Dan Theodorescu - US grants

DESCRIPTION: (adapted from the investigator's abstract) Up to 40% of patients presenting with "superficial" (i.e. non muscle invasive) bladder cancer develop the muscle "invasive" life threatening form of the disease during subsequent follow up. In clinical studies, over expression of the epidermal growth factor receptor (EGFR) and the Ras oncogene have been associated with this phenotypic tumor transition. Using a powerful orthotopic invasion assay coupled with gene transfer techniques, the applicant's previous work in human bladder cancer has suggested a causal link between EGFR and Ras gene over expression, and the invasive phenotype. However the molecular pathways by which these two genes effectively trigger or facilitate the invasive process is incompletely understood. Preliminary and published data implicate Ras and phosphotidylinositol 3-kinase (PI3K) in this process and suggest that these molecules may carry EGFR mediated signals downstream to Rho family GTP binding proteins, which in turn control cellular motility and hence contribute to the invasive phenotype. The applicants propose to continue these studies and begin to elucidate the EGF mediated pathways leading to enhanced tumor motility in vitro and the impact this has on tumor invasion in vivo, by testing the following hypotheses: 1) EGFR over expression can confer the invasive phenotype to superficial bladder cancer, 2) when EGFR confers the invasive phenotype, this is mediated via both Ras and PI3K, 3) activation of Rho family proteins is necessary in order for EGFR mediated signals to induce invasion and serve to integrate signals from both Ras and PI3K in this process. To test these hypotheses they propose the following Specific Aims: 1) Alter the levels of EGFR in bladder cancer cell lines by over expressing wild type EGFR in superficial cell lines and suppressing EGFR expression in invasive cell lines, 2) Modulate PI3K and Ras activity in cells over expressing EGFR by using dominant negative and constitutively active PI3K and Ras constructs and 3) Determine if Rho family GTP binding proteins are effectors of EGF induced motility and invasion. They conclude that because their orthotopic assay closely mimics the behavior of bladder cancer in humans, successful completion of these specific aims will provide biologically relevant molecular information on the signalling pathways regulating EGF stimulated bladder cancer invasion in vivo and may provide a foundation for the development of novel therapies that interfere with this process in patients with superficial bladder cancer.

Background and Significance: The pattern of cancer metastasis was initially appreciated by Paget, who proposed the "seed (cancer cell) and soil (organ of metastasis)" hypothesis to explain the observed organ tropism of this phenomenon. In this regard, prostate cancer (CaP) has a strong predilection for bone metastasis with other sites being only rarely involved. However, even once lodged in a distant organ, a cancer cell cannot grow >2mm without the process of angiogenesis. Therefore, since the "seed" is the same in all organs, the "soil" must be different in each organ, in order to permit the metastatic cells to grow. Taken together, these observations form the basis of our Guiding Hypothesis can be stated as: the underlying reason for organ tropism of CaP is an organ specific induction of angiogenesis. For example, CaP cells may interpret abnormal cellular environments such as a metastatic site, differently than a normal cell. While a normal prostate epithelial cell thrives in the prostate microenvironment, it does not in the bone stroma. Conversely, a CaP cell may thrive in both microenvironments but not in that of liver tissue, explaining in part the organ tropism of CaP metastasis. We have recently discovered, that the angiogenic factor VEGF can also be regulated by cell surface contact in CaP. Focal adhesion kinase (FAK), Src, PI3K, Raf and MEK were found to be key effectors in this Ras- independent signaling pathway. This novel cell surface mediated signaling pathway may constitute an important way which malignant cells employ to enhance their angiogenic potential and may explain the organ specificity of CaP metastasis. Because of the potential therapeutic applications that inhibition of this process may have on patients with CaP, we propose to further delineate and dissect the mechanisms underlying VEGF induction by cell surface contact by testing the following Hypotheses: 1) Organ specific VEGF induction is a key determinant of organ tropism of CaP growth and metastasis in animal models; 2) FAK, Src, PI3K, Raf, Rap1 and MEK signaling networks are responsible for organ tropic CaP VEGF induction in vivo, 3) Novel intracellular signaling networks and promoter motifs are responsible for cell surface mediated VEGF induction. To test these hypotheses and additionally to lay the ground work for translational connections to human disease, we propose studies with the following Specific Aims: 1) Determine if VEGF induction in metastatic CaP cells is organ specific and whether VEGF is a mediator of organ tropism of CaP metastasis in animal models; 2) Determine the complexity of FAK, Src, PI3K, Raf, Rap1 and MEK signaling networks and their relevance in either organ specific VEGF induction in vivo or organ tropism of metastasis; 3) Discover novel intracellular signaling networks and promoter motifs responsible for cell surface mediated VEGF induction. Conclusions: Successful completion of these specific aims will provide biologically relevant molecular information on the signaling pathways regulating VEGF induction and may both enhance our understanding of the organ specificity of prostate tumor metastasis and provide a foundation for the much-needed development of novel therapies that interfere with this process in patients with this lethal and far too prevalent disease.

[unreadable] DESCRIPTION (provided by applicant): Background and Significance: 40% of patients presenting with "superficial" (non-muscle-invasive) bladder cancer develop the "invasive" life-threatening form of the disease during follow up. In clinical studies, overexpression of Epidermal Growth Factor Receptor (EGFR), Ha-Ras mutation and loss of tumor suppressor gene PTEN have been associated with this phenotypic tumor transition. However, the exact molecular pathway by which these genes effectively trigger or facilitate the invasive process is incompletely understood. Our original R29 hypothesized that EGFR signaling enhances bladder tumor motility in vitro and invasion in vivo and intended to determine the signaling pathways used by EGFR in this process. Since funding of the R29 in 9/97, we have made the following important observations which support the original hypothesis and address the aims of the original application: 1) EGFR and Ras inhibition diminished the motility of invasive bladder cancer cells; 2) EGF stimulates motility in non-invasive cells via PI3K and this requires activity of Rho and Ras effector Rat; 3) In non-invasive cells, baseline RalA activity is low while invasive cells have constitutively higher activation; 4) Invasive cells have low levels of RhoGDI2 expression. Reconstitution of this gene leads to diminished motility and activity of RalA but not RhoA suggesting this gene may be the first RalGDI identified to function as an invasion suppressor; 5) Inhibition of PI3K activity via PTEN reconstitution in invasive cells with inactive PTEN, results in an inhibition of orthotopic invasion in vivo and a decrease in RhoA activity. Since the overall biology of both Ral and RhoGDI2 is poorly understood, but might be critical for regulating tumor invasion in patients with bladder cancer, we propose the Guiding Hypothesis that EGF mediates bladder tumor invasion via Ral activation. We will test this hypothesis with a matrix of technologies ranging from basic biochemistry to clinical oncology to address Ral biology in human bladder cancer. These include: 1) unique paired human bladder cancer cell lines with different invasive abilities; 2) a novel organotypic bladder model allowing in vitro study of tumor invasion; 3) an orthotopic assay evaluating the effects of candidate molecules on in vivo bladder cancer invasion; 4) transgenic and knockout mice with appropriate genetic and phenotypic profiles; 5) a human tissue bank with pathologically and clinically well characterized frozen specimens. Specific Aims: 1) Determine the role and pathobiology of Ral in bladder cancer invasion in organotypic, murine orthotopie and human tumor studies; 2) Determine the regulators of Ral activation (RhoGDI2, etc..) and their effect on intracellular Ral localization and bladder cancer nfigration and invasion; 3) Determine the protein complexes associated with Ral in vitro and in vivo, including those found in human cancer. Conclusion: Completion of these specific aims will provide biologically relevant molecular information on the signaling pathways regulating bladder cancer invasion in vivo and lead to the rational development of diagnostic and prognostic tools predicting the development of invasive disease and therapies to interfere with this process in patients with superficial bladder cancer.

DESCRIPTION (provided by applicant): The median survival time of men with prostate cancer that progresses to androgen independence is approximately two years. The goal of our Program is to elucidate the alterations in signal transduction and associated changes in gene expression that underlie prostate progression to the androgen independent state. Our Program brings together productive and experienced investigators at the University of Virginia and the University of Colorado with the complementary expertise needed to fulfill the stated goals. Our team includes experts in basic and clinical aspects of human prostate cancer biology (D. Theodorescu; Colorado), signal transduction and the androgen receptor (B. Paschal; Virginia), and microRNA regulation (A. Dutta; Virginia). Project 1 will determine how hypoxic signals are sensed by post-translational mechanisms and transduced into changes in gene expression that promote prostate cancer progression, including metastasis. Project 2 will use new mouse models to determine how kinases downstream of PI-3 kinase cooperate to drive prostate tumorigenesis. Project 3 will determine how changes in microRNA profiles regulate cell proliferation and prostate cancer progression to androgen independence. The three Projects are supported by three Cores that have a strong track record of providing support for Program members. Core A (Administration; Director, B. Paschal) will enhance productivity by facilitating communication between the Virginia and Colorado sites, and by fulfilling biostatistical needs (Biostatistician, M. Conaway) of the Program. Core B (Transgenic Models and Animal Imaging; Director, David Wotton) is directed by a mouse genetics expert who will develop genetically engineered murine models for prostate tumorigenesis, and assist with xenograft production. Core C (Tissue Analysis; Director, H. Frierson) will perform histological and immunohistochemical analysis of mouse and human prostate. Our team of basic and clinician scientists has a track record of collaboration and a shared vision of defining prostate cancer progression mechanisms. The long-term objective is to translate our understanding of prostate cancer progression mechanisms into the identification of new drug targets and pre-clinical models that recapitulate key aspects of the human disease.

ADMINISTRATIVE OFFICE AND SENIOR LEADERSHIP (Admin Core-850) ABSTRACT Under the leadership of Dan Theodorescu, MD, PhD, the University of Colorado Cancer Center (UCCC) Senior Leadership group and Administrative Office (AO) develop the strategic vision, policies and procedures to promote cancer research across the UCCC member institutions and external constituencies. Scientific and administrative leadership oversee the UCCC activities in order to promote collaborative multidisciplinary/transdisciplinary research through the integration of basic, clinical, translational and population- based research across the UCCC. The support of cancer training and education across the UCCC is also a responsibility of the leadership group. Senior leadership comprises the following individuals: Andrew Thorburn, PhD, Deputy Director; Cathy Bradley, PhD, Associate Director for Population Sciences Research; James DeGregori, PhD, Associate Director for Basic Research; S. Gail Eckhardt, MD, Associate Director for Translational Research; Thomas Flaig, MD, Associate Director for Clinical Research; John Tentler, PhD, Associate Director for Education; and Mark F. Kochevar, MBA, Associate Director for Administration and Finance. Mr. Kochevar is responsible for constructing and maintaining the administrative infrastructure that supports and coordinates the activities of the UCCC. The UCCC AO is responsible for: 1) research administration, including oversight of the grant portfolio and CCSG-related activities; 2) financial management, including post-award and overall UCCC fiscal activities: 3) human resources management; 4) communications, including external public relations, publications, website and internal communications; and 5) general management activities.

DESCRIPTION (provided by applicant): Bladder cancer kills 13,000 Americans each year, but very few research projects target this disease. Most of these deaths are due to metastatic spread, commonly to the lungs. A fuller understanding of the molecular mechanisms driving growth and dissemination of bladder cancer is likely to provide new therapeutic opportunities. RalA and RalB, two homologous GTPases play an important role in growth and lung metastasis in xenograft models of human bladder cancer. Furthermore, these paralogs are activated by phosphorylation at sites regulated by Aurora-A and PKC, two kinases associated with human bladder cancer growth and progression. To define signaling pathways downstream of Ral, genome-wide analysis of Ral dependent gene expression was carried out and revealed that RalA and RalB regulate the expression of CD24, a GPI-linked glycoprotein and biomarker of metastasis development in bladder cancer. Moreover, this data identified the zinc finger transcription factor RREB1 as a putative regulator of CD24 expression and RalBP1, an effector of both Ral paralogs, as a regulator of RREB1 activity. Based on this data, we formulate the Guiding Hypothesis that RalA and RalB regulate a new downstream signaling pathway consisting of RalBP1, RREB1 and CD24 via which they contribute to bladder cancer growth and lung metastasis and furthermore, that biomarkers of RalA and RalB activation such as phosphorylation or transcriptional signatures are prognostic for development of bladder cancer metastasis in patients. Three Specific Aims test this hypothesis: In Aim 1, the role of RalA and RalB phosphorylation in bladder cancer growth, migration and lung metastasis will be determined using phospho inactive mutants. The prognostic relevance of Ral paralog phosphorylation to metastasis development will be evaluated in tissues from patients with locally advanced bladder cancer. Aim 2 will determine how RalBP1 regulates RREB1 activity and how RREB1 in turn, regulates CD24 expression. Since genome-wide profiling of Ral dependent gene expression led to the discovery of CD24, we will use tissues from Aim 1 and advanced computational tools to generate paralog specific signatures of Ral expression. The ability of these signatures to predict the development of metastatic disease in patients will be evaluated. In Aim 3 we evaluate the requirement of CD24 for bladder carcinogenesis and progression. A CD24 knockout mouse will be used to study the necessity of CD24 in chemically induced bladder carcinogenesis and/or subsequent invasion and metastasis of resultant tumors. A separate study will test whether anti-CD24 antibodies can inhibit growth of established bladder cancer lung metastases. Establishing the biological relevance of Ral phosphorylation to bladder cancer lung metastasis and the regulatory connections between RalBP1, RREB1 and CD24 provide opportunities for therapy. Translational studies associated with this mechanistic work provide biomarker predictors for metastasis development in patients while a preclinical study provides a foundation for the near term development of novel therapies directed at established metastatic disease. PUBLIC HEALTH RELEVANCE: Bladder cancer kills 13,000 Americans each year, but few research projects are targeted to this disease. For most of these patients, the cause of death is attributable to metastatic spread, commonly to the lungs. The goal of this project is to understand the mechanisms that underlie lung metastasis in human bladder cancer and use this knowledge to predict and treat this condition in patients.

DESCRIPTION (provided by applicant): Locally advanced bladder cancer develops into lethal metastatic disease, commonly to the lung, in half of all patients. Using both clinical and laboratory methodologies, we identified a new molecular pathway that regulates metastatic lung colonization (growth of micrometastases into clinical disease), components of which are altered in human disease. This pathway involves RhoGDI2 (ARHGDIB, Ly-GDI), c-Src, Rac1, endothelin-1 and versican. Microarray profiling of lineage related human bladder cancer cell lines of differing lung metastatic ability coupled with analysis of human bladder cancers at different stages identified RhoGDI2 as a candidate inhibitor of metastatic colonization. We subsequently found decreased protein expression of RhoGDI2 is an independent predictor of metastasis development in patients with bladder cancer. We also found that c-Src regulates this pathway in part by phosphorylating RhoGDI2, which increases its potency as a suppressor of lung colonization. Consistent with these data, c-Src expression decreases as a function of bladder tumor stage, and this decrease appears mutually exclusive with reduced RhoGDI2 expression. RhoGDI2, like other RhoGDIs, binds and inhibits Rho family GTPases. Investigation of these proteins yielded the surprising result that inhibition of metastases by RhoGDI2 correlated with activation of Rac1. In concurrent work, aimed at finding effectors of this pathway, gene array studies led us to identify endothelin-1 and versican as mRNAs downregulated by RhoGDI2 and upregulated in high stage human bladder cancer. Both are known macrophage chemoattractants and depletion of either protein reduced macrophage migration toward metastatic bladder cancer cells. RhoGDI2 re-expression in bladder cancer cells was also associated with decreased macrophage infiltration of lung metastases in mice. Since tumor associated macrophages (TAMs) have been found to promote metastatic colonization in many types of cancer, recruitment of macrophages by endothelin-1 and versican may mediate enhancement of metastatic growth in cells with decreased RhoGDI2. Hence, our Guiding Hypothesis is that active RhoGDI2 inhibits metastatic colonization by downregulating endothelin-1 and versican expression, which reduces tumor associated macrophage recruitment to the metastatic site. Specific Aims are proposed to test this hypothesis, develop novel prognostic tools for advanced bladder cancer and identify new targets for therapy in patients. Aim 1: Evaluate the role of RhoGDI2 as a regulator and predictor of metastasis. Aim 2: Elucidate the mechanism by which RhoGDI2 activates Rac1 to inhibit metastasis;Aim 3: Evaluate effectors of metastasis suppression by RhoGDI2. Completion of these aims will contribute clinically useful knowledge as well as providing mechanistic insights into a novel pathway that suppresses bladder cancer metastasis. Doing so will also provide a new paradigm of how metastasis suppressor proteins can function by affecting innate immunity and facilitate translational approaches aimed at development of rational therapies for patients with bladder cancer. PUBLIC HEALTH RELEVANCE: Bladder cancer kills 13,000 Americans each year and is associated with a cost per patient from diagnosis to death of ~$150K, the greatest of any cancer but, unfortunately, disproportionately few research resources are targeted to this disease. For most of these patients, the cause of death is attributable to metastatic spread, commonly to the lungs. The goal of this project is to understand the mechanisms that underlie lung metastasis in human bladder cancer and use this knowledge to predict and treat this lethal condition in patients.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There has been controversy related to the management of non-muscle invasive bladder cancer, which invades the lamina propria (pT1). This stage of bladder cancer has a significant propensity for recurrence and progression. We need to further understand the role of immunotherapy and/or whether this form of therapy should be optimized in order to improve therapeutic intervention of early stage bladder cancer, especially in those patients with recurrence of their tumor in the context of adjuvant BCG therapy. On the basis of the ability of interferon-inducible CXC chemokines to promote Th1 immunity and inhibit angiogenesis, we have coined the term, "immunoangiostasis" for their potential biological role in promoting tumor regression. Recently, we have identified the importance of the biology of immunoangiostasis in mediating tumor regression related to renal cell carcinoma. In this proposal, we hypothesize that the biology of immunoangiostasis is critical to the full success of adjuvant immunotherapy with BCG in patients with non-muscle invasive bladder cancer. Moreover, we postulated that in patients who fail to respond to this type of adjuvant therapy,, failure is due to their inability to manifest a full immunoangiostatic effect to their tumor. This latter concept would also suggest that there may be immunoangiostatic mechanisms that could be further optimized in order to fully mediate immunoangiostasis in these tumors. The protocol submitted for review will be used to determine whether the expression of interferon-inducible CXC chemokines in patients with non-muscle invasive bladder can be used as a biomarker to predict those patients that will respond vs. patients that will fail to respond to immunotherapy.

DESCRIPTION (provided by applicant): The University of Colorado Comprehensive Cancer Center (UCCC) is the only NCl-designated Cancer Center in Colorado serving a primary catchment area of Colorado and Wyoming with a population of about 5.5 million. The mission of the UCCC is to discover, develop, and deliver breakthroughs in the diagnosis, treatment, and prevention of cancer with the underlying theme of personalized care woven throughout the enterprise. Structured as a matrix center within the Univ. of Colorado School of Medicine, the UCCC is an NCI recognized consortium encompassing Univ. of Colorado Boulder, Colorado State University, National Jewish Health, Univ. of Colorado Hospital, the Children's Hospital, Denver Health System, Kaiser Permanente of Colorado and the Denver Veterans Administration Medical Center. The consortium institutions have made commitments to cancer medicine of approximately $101 million since 2005. There are 248 full members (nearly all NIH funded cancer researchers in the state of Colorado) who participate in six research programs including: Cancer Cell Biology; Molecular Oncology; Developmental Therapeutics; Hormone-Related Malignancies; Lung, Head & Neck Cancer, and Cancer Prevention and Control. During the past five years, peer-reviewed cancer-related funding reached $102 million and 3,181 publications (35% collaborative) were produced. Our innovation in research is exemplified by our members' lead roles in a lung cancer SPORE, LIVESTRONG Center of Excellence, biomarker development (ALK rearrangement, EGFR) for individualized therapy by our Pathology Shared Resource and development of a top Animal Cancer Center. Annual accrual to therapeutic trials increased from 595 to 1,044 which represent 27% of newly diagnosed patients. Research is supported by 13 Shared Resources located across the consortium and accessible to all UCCC members. These resources leverage NIH investments in our Colorado Clinical and Translational Science Institute (CCTSI) to provide cost efficiencies by avoiding duplication and enhancing critical mass. Development activities focused on strategic recruitments and pilot and seed grant programs have led to a return on investment of $11 million in direct cancer research grant funding. During the next five years, our Strategic Plan includes development of programs in Metastasis and Tumor Microenvironment, Cancer Stem Cells, Survivorship and Health Disparities and a Human Research Imaging Shared Resource which will cross species and enhance our drug development pipeline and personalized therapy clinical trials. Additional clinical and research facilities in construction, will allow us to mainain our upward research and clinical growth trajectory.

The last major advance in the treatment of metastatic bladder cancer (BC) took place in 1997 with the advent of gemcitabine. Despite this advance, visceral metastases are usually fatal. The overall goal of the proposed studies is to develop small molecule inhibitors that block a critical node in the metastatic process. We found that Rai GTPases serve as the molecular switches of a therapeutically tractable signaling pathway that allows UC cells to grow in the lung, the most common visceral metastatic site. The clinical significance of this pathway and validity of Rai as a therapeutic target is supported by finding that high Rai expression in tumors places patients at higher risk for metastasis and the requirement of Rai expression for lung metastasis to occur in animal models of UC. Our Guiding Hypothesis for this application is that small molecules targeting Rai provide effective therapy for metastatic UC. With support from the MD Anderson Bladder SPORE Developmental Research Program (DRP), we evaluated >500K compounds for their ability to bind RalA or RalB in computational and combinatorial screens and selected 99 hits. These were evaluated in a series of secondary assays allowing us to select Rai Binding Compound (RUC)8 and 10 to be pursued in this application. RUC8 and 10 were selected because they: 1) inhibit RalA to RalBPI binding in human UC cells and RalA induced spreading in murine embryo fibroblasts; 2) inhibit in vitro monolayer growth (IC50 0.5-1.9 pM) of human UC cells; 3) bind RalB directly by nuclear magnetic resonance (NMR) spectroscopy; and 4) have good pharmacokinetic (PK) properties in mice (Cmax 1.3-23 pM, T1/2 3.7-4.6 hrs). To develop this novel class of agents we propose the following Specific Aims: Aim 1: Characterize higher potency 2^* generation compounds based on RUC8 and 10 using medicinal chemistry, computational fragment-based design, and similarity search of chemical databases. In the unlikely situation that higher potency compounds are not found in Aim 1, we will pursue Aim 2 and 3 using RUC8 and 10, given their adequate IC50 and in vivo PK. Aim 2: Evaluate 2 generation compounds for their in vivo therapeutic efficacy in novel human UC models of visceral metastasis. Aim 3: Develop predictive biomarkers of response to antlRal therapeutics in human tissues that will position us for Phase 1 trials by end of this project. Documented interest by Astra Zeneca in our work improves overall chances for success in translating our novel Rai inhibitors into the clinical setting as anticancer therapeutics.

The overall goal of the Tissue Analysis Laboratory (the Core) is to provide technical and professional support for each Project. The Core at the University of Virginia has been in place for the past decade of the two previously funded prostate POI programs. All assessments will be made by two highly skilled, experienced genitourinary pathologists with expertise in examining mouse and human prostate tissues and in immunohistochemistry. Dr. Frierson will provide overall direction ofthe Core laboratory at the University of Virginia, while Dr. Lucia will lead the laboratory at the University of Colorado. The Core will have a large impact on the POI. as it provides tissue examination for in vivo models that compliment in vitro experiments. Comparative analysis of murine prostate cancers and clinical human prostate cancer specimens from different patient cohorts from two different laboratories will impact the field by clarifying signaling pathways in prostate cancer progression. The Core will have 3 Aims: Aim 1. Histologic analysis of cell cultures, xenografts, mouse models, and clinical human prostate tissue specimens. Specimens will be processed as archival (formalin-fixed, paraffin-embedded) samples or as frozen sections. In addition, Drs. Frierson and Lucia will use their expertise as pathologists to examine the prostate glands of transgenic mice to determine histopathologic changes induced by the genetic modifications. Aim 2. Immunohistochemical analysis and optimization of antibodies, specifically to determine their utility in formalin-fixed, paraffin-embedded material and. if necessary, in frozen sections. Drs. Frierson and Lucia will use the avidin-biotin immunoperoxidase technique and their immunohistochemical expertise to determine optimal reagent conditions and antibody staining in cell cultures, xenografts, mouse models, and clinical human prostate cancer specimens. Aim 3. Selection, preparation, and procurement of cells for laser microdissection. Drs. Frierson and Lucia have the ability to perform laser microdissection of prostate glands from human prostate tissues, transgenic mice, and xenografts for molecular studies

The Core will perform two major functions: We will assist with the generation and analysis of xenograft tumor models, and generate novel genetically engineered mouse models for prostate tumorigenesis. The generation and analysis of xenografts tumor models is time consuming and requires specific technical expertise. We will provide technical assistance and training to facilitate the use of xenograft models. A major bottle-neck in the testing and generation of better mouse models of prostate cancer is combining standard genetic models with specific additional mutations. We will generate experimental animals in the Core, thereby removing much ofthe initial burden of complex transgenic experiments from the individual Projects within this Program. This will facilitate the use of such genetic models, and will minimize cost and delays due to lack of access to genetic models and lack of resources. Specifically, in support of the Aims of the three Projects within the Program we will: 1) Provide technical support and training for in vivo xenograft experiments and tumor imaging. Members ofthe Core will be involved in the planning and initiation of xenograft experiments, and will assist with in vivo imaging, induding Xenogen imaging and X-ray imaging of metastases to bone. 2) Generate novel combinations of genetic alterations in mice to address roles in prostate tumorigenesis. We will combine novel conditional mutations in Rala and Ralb with prostate specific deletion of the Pten tumor suppressor. Additionally, we will analyze a series of prostate specific PKN1 transgenes, and combine the most informative with a transgene in which constitutively active AKT1 is expressed in the prostate. 3) Generate mice with a targeted Pkn1 conditional null allele. Mice will be generated from ES cells with a targeted conditional mutation in the Pkn1 gene, and prostate-specific deletion of Pkn1 will be combined with the Pten null mutation, 4) Transfer transgenic prostate tumor models to a pure FVB strain background. All mutations will be transferred to a pure FVB strain background to simplify the analysis of prostate specific tumor phenotypes. By providing these services, we will allow the Program to begin to analyze prostate cancer progression more effectively using animal models.

The Administrative Core A will facilitate interactions between Program members and provide biostatistical support for Projects and Cores. Communication between POI members is critical for success of the Program. Effective communication will promote collaboration and synergy between projects, and accelerate the pace of discovery. Regular internal and external review of scientific progress and evaluation of cores will promote research excellence and ensure that projects are provided with the highest quality services. Application of appropriate statistical tools is essential for rigorous evaluation of hypotheses and for improving experiment design. Functions ofthe Administrative Core include promoting communication and collaboration within the Program by supporting web-based communication between the University of Virginia and the University of Colorado, organizing regular scientific meetings, and providing administrative support to Project Leaders and Core Directors. The Core will also oversee the review of scientific quality and fiscal management of the Projects and Cores. Another key activity of the Core is to provide statistical support and instruction for the Program. This involves assisting in the design and analysis of in vitro and in vivo studies, development and application of novel statistical methodologies in support of emerging Program objectives, and provision of training in statistical methods.

MicroRNAs are 17-24 nucleotide-long, single-stranded RNA molecules that profoundly impact the gene expression program of a cell by annealing to and suppressing the expression of many target genes. Changes in expression of specific microRNAs and other types of short RNAs are involved in different types of malignancies, including prostate cancer. We know that progression of prostate cancer is accompanied by changes in the gene expression program, but the role of microRNA and other short RNAs in progression have not been studied. In this proposal we will clone and sequence Solexa/lllumina libraries of small RNAs from prostate cancer cells to identify microRNAs and other short RNAs that are (a) regulated by PTEN loss, (b) regulated by overexpression of PKN1 and (c) correlated with progression to androgen independence. We will also follow the changes in known microRNAs by hybridization to locked-nueleic acid microarrays. These results will identify new short RNAs involved in prostate cancer progression and test the hypothesis that specific molecular genetic lesions are correlated with specific microRNA changes during the progression of prostate cancer. We will determine whether microRNAs repressed during prostate cancer progression contribute to the phenotype of progression, both in vitro and in vivo, starting with four microRNAs (in two families) that have already been validated in preliminary results. Finally, we will identify targets of these microRNAs whose de-repression during cancer progression leads to worsening ofthe malignant phenotype. We will identify the targets of two key microRNA families involved in cancer progression by a novel combination of experiments and computational target prediction, starting with eight targets that have already passed the experimental filters. Thus new technologies, short RNA cloning and ultrahigh-throughput sequencing, and new combinations of assays for identification of relevant targets will be applied to the molecular analysis of prostate cancer progression, and the function of key microRNAs In this process will be dissected. The results are expected to open new avenues of research, therapy, and diagnosis of prostate cancers.

Somatic mutations and deletions in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been identified in a variety of cancers, and in prostate cancer PTEN alterations occur in at least 30% of primary cancers and 63% of metastatic cancers. Since reduced PTEN activity enhances Pl-3 kinase-dependent pathways that promote tumorigenesis. defining the pathways that operate downstream of Pl-3 kinase signaling is essential for understanding transformation mechanisms and for identifying new therapeutic targets. Expression of activated AKT in transgenic mice is not sufficient to phenocopy PTEN loss in prostate, thus additional effectors of Pl-3 kinase signaling are required for prostate cancer to progress beyond prostate intra-epithelial neoplasia (PIN). We hypothesize that the Protein Kinase C-related kinase 1 (PRK1; termed PKN1 after the gene name) is a Pl-3 kinase effector whose properties suggest it cooperates with AKT to transform prostate cancer cells. PKN1 is a direct substrate of PDK1, is over-expressed in human prostate cancer and promotes proliferation in xenograft models, and it modulates androgen receptor (AR) activity by acting as a histone H3 kinase. We show that conditional expression of constitutively active PKN1 in mouse prostate is sufficient to generate murine PIN at 22 weeks. In Aim 1 we use transgenic mice to characterize prostate cancer phenotypes resulting from PKN1 expression, alone and in combination with AKT. We will determine the role PKN1 plays in transducing tumorigenic effects caused by loss of PTEN. In Aim 2 we use xenograft and cell culture approaches to determine how PKN1 stimulates tumor growth by exploring its function as a cleavage furrow kinase, and as a factor that modulates the expression and activity of cell cycle regulators. In Aim 3 we test models for how PKN1 kinase activity promotes gene expression. Including PKN1 phosphorylation of AR and PKN1 generation of specific epigenetic marks. Successful completion of these aims will provide new insights into PTEN/PI-3 kinase signal transduction mechanisms including how PKN1 promotes prostate cell proliferation and how PKN1 regulates AR. We will generate new pre-clinical models of prostate cancer that recapitulate molecular events that occur with loss of PTEN

We found that expression and activity of RalA & RalB are induced by hypoxia and androgen deprivation (AD) in vitro in parallel with CD24 expression. We determined that a hypoxia-Inducible factor 1a (HlF1a) response element in the 5' region ofthe CD24 gene underlies this effect. Since H!F1a expression is necessary for metastasis in prostate cancer (PCa) and its translation is regulated by mTOR, we evaluated HlF1a protein expression and discovered that expression of RalA and RalB Is required for maximal HlF1a response in hypoxia and AD. We also found that, like HlF1a, RalA undergoes proline dehydroxylation during hypoxia and this promotes engagement with its effector, phospholipase D (PLD), a regulator of mTOR. Further, we identified the elF3b translation initiation component that binds to mTOR. as a direct RalB binding partner. Hence propose the hypothesis that in PCa. Ral GTPases are oxygen and androgen biosensors and their activity allows metastatic PCa cells to overcome the natural and therapeutic adversity of hypoxia and AD by maintaining mTOR-mediated translation of HlF1a. Specific Aims will test this hypothesis: Aim 1 will investigate the role of post-translational Ral modifications on HlF1a expression. We will map proline hydroxylation sites in RalA and determine their role in RalA activity and HlF1a expression. We will also evaluate the role of RalB phosphorylation on HIF1a expression, since we have discovered that RalB is activated by phosphorylation at S198 by PKC, a hypoxia activated kinase. In Aim 2 we determine the nature of Ral expression on global and HlF1a translation and assembly and activity of cap-dependent translational complexes in hypoxia and AD. We also examine these characteristics in RalB mutants deficient in interaction with elF3b. Aim 3 will determine the requirement for Ral in PCa using human xenografts and novel transgenic models. Gene expression signatures associated with post-translational modifications of Ral will be evaluated in tumors from patients treated by prostatectomy to determine their ability to predict recurrence. Our project provides a new paradigm by showing Ral GTPases are oxygen and androgen sensors that regulate HlF1a while integrating in the P01 by shared aims with other projects and use of all cores.

? DESCRIPTION (provided by applicant): CD24, a GPI-linked plasma membrane protein, is a major yet understudied biomarker of poor patient outcomes in many cancer types. CD24 was long believed to lack the ability to drive aggressive tumor phenotypes. We challenged this view by showing CD24 drives bladder tumor growth and metastasis, revealing CD24 as a therapeutic target. However, anti-CD24 monoclonal antibody (mAb) therapy only led to moderate reduction in tumor growth and lung metastasis. Interestingly, we found shRNA and mAb both depleted cell surface CD24 similarly, yet shRNA reduced tumor growth and metastasis and intracellular CD24 levels more profoundly. We also discovered CD24 in the nucleus (nCD24). nCD24 is chromatin bound, and its level is reduced more by shRNA than by mAb. nCD24 drives bladder cancer cell growth and gene expression, while high patient tumor levels are associated with poor outcome. Since the moderate impact of mAb therapy may be due to an inability to deplete nCD24, we searched for ways to reduce total cellular CD24 and discovered that androgen receptor (AR) stimulation induces CD24 expression. Concurrent work defining the mutational landscape of bladder cancer found CBP and its paralog p300 are commonly mutated in tumors of low stage, grade and CD24 expression. Importantly, in their wild type form these bind AR and enhance its activity while depletion of CBP or p300 reduces bladder cancer cell CD24 expression and in vitro growth. Here we propose the Hypothesis that WT CBP/p300 and androgen drive metastasis via induction of CD24, which in turn regulates effectors of this process; and thus enhancing CD24 depletion therapy will improve tumor control. Three Specific Aims will test this hypothesis: 1) Investigate the relationship of CBP/p300 to CD24, tumor growth and patient outcome: The biological and prognostic relevance of CBP/p300 mutations or expression is unknown in any cancer. Using human tumor specimens and human bladder cancer cells lacking CBP/p300, we test the hypothesis that WT CBP/p300 expression is a poor prognostic marker for patients and, using animal models, that this is driven in part by CD24 and AR. 2) Determine the effectors of CD24. Finding nCD24 drives tumor growth and gene expression is a paradigm-shifting explanation of how CD24 can impact cancer behavior without an intracellular domain and why mAb therapy is suboptimal. We use ChIP-seq, transcriptional profiling, animal models and human specimens to test the hypothesis that nCD24 induces genes that drive bladder tumor progression. 3) Enhance the effectiveness of CD24 depletion therapy. CD24 depletion by si/shRNA or mAb leads to tumor regrowth in vivo despite continued CD24 suppression. Here we test the hypothesis that acquired and intrinsic resistance of cancer cells to CD24 depletion is due to specific targetable genes. We apply a synthetic lethal screen to identify those responsible for intrinsic resistance and study RCHY1, a gene we found induced in CD24 shRNA depleted bladder cancer cells as a promising candidate driver of acquired resistance. Impact: Our work will provide clinically relevant biomarkers and therapeutic targets while informing CBP/p300 and CD24 biology fields.

? DESCRIPTION (provided by applicant): Bladder cancer kills 15,000 annually and because of few therapeutic advances there is a need for innovation. This project has discovered suppressors of bladder tumor growth and metastasis and shown that low expression of AGL, an enzyme involved in glycogenolysis, and RhoGDI2, an inhibitor of multiple GTPases, is associated with cancer recurrence and death. The Goal of this continuation project is to understand how reduced AGL levels result in aggressive bladder cancer and to translate this knowledge into novel therapies. We found bladder cancer cells with low AGL express elevated levels of Hyaluronic Acid (HA) synthase 2 (HAS2), and its extracellular product HA, and have activated RhoC and Rac1, known drivers of tumor progression. We demonstrated RhoC/Rac1 activity is suppressed by RhoGDI2, linking the actions of the two metastasis suppressors. Cells with low AGL also exhibit increased glycolysis, glucose import and autophagy. When either glucose import or autophagy are blocked, low AGL tumor cells show greater reduction of in vitro growth than cells with higher AGL levels, exposing a vulnerability of these aggressive cells. These data support an innovative Hypothesis: Tumor cells with low AGL generate growth and metastatic signals via production of HA, activation of its receptors CD44 and RHAMM, and downstream effectors such as RhoC and Rac1. For these signals to be actualized, sufficient energy needs to be provided via enhanced glucose import and autophagy. Three Specific Aims will test this hypothesis. 1) Evaluate impact of HA signaling on aggressiveness of bladder cancers with low AGL. We will determine if HA drives tumor progression via its receptors CD44 and RHAMM and effectors RhoC and Rac1 that are inhibited by RhoGDI2. Experiments will use RNAi and receptor inhibition in human xenografts. 2) Determine role of the AGL signaling network in bladder cancer development and progression. We will evaluate the independent predictive value of tumor suppressor genes and components of the AGL signaling network in a large panel of annotated human tumors, to define a predictive multigene biomarker signature and possible new therapeutic targets. We will use full and conditional (bladder urothelium) AGL knockout mice in a chemical carcinogenesis model that generates spontaneous visceral metastases to test whether lack of AGL makes urothelium more susceptible to carcinogenesis and resulting tumors more aggressive. 3) Target molecular vulnerabilities of bladder cancers with low AGL expression. Here we test whether blocking glucose import and/or autophagy reduces in vivo bladder tumor growth. We will use shRNA and pharmacologic agents already in preclinical/clinical testing. We will screen shRNA libraries to metabolic and autophagy genes in cells with low AGL to discover novel genes essential to the aggressive tumor phenotype, with the expectation that some of these will become therapeutic targets. We will evaluate combined inhibition of HA signaling and energy production pathways with the expectation this will have synergistic detrimental effects on tumor progression. Impact: This project will lay the foundation for future biomarker-driven personalized clinical trials that target critical vulnerabilities in bldder cancer while informing us on how AGL executes its unanticipated role in cancer.

STRUCTURAL BIOLOGY SHARED RESOURCE (Core-405) ABSTRACT Overview: The Structural Biology Shared Resource (SBSR) aims to facilitate and promote the application of macromolecular structural biology methods in cancer research for UCCC members. SBSR meets these goals by providing UCCC members instrumentation and expertise in X-ray Crystallography (X-ray) and Nuclear Magnetic Resonance Spectroscopy (NMR). Examples of projects studied in the SBSR fall into three main topic areas: Dynamics of cancer target activity, gene regulation, chromatin and epigenetics, and molecular targeting for the development of novel anti-tumor agents. Equipment: The SBSR capabilities have expanded significantly over the last 5 years through institutional support and the NIH Shared Instrument Grant Program. As a result, the X-ray facility has acquired a new in-house data-collection system that consists of a Rigaku MicroMax-007 X- ray generator, PILATUS3 R 200K Hybrid Pixel Array Detector with VariMax Optics and Oxford cryo-cooling systems. It also has a Rigaku/MSC robotics system for production of crystallization screens, drop setting and plate imaging. The NMR facility is equipped with Agilent/Varian 500, 600 and 900 MHz spectrometers at the Anschutz Medical Campus and an Agilent/Varian 800 MHz spectrometer at the UCB campus. Services: The SBSR provides expertise and access to highly specialized instrumentation for X-ray crystallography and NMR based structural studies of biomolecules relevant in cancer biology. Consultation and Education: SBSR personnel provide advice on sample preparation, data collection, structure determination, and data presentation for publication. Hands-on instrument training is important function of SBSR staff. Management: The SBSR is an institutional core managed by the institution as part of the Structural Biology and Biophysics Core Facilities. CCSG funding represents 34% of the annual operating budget. The remaining support comes from institutional support (50%) and user fees (16%). In regard to UCCC, SBSR is overseen by the Associate Director for Basic Research. Use of Services: Since July 2011, 54 investigators have used the services. Thirty-five percent of users were UCCC members, representing five of the six UCCC Research Programs and resulting in 55 peer- reviewed publications. Future Directions: The SBSR has several primary future directions that will enhance the SR and UCCC member cancer research: 1) Outreach to UCCC members from non-structure labs to create awareness of the opportunities SBSR technology offers and provide training in such technology; 2) incorporate into SBSR the new Cryo-Electron Microscopy resources that are currently being developed; 3) Collaborate with PMTSR to develop a comprehensive protein expression and purification service; 4) Initiate lifecycle replacement of several NMR resources.

TISSUE BIOBANKING AND PROCESSING SHARED RESOURCE (Core-745) ABSTRACT Overview: The Tissue Biobanking and Processing Shared Resource (TBPSR) supports translational and clinical research projects of UCCC members by providing access to high-quality tissue and body fluid specimens that are associated with clinicopathologic and outcomes data. Equipment: The TBPSR has a wide variety of state of the art equipment for the procurement, processing, storage, and analysis of biospecimens. Services: The TBPSR provides services in three main areas 1) procurement, processing, archiving, and distribution of fresh and formalin-fixed tissues and body fluids, 2) specimen characterization, quality control, and linkage with pathological and clinical annotation, 3) special processing and auxiliary services including tissue microarray construction, laser capture microdissection and computer-assisted imaging/ image analysis. The TBPSR collaborates with the Cancer Clinical Trial Office and Health Data Compass to provide consents and detailed clinical and pathologic data correlated with distributed specimens. Consultation and Education: The TBPSR directors and staff consult with UCCC members to discuss protocol, project and grant development. The TBPSR directors provide educational sessions on biorepository and specimen management to researchers, students, and clinicians. Management: The TBPSR is a division of the larger Biorepository Core Facility, an institutional core managed by the institution. TBPSR is overseen by UCCC Associate Director for Population Sciences Research. Use of Services: Over the current funding period 64 UCCC members have used the services, representing all 6 Programs and resulting in 55 peer reviewed publications. CCSG funding represents 7% of the annual operating budget. The remaining support comes from the CCTSI grant (6%), other peer-reviewed grants (61%), institutional support (13%) and user fees (13%). Future Directions: The TBPSR proposes the following initiatives that will enhance the SR and UCCC member cancer research: 1) Implement the iLab® system for management of the TBPSR; 2) Expand integration with the Health Data Compass. Compass is an integrated enterprise health data warehouse of all patient data obtained from consortium institutions (UCD, UCH, CHCO) which also provides analytic services designed to transform data-driven processes in clinical research, molecular discovery, and precision medicine; 3) Integrate with the Oncology Research Information Exchange Network (ORIEN), a consortium of academic medical centers utilizing a common protocol for tracking patient molecular, clinical and epidemiological data that follows the patient throughout his or her lifetime. This will expand the scope of the TBPSR locally and also help advance cancer research and care nationally.

PROTOCOL REVIEW AND MONITORING SYSTEM (Core-361) ABSTRACT The Protocol Review and Monitoring System (PRMS) of the University of Colorado Cancer Center (UCCC) is an integral component of the clinical research performed at the UCCC consortium. This component was disapproved in 2011 and subsequently re-engineered by a new chair, Jonathan Gutman MD, Associate Professor in the Department of Medicine and a clinical investigator in allogeneic stem cell transplantation for hematologic malignancies. These efforts led to re-approval by the NCI in June 2015. PRMS functions for cancer clinical trials across the UCCC consortium include: 1) review of scientific merit prior to submission to an IRB; 2) assessment of study priority and feasibility, and 3) monitoring of intervention trials for accrual and scientific progress. The PRMS collaborates with, but operates independently of the IRB and the UCCC Data and Safety Monitoring Committee (DSMC). The PRMS is comprised of two committees, the PRMS-Executive Committee (PRMS-EC) and the PRMS-Scientific Review Committee (PRMS-SRC). Members of these committees reflect the breadth and diversity of disciplines of UCCC. The PRMS-EC and PRMS-SRC meet on alternating weeks resulting in efficient disposition and flow of protocols. The PRMS-EC assesses prioritization and feasibility of protocols and grants expedited approval if appropriate. Evaluation of scientific merit is performed by the PRMS- SRC. New protocol submissions to PRMS are reviewed with differing levels of intensity depending on the type of protocol. Institutional and industry intervention trials receive the most comprehensive review before being approved to submit to the IRB. Once a protocol is activated, the PRMS-EC monitors scientific progress. PRMS- EC membership includes the PRMS chair, deputy chair, PRMS administrator and all disease site leaders. Inclusion of disease site leaders ensures that larger scope issues ? prioritization, feasibility, and progress ? are continuously revisited by those responsible for leading clinical research in their respective areas. The PRMS- SRC provides focused, well documented scientific reviews of institutional and industry intervention protocols. PRMS-SRC membership includes the PRMS chair, deputy chair, PRMS administrator, biostatisticians and a diverse group of investigators from all oncologic disciplines. Since 2013, PRMS has reviewed and prioritized 397 intervention studies. 25% (98) were NCTN or external peer reviewed studies that received a feasibility and prioritization review only. The remaining 75% were institutional (45) and industry (255) studies. Of the institutional studies, 7 (16%) were disapproved. During this same period 67 six month low accrual warning letters, 39 ten month low accrual warning letters, and 42 twelve month low accrual warning letter were issued for intervention studies and 35 were closed by PRMS for a lack of progress or closed voluntarily by the PI following receipt of letters from PRMS. During the next grant period PRMS will continue to apply a careful evaluation of feasibility and prioritization and perform a rigorous review of scientific merit to ensure that UCCC investigators open studies that are scientifically meritorious, feasible to complete, and meet the priorities of the UCCC.

PROTEIN PRODUCTION/ MONOCLONAL ANTIBODY/ TISSUE CULTURE SHARED RESOURCE (Core-544) ABSTRACT Overview: The Protein Production, Monoclonal Antibody, Tissue Culture Shared Resource (PMTSR) supports basic and translational research projects of the UCCC members by providing authenticated cell lines, producing monoclonal antibodies and recombinant proteins, and providing real time live cell imaging of cultured cells. Equipment: In 2015, we acquired an Essen BioScience IncuCyte, which offers real time imaging of living cells to quantitate cell proliferation, apoptosis, cell migration, chemotaxis and gene expression in an automated, high throughput manner. This instrument offers new technical advantages that were not previously available to our investigators. Services: The PMTSR has assembled a collection of over 200 authenticated human cancer cell lines, which are available to UCCC members at a reduced price relative to commercial services, to encourage the responsible use of established cell lines in biomedical research. Investigators can use the PMTSR to prepare large volumes (0.5 to 10 liters) of cells for in vitro and in vivo biological studies. For over 25 years, the PMTSR has produced hybridomas and purified monoclonal antibodies for use in basic and preclinical studies. Recombinant proteins are produced using baculovirus expression systems or in mammalian (293) cells. Consultation and Education: The PMTSR has advocated for the use of authenticated cell lines in cancer research by providing seminars outlining the impact of contaminated/misidentified cell lines upon biomedical research and by providing information on cell authentication services to UCCC members. A number of seminars have been offered regarding the use and technical abilities of the IncuCyte Bioimaging system. The PMTSR director and staff routinely consult with UCCC members on projects involving offered services and technologies. Management: The PMTSR is a UCCC SR managed by the UCCC and is overseen by the Associate Director for Basic Research. CCSG funding represents 31% of the annual operating budget. The remaining 69% support is from user fees. Use of Services: Since July 2011, 157 investigators have used the PMTSR services. Fifty- seven percent of users were UCCC members, representing all UCCC Programs and resulting in 65 peer- reviewed publications. Future Directions: 1) Expand our collection of authenticated cancer cell lines and continue to make them available to UCCC members at reduced prices as a means to ensure compliance with new NIH and journal policies. 2) Collaborate with Structural Biology Shared Resource (SBSR) to develop service offering on-demand purification of recombinant proteins. 3) Support the expanded use of the IncuCyte in cancer research. 4) Contribute to international efforts to develop platforms for murine cell line authentication and when available, provide these authenticated lines to UCCC members.

PROJECT SUMMARY / ABSTRACT The University of Colorado Cancer Center (UCCC), in its 29th year of NCI designation, has built on its ?excellent? overall rating in 2011, with major scientific advances and national leadership. Serving the catchment area of the State of Colorado, UCCC, a matrix within the School of Medicine, is a multidisciplinary research consortium of the University of Colorado at Denver and Boulder, Colorado State University and clinical affiliates University of Colorado Hospital, Children?s Hospital Colorado and the Denver VA. The UCCC achieves its mission to discover, develop, and deliver breakthroughs in cancer science through interdisciplinary collaborative research. UCCC discoveries with potential to change cancer medicine include: 1) role of COX-2 in driving progression of in situ disease to breast carcinoma with a prevention trial being developed; 2) molecular basis of radiation mucositis translated into treatment strategies for survivors; 3) ?first in class? inhibitor of Ral, a previously deemed ?undrugable? GTPase known to drive many pancreatic, colon and lung cancers, now in commercial development; 4) role of androgen receptor in breast cancer, leading to a DOD Clinical Translational Research Award and national trial of oral antiandrogens in triple negative cancers; 5) showing genotype-directed therapy in lung cancer leads to better survival; 6) novel therapeutic combinations in Investigator Initiated Trials (IIT) in multiple cancers, developed from synthetic lethal functional genomic screens. The UCCC?s national leadership and recognition include our Lung SPORE, LAPS U10 and ET-CTN UM1 grants supporting NCTN and CTEP trials, a SPECS U01 grant, election into the NCCN, and rise to 15th adult cancer program in USNWR. The UCCC has 196 (28 new) Full members, representing 96% of all NCI-funded PIs in CO, in 6 programs: Molecular Oncology (MO); Cancer Cell Biology (CCB); Developmental Therapeutics (DT); Hematologic Malignancies (HEME); Lung, Head & Neck Cancer (LHN), and Cancer Prevention and Control (CPC). Since July 2011, 2,636 publications (22% inter- and 25% intra-programmatic) were produced with 15% having high (>9.6 IF) impact (13% previously), 88 patents issued, 26 licenses negotiated (104% and 116% increase), 15 startup companies founded and 47 intervention IITs opened. In 2015, members held $52M in direct peer-reviewed cancer funding (24% increase in # of NCI grants), accrued 1499 subjects (52% increase) to intervention trials and 706/3946 newly registered patients (18%) to intervention treatment trials. 10 Shared Resources that leverage institutional and NIH investments support UCCC research. Skillful management, recruitments, a $74M investment and 3 new endowed chairs, addressed NCI site visit and EAB concerns. Future directions include multidisciplinary initiatives to promote healthy living, tackle resistance to targeted agents, enhance immunotherapy, leverage comparative oncology, expand cancer bioengineering and develop a statewide clinical research network. CCSG renewal and new institutional commitments of $65M and 20K sf of space will allow the UCCC to maintain its upward trajectory of high impact contributions and help reduce the cancer burden in its catchment area and worldwide.

PLANNING AND EVALUATION (Core-453) ABSTRACT Overview: The University of Colorado Cancer Center's (UCCC) Planning and Evaluation (P&E) activities, under the leadership of Dan Theodorescu MD PhD, are focused on the comprehensive strategic review of the UCCC and its Research Programs, Shared Resources (SRs), Clinical Research, and associated operations to ensure the optimization of UCCC's activities and the recognition and pursuit of particular opportunities. The P&E component is conducted in a multi-faceted approach that includes the review and assessment by the External Advisory Board (EAB), several key internal advisory committees, and the Senior Leadership of the UCCC. These activities are augmented by specific ad hoc individual and group external reviewers along with key input from UCCC members through UCCC organizational structures. Following the NCI Site Visit of the UCCC in June 2011 and receipt of the detailed CCSG review, UCCC Senior Leadership and Research Program Leaders initiated a series of meetings to identify key areas for development and recruitment in addition to those strategic directions outlined in the Director's Overview. Key areas were identified as recruitment of an Associate Director for Population Sciences Research, the reconstruction of the Protocol Review and Monitoring System (PRMS) which had been disapproved and implementation of better educational opportunities and infrastructure (new clinical trial management system (CTMS)) in support of clinical investigation. The UCCC External Advisory Board (EAB) played a critical role in this strategic planning and evaluation activities through annual visits and written feedback. Accomplishments: Since 2011 such P&E activities have led to the successful accomplishments of many of the strategic directions outlined in the Director's Overview in addition to the key areas outlined above. In the next funding period, the P&E component proposes the following Specific Aims: 1) Creation of appropriate external and internal advisory bodies to ensure strategic development, expert guidance, and rigorous review of UCCC research programs and activities; and 2) Development of processes for continuous review and improvement of UCCC programs to ensure efficiency, effectiveness, and concordance with UCCC strategic initiatives.

PHARMACOLOGY SHARED RESOURCE (Core-529) ABSTRACT Overview: An important component of drug-based studies is the assessment of drug exposure in both preclinical and clinical studies. The Pharmacology Shared Resource (PharmSR) provides a mechanism by which University of Colorado Cancer Center (UCCC) investigators can plan and carry out pharmacokinetic (PK) and pharmacodynamic (PD) studies in consultation with expert investigators. Equipment: Quantitative analytical methods offered include high performance liquid chromatography (HPLC) with UV/Vis and fluorescent detection, LC tandem mass spectrometry (LC/MS/MS), and other biochemical analysis methods (e.g. ELISA, enzymatic). Institutional support led to the purchase of an AB SCIEX QTRAP? 6500 LC/MS/MS system during the last funding period, providing state of the art technology to UCCC investigators. Services: Services offered by the PharmSR include: (1) consultation on PK study design; (2) development, validation and implementation of appropriate analytical assays for drugs in biological fluids and tissues; and (3) PK and PD modeling and mathematical analysis of analytical data. PK modeling services include systems based approaches (non- compartmental modeling), compartmental modeling, Physiologically-Based PK analyses (PBPK) and population approaches. Consultation and Education: The PharmSR provides consultation to help members with limited pharmacology or PK background to design appropriate experiments and analyze PK data. Consultations provided to members roughly equates to approximately 5 letters of support being requested annually for grant submission proposing use of the PharmSR. Management: The PharmSR is located at CSU and is an institutional core jointly managed by the UCCC and CSU. CCSG funding represents 24% of the annual operating budget. The remaining support comes from the CCTSI grant (13%) and user fees (63%). PharmSR is overseen by the Associate Director of Translational Research. Use of Services: Since July 2011, 55 investigators have used the PharmSR services. Thirty-six percent (20) were UCCC members representing four of the six UCCC programs and generating 20 peer-reviewed publications. Future Directions: The Pharm SR is currently establishing multiplex assays to directly quantitate panels of ATP-binding cassette (ABC) transporter proteins and drug metabolizing enzymes (P450s) from in vitro, preclinical and patient specimens. The goal is for the PharmSR to offer these focused proteomic assays to quantitate proteins relevant to drug development and mechanism of action.

MOLECULAR PATHOLOGY SHARED RESOURCE (Core-957) ABSTRACT Overview: The Molecular Pathology Shared Resource (MPSR) consists of 3 thematically related and operationally coordinated business units (Histology, Cytogenetics and Pre-analytical Evaluation and Molecular Testing (PEMT)). MPSR supports translational and clinical research projects of UCCC members by providing clinical-grade services and molecular assays for analysis of pre-clinical and patient samples at reduced prices, greatly facilitating cancer research. Equipment: Major equipment: Histology unit has tissue processors and embedding stations, microtomes and automated staining equipment; Cytogenetics unit has high-quality Zeiss fluorescence microscopes equipped with last generation cameras and automated interference filter wheels; PEMT has Next Generation Sequencers (NGS), capillary electrophoresis, real-time PCR among others. Services: MPSR offers pathologist examination of tissue samples, tumor sample microdissection, standard and customizable histologic tissue processing and staining, cytogenetic techniques (ploidy analyses, G-banding and spectral karyotyping, metaphase FISH with single locus and painting probes, interphase FISH using 2-target to 6-target multiplexing), and a wide range of nucleic acid-based assays (Sanger sequencing, next-generation sequencing, quantitative PCR, micro-satellite instability testing). Activities in the Histology and PEMT units are performed in a CLIA-certified, CAP-inspected environment by dedicated technologists, resulting in clinical-grade data while those in Cytogenetics are GLP. All data is provided in an analyzed format and include bioinformatic analysis of complex data sets, coupled with comprehensive interpretation of results and illustrative documentation. The MPSR also supports and aids in the design, implementation, and validation of custom assays both for specific investigator use and for wider use by the UCCC community. Consultation and Education: The MPSR offers consultation, as well as one-on-one and group educational sessions for all services and assays, for both assay design and data analysis. Instructional sessions are offered to UCCC members, in order for users to precisely understand which assays may best serve their individual translational research needs. Management: The MPSR is a UCCC shared resource managed by the Cancer Center. CCSG funding represents 21% of the annual operating budget. The remaining support comes from user fees (79%). MPSR is overseen by UCCC Associate Director for Translational Research. Use of Services: Since July 2011, 147 investigators have used the services. 78% percent of users were UCCC members, representing all 6 Programs and resulting in 118 publications. Future Directions: The MPSR has a number of future directions that will enhance the SR and UCCC member cancer research: 1) The PEMT unit will significantly expand NGS capabilities, increasing the number of genes analyzed in panels and the types of alterations that can be detected; 2) The Cytogenetics unit will introduce RNA FISH capabilities and automated scanning and computer-assisted analyses for FISH in cell suspensions and tissue sections.

MOLECULAR ONCOLOGY PROGRAM (Project-368) ABSTRACT Overview and Goals: The goal of the Molecular Oncology (MO) program is to harness the power of basic science approaches, from biochemistry to functional genomics, to elucidate molecular mechanisms of cancer evolution and progression, with the ultimate goal of enabling effective strategies for cancer prevention, diagnosis and treatment. The expertise of program members is broad and deep, with major strengths in the control of gene expression, epigenetic regulation, DNA repair and damage responses, telomeres, pathways controlling cell fate and elucidation of cancer-relevant molecular structures. Research Highlights: The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). A multidisciplinary team including members at UCB consortium site showed HIF1A employs Mediator-associated kinase CDK8 to stimulate RNAPII elongation. These results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia, and which may lead to novel therapeutic approaches (Cell, 2013). Program Activities: To accomplish this goal, the MO co-leaders employ resources provided by the UCCC to orchestrate intra- and inter-programmatic collaborations through organization of annual retreats and periodic technology forums, as well as routine chaperoning of transdisciplinary collaborations. Enabled by UCCC support, the co-leaders identify and evaluate novel technologies essential to catalyze new research by MO members through the creation and expansion of Shared Resources (SR), and by providing pilot funding to use these technologies, while leveraging resources and research strengths of consortium institutions across the state of Colorado. Through coordinated transdisciplinary relays from MO members to investigators in translational and clinical research programs, the discoveries made in this program move from bench to preclinical investigations and investigator-initiated trials (IITs), which will ultimately improve diagnosis, treatment and prevention of cancer. Members: The program has 43 Full and 20 Associate members with 89 grants encompassing $3.3M NCI and $7.5M of other cancer peer-reviewed research grant funding in 2015. Members are distributed across 20 basic science and clinical departments in the SOM and SOP at UCD as well as at UCB, CSU and at non-consortium institutions. MO members published 630 cancer-focused publications since 2011 of which 30% were inter- and 13% intra-programmatic. Future Directions: We plan to enhance current strengths in functional genomics and epigenetics, develop new capabilities in metabolomics, proteomics and cryo-electron microscopy, and promote interactions with programmatically relevant organizations at UCD such as the Linda Crnic Institute for Down Syndrome and the Division of Biomedical Informatics and Personalized Medicine (BIPM). These efforts will advance the discovery of basic cancer processes and their translation into improved cancer prevention and treatment.

LUNG, HEAD & NECK CANCERS PROGRAM (Project-534) ABSTRACT Overview and Goals: The Lung, Head and Neck Cancer (LHN) Program unites scientists focused on malignancies of lung and head and neck with the goal to decrease the incidence, morbidity and mortality of lung and head and neck cancer. Lung cancer is the most common cause of cancer death in the UCCC catchment area while HNC causes significant morbidity and mortality. The smoking rate in Colorado is lower than the national average but still causes the majority of LHN cancers, however, non-smoking related cancers in young, particularly female, patients are rising. Thus, research in the LHN program focuses on tumors caused by tobacco as well as other etiologies (non-smoking related driver mutations and human papilloma virus (HPV)). Program research falls into 3 areas: Risk, Early Detection and Chemoprevention; Tumor Genetics and Biology; and Experimental Therapeutics. Research Highlights: Research initiatives directly translated to our catchment area include development and validation of biomarkers to inform management of CT detected lung nodules; investigation of the biology of premalignant lesions to define risk of progression to cancer; chemoprevention of lung cancer and HNC; discovery of targetable mutations with translation to clinical trials; defining tumor vulnerabilities and mechanisms of resistance; developing biomarkers to provide personalized treatment; discovering the pathogenesis of HPV-associated HNC; and translating new approaches to decrease treatment side effects and improve survivorship. Program Activities: The program fosters interactions through seminars, retreats and training bringing together basic and clinical scientists in collaborative projects. Pilot grants through the UCCC, Lung SPORE and developing HNC SPORE support promising research initiatives. UCCC Shared Resources that are highly utilized by program members include Flow Cytometry, Genomics, Molecular Pathology, Biostatistics/Bioinformatics and Tissue Biobanking and Processing. Program leaders and members mentor young investigators through Career Enhancement Programs in the Lung and developing HNC SPOREs; the UCCC K12; the VA Career Development Program; the NCI T32 developed and led by the Program Leaders; and an NHLBI Pulmonary Sciences T32. Members: The LHN program is comprised of 22 Full and 27 Associate members, from 2 consortium institutions (UCD and VA) and appointed in the Schools of Medicine, Dental Medicine and Public Health at UCD. LHN members hold $3.3M ($1.3M NCI) in peer-reviewed cancer research funding in 2015 and produced 362 cancer-focused publications since 2011, of which 49% were inter- and 30% intra-programmatic, exemplifying the highly interactive nature of the LHN program. Future Directions: The LHN program will further enhance early detection, prevention and biomarker research in lung cancer and HNC and improve the integration of clinical and molecular diagnosis and targeted therapies for LHN cancer patients. We also intend to enhance research in HPV-related HNC and encourage increased research in immunotherapy.

HEMATOLOGIC MALIGNANCIES PROGRAM (Project-265) ABSTRACT Overview and Goals: Hematologic malignancies represent some of the most aggressive forms of cancer, and in many cases, current therapeutic options are very limited. Thus, the goal of the new Hematologic Malignancies (HEME) Program, which was formally established in early 2015, is to define key biological features of leukemia and related blood cancers and translate these into improved therapeutics. HEME was formed in 2015 based on the vision of the UCCC leadership with concurrence of our EAB and leverages the enormous growth in the hematologic malignancies community at AMC over the last 5 years. The major scientific strengths in HEME focus on the epigenetic regulation of cellular processes, key molecular events occurring as normal cells transition to malignant states, metabolic processes that define tumor-specific properties, and characterizing malignant stem cells. Research Highlight: Recently members discovered that germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia opening up novel avenues for prevention and assessment of cancer predisposition risk in such patients (Nat Genet, 20151). Program Activities: To accomplish this goal, HEME co-leaders employ resources provided by the CCSG to orchestrate intra- and inter-programmatic collaborations through organization of annual retreats and periodic technology forums, and routine chaperoning of transdisciplinary collaborations. Program members utilize Shared Resources (SR) for preclinical mouse models and employ patient-derived specimens and tumor models as a means to evaluate candidate therapies. In addition, work across the consortium with CSU/FACC on spontaneous disease models in companion animals complements an extensive adult and pediatric human clinical trials portfolio at that seeks to advance multiple targeted therapies across all ages. Members: The HEME program is comprised of 24 Full and 11 Associate members with 65 grants and $1.4M NCI and $1.3M other peer-reviewed cancer research grant funding in 2015. The group of multidisciplinary investigators includes the entire spectrum of pediatric and adult blood cancer research groups across the UCCC consortium. 86% of members (30) are located in 10 clinical and basic science departments at UCD and CSU; and the remainder are at non-consortium institutions. From program inception (7/2014) the group produced 77 cancer-focused publications, of which 32% were inter- and 13% intra-programmatic. Future Directions: We expect the HEME program to expand substantially. Ongoing recruitment efforts include clinical research leadership roles in immunotherapy, pediatric BMT, myeloma, and expertise in adolescent young adult (AYA) populations. In addition, growth in immunology, pharmacology and molecular biology is expected to further strengthen our basic science efforts. For this dynamic program, we will focus on supporting interdisciplinary and translational studies, collaborative projects, training and mentoring of junior investigators, and the development of key resources required for laboratory and clinical research.

GENOMICS SHARED RESOURCE (Core-298) ABSTRACT Overview: The Genomics Shared Resource (GSR) offers UCCC members high-quality and cost-effective services and technical support for high-throughput genomic and proteomic investigation to study cancer pathogenesis, therapeutics, genetic susceptibility and biomarker development. Equipment: The GSR houses equipment to support DNA next-generation sequencing (NGS) (Illumina HiSeq 2000, 2500 and 4000, Illumina MiSeq, and LifeTech IonPGM); DNA microarray analysis (Affymetrix GeneChip and GeneTitan systems; Illumina iScan system; and Agilent SureScan Microarray system); single-cell genomics (Fluidigm C1; Juno; Access Array and Biomark HD Systems); proteomics (Somalogic SOMAscan); and Affymetrix, Agilent and Illumina microarray and sequencing software. Services: The GSR offers methodologies for quantitative assessment of DNA/RNA/Protein including evaluation of human and murine cells harboring shRNA and CRISPR libraries UCCC members have obtained from the Functional Genomics Shared Resource (FGSR). Our capabilities include genome-wide analyses of DNA mutations and RNA expression from tissues to single cells. With these capabilities, the GSR provides: 1) Genome-wide gene variations, gene expression and transcriptome analysis, epigenetics and cytogenomics using NGS and gene microarrays; 2) Single cell genomic analysis; 3) Quantitate selected genes/panels using real time PCR and digital PCR; 4) Proteomic quantitative analysis. Consultation and Education: The GSR provides consultation for designing and analyzing reliable genomics and proteomic experiments and helps keep members up to date as technology advances through seminars and hands-on training. Management: The GSR is an institutional core managed by the UCCC, and is overseen by the Associate Director for Basic Research. Use of Services: Since July 2011, the GSR has provided services to 313 investigators. Forty-seven percent of users (148) were UCCC members, representing all 6 Programs and resulting in 155 peer reviewed publications. CCSG funding represents 5% of the annual operating budget. The remaining support comes from the University of Colorado NIH funded CTSA (Colorado Clinical and Translational Sciences Institute (CCTSI)) grant (4%), and user fees (91%). Future Directions: The GSR has three main future directions that will enhance the SR and UCCC member cancer research: 1) Optimize single cell genomics; 2) Enhance aptamer-based proteomics; 3) Adopt single molecule sequencing.

FUNCTIONAL GENOMICS SHARED RESOURCE (Core-564) ABSTRACT Overview: Functional Genomics explores gene and protein function at the individual gene level and on a genome-wide scale. The Functional Genomics Shared Resource (FGSR) provides UCCC members access to tools that investigate gene function on a genome-wide scale; develops novel protocols and offers expertise for the use of Functional Genomics tools; and provides a forum for scientific exchange. By promoting collaboration in Functional Genomics, the FGSR serves to catalyze discoveries by UCCC members. Equipment: Through continuous assessment of novel technologies and UCCC members' needs, the FGSR has acquired diverse tools, including genome-wide short hairpin RNA (shRNA), Open Reading Frame (ORF) and CRISPR libraries. Today, our human shRNA collection contains 176,283 clones targeting >22,000 unique genes, while our mouse collection contains 138,538 clones targeting >21,000 unique genes. Our ORF library contains 15,744 plasmids for protein overexpression corresponding to 13,082 unique human genes. Our pooled gene knockout CRISPR libraries contain an average of 6 guide RNAs (gRNAs) per gene to inactivate ~18,000 human genes and ~15,000 murine genes. The transactivating CRISPR library contains similar numbers of gRNAs to induce activation of ~18,000 human promoters. Our arrayed CRISPR library has 2 gRNAs per gene for 16,190 human genes. Services: The FGSR assists members in all aspects required for the effective use of the tools provided, preparing bacterial glycerol stocks, providing plasmids for entire libraries or individual clones, and preparing lentiviral particle suspensions. The FGSR also works with client labs to create `custom panels' targeting gene collections of interest. Consultation and Education: The FGSR personnel assist members on a daily basis with experimental design and troubleshooting. The FGSR website hosts a large number of very detailed protocols and bibliographic sources that are updated constantly. The FGSR also works with program leaders to host `technology forums' where UCCC members are updated on the latest developments and applications in the field of Functional Genomics. Management: The FGSR is managed by the UCCC, and is overseen by the Associate Director for Basic Research. Use of Services: Since July 2011, 158 client laboratories have used the services. Seventy-seven percent of these groups (122) were UCCC members, representing all 6 Programs and resulting in 65 peer-reviewed publications. CCSG funding represents 41% of the annual operating budget. The remaining support comes from user fees (59%). Future Directions: We will continue to acquire the latest tools in the Functional Genomics field, such as novel CRISPR-based technologies for genome editing. We will also expand our service and education portfolio via technology forums and webinars.

FLOW CYTOMETRY SHARED RESOURCE (Core-731) ABSTRACT Overview: The Flow Cytometry Shared Resource (FCSR) supports translational and clinical cancer research projects of UCCC members with flow cytometric analysis, high-speed cell sorting, multiplexed fluorescent microsphere assays, and cell counting services. Equipment: FCSR houses three high-speed sorters, four analyzers (3,000 and 8,000 hours/year of cell sorting and analysis capacity, respectively), a new CYTOF 2 mass cytometer, a Luminex Magpix system (1,500 hours/year capacity), and a cell counter. Services: FCSR provides a wide range of assays particularly pertinent to cancer research, including cell cycle, cell proliferation, apoptosis, cell viability, cell signaling, multiplexed cytokine quantitation, stem cell detection, fluorescent protein analysis, and cell phenotyping along with cell sorting. Consultation and Education: FCSR provides consultation to help those members with limited flow cytometry background to design appropriate experiments. In addition, regular educational sessions are held to inform UCCC members and the investigators in their laboratories about the fundamentals of flow cytometry along with information on new techniques and FCSR capabilities. Many investigators also call FCSR on an ad hoc basis for advice on performing experiments. The FCSR also offers assistance and collaboration in the development of new methods. Management: This resource is an institutional core managed by the UCCC and is overseen by the Associate Director for Basic Research. Use of Services: FCSR is among the most highly used of the UCCC shared resources. Since July 2011, 266 investigators have used the services. Sixty-two percent of users (164) were UCCC members, representing all six programs and resulting in 160 peer-reviewed publications. CCSG funding represents 19% of the annual operating budget. The remaining support comes from a NIAMS P30 grant (1%), institutional support (4%) and user fees (75%). Future Directions: FCSR will continue to play a central role in the research conducted by UCCC members by continuing to provide outstanding flow cytometry related services and education and developing cross-disciplinary collaborations with other shared resources. In addition, the FCSR has several future directions that will enhance the SR and UCCC member cancer research including: 1) Raise awareness and promote use of CyTOF2 instrument; 2) Acquire a four- or five-laser analyzer that can detect at least 14 colors and evaluate samples from 96-well plates.

EARLY PHASE CLINICAL RESEARCH SUPPORT (Core-030) ABSTRACT The Early Phase Clinical Research Support (EPCRS) component provides data management support to conduct novel early-phase clinical trials designed from research findings of UCCC members. The overall objective of the EPCRS is to promote innovative clinical trials in the UCCC catchment area (the State of Colorado), to facilitate inter- and intra-programmatic translational collaboration within UCCC, and to support the development of novel cutting-edge therapies and strategies to prevent and treat cancer. These clinical trials are hypothesis-driven, high-priority, innovative, Pilot (feasibility) or Phase 0/I institutional trials focused on early phase testing of an agent or device for the diagnosis, prevention, detection or treatment of cancer. Expected outcomes include subsequent later stage clinical testing through the National Clinical Trials Network (NCTN), the Phase II mechanism of the Experimental Therapeutics Clinical Trials Network (ET-CTN), or in conjunction with independent peer-reviewed grant support or industry funding. Although EPCRS was not funded over the last cycle due to disapproval of our PRMS, UCCC has continued to conduct early phase trials which adhere to the NCI eligibility requirements of this mechanism and through our NCI UM1 Southwest Early Clinical Trials (SECT) Consortium (UM1CA186688) with MD Anderson Cancer Center, as well as institutional and industry support, funding has been available to translate bench observations made by our translational UCCC members, to the bedside. Going forward a number of changes have been instituted to further ensure that only the highest quality early clinical science is carried out at UCCC. These include an Investigator-Initiated Trial (IIT) structure, that includes an IIT Incubator (IIT-I) meeting chaired by Eckhardt that brings together a multidisciplinary group of investigators to discuss and promote hypothesis-driven early trials, as well as an IIT Review Committee (IIT-RC) chaired by Flaig and Eckhardt that is charged with ensuring that all components of a concept such as the scientific rationale, statistics, data management, budgeting and finance are assembled to enhance feasibility and to enable prioritization for funding decisions. In addition to the requested CCSG funds, UCCC will provide $250K/yr for early-phase clinical trials that will facilitate expansion of IITs and provide greater opportunity for the translational science of UCCC programs to impact patients in our catchment area. Our future directions include: 1) Establishing processes that stimulate direct collaborations between the UCCC clinical disease groups and the UCCC Research Programs; 2) Working with preclinical scientists in the UCCC to enable the development of novel animal models that can be used provide the rationale for hypothesis-driven early clinical trials; 3) Evaluating challenges regarding participation among racial/ethnic and socioeconomically underserved populations in early clinical trials; 4) Interacting with major UCCC wide strategic initiatives that involve early clinical investigation.

DEVELOPMENTAL THERAPEUTICS PROGRAM (Project-608) ABSTRACT Overview and Goals: The goal of the Developmental Therapeutics (DT) Program is to reduce the cancer burden through identification, development and testing of novel anticancer therapies and approaches. DT accomplishes this goal by hosting members with the required scientific expertise in the following areas: Target Inhibition and Companion Biomarkers, Drug Delivery, Radiation Biology and Delivery, Pharmacology, Stem Cells and Tumor Microenvironment, Immunotherapy, Preclinical Models and Imaging, Comparative Oncology and Clinical Trials. The program integrates this rich expertise into four major interdisciplinary focus groups (Drug Discovery, Preclinical Development, Early Clinical Development, and Delivery) that are linked by an overarching thematic emphasis on biomarkers and precision/ personalized medicine. Research Highlight: An example of the way the DT program takes laboratory studies forward to clinical testing is work from multiple DT members on Trametinib, a MEK1/MEK2 inhibitor, in patients whose tumors acquired resistance to BRAF inhibitor (J Clin Oncol, 20131; Lancet Oncol, 20122). Program Activities: Through stand-alone and intra-programmatic retreats, monthly meetings and a new grants program, the DT program promotes the transition of early findings through the therapeutic development process across the entire UCCC. For example, preclinical studies are conducted using novel model systems, pharmacology, and functional imaging allowing for the discovery of the appropriate biomarkers and patient selection criteria for incorporation into early clinical trials of targeted agents being tested pre-clinically. Through the establishment of close interactions between basic research laboratories, clinical scientists, the NCI, and the pharmaceutical industry, the DT leadership nurtures rapid development of new treatments and biomarkers. Members: The DT program has 111 members including 57 Full and 54 Associate members. The membership represents all 3 academic consortium institutions (University of Colorado Denver (UCD), University of Colorado Boulder (UCB), Colorado State University (CSU)) and are located in six different schools or colleges (Schools of Medicine, Pharmacy, Public Health, Liberal Arts at UCD; College of Arts and Sciences at UCB; College of Veterinary Medicine and Biomedical Sciences at CSU), and 18 departments. Current research funding is $18M. Peer-reviewed funding is $5.4M with $2.8M from the NCI and $2.6M from other peer sponsors. The DT Program produced 1,108 cancer-focused publications since the last review, of which 39% were inter- and 29% intra-programmatic, demonstrating the collaborative nature of the program. Future Directions: The program will enhance investigator initiated clinical trials (IITs) capitalizing on new investments by the UCCC that increase support for regulatory submissions and data management of IITs. The DT program will continue preclinical development of our own novel agents conceived within our program (e.g. SVC112, ONK101, neoamphimedine), and promote the identification of novel and efficacious drug combinations based on tumor molecular characteristics, biomarkers, and clinical testing of these therapies.

DEVELOPMENTAL FUNDS (Core-065) ABSTRACT Overview and Goals: CCSG Developmental Funds are used with the goal of supporting the pursuit of new scientific opportunities that, when properly selected, provide a significant boost to UCCC research activities. In the current grant period, CCSG Developmental Funds have been used in combination with philanthropic funds to promote strategic priorities of the UCCC in personalized medicine and population health by supporting two projects. The first project is the Human Tumor Model-based Databank (HTMD). The HTMD has two goals: 1) Assist UCCC members to establish their own Patient Derived Xenografts (PDX) and 2) house a central database of all `omics information on PDX so evaluated that can be used for integrative analyses and queried remotely by UCCC members seeking to study tumors with specific genomic, transcriptomic or proteomic markers. The second project was aimed at nurturing growth of survivorship research at the UCCC via support for a staff investigator, Jean Kutner MD, charged with promoting this area in our Cancer Prevention and Control (CPC) program. Scientific Progress: With the goal of promoting translational research, UCCC leadership invested in the development of the HTMD. In the last 5 years established over 250 unique patient derived xenografts (PDX). Over 100 of these have been `omically profiled and are available for use and data query by UCCC members. This success allowed us to obtain a CCSG administrative supplement both promoting this UCCC program and benefitting the nation by contributing our PDXs to the NCI repository. The PDXs have been used in 24 publications and have lead or supported the initiation of clinical trials, some highlighted in our Program and SR write-ups. In the same time frame, Kutner formed a UCCC Special Interest Group (SIG) for cancer survivorship research that led to: 1) opening of eight survivorship research protocols; 2) a collaborative pilot program with the Anschutz Health and Wellness Center (AHWC) to enroll cancer survivors in a supervised, exercise program to measure the effect of an exercise regimen on quality of life and outcomes. A number of publications (33) and grants ($1.35M in 2015) can be attributed to Kutner's leadership of the SIG. Future Directions: In the next 5 years we aim to use CCSG Developmental Funds to support pilot grants with cancer relevant organizations across the UCCC consortium in a cost-shared manner with the objective to develop collaborative and synergistic scientific projects that promote exceptional research across UCCC and leverage existing institutional strengths. Projects will be selected based on their potential to exploit unique research ideas and capabilities and translate these into innovations that are likely to lead to external funding and subsequent major advances in cancer medicine. We will monitor and evaluate success of this pilot grant funding based on measurable outcomes (publications, grants, trials). In summary, this program will leverage institutional strengths and financial resources to establish and/or deepen inter and intra-institutional relationships and promote future sponsored research grant applications and cancer research innovations.

CLINICAL PROTOCOL AND DATA MANAGEMENT (Core-176) ABSTRACT Overview: The Clinical Protocol and Data Management (CPDM) provides central management and oversight functions for coordinating, facilitating and reporting on clinical and population sciences trials across the UCCC consortium which serve our catchment area of the State of Colorado. The CPDM is led by Thomas Flaig MD, Associate Director for Clinical Research and supported by Colleen Kellackey RN, Consortium Clinical Research Director. The UCCC Data and Safety Monitoring Plan (DSMP) was approved by the NCI in October, 2014. The Data Safety and Monitoring Committee (DSMC) provides a robust structure for auditing clinical trials across the consortium. DSMC's oversight is prioritized for high-risk Phase I and Phase II protocols. Accomplishments: Since 2011, the CPDM substantially improved clinical research support as follows: 1) CPDM led an institutional- wide effort to select and implement the OnCore® commercial clinical trial management system (CTMS) integrated with the UCD clinical data repository (Health Data Compass). OnCore was activated July 2015; 2) CPDM leadership implemented an acuity tool to right-size the clinical research staff for the adult Cancer Clinical Trials Office (CCTO); 3) CPDM increased its support for institutional trials by adding staff; and the 4) the DSMC auditing capacity was increased by 2 auditors. Quality Control and Training: An education coordinator position was added to the CPDM. In addition to outlining and coordinating training and orientation for data managers, nurses and new faculty, the coordinator works closely with the DSMC to review trends from internal and external audits. Based on these findings, the coordinator works with the CPDM leadership to develop focused educational outreach to clinical investigators and recommends policy updates to improve the quality and compliance of clinical research. Trials and Catchment Area Demographics: The CPDM oversees an annual average of 382 clinical research studies. In FY2015 1,499 patients were accrued to intervention studies and 706 were accrued to intervention-treatment studies representing 18% of the analytic cases reported by UCH, CHCO and the VA. Patients accrued to clinical trials come from 61 of 64 Colorado counties. Based on data from 2015: 1) Although approximately half the cancer population is female, 58% of the accrued patients were female, representing a substantial increase from 2012 (49%) and 2) 3% and 5% of patient accruals were African American and Hispanic, respectively. UCCC continues to enroll women and African Americans in rates that are consistent with the cancer incidence in these specific populations and has a plan to increase Hispanic enrollment to at least 10%, consistent with the cancer incidence in the population. Taken together, these data demonstrate substantial progress in the CPDM and further demonstrate the UCCC's commitment and success in providing safe and equitable clinical investigative opportunities to Colorado's cancer patients.

CANCER PREVENTION AND CONTROL PROGRAM (Project-018) ABSTRACT Overview and Goals: The goal of the Cancer Prevention and Control (CPC) Program is to develop and implement ways to reduce cancer risk and enhance outcomes for cancer survivors across Colorado. The scientific expertise of CPC members spans laboratory to population research and includes disciplines from basic science to behavioral science and health services research. CPC members conduct investigations into how factors that affect cancer risk, diagnosis, treatment, and outcomes can be modified to reduce the burden of cancer in Colorado and beyond. The CPC program has three specific aims: 1) find new ways to prevent cancer, 2) find new ways to deliver cancer prevention services, and 3) improve cancer survivorship. Within the Colorado catchment area, health disparities research cuts across each of our aims as CPC members focus research on the growing Hispanic population, the medically underserved, and cancer survivors. CPC investigators comprehensively explore both fundamental and applied questions, as evidenced by a very diverse funding portfolio, collaborative publications, and clear impact on cancer within our catchment area. Research Highlights: CPC research has led to advancements in primary and clinical preventive services, statewide policies, and improved cancer survivorship. CPC investigators studied promising agents in pre-clinical studies, identified breast cancer risk factors in Hispanic women, developed and operated the Colorado Colorectal Screening Program, made advancements in the understanding of obesity and breast cancer, and affected cancer prevention and control policy across the state. Program Activities: Investments in pilot funds, mentorship, and program planning initiatives since the last review cycle have led to a vibrant CPC program with many new research initiatives. In July 2015, Cathy Bradley PhD, was recruited to be the UCCC Associate Director for Population Sciences Research, with nearly $6M in resources to invest in cancer prevention research at the UCCC. Members: The CPC Program is comprised of 26 Full and 35 Associate members from all 3 academic consortium institutions, 9 schools and centers, and 25 academic departments within the University of Colorado Denver, Colorado State University, and University of Colorado Boulder. Since 2011, CPC members produced 479 cancer-related publications of which 154 were collaborative (18% inter-programmatic and 21% intra- programmatic). In 2015, CPC members held 79 grants (increase of 18%), totaling $10M; $2.3M from the NCI and $3.6M from other peer-reviewed sources. Future Directions: The CPC program is infused with substantial monetary investments, stimulating new pilot work, and engaging in several active recruitments including a co- program leader who will be supported by an endowed chair, and at least three other new CPC faculty members in epidemiology, behavioral health, and/or health services research. We aim to leverage these new resources to enhance the translation of our chemoprevention research into clinical trials, expand our health services and population sciences research portfolio, and further improve our research activities in survivorship.

CANCER CELL BIOLOGY PROGRAM (Project-113) ABSTRACT Overview and Goals: Accumulation of defects in the regulation of cell behavior results in uncontrolled proliferation, immune evasion, invasiveness and metastasis. Understanding these mechanisms will provide new diagnostic markers and therapeutic targets. The major goal of the Cancer Cell Biology (CCB) Program is to foster and improve research focused on dissecting the cellular regulatory functions that establish and maintain this malignant phenotype and to apply this knowledge to translational and clinical investigations. CCB members have expertise in many areas and disciplines: Cell Cycle Regulation, Apoptosis and Autophagy, Developmental Biology and Stem Cells, Immunotherapy/Immunology, Signal Transduction and Tumor Microenvironment and Metastasis. This deep and diverse expertise results in collaborations, enhanced training and facilitation of technological innovations through UCCC Shared Resources (SR). Research Highlight: A multidisciplinary team including members at the UCB consortium site mapped the cell cycle phosphoproteome of the yeast centrosome. This molecular resource will provide foundational knowledge about the cell cycle in cancer and other diseases (Science, 20111). Program Activities: To accomplish its goal, the CCB program co-leaders employ resources provided by the UCCC to foster interactions by organizing retreats, mentoring programs, and weekly seminars attended by program members, students, fellows, and non-program faculty. Our collaborative publications and grants demonstrate the success of our endeavors. Furthermore, key members of the CCB Program have collaborated effectively with other programs, resulting in joint grant awards and submissions. Members: The program has 43 Full members with $2.2M in grant funding from NCI and $5.9M in other peer-reviewed research grant funding in 2015. Members are from 5 basic science (21%) and 7 clinical (51%) departments in the SOM, from the School of Dental Medicine (5%), and the School of Public Health (1%) at AMC; and the College of Liberal Arts and Sciences (2%) at the downtown campus. Thirteen percent of members are at UCB; 2% at CSU; and 5% are at non-consortium institutions. Program members published 690 cancer-relevant publications in the previous grant period of which 41% were inter- and 17% were intra-programmatic. Future Directions: We will enhance our high degree of productivity and collaborative science by continuing to support fundamental research in cell biology, by guiding these fundamental discoveries into the clinic and by leveraging the scientific strengths found in the consortium institutions unique to the State of Colorado. Specifically, we will accomplish this goal by enhancing the training and mentoring of students, fellows and junior faculty, by increasing the number of novel cancer biology and mechanism discoveries that lead to collaborative scientific studies and are translated into clinical applications, and by providing access to new technologies and innovative experimental models of cancer.

BIOSTATISTICS / BIOINFORMATICS SHARED RESOURCE (Core-667) ABSTRACT Overview: The University of Colorado Cancer Center (UCCC) Biostatistics and Bioinformatics Shared Resource (BBSR) provides biostatistical and bioinformatics leadership and analytic support for laboratory- based cancer research, experimental therapeutic studies and clinical trials, as well as population-based studies (e.g., epidemiological, health outcomes and disparities research in our catchment area), to UCCC members. Expertise: Substantial expertise is available for the analysis of clinical trials, statistical genetics, next- generation sequencing, predictive modeling, cancer-focused bioinformatics, and health services research. Services: BBSR support of UCCC members begins with study design to address research questions, along with sample size determination and analytical plans, as needed for grant and clinical trial development and protocol submission. BBSR support also includes data and safety monitoring of clinical trials analysis, and assistance with manuscript preparation in relation to data interpretation and scientific conclusions. The BBSR actively participates in the Protocol Review and Monitoring System (PRMS) and Data and Safety Monitoring Committees (DSMC). The BBSR collaborates with other UCCC Shared Resources (particularly Genomics and the Molecular Pathology Shared Resources) in research studies and the development of new bioinformatic pipelines. Consultation and Education: Faculty biostatisticians and bioinformaticists also provide educational opportunities, through one-on-one training in biostatistics and bioinformatics, and are engaged in consultation with investigators and in the development of novel methods and tools that directly enhance UCCC member research. Management: The BBSR is comprised of two distinct components ? biostatistics and bioinformatics. Biostatistics is a UCCC Shared Resource while bioinformatics is an institutional core. Both are managed by UCCC; and both are overseen by the Associate Director for Population Sciences. Use of Services: Since July 2011, 252 investigators have used BBSR services. Sixty-seven percent of users (169) were UCCC members, representing all 6 Programs and resulting in 112 peer-reviewed publications. CCSG funding represents 25% of the annual operating budget. The remaining support comes from institutional support (43%), the CCTSI (4%) and user fees (28%). Future Directions: In the next 5 years, the BBSR will increase bioinformatics staff to accommodate the predicted growth of research projects that generate high-dimensional data. Future hires in bioinformatics will leverage resources with the newly-formed Division of Biomedical Informatics and Personalized Medicine (BIPM). Biostatistics staff hires will also be needed to accommodate planned growth in Population Sciences and clinical research including trials. Supporting the initiatives that form the Future Directions in the Director's Overview, the BBSR anticipates playing an important role in the Overcoming Resistance to Targeted Agents, the Promoting Healthy Living Initiative and the Front Range Clinical Trials Network, the latter requiring biostatistical expertise in multisite cancer clinical trial design.

ANIMAL IMAGING SHARED RESOURCE (Core-441) ABSTRACT Overview: The objective of the Animal Imaging Shared Resource (AISR) is to support cancer research by providing comprehensive high-end imaging services, validation of novel contrast agents, collaboration and training in animal imaging, study design, quantitative image analysis and translation of imaging technologies to human clinical trials. The AISR was created in collaboration with and is jointly supported by the University of Colorado NIH funded CTSA (Colorado Clinical and Translational Sciences Institutes (CCTSI)). Equipment: The AISR has a Bruker 4.7 Tesla MRI/MRS scanner, an IVIS200 bioluminescence imager, and a Siemens Inveon microPET/CT. In the past five years, all acquisition and post-processing imaging software for each scanner has been upgraded. A new PET/CT suite has also been built to accommodate 18F- and, especially, long half-life radioisotope (64Cu, 124I) studies. In addition, the AISR has multiple high-resolution Bruker NMR spectrometers (300, 400, 500 MHz) for ex vivo discovery of novel metabolic biomarkers and potential translation into metabolic imaging protocols. Services: The AISR offers full animal imaging services, including study design assistance, IACUC approvals (all imaging SOPs are in place), handling of animals during acquisition, image acquisition and multimodal image analysis, as well as preparation of grant proposals, study reports and publications. In addition to anatomical imaging protocols, the AISR has established and validated various advanced imaging protocols (both pre-clinical and translational) for tumor physiology and microenvironment such as diffusion-weighted imaging (DWI), dynamic contrast-enhanced MRI (DCE-MRI), vessel-size imaging (VSI), iron oxide nanoparticle T2-MRI, and 18F-fluoroestradiol FES-PET. Consultation and Education: The AISR offers advanced imaging, image analysis and metabolomics courses and workshops for faculty, students and staff 2-3 times per year. Management: The AISR is an institutional shared resource managed by the UCCC. CCSG funding represents 27% of the current annual operating budget. The remaining support comes from the CCTSI grant (10%), institutional support (12%) and user fees (51%). AISR is overseen by the UCCC Associate Director for Translational Research. Use of Services: Since July 2011, 123 investigators have used the services. Fifty-four percent of users were UCCC members, representing all 6 Programs and resulting in 72 peer-reviewed publications. Future Plans: A new animal SPECT/CT scanner (Mediso) will be installed in 2016. A coil and hardware upgrade of the existing 4.7 Tesla MRI scanner and a purchase of a new 9.4 Tesla Bruker BioSpec MRI scanner will take place in 2016-2018. A new optical bioluminescence/ fluorescence scanner will also be added in 2016-2018. Purchase of a new imaging-guided (CT) X-ray irradiator is currently under negotiation. AISR will play an important role in Advancing Cancer Immunotherapy, one of the 6 cross-cutting strategic research initiatives in the Director's Overview (Part III).