David J. McConkey - US grants

DESCRIPTION The proposal is designed to establish how oxidative stress caused by bleomycin treatment leads to macrophage apoptosis and lung fibrosis. The investigators postulate that this apoptotic response causes impaired clearance of lung debris and release of inflammatory cytokines, particularly IL1 beta. They present three specific aims to test the hypothesis. Aim 1 is an in vitro study using peritoneal macrophages to determine the involvement of oxidative stress in bleomycin-induced apoptosis. The second aim is an in vivo study using p53 knockout animals to test the role of this protein in apoptosis and concomitant bleomycin-induced fibrosis. Third is to define the role of IL1 beta and interleukin converting enzymes in lung fibrosis.

DESCRIPTION (adapted from investigator's abstract): While the PI and others in the field have demonstrated a requirement for altered intracellular calcium levels in the regulation of apoptosis, little data are available regarding the intracellular targets for this factor. A calcium regulated protease has been identified by the PI from the nucleus that appears to be required for both lamin B degradation and DNA fragmentation. The PI has also demonstrated that there is a caspase cleavage consensus sequence in the IP3 receptor. This proposal is designed to: 1) identify the calcium-activated protease required for thymocyte apoptosis; 2) determine if the anti-apoptotic protein bcl-2 regulates this proteases and 3) determine if the type 1 IP3 receptor is required for apoptosis.

Agents that target tumor vasculature have tremendous potential as anti- cancer therapies because they do not appear to induce drug resistance. However, these agents are cytostatic rather than cytotoxic, which poses a unique set of problems for the evaluation of drug efficacy because classical measures of tumor (response" may not be valid. It will be especially important to identify their biological mechanisms of action and to develop new methods to detect these effects in primary patient tissue specimens. To this end, we have developed a technique that allows for the detection of dying tumor endothelial cells, and our preliminary results strongly suggest that anti-angiogenic agents induce apoptosis in tumor endothelial cells. The overall goal of the present studies is to determine whether endothelial cell apoptosis is a sensitive marker of efficacy in tumors treated with anti-angiogenic therapies can distinguish patients who respond to these drugs from those who do not. To this end, we propose to: (1) Define the role of specific "survival" pathways in the maintenance of endothelial cell viability in vitro. Cells will be exposed to growth factor withdrawal or VEGF receptor antagonists, and effects on signaling pathways previously implicated in cell survival (i.e. AKT) will be evaluated. We will also isolate mRNA from these cells and analyze changes in apoptosis-associated gene expression following VEGF withdrawal. (2) Determine the role of endothelial cell apoptosis in orthotopic tumor models. Nude mice bearing human pancreatic, colon, or prostate tumors will be treated with investigational agents, and effects on tumor endothelial cell apoptosis will be measured CD31/TUNEL staining. (3) Characterize the role of endothelial cell apoptosis in the effects of anti-angiogenic therapies in patients. Levels of endothelial cell apoptosis in patients treated with anti-angiogenic agents will be correlated with tumor blood flow changes and radiographic measurements of response. These studies will allow us to rapidly determine whether tumor endothelial cell apoptosis can be used as a surrogate for clinical response in patients treated with this class of novel anti-cancer agents.

1-(2-Oxo-4-amino-1,2-dihydropyrimidin-1-yl)-2-deoxy-2,2-difluororibose; 2',2'-DFDC; 2',2'-difluoro-2'-deoxycytidine; 2',2'-difluorodeoxycytidine; 2'-deoxy-2'-difluorocytidine; 2'Deoxy-2',2'-Difluorocytidine; 2,2 difluorodexoycytidine; 20S Catalytic Proteasome; 20S Core Proteasome; 20S Proteasome; 20S Proteosome; 3-carboxypropionyl-alanyl-alanyl-alanyl-4-nitroanilide; 3-carboxypropionyl-trialanine-p-nitroanilide; APO2; Advanced Cancer; Advanced Malignant Neoplasm; Agents, Cytostatic; Antibodies; Apoptosis; Apoptosis Pathway; Apoptotic; Assay; Athymic Nude Mouse; BCG; BCG (Connaught); BCG (Pasteur); Bacillus Calmette-Guerin; Bioassay; Biologic Assays; Biologic Marker; Biologic Products; Biological Agent; Biological Assay; Biological Markers; Biological Products; Bladder; Bladder Neoplasm; Bladder Tumors; Blood; Body Tissues; Bortezomib; Bypass; CAP20; CAP20 protein; CAP4 protease; CASP8 Protein; CDK; CDK Inhibitor Protein; CDK-Interacting Protein 1; CDK2-associated protein 20 kDa; CDKI Protein; CDKN1; CDKN1 protein; CDKN1A; CDKN1A Protein; CDKN1A gene; CIP-1 Protein; CIP1; Cachectin; Cachectin-Tumor Necrosis Factor; Calmette-Guerin Bacillus; Cancer Treatment; Cancer cell line; Cancer of Bladder; Cancer of Urinary Bladder; Cancers; Carcinoma Cell; Carcinoma, Transitional Cell; Caspase-8/Flice; Cdk2 inhibitor protein; Cell Death; Cell Death, Programmed; Cell Line; Cell Lines, Strains; Cell-Death Protease; CellLine; Cells; Cessation of life; Chemicals; Cip1 protein; Clinical Trials; Clinical Trials, Phase I; Clinical Trials, Unspecified; Clinical Trials: Cancer Treatment; Combined Modality Therapy; Complement; Complement Proteins; Cyclin Kinase Inhibitor; Cyclin-Dependent Kinase Inhibitor; Cyclin-Dependent Kinase Inhibitor 1A; Cyclin-Dependent Kinase Inhibitor 1A Gene; Cyclin-Dependent Kinases; Cyclin-Dependent Protein Kinases; Cystectomy; Cytostatic Drugs; Cytostatics; DR4; DR5; Data; Death; Defect; Difluorodeoxycytidine; Disease; Disorder; Dose; Doxorubicin/Gemcitabine; Drugs; EC 2.7; ENPT; Early-Stage Clinical Trials; End Point; EndPointCode; Endpoints; Enrollment; Event; FADD-Like ICE; FADD-homologous ICE/CED3-Like Protease; FLICE protein; Family; Funding; Future; Genes, p53; Genome, Human; Goals; Heterograft; Histones; Hour; Human; Human Genome; Human, General; ICE-like protease; IFN; Immunomodulators; In Situ Nick-End Labeling; In Vitro; Incubated; Induction Therapy; Infection; Institution; Interferons; Invasive; KILLER; KILLER/DR5; Killings; Kinases; L-Alaninamide, N-(3-carboxy-1-oxopropyl)-L-alanyl-L-alanyl-N-(4-nitrophenyl)-; Laboratories; Ligands; MACH protein; MDA 6; MGC9365; Macropain; Macroxyproteinase; Malignant Bladder Neoplasm; Malignant Cell; Malignant Epithelial Cell; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Malignant Neoplasms; Malignant Tumor; Malignant Tumor of the Bladder; Malignant neoplasm of urinary bladder; Man (Taxonomy); Man, Modern; Mch5 protease; Measures; Mediating; Medication; Messenger RNA; Methods; Mice, Athymic; Mice, Nude; Molecular; Molecular Marker; Multicatalytic Proteinase; Multimodal Therapy; Multimodal Treatment; Multimodality Treatment; Muscle; Muscle Tissue; N-succinyl-L-trialanine p-nitroanilide; NEOADJ; Neoadjuvant; Neoadjuvant Therapy; Neoadjuvant Treatment; Normal Tissue; Normal tissue morphology; Nude Mice; Operation; Operative Procedures; Operative Surgical Procedures; Organ; P21; P53; PS-341; PS341 cpd; Pathway interactions; Patients; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacodynamics; Phase; Phase 1 Clinical Trials; Phase I Clinical Trials; Phase I Study; Phosphotransferases; Pic-1 protein (cyclin); Primary Neoplasm; Primary Tumor; Process; Production; Prosome; Proteasome; Proteasome Endopeptidase Complex; Proteasome Inhibition; Proteasome Inhibitor; Proteins; Proteosome; RNA, Messenger; RNA, Small Interfering; Receptor Protein; Recombinants; Reproduction spores; Research; Residual Tumors; Resistance; Reticuloendothelial System, Blood; Role; SAHA; SANA; SCHED; SDI1; SLAPN; SYS-TX; Sampling; Schedule; Science; Sentinel; Signature Molecule; Small Interfering RNA; Spores; Staining method; Stainings; Stains; Standards; Standards of Weights and Measures; Suberoylanilide Hydroxamic Acid; Suc-Ala(3)-NA; Suc-Ala(3)-Nan; Suc-Ala-Ala-Ala-p-nitroanilide; Surface; Surgical; Surgical Interventions; Surgical Procedure; Systemic Therapy; TNF; TNF (unspecified); TNF Alpha; TNF Receptor Ligands; TNF protein, human; TNF superfamily, member 2 protein, human; TNF-2 protein, human; TNF-alpha; TNFA; TNFRSF10A; TNFRSF10A gene; TNFRSF10B; TNFRSF10B gene; TNFSF2 protein, human; TP53; TP53 gene; TRAILR-1; TRAILR1; TRAILR2; TRICK2; TRICK2A; TRICK2B; TRICKB; TRP53; TUNEL; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxicities; Transfection; Transitional Carcinoma; Transitional Cell Carcinoma; Transitional Cell Carcinoma of the Urinary Tract; Transphosphorylases; Transplantation, Heterologous; Tumor Cell; Tumor Necrosis Factor; Tumor Necrosis Factor Family Protein; Tumor Necrosis Factor-alpha; Tumor Necrosis Factor-alpha (macrophage-derived); Tumor Necrosis Factors; Tumor Protein p53 Gene; Tumor Tissue; Tumor Volume; Tumors, Residual; Unresectable; Up-Regulation; Up-Regulation (Physiology); Upregulation; Urinary Bladder Malignant Tumor; Urinary Bladder Neoplasm; Urinary Bladder Tumor; Urinary System, Bladder; Urinary System, Urine; Urine; Urothelial Carcinoma; Velcade; Volume, Tumor; Vorinostat; WAF-1 Protein; WAF1; WAF1 CIP1; WAF1 protein; Week; Work; Xenograft; Xenograft procedure; Xenotransplantation; ZTNFR9; anticancer therapy; base; biomarker; biopharmaceutical; biotherapeutic agent; cancer cell; cancer therapy; caspase; caspase-8; cdk Proteins; cdn1 protein; chemotherapeutic agent; clinical investigation; combination therapy; combined modality treatment; combined treatment; cultured cell line; cyclin-dependent kinase Inhibitor p21; cystein protease; cystein proteinase; cysteine endopeptidase; cytokine; cytotoxic; dFdC; dFdC/DOX; dFdCyd; design; designing; disease/disorder; drug/agent; enroll; experiment; experimental research; experimental study; gemcitabine; gene product; human TNF protein; in vivo; inhibitor; inhibitor/antagonist; knock-down; mRNA; malignancy; mda-6 protein; multicatalytic endopeptidase complex; multimodality therapy; necrocytosis; neoplasm/cancer; neoplastic cell; novel; oncoprotein p21; p21 cell cycle regulator; p21 cyclin kinase inhibitor; p21(cip1); p21(waf1-cip1); p21-WAF1; pathway; peripheral blood; phase 1 study; phase 1 trial; phase I trial; preclinical study; protein p21; protocol, phase I; receptor; receptor expression; research study; residual disease; resistant; response; senescent cell-derived inhibitor protein 1; siRNA; social role; suberanilohydroxamic acid; succinyl-alanyl-alanyl-alanine-p-nitroanilide; succinyl-trialanine-4-nitroanilide; succinyl-trialanine-p-nitroanilide; succinyltrialanine-p-nitroanilide; surgery; terminal nick end labeling; tumor; tumor necrosis factor (unspecified); tumor necrosis factor, human; tumor necrosis factor-2 protein, human; tumor necrosis factor-alpha promoter allele-2 protein, human; urinary bladder

PROJECT SUMMARY Proteasome inhibitor (PI)-based combination chemotherapy is currently being evaluated for the treatment of pancreatic cancer and other solid malignancies. Our hypothesis is that PIs cause endoplasmic reticular (ER) stress in cancer cells and this stress mediates cell killing. Furthermore, we have obtained preliminary evidence that the effects of PIs on cell death are highly heterogeneous and are linked to whether or not they induce phosphorylation of eIF2¿, a component of the unfolded protein response (UPR) that mediates suppression of global protein synthesis. Specifically, PIs promote strong phosphorylation of eIF2¿ phosphorylation in the cell lines that are relatively resistant to PI-induced apoptosis, but they fail to do so (or attenuate translation) in the cell lines that are most sensitive. Identifying the biochemical basis for this heterogeneity could enable the prospective identification of tumors that are most likely to respond to PI-based combination chemotherapy and should yield new targets for therapeutic intervention. We also wish to better define the molecular mechanisms involved in the apoptosis that is induced by one of the most promising PI-based combination regimens, namely, the combination of bortezomib plus histone deacetylase (HDAC) inhibitors. We have obtained good preliminary evidence that HDAC inhibitors promote proteasome inhibitor-mediated apoptosis by disrupting cytoprotective structures known as aggresomes that appear to function to alleviate ER stress. Gene silencing studies have demonstrated that the HDAC responsible for aggresome disruption is HDAC6, and it is possible that more selective HDAC6 inhibitors will yield comparable or better tumor cell killing than pan HDAC inhibitors (like SAHA) with less toxicity. To directly test our hypotheses we propose the following Specific Aims. (1) Define the molecular mechanisms that control bortezomib-induced phosphorylation of eIF2¿. We will test the hypothesis that PIs inhibit PERK activation by inducing the expression of a molecular chaparone (HSP70?) that blocks PERK homoaggregation in drug-sensitive cells; (2) Determine role of ER stress in PI-induced apoptosis. Here we will assess the contributions of ROS, Ca2+, JNK, Noxa, and caspase-4 ot PI-induced apoptosis; (3): Determine the toxicity and anti-tumor efficacy of combination therapy with PIs and HDAC inhibitors in xenografts. We will compare the effects of combination therapy with PIs plus SAHA (a pan HDAC inhibitor), tubacin (HDAC6-selective), or SNDX-275 (type I HDAC-specific) in vitro and in orthotopic tumors derived from sensitive and resistant cell lines. We will also investigate whether or not pharmacodynamic markers of drug-target interaction and biological response can be measured in the peripheral blood of these animals and apply these methods to measure the effects of therapy with bortezomib plus SAHA within the context of a Phase II clinical trial in patients with pancreatic cancer.

The Administrative Core plays a critical organizational role in the success of the SPORE. Dr. Colin Dinney, the Core Director and SPORE Principal Investigator, will facilitate the functioning and interactions of all the projects and the cores. Drs. David McConkey and William Benedict as the Co-Directors of the Administrative Core will co-chair the Executive Committee that is composed of all project Co-Leaders and Core Directors. The SPORE Grant Program Administrator will work closely with Drs. Dinney, McConkey and Benedict to schedule all meetings with investigators and provide timely and effective communication with investigators both within and outside The University of Texas M. D. Anderson Cancer Center. The specific responsibilities of the Administrative Core are: (1) To monitor the research activity of the SPORE; (2) To promote integration and communication between the SPORE and the MD Anderson Multi-disciplinary Research Program in Bladder Cancer (MRP) and the Cancer Center Support (Core) Grant; (3) To monitor scientific integrity and overall compliance with all governmental and NCI regulations and requirements and to coordinate quality assurance including data quality control in conjunction with the Biostatistics and Specimen and Clinical Bioinformatics Core; (4) To provide administrative support for the Developmental Research and Career Development Programs; (5) To convene all necessary meetings, including those of the Executive Committee, Internal Scientific Advisory Committee, and External Scientific Advisory Committee; (6) To oversee expenditures and maintain budget infonnation; (7) To communicate and consult frequently with the NCI Project Leader and other staff and prepare all necessary internal and external reports; (9) To establish and maintain electronic communication among all SPORE investigators, including e-mail and a Web page describing SPORE activities, projects, and protocols; (8); To coordinate the activities of the Bladder Cancer Support Team (patient advocacy group); (9) To establish and monitor policies for the recruitment of women and minorities into this program and into any clinical intervention that should arise out of the SPORE; (10) To encourage communication with other groups interested in bladder cancer translational research; (11) To encourage Pharma/Biotech interaction with the SPORE Program.

Funding for bladder cancer (BC) research has fallen behind that provided for other malignancies. This is especially the case for junior individuals. Therefore our SPORE In BC has provided one of the unique resources for seed funding available to promising young laboratory and clinical scientists with a special interest in pursuing independent translational research in BC. The overall goal of the Career Development Program (CDP) is to support and mentor when appropriate the scientific careers of these individuals to expand the number of such talented young investigators. Funding for bladder cancer focused research has lagged behind support provided for other malignancies. The specific aims of the CDP within this Bladder Cancer SPORE renewal builds on the strengths of our current SPORE which included. Recruiting innovative entry-level and junior scientists into The University of Texas MD Anderson Cancer Center Bladder Cancer SPORE to enhance its overall translational BC research capability. Providing the opportunity for individuals with previous experience in the research and treatment of cancer at other disease sites who wish to develop expertise in BC. Promote the careers of the selected scientists by facilitating the development of skills necessary to be productive independent investigators in BC translational research. Train clinical oncologists and basic scientists who can rapidly translate basic observations in cellular and molecular biology into clinically applicable BC research programs and the design of protocols.

Few funding opportunities exist nationwide for pilot projects that are focused on bladder cancer (BC). Furthermore, as the only program of its kind in the country, the MD Anderson BC SPORE must reach out directly to other translational scientists who are or may become interested in BC research by fostering interinstitutional collaboration. To address these important needs, the SPORE Developmental Research Program has evolved from a structure that largely supported projects based at MD Anderson to one that seeks to establish collaborations with groups at other institutions and establish flexible and nimble processes for identifying and rapidly funding them. To this end, we propose the following Specific Aims in this renewal application: 1.To identify potentially high impact emerging translational research projects focused on urothelial cancer,preferably at other institutions; 2. To provide seed funding for these projects for 1-2 years; 3. To incorporate the most successful as full projects within the SPORE.

Tumors that express mutant protein kinases are usually dependent upon them for growth and survival. Activating mutations in FGFR3 occur in over half of low-grade non-muscle invasive bladder cancers (BCs) and in a quarter of muscle-invasive tumors, and small molecule and antibody-based FGFR3 inhibitors have exhibited potent growth-inhibitory activities in some BC cell lines and xenografts in preclinical studies. However, clinical translation of these observations has not occurred, in part because dose escalation trials have revealed that FGFR inhibitors produce some toxicity, and whether the extent of target inhibition at non-toxic doses is sufficient to produce apoptosis and/or growth arrest is not clear. We have assembled a collaborative group involving the GU Cancers team at Astra-Zeneca and Dr. Margaret Knowles (University of Leeds, UK) to conclusively determine the value of FGFR3 as a therapeutic target in BC. Our approach will be to use our unique panel of cell lines and xenografts to (1) isolate biomarkers that predict FGFR3 dependency better than FGFR3 mutational status alone and (2) develop pharmacodynamic approaches to determine the extent of tumor FGFR3 pathway inhibition and correlate it with biological response. We will also explore the effects of the novel tumor suppressive forerunner gene ARL11 on Ras pathway activation and define the relationships between ARL11 downregulatlon, FGFR3 and Ras mutational status, and Ras pathway activation in primary tumors, studies that are based on novel findings obtained in Project 1. We will then perform a neoadjuvant clinical trial to determine whether the doses of AZD4547 that can be safely achieved in patients produce sufficient target inhibition to cause apoptosis and/or growth arrest in primary tumors. This methodical approach will provide the strong mechanistic information required for the intelligent design of subsequent Phase II studies in low-grade and muscle-invasive BCs as well as in hematological and other tumors.

DESCRIPTION (provided by applicant): The overall goal of this University of Texas MD Anderson Cancer Center SPORE in Genitourinary Cancer is to facilitate innovative translational research in the prevention, detection, and treatment of this disease leading to the elimination of bladder cancer (BC) as a major health problem. We have invested in several major translational research themes which include: the development of non- or minimally-invasive markers for early detection of BC in high-risk individuals or for surveillance and the early detection of recurrence in those with the disease, the identification of inherited factors that contribute to increased or decreased risk of developing BC, the elucidation of the molecular events (genetic and epigenetic) that mediate the earliest stages of urothelial neoplasia, the determination of whether activating mutations and gene amplifications present in BCs drive cancer progression and serve as potential therapeutic targets, the identification of molecular and biological markers that can be used to distinguish favorable from unfavorable biology in non-muscle Invasive and more advanced disease that direct us to optimal therapy (personalized medicine), the development of novel therapeutic approaches for non-muscle Invasive BC that are alternatives to bacillus Calmette-Guerin (BCG) and/or are effective in BCG-refractory disease, and the Identification of novel agents for the treatment of metastatic BC. To achieve these goals, our SPORE has assembled clinicians and basic scientists including urologists, medical oncologists, pathologists, molecular epidemiologists, molecular and cell biologists, biostatisticians, and experts in development of new technologies and informatics. The SPORE includes 5 inter-related projects that deal with 1) early detection of BC, 2) risk assessment for BC 3) biology and therapeutic targeting of the fibroblast growth factor receptor -3, 4) therapeutic targeting of Ral GTPases, and 5.) the development of adenoviral mediated gene therapy for refractory tumors. These projects are supported by 3 Cores: (A) Administrative; (B). Biostatistics and Bioinformatics; and (C) Pathology & Data Management. All of the scientific projects are translational in nature; focus on human BC; involve clinical and basic investigators and biostatisticians; interact with the other projects; and utilize Core resources. Innovative Developmental and Career Development Projects have brought new investigators into and stimulated the SPORE that are represented in each of the major projects. Achievement of the aims and objectives of this proposal will result in a major decrease in the incidence, morbidity and mortality of BC.