderson Multidisciplinary Research Program (MRP) awards, and three MD Anderson/Baylor Collaborative Multidisciplinary Research Projects. Some $435,000 of CCSG support for faculty recruitment has leveraged $4.5M in MD Anderson start-up packages and more than $11.6M in new extramural grants. MRP awards provide seed funding of $250,000 over 3 years for groups of three or more center members to support planning and development of cores and projects, and to obtain preliminary data for submission of an application for a P01, SPORE or CPRIT multi-investigator grant. Since the inception of MRP awards in 1997, MD Anderson has invested $7.6 million in these seed grants, associated with external peer-reviewed funding of more than $121 million, a 16-fold return on investment Since the last renewal, 8 proposals supported with $535,955 from CCSG Development Funds and matched approximately 2:1 with institutional funds, have leveraged $7,433,633 in peer-reviewed funding. Three Baylor/MD Anderson Collaborative MRPs supported with $92,518 in CCSG Development Funds resulted in a P01 award in 2011 of more than $13M over 5 years to Drs. E. Shall (MD Anderson) and C Bollard (Baylor) for their studies on improving cord blood transplantation in patients with cancer. Development funds supported establishing the Translational and Analytical Chemistry Core, the Characterized Cell Line Core, and the Functional Proteomics Reverse Phase Protein Array Core, which in aggregate have provided services to 273 center members and resulted in more than 200 publications. The Mutant Mouse Pathology Service has become part of the Research Animal Support Facility. In the current CCSG application. Development funds are requested to continue support for faculty recruitment and fund additional MRP awards. Support is also requested for three developing shared resources: the shRNA and Open Reading Frame Core, the High Throughput TALEN Engineering Core, and eHealth Technology. A competitive internal peer-review mechanism is also proposed for the allocation of Development Funds to provide support for establishing new methods and implementing new technologies within the shared resources.

PROJECT SUMMARY (See instructions): Administration of the CCSG is consolidated in the Office of Translational Research under the direction of the CCSG Co-PI, Dr. Robert Bast, who reports to the Pl, Dr. Ronald DePinho, for matters relating to the CCSG. Administrative support is provided in the following areas: (1) business operations and financial management of shared resources; (2) central organization and coordination for the scientific and technical aspects of shared resource operations; (3) facilitation of programmatic interactions and engagement; (4) facilitation of communication between center director, program leaders, shared resource directors, and program members; (5) development, management, and maintenance of databases for program funding, program membership, program member publications, program member biographical information, and shared resource utilization; (6) allocation and monitoring of the use of CCSG development funds; (7) coordination and writing of CCSG progress reports and competitive renewal applications; (8) organization of CCSG Executive Committee and annual External Advisory Board meetings. The 5 administrative staff supporting the CCSG have extensive scientific, management, and administrative skills and experience. Alan McClelland, PhD, is responsible for analysis, planning and facilitation of all CCSG funded activities and reports directly to Dr. Bast. Dr. McClelland manages, directs, and oversees the overall administration of the CCSG award. Katherine Stemke Hale, PhD, is responsible for scientific, technical, and operational oversight, coordination and strategic planning for all institutional shared resources. Holly Stephenson maintains program membership data, assigns grant funding to programs as directed by the Pl, prepares and submits progress reports and cancer center summaries, organizes the logistics of external advisory board visits, and provides administrative support for the preparation of the CCSG competitive renewal application. Audrey Jones is responsible for managing all aspects of CCSG finances, including budgeting, allocation, monitoring, and reporting for all sources of funds that support CCSG-funded infrastructure, including shared resources. Carolyn Duff works with the Faculty Information System (PIS), a database system used for CCSG publication data management and reporting, and is also responsible for updating MD Anderson internal and external websites for CCSG programs and shared resource content.

PROJECT SUMMARY (See instructions): The Bioinformatics Shared Resource (BISR) provides consultation, collaboration, and support for researchers and core facilities throughout MD Anderson in the statistical analysis and biological interpretation of data from high-throughput pre-clinical technologies. The BISIR has specialist expertise in the bioinformatics of all types of microarrays, next-generation sequencing, mass spectrometry, and flow cytometry. The consultation and collaboration services of the BISR (in hours) have grown 3.1 fold over the past 5 years. The BISR uses a heterogeneous computing environment supported across Windows, Unix/Linux, and Mac OS X operating systems, with access to more than 300 terabytes of in-house storage space for home directories, research data, and data mirrors. It accesses in-house parallel computing capability through a 48-processor Cray XD1 HPC cluster and a 290-processor distributed computing Condor pool of over 160 Windows workstations (each with >2GB of memory) and 8 servers (ranging from 4GB to 16GB of memory). BISR services have been used over the past 5 years by 301 researchers 92% of whom are peer-reviewed cancer center members. Publications cited using the BISR have appeared in Nature, Science, N Engl J Med and J Clin Oncol. Annually, MD Anderson has provided institutional support to the BISR in the amount of $1,244,846. The BISR is requesting funding from the CCSG in the amount of 3% of its total operating budget. In the last 5 years, the BISR has (1) recruited 9 very strong new faculty members from top institutions plus 2 more with joint appointments. It will continue to recruit top bioinformatics faculty and will encourage their participation in multidisciplinary collaborative research to complement their investigator-initiated research; (2) recruited 8 new statistical analysts and 3 biocatalysts to support projects around the institution; (3) created a cadre of 14 postdoctoral fellows, who participate in collaborative research; (4) recruited 4 new programmer/software engineers plus 6 on contract to provide programming strength for its support functions. It will continue to seek talented computer specialists; (5) established a popular hands-on workshop series on bioinformatics tools for MD Anderson biologists and clinical researchers (>500 attendees for 2-hour sessions to date). The BISR will continue to encourage the professional growth of ail of its members through advanced education In bioinformatics and computational skills. It will continue to improve its emerging status as one of the world?s leading bioinformatics groups as we pursue MD Anderson's mission: Making cancer history.

PROJECT SUMMARY (See instructions): The Biostatistics Resource Group (BRG) provides biostatistical collaboration, consultation, and quantitative research resources to clinical, laboratory, and prevention scientists at MD Anderson who are engaged in the planning, analysis, quality assurance, and interpretation of research studies. The BRG has pioneered the use of the Bayesian approach in adaptive clinical trials, particularly, in the Phase I and Phase II settings. The consultation and collaboration services of the BRG (in hours) have grown by 10% over the past 5 years. The primary equipment and technology of the BRG is a well-supported computing environment that extends across varied operating systems: Windows, Unix/Linux, and Mac OS X; providing access to more than SO non-standard desktop, client, and server applications; more than 300 terabytes of in-house storage space for home directories, research data, and data mirrors; and to in-house parallel computing capability through a 48-processor Cray XD1 HPC cluster and a 290-processor distributed computing Condor pool composed of over 160 Windows workstations (with at least 2GB of memory) and 8 servers (ranging from 4GB to 16GB of memory). BRG services have been used by 1070 researchers over the past five years, with 79% of the use by peer-reviewed cancer center members. The BRG is requesting funding from the CCSG in the amount of 9% of the total operating budget. Publications cited using the BRG have appeared in several high impact journals such as JAMA, J Clin Oncol and J Natl Cancer Inst. Annually, MD Anderson has provided institutional support to the BRG in the amount of $3,952,162. The BRG will continue to attract highly-qualified biostatistics faculty and to encourage their participation in multidisciplinary collaborative research; to hire and train highly-qualified statistical analysts and to encourage their professional growth through advanced education in statistics methodology and the application of computational tools; to instruct and mentor talented graduate and postdoctoral researchers; and to design innovative biostatistical approaches that promote discovery in cancer research. The BRG will continue to be the world's leading group in the development and application of Bayesian methodology for medical research.

Cancer Center; Cancer Center Support Grant; Grant;

Award; Cancer Center; Clinical Investigator; Grant; Leadership; Malignant Neoplasms;

. PROJECT SUMMARY (See instructions): The Characterized Cell Line core (CCLC) was established in 2008 with the purpose of preserving and distributing cell lines developed at MD Anderson. The initial focus was on providing well-characterized cell lines so that researchers could choose the correct cell line for their research. Services were expanded almost immediately to include human cell line validation and Mycoplasma testing. Cell line validation is done by short tandem repeats (STR) profiling. To avoid duplicating equipment and effort, the polymerase chain reaction (PCR) fragmentation reactions are run by the Sequencing and Microarray Facility on an Applied Biosystems 3730 XL genetic analyzer. The CCLC has developed a novel STR matching algorithm that can take into account variations due to cell line cross-contamination and to genomic instability. CCLC has also established a proprietary database with STR profiles from over 1000 unique cell lines as well as over 2000 public STR profiles. The CCLC also offers mutational analysis of human cell lines as another method to ensure cell line authenticity. Mutational analysis is done using a Sequenom MassARRAY, which runs a primer extension based method to determine sequence information on a single base. The CCLC has developed a custom somatic mutation panel enabling incorporation of new somatic mutations as they are published in the literature, rather than waiting for a new somatic mutational panel from Sequenom. The CCLC also offers custom-designed panels. Since 2008, the CCLC has been used by 128 Center members, representing all 19 CCSG programs. The institution has supported this core with funding of $99,692 for capital equipment, including incubators, a -80? C freezer, PCR machines, a plate reader, and a Qiacube robot. The CCLC has facilitated publication of 53 reports since 2008, with 58% in journals with an impact score >5 and 15% in journals with an impact factor >10. Publications cited using the CCLC have appeared in several high impact journals such as Nat Gen, Cell, Cancer Cell and J Natl Cancer Inst. Peer-reviewed investigators account for 91% of the utilization, and 34% of the total costs are requested from the CCSG. This will enable expansion of services and additional testing of cell lines to identify cross-contamination between species as well as to test for potential virus contamination. Future work will focus on expanding the number of cell lines available to MD Anderson researchers, expanding characterization of cell lines to include whole genome/exome sequencing approaches, and improving methods to detected cross contamination especially intra-species cross-contamination.

The Clinical & Translational Research Center (CTRC), established in 1990 and rated Outstanding in the 2008 CCSG review, focuses on new drug development. This comprehensive 18-bed combined treatment and laboratory unit serves as the primary site for personalized, protocol-based, innovative clinical and translational research. Services provided are research infrastructure and oversight as well as 24-hour phlebotomy/lab support for pharmacologic testing. Other services include specialized processing for DNA and RNA analysis, real-time internet-based tracking of audit time-points, Ei

The Clinical Trials Support Resource (CTSR) provides infrastructure support for all aspects of clinical protocol research at MD Anderson. The services of this shared resource encompass submitting, activating, and closing single and multi-centered clinical trials; educating research staff on human subjects/clinical research; auditing and monitoring active clinical trials; and ensuring regulatory compliance of IND studies. The online data management system is maintained by the CTSR. The CTSR complements the activities of the Protocol Review and Monitoring System by supporting the electronic protocol submission and review processes, and the CTSR will manage the transition from the current electronic protocol management system to a new platform called eResearch, which will go live in early 2013. The CTSR is supported by 106 staff members (of which 8 full-time employees receive full or partial support from the CCSG) under the direction of Dr. Aman Buzdar, Vice President for Clinical Research Administration. During the past 5 years, the number of new protocols managed by CTSR has increased by 4% from 4,632 in fiscal year (FY) 2007 to 4,818 in FY 2011. Overall, trials led by MD Anderson investigators contributed to the FDA approval of 19 drugs for initial or new indications. New patient registrations have increased from 28,762 in FY 2007 to 46,262 in FY 2011, and the number of patients enrolled in therapeutic clinical trials has increased by 18%, from 6,219 in FY 2007 to 7,558 in FY 2011. During the last grant year, the funds used to support the CTSR salaries were $442,190 (9%) from the CCSG, $812,112 (16%) from user fees, and $3,742,322 (75%) from MD Anderson. In the next award cycle, the level of support from the CCSG, user fees, and MD Anderson is projected to be closer to 4%, 7%, and 89%, respectively. During this past 5-year funding period, CTSR resources were used by 934 principal investigators affiliated with all 19 CCSG programs. Peer reviewed investigators account for 61% of the utilization and 4% of total costs are requested from the CCSG. Publications cited using the CTSR have appeared in several high impact journals such as N Engl J Med and J Clin Oncol. Future plans include implementation of eResearch, which will serve as the new institutional clinical data enterprise system; coordination and modification of inter-departmental operational processes to further reduce time from submission to activation of clinical trials to 60 days; and further enhancements to the human subjects protection training programs.

The Monoclonal Antibody (MAb) Facility (MAF) provides non-commercially-available antibodies to MD Anderson Cancer Center investigators for specific applications. The MAF serves basic, translational and clinical researchers across the institution and is currently developing projects in each of these three areas. Although the services are based on traditional murine hybridoma technology, the MAF offers custom immunization strategies and support for functional screening in order to produce unique antibodies suitable for novel applications. The MAF occupies 726 sq. ft. in 1SCRB, home to the Center for Cancer Immunology Research (CCIR) on the South Campus. This is a state-of-the-art facility for immunology research that provides a platform for integrating basic and clinical research programs. The CCIR is equipped with customized laboratory services, centralized tissue culture rooms, liquid nitrogen tank rooms, and glassware washing and sterilization facilities, all of which are available to the MAF. The MAF has a tissue culture laboratory (SCR 4.2158), a protein chemistry area (SCR 4.2220), and space in the South Campus Vivarium that is a component of the Research Animal Support Facility shared resource. In the last 5-year period, the MAF developed three MAbs that have potential for clinical development as therapeutic agents, and several additional candidates are in preclinical assessment. Several antibodies produced by the MAF have been licensed or are in the process of being licensed for commercial development. MD Anderson members with peer-reviewed funding accounted for 97% of the usage of the resource and 30% support is requested from the CCSG. Since 2007, the MAF has supported the research of 40 MD Anderson investigators with peer reviewed funding representing 16 CCSG Programs, compared to 13 investigators in the previous grant period. Hybridoma production increased from 44 to 79 projects (a 178% increase), and total services provided increased more than 300% during this grant cycle. Publications cited using the MAF have appeared in Nature, PNAS, Blood and Nature Medicine. Future plans include the purchase of a bioreactor for large scale production to support the increasing demand for the quantities of MAbs required for preclinical development Novel methods of immunization including DNA expression will be explored. The facility also plans to explore the direct generation of fully human antibodies using humanized mice or using Phage display scFv libraries as a more rapid and direct strategy to produce antibodies for future clinical applications of newly-discovered markers.

. PROJECT SUMMARY (See instructions): The High Resolution Electron Microscopy Facility (HREMF) was established in 1997 to provide a resource to investigators for high resolution imaging of cells, tissues, and nanomateriais. The mission of the HREMF is to provide scanning and transmission electron microscopy services to members of MD Anderson. The HREMF occupies 536 square feet in the R.E.

PROJECT SUMMARY (See instructions): The Genetically Engineered Mouse Facility (GEMF) generates mouse models for MD Anderson Cancer Center members and offers essential services for archiving important models and receiving novel models from outside sources. The facility provides gene-targeting in mouse embryonic stem cells, blastocyst injection services, pronuclear injection services, embryo and sperm cryopreservation services, rederivation services, in vitro fertilization (IVF), generation of novel mouse embryonic stem cells, and various resources for generating DNA constructs and generating and testing conditional GE mice. In the past 5 years, over 1,200 animal lines were generated by either pronuclear injection or blastocyst injection; over 200 clones were generated by gene-targeting; more than 15,000 embryos were frozen; more than 3,500 straws of sperm were archived, and more than 200 mouse lines were cleaned through rederivation. In addition to knockout mice, the facility has made mouse strains with unique fusions, mutations or protein isoforms to further elucidate genetic effects. Utilization of GEMF services has remained steady at 200-300 procedures per year between 2007 and 2011. The GEMF has seen increases in services such as rederivation, used as more models are brought into the institution, and the mouse archiving services of embryo and sperm cryopreservation. The GEMF has provided services to members of 18 CCSG programs during the last 5 years, serving over 100 Cancer Center members. Peer-reviewed investigators account for 89% of the utilization and 51% of total costs are requested from the CCSG. Publications cited using the GEMF have appeared in Nature, Science, Cell Stem Cell, PNAS and Nat Med. Institutional support for the facility includes over $120,000 in funding for capital equipment since 2007. Future plans include working with the newly established Tai Effector Nucleases facility that can be used to quickly generate targeted animal models. The GEMF is exploring cryopreservation protocols that are more effective at producing high percentage IVF-capable sperm, and the use of IVF to quickly and efficiently produce large numbers of embryos.

PROJECT SUMMARY (See instructions): Established in 2008 as an emerging core facility, the Functional Proteomics Reverse Phase Protein Array (RPPA) Core provides cancer center members with access to a powerful, high-throughput, quantitative cost effective antibody-based assay to characterize basal or ligand-induced protein expression and modification, and time-resolved responses appropriate for systems biology analysis. The RPPA provides information to integrate the consequence of genetic aberrations in cancer, to validate therapeutic targets and to evaluate drug pharmacodynamics. MD Anderson provides 640 square feet of space, additional salary support, and administrative support through the Department of Systems Biology. Services include providing standard operating procedures to investigators for extraction of protein from cells or tumor tissue, processing protein extracts received from investigators, robotic arraying on nitrocellulose-coated slides, and probing with antibodies validated by the RPPA core. Major equipment purchased in part with institutional funding of $320,000 includes a Tecan robotic liquid handling system for serial dilution of cell/tumor lysates and sample transfer, two Aushon 2470 arrayers for printing of cellular/tumor lysates onto nitrocellulose-coated slides and two Dako Universal Staining systems for probing slides with antibodies. Data are analyzed using customized software (MicroVigene and Supercurve Fitting) to determine signal intensity, curve construction, and relative protein concentration. Importantly, the facility validates antibodies to expand the available antibody repertoire. The facility established qulity control processes to improve the quality and accuracy of the RPPA data sets. Since 2008, the facility has processed 47,306 samples from 179 users with a panel of 150 to 180 antibodies. Center members with peer-reviewed funding account for 78% of users, and 20% of total costs are requested from the CCSG. Service utilization grew by an average 14% per year over the past 4 years and 40% over the last 4 years. Publications cited using the RPPA have appeared in high impact journals such as Nature Medicine, Nature Genetics, PNAS and Cancer Cell, etc. In the future, the RPPA Core plans to 1) expand the validated antibody repertoire; 2) optimize assay conditions for laser-aided micro-dissected or paraffin-embedded tissue samples; 3) explore RPPA applications for body fluids; 4) provide forward phase protein array; and 5) develop technology for kinase affinity capture.

. PROJECT SUMMARY (See instructions): The Flow Cytometry and Cellular imaging Facility (FCCIF) was established to provide access to state-of-the art cell analysis technology for MD Anderson investigators, and provides cell sorting, analytical flow cytometry, cellular imaging and custom monoclonal antibody (mAb) conjugations to its users. The Core provides researchers with technical expertise in instrument operation, assay development, data acquisition and various analysis techniques. Analytical flow cytometry is an indispensable tool for the study of all aspects of cell biology, including protein expression, cell proliferation and differentiation, cell signaling pathways, enzyme activity, gene regulation, ceil lineage, apoptosis, autophagy and chemotherapeutic resistance. The Core has recently acquired a DVS CyTOF Mass Cytometer, enabling the detection and characterization of up to 100 molecular markers at the single cell level. This instrument represents a transformational technology enabling the detection and characterization of rare and mixed cell populations on the single cell level. Cell sorting. Cell isolation for culture and further characterization is performed via droplet-based sorting, which isolates a wide variety of cells based on combinations of antibody-based stains, fluorescent protein expression, and viability indicators. Imaging. The Core offers researchers tools and techniques for image acquisition, SD-reconstruction, and time-series observation as well as a variety of image processing and analysis functions via laser scanning cytometry and confocal microscopy and also offers multispectral, epifluorescent, and colorimetric microscopy. Custom mAb conjugations. Antibody conjugation is a new service of the FCCIF that provides conjugates with fluors and tags that are not commercially available. The FCCIF uses 24 major instrument systems supporting the research of-345 investigators who hold 13 POIs, 112 ROIs, and 9 P50 SPOREs. Peer-reviewed investigators account for 94% of the utilization, and 35% of total cost is requested from the CCSG. Over the past 5 years, the FCCIF has performed more than 50,000 hours of service, representing a 125% increase over the prior grant period. Over the past 5 years, the FCCIF has facilitated publication of 408 reports, with 67% in journals with an impact factor >5 and 22% with an impact factor >10. In the future, the FCCIF will continue to develop the use of the current instrumentation, including the DVS Sciences CyTOF, and novel analysis programs, including the SPADE algorithm. Older equipment will be replaced, and an Amnis Image Stream, a Vectra 2 automated multispectral imaging system and single-cell analysis systems such as Fluidigm's BioMark may be added.

Abstract ? Core D (Administrative Core) The Administrative Core (Core D) will be responsible for the overall administration of the P01 program. It will provide clerical support, meeting organization, data management and planning/evaluation services to this Program. The responsibilities of Core D include the following aims: (1) Monitoring research progress in the P01 program via monthly scientific meetings and quarterly progress updates; (2) Providing scientific oversight and research direction both from the Core D leader, Dr. DePinho, and also from the experienced membership of this program's Scientific Advisory Board; (3) Fostering communication and collaboration between P01 program participants by facilitating meetings (in person and via the internet), managing a annual program retreat, and maintaining a website for program data sharing and communication; (4) Providing fiscal oversight and resource allocation including the review accounting summaries to ensure fiscal management, enabling subcontracts, and establishing cost centers and reporting practices; and (5) Facilitating data and reagent sharing and dissemination.  

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Abstract - Project 1 (Targeting Metabolic Dependencies in PDAC) Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with a median survival of less than 6 months and a dismal 5-year survival rate of 7%. The highly malignant nature of PDAC is largely the result of driving oncogenic Kras mutations (Kras*) in >90% of tumors, as well as the heterogeneous nature of the disease at both the genomic and cellular levels. To date, no drug directly targeting Kras* has reached the clinic, and inhibitors of Kras* effector pathways in clinical trials have achieved only minimal responses followed by relapse of aggressive disease. Furthermore, immune targeting of PDAC has so far been unsuccessful. Thus, a critical need remains to identify new therapeutic vulnerabilities in PDAC. In our previous grant cycle, Project 1 and our P01 team established a role for Kras* in tumor maintenance in vivo wherein it controlled key metabolism enzymes supporting cancer-relevant anabolic processes that, in turn, are required for Kras*-driven PDAC maintenance. Using our inducible Kras* PDAC model, we also identified a subset of tumor cells with tumor-initiating cell (TIC) properties that can survive Kras* extinction and may lead to tumor recurrence following oncogene ablation. One of the hallmarks of these Kras* extinction-resistant cells (KRCs) is the shift from aerobic glycolysis to mitochondrial oxidative metabolism to sustain cell viability. Our P01 team further demonstrated that, while PDAC exhibits high basal autophagy, autophagic flux in KRCs was further enhanced to supply substrate for mitochondrial oxidative phosphorylation (OXPHOS), and targeting autophagy or OXPHOS effectively eliminated KRCs to prevent tumor relapse. Therefore, the collaborative work from our P01 team strongly suggests that effective therapeutics for PDAC should target not only players essential for Kras*-dependent tumor maintenance, but also pathways required to maintain KRCs. These data are the basis for an initiative begun during the last grant cycle to develop a novel OXPHOS inhibitor compound in PDAC, IACS-10759, at the Institute for Applied Cancer Science. In this next cycle, Project 1 will continue our efforts to better define the metabolism programs that sustain Kras*-dependent tumors as well as KRCs and to explore the translational potential of targeting metabolic processes, including targeting OXPHOS with IACS- 10759. Project 1 will work closely with the Cores, which have extensive expertise in pathology, preclinical therapeutics, and computational biology, and will be highly integrated with Project 2 to characterize the role of autophagy-regulating pathways. Our studies will also integrate with Project 3, using our inducible Kras* mouse model to explore the effects of Kras*-dependent and ?independent metabolism programs on tumor immunity and response to immune checkpoint therapy. The knowledge gained from these highly integrated studies aims to inform future clinical trials opportunities.  

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Program Abstract - Overall The goal for this renewal application is to a) elucidate oncogenic Kras (Kras*)-regulated metabolic pathways mediating pancreatic ductal adenocarcinoma (PDAC) tumor maintenance, b) define collateral metabolic dependencies, and c) establish how interventions targeting these processes influence tumor immunity, in order to guide the design of clinical trials with existing drugs and to identify new therapeutic points of attack. Our P01 program comprises 3 highly interdependent and collaborative projects and 4 essential cores with the goals of conquering PDAC through targeting metabolic vulnerabilities in conjunction with immunotherapy. Project 1 (DePinho with Draetta) has demonstrated that Kras* extinction in PDAC leads to pronounced tumor regression that involves critical functions of Kras* in metabolic reprogramming. We have also identified Kras*-extinction resistant cells (KRCs), which show dramatic adaptive metabolic changes (in OXPHOS and autophagy) allowing survival upon Kras* inactivation. These studies thereby provide benchmarks for successfully targeting Kras* in vivo and predicting resistance mechanisms that may be encountered in the clinic. Thus, the goal of Project 1 is to kill the bulk Kras*-dependent tumor cells through identification of metabolic targets essential for Kras*-mediated PDAC maintenance and to define methods to eliminate KRC through inhibiting autophagy and OXPHOS survival mechanisms. Project 2 (Bardeesy with Kimmelman and Cantley) has discovered that PDAC is dependent on lysosome-dependent nutrient scavenging pathways for metabolic homeostasis and tumor growth, and has identified a transcriptional program that activates these processes. The goal of Project 2 is to decipher how lysosomal scavenging supports PDAC growth and how cancer cells can escape their dependence on these processes, thereby informing improved therapeutic approaches. Project 2 will identify the metabolic outputs of lysosomal mediated recycling pathways, establish which of these outputs play roles in PDAC growth, and explore metabolic escape pathways in order to identify novel therapeutic combinations synergizing with lysosomal inhibition. Project 3 (Kalluri with Allison) has defined profound alterations in the tumor microenvironment, including a prominent CD8 T cell infiltration following Kras* extinction in PDAC. Project 3 will define the immune profiles throughout the genesis, regression and recurrence of PDAC and will determine the causal role of CD8 and CD4 cells in PDAC regression, as well as explore new opportunities to test the efficacy of checkpoint blockade therapy and assess the associated adaptive mechanisms underlying immune suppression. Furthermore, Project 3 will determine the direct impact of metabolically targeted therapy on tumor immunity and define effective methods to combine such therapies with immune checkpoint blockade therapy. Highly innovative cores for Pathology (Maitra), Preclinical Therapeutics (Benes), Computation (Futreal), and an Administrative Core will enable these Projects.

Project Summary Pancreatic cancer remains the most lethal disease with no effective therapeutics. We have recently made conceptual advances in targeting signature genomic deletions, a hallmark of human cancers. We demonstrated earlier that passenger deletions could confer cancer cell specific vulnerabilities, which we termed ?Collateral Lethality?. We deployed this concept in targeting the SMAD4 deletion, which occurs frequently in pancreatic cancer, and have identified malic enzyme (ME) 3 as a target for collateral lethality in SMAD4-deleted pancreatic tumor cells harboring adjacent deletion of malic enzyme 2. We unexpectedly discovered that mitochondrial malic enzymes are required for the uptake of branched chain amino acids (BCAAs) in pancreatic cancer, which has been implicated as a diagnostic plasma marker for early detection of pancreatic cancer. Our overall goals are: to validate ME3 as a therapeutic target for pancreatic cancer in vitro and in PDX models (Aim 1) and in vivo using chimeric PDAC model (Aim 2); and to identify useful therapeutic agents that specifically target ME2 deleted cells (Aim 3). Our goals align well with the NCI directive on ?Scientific Framework for Pancreatic Ductal Adenocarcinoma.?

Abstract/Summary This proposal aims to dissect the actions of oncogenic KRAS (Kras*) its circuitry in controlling CRC immune biology with the goal of illuminating effective therapeutic strategies for testing in CRC patients. An array of molecular and pathobiological analyses comparing Kras* `on' versus Kras* `off' states in our mouse model of human CRC has revealed that Kras* drives and maintains invasive and metastatic disease, with Kras* expression correlating with a significant increase in myeloid derived suppressor cells (MDSCs) and decrease in killer T-cells. Preliminary mechanistic studies have shown that Kras* activates TGF?, which in turn represses IRF2 (a master interferon regulatory factor), resulting in suppression of interferon response. The interferon network normally functions to promote anti-tumor responses. Thus, our overall goal in this study is to evaluate two hypotheses: (1) that Kras*/TGF?-mediated repression of IRF2 creates an immune suppressive tumor microenvironment enabling cancer progression, and (2) that Kras*-driven immune suppression may provide a basis for the de novo resistance to immune checkpoint blockade (ICB) therapy observed in the majority of CRC patients. To achieve these goals, we propose the following Specific Aims: In Aim 1, we will characterize the immune suppressive cell subtypes driven by Kras* in primary CRC utilizing our novel CRC mouse model, and evaluate the effects of Kras* mutation on MDSC and TAM activities to identify the signaling molecules governing immune suppression in these tumors. In Aim 2, we will determine the mechanism by which TGF? suppresses IRF2, and identify the immune circuits regulated by IRF2 in Kras*-driven CRC that may contribute to an immune suppressive tumor microenvironment enabling cancer progression. In Aim 3, we will investigate whether the neutralization of key Kras*-regulated targets in CRC can reverse primary resistance to immune checkpoint blockade therapy. Collectively, this proposal aims to identify novel combinations to improve the ICB sensitivity in Kras* CRC.

Project 2 -­ Abstract/Summary    Our  mechanistic  work  has  established  general  principles  of  metabolic  reprogramming  in  PDAC,  revealing  therapeutic  insights that show dramatic effects  in preclinical  models  (alanine  metabolism  blockade)  as  well  as  benefit in the clinic (autophagy inhibition). In this next cycle, we seek to address two key questions to enable us  to  most effectively define and  exploit  metabolic  vulnerabilities  in PDAC.  First, how  do  the  different  concurrent  cancer gene mutations that define PDAC subsets influence cancer cell metabolism and the metabolic interplay  with  the  TME?  Secondly,  what  role  does  regulation  of  mitochondria,  organelles  central  to  integration  of  metabolism and innate immune signals, play in metabolic reprogramming and cancer-­stroma cell interactions in  genetic subsets of Kras* PDAC? By developing a series of novel GEM models and patient-­derived KRAS* PDAC  models  representing  major  genotypes,  we  have  identified  pronounced  genetically-­driven  differences  in  tumor  cell metabolism and cancer-­immune cell interactions. We hypothesize that cooperating genetic alterations drive  distinct  metabolic  programs  and  associated  vulnerabilities  in  cancer  cells.  Moreover,  we  predict  that  immune  differences  will  further  influence  cancer  cell  metabolism  in  these  distinct  genetic  settings.  To  address  these  hypotheses,  we  propose  the  following  aims:  #1  Determine  the  influence  of  major  PDAC  gene  mutations  on  metabolic  programs;?  #2  Assess  role  of  the  autophagy/lysosome  system  on  tumor-­TME  crosstalk  in  genetic  subsets of PDAC;? and, #3 Investigate mitochondrial regulation and relationship to metabolism and inflammatory  signaling in PDAC subsets. 

Core D (Administrative Core) -­ Abstract/Summary.  The  Administrative  Core  (Core  D)  will  be  responsible  for  the  overall  administration  of  the  P01  program.    It  will  provide  clerical  support,  meeting  organization,  data  management  and  planning/evaluation  services  to  this  Program. The responsibilities of Core D include the following aims: (1) Monitoring research progress in the P01  program  via  monthly  scientific  meetings and  quarterly  progress  updates;?  (2) Providing  scientific oversight  and  research direction both from the Core D leader, Dr. DePinho, and also from the experienced membership of this  program?s  Scientific  Advisory  Board;?  (3)  Fostering  communication  and  collaboration  between  P01  program  participants by facilitating meetings (in person and via the internet), managing an annual program retreat, and  maintaining a website for program data sharing and communication;? (4) Providing fiscal oversight and resource  allocation  including the  review of  accounting  summaries  to ensure  fiscal  management,  enabling  subcontracts,  and  establishing  cost  centers  and  reporting  practices;?  and  (5)  Facilitating  data  and  reagent  sharing  and  dissemination to the research community.   

Project 1 -­ Abstract/Summary  Oncogenic  KRAS  (KRAS*)  is  universally  present  in  >90%  of  human  pancreatic  adenocarcinoma  (PDAC)  and  functions as the dominant driver for tumor development. The central theme of Project 1 has been the elucidation  of KRAS* function in PDAC and translate of these insights into effective therapies. In our previous grant cycles,  Project 1 and our P01 team have established the essential role for KRAS* in PDAC maintenance, prompting the  development  of KRAS*-­targeted exosome-­based RNAi  therapy  from  Project  3 that  has  been  translated  into  a  first-­in-­human  trial.  Collaborative  work  from  Project  1  and  Project  2  has  not  only  elaborated  the  regulation  of  anabolic metabolism and nutrient salvage pathways that are essential for KRAS*-­driven tumor maintenance, but  also identified molecular and metabolism mechanisms leading to the resistance to KRAS* depletion. While these  studies elucidated the cancer cell-­autonomous regulation of KRAS*-­dependency in PDAC, recent studies from  Projects  1  and  3,  further  demonstrated  the  profound  impact  of  KRAS*  on  the  remodeling  of  the  tumor  microenvironment  (TME),  a  major  hallmark  of  PDAC  and  barrier  for  effective  therapies,  including  immunotherapy. Specifically, we discovered a shift of myeloid infiltration in TME toward macrophages following  KRAS*  extinction,  which  produce  TGFb? to  support  Kras*-­independent  cancer  cell  survival  growth.  In  depth  analysis  of  the  TME  further  identified  alternative  immune  checkpoints  and  demonstrated  the  importance  of  myeloid  cells  in  immune  suppression,  leading  to  the  development  of  novel  immunotherapy  regimens  with  unprecedented  prolongation  of  survival  in  preclinical  models.  In  this  treatment  study,  we  discovered  a  similar  shift  toward  monocytic  myeloid  lineage  which  may  contribute  to  the  resistance  to  combinatory  immune  checkpoint  inhibition.  Therefore,  in  this  next  cycle,  Project  1  will  focus  on  elucidating  the  crosstalk  between  KRAS* and the TME, focusing on monocytic myeloid cells, with the goal of translating these mechanistic insights  into effective new  therapies.  To achieve this  goal,  we  will  work  closely  with our  innovative  Cores, which  have  extensive  expertise  in pathology,  mouse  modeling  and  computational biology, to  conduct  a  systematic  in  vivo  genetic  validation  of  the  myeloid-­dependent  KRAS*  escape  mechanism.    These  studies  will  be  followed  by  experiments  to  characterize  the  myeloid-­mediated  resistance  to  the  alternative  immune  checkpoint  inhibition  regimen  we  have  discovered.  In  the  end,  we  will  evaluate  the  anti-­tumor  potential  of  blocking  such  myeloid-­ mediated  resistance  mechanisms  in  combination  with  novel  immune  checkpoint  inhibitions.  Our  study  will  be  highly  integrated  with  Project  2  to  characterize  the  impact  of  signature  PDAC  mutations  on  TME-­mediated  KRAS* escape  mechanisms.  In addition,  outcome  from  the proposed  studies  will  be  highly  complementary  to  the  preclinical  studies  of  Project  3  to  deplete  KRAS*  in  cancer  cells  with  exosome-­based  RNAi  therapy,  in  combination  with  rationally  designed  immune  targeting  strategies  informed  by  findings  from  Project  1.  The  knowledge gained from these highly integrated studies aims to inform future clinical trial opportunities.