Anton Wellstein - US grants

Inhibition of Heparin-Binding Growth Factors. Sustained prostate cancer growth and metastasis requires paracrine signals between the tumor cells and the normal surrounding host tissue. One crucial function of these signals is to recruit endothelial cells and thus new blood vessels for the nourishment of the expanding tumor mass. This proliferation and migration of endothelial cells in the vicinity of progressing tumors con- trasts with the extremely low turn-over rate of endothelial cells in the healthy adult. Thus, a selective blockade of the tumor-induced endothelial cell proliferation should inhibit tumor growth and potentially metastasis with only few adverse effects. We found that the most effective endothelial cell growth factors released from prostate cancer cells in vitro are heparin-binding growth factors (HBGFs) and we have therefore focused our search for inhibitors on heparin-like polysulfates. We have demonstrated that HBGF action in vitro can be blocked by a structural analogue of heparin: pentosanpolysulfate (PPS). Furthermore, the growth of human prostate cancer cell lines into tumors in athymic nude mice can be inhibited by the treatment of the animals with PPS. PPS was effective against tumors derived from in vitro PPS-sensitive and from in vitro PPS-resistant tumor cell lines. This data suggests that PPS blocks the hosts' reaction to the HBGF(s) released from the tumor cells. We propose the following studies: 1. To study the efficacy of PPS in a Phase II trial with prostate cancer patients. In an ongoing Phase I trial with PPS in patients with advanced cancer, we will determine a dose schedule that is safe and maintains continuously high concentrations of biologically active drug in the patients. 2. To develop new synthetic heparinoids as HBGF-inhibitors in vitro and in vivo. In particular, to find analogues with improved therapeutic index. 3. To determine to what extent endothelial cell proliferation in normal, hypertrophic and cancerous prostate tissue can serve as an indicator of the disease state, prognosis of the patient and responsiveness to therapy. 4. To probe for expression of known HBGF genes in normal, hypertrophic and cancerous prostate tissue as potential markers of the progression and prognosis of the disease as well as therapeutic response to the HBGF-targeted therapy. An important aspect of our studies is to analyze the results according to the ethnic origin of the patients. This should enable us to detect differences in the biological and molecular markers and thus understand differences in the prognosis and therapeutic responsiveness of prostate cancer.

Mammary Carcinogenesis: The Role of Pleiotrophin. Breast cancer growth and metastasis requires growth factor signals between the tumor cells and the normal surrounding host tissue. We propose that the secreted polypeptide growth factor pleiotrophin (PTN) plays a major role in mammary carcinogenesis as well as in the growth and metastasis of breast cancer. This hypothesis is based on the biological effects of PTN expression in selected tumors models, the high levels of PTN mRNA in 60% of tumor samples from breast cancer patients and on its upregulation during carcinogen-induced rodent mammary cancer. Furthermore, PTN is highly expressed in immature mouse mammary glands that are sensitive to carcinogens and is downregulated permanently in mice that have undergone pregnancy and are protected against carcinogenesis. Finally, the activity of PTN on endothelial cells in vitro suggests that PTN can serve as an angiogenesis factor during tumor progression. In support of this role of PTN as an angiogenesis factor, reduction of constitutively expressed endogenous rib mRNA in human tumor cell lines by PTN-targeted ribozymes reduced tumor growth and metastasis even when in vitro growth of the tumor cells remained unaffected. Based on these preliminary studies, we wish to elucidate the role of PTN in breast cancer progression and in mammary carcinogenesis with the ultimate goal to develop better prognostic markers as well as novel therapeutic strategies. Under aim 1 we will study whether serum levels of PTN in breast cancer patients can predict long term outcome of their disease an-or the response to therapy. We will use samples from patients with over 10 years of follow-up from our serum bank core facility for this analysis. Under aim 2 we will study to what extent hormonal regulation affects PTN expression in mammary gland maturation and during mammary carcinogenesis in rodent models. We will study the influence of steroid hormones and retinoid acid receptor ligands on PTN expression and on mammary gland biology or carcinogenesis in animals and in organ culture. Under aim 3 we will study to what extent sustained high levels of expression of rib will affect mammary gland maturation, hormonal responsiveness and susceptibility to carcinogens at different stages of mammary gland maturation. To achieve this, we will express PTN as a transgene in mice under a tetracycline-regulated promoter. We will study the effects of a challenge of the transgenic animals (and of organ explants) with hormonal stimuli as well as with carcinogens. Under aim 4 we will study to what extent reduction of endogenous PTN expression will affect mammary gland physiology and pathology. We will use ribozyme-targeting of rib to achieve this goal. We will generate mice expressing PTN-targeted ribozymes under the control of a tetracycline-regulated promoter and study spontaneous gland maturation during pregnancy as well as the effects of hormonal and carcinogen challenges with the endogenous PTN suppressed by the ribozyme transgene. Under aim 5 we will develop gene transfer vectors to target rib expression with ribozymes in vivo. We will use adenovirus- and herpes virus-based vectors for gene transfer.

Acidic or basic fibroblast growth factors (FGF) are present at significant concentrations in most normal tissues in the adult, can stimulate angiogenesis and thus have the potential to play an important role during tumor growth and metastasis. However, both of these FGFs are immobilized in an inactive state on the extracellular matrix and it is only poorly understood how they are solubilized and activated to reach their extracellular receptors. One mechanism through which these growth factors can be mobilized is by binding to a secreted binding protein for FGF (BP) that was described by Wu et al in 1991. In recent work from our laboratory, we detected high levels of BP mRNA in majority of squamous cell cancer (SCC) samples from patients and in SCC cell lines in culture. On the other hand, we did not detect BP mRNA in normal adult human tissues or in normal adult rodent tissues as well as in a series of cultured cell lines that were not of squamous origin. In contrast with the lack of expression of BP in adult tissues, we found BP mRNA highly expressed in murine embryonic squamous epithelia of the lungs and skin during late gestation. In functional studies with non-tumorigenic cells (SW-13), we demonstrated that expression of BP can mobilize and activate bFGF leading to tumor growth and angiogenesis of the BP- transfected cells. Furthermore, in an SCC cell line expressing high levels of BP mRNA, reduction of BP expression using BP-targeted ribozymes reduced tumor growth and angiogenesis of xenografts of these cells in athymic nude mice. This suggests a potentially rate- limiting role of this protein for SCC tumor growth in vivo. In addition, we found that retinoids downregulate BP mRNA rapidly through a posttranscriptional mechanism. We propose the following experiments to study the role of BP in tumor growth as well as the mechanism(s) and therapeutic significance of its downregulation by retinoids: Under AIM 1, we will express BP in cell lines which have a low tumorigenic potential and are negative for BP and study phenotypic changes. The expression of BP will be under the control of a tetracycline- regulated promoter that allows to regulate transfected BP in vitro or in vivo by administration of tetracycline. Under AIM 2, we will use molecular targeting of BP mRNA with ribozymes to elucidate the contribution of BP to tumor growth of cell lines that express BP. Under AIM 3, we will study the mechanism(s) of retinoid regulation of BP with respect to receptor subtype and the retinoid- dependent target site in the BP mRNA. Furthermore, we will study to what extent downregulation of BP by retinoids contributes to the effect of these drugs on SCC tumors.

[unreadable] DESCRIPTION (provided by applicant): The secreted growth factor pleiotrophin (PTN) acts on endothelial as well as epithelial cells and fibroblasts and it can drive tumor growth, angiogenesis, invasion as well as metastasis. Recently, we identified a receptor for PTN, the orphan tyrosine kinase anaplastic lymphoma kinase, ALK that shares homology of its kinase domain with the insulin receptor family. ALK and PTN are overexpressed in a majority of human cancers samples whilst a series of normal tissues showed no detectable receptor expression. Ribozyme-mediated reduction of ALK in glioblastoma and other tumor cells resulted in a "gene dose"-dependent reduction of xenograft tumor growth in mice and supports ALK as a valid target for drug discovery. Recently we identified small-molecule drugs that inhibit the PTN-stimulated ALK kinase in cultured cells at nanomolar concentrations and we used homology modeling to generate a 3D model of the protein with these drug candidates. We hypothesize that it will be possible to identify novel selective inhibitors and propose the following aims: Aim 1, Lead identification: We will identify in silico novel ALK kinase inhibitors using de novo drug design, library screening and virtual combinatorial library generation. The best candidates will be synthesized for the biologic assays. Aim 2, to study the inhibition of PTN-stimulated ALK kinase activity in comparison to other growth factor-induced receptor kinase activity by these candidate inhibitors. Additional molecular modeling using these data will then be used to design and optimize inhibitors. Aim 3, Lead optimization: To improve lead compounds emanating from the biologic assays by further refinement of the molecular modeling of drug / protein interaction. These new inhibitors will then be assayed against PTN-stimulated ALK kinase activity in intact cells. Aim 4: To study the efficacy and potency of the best inhibitors on cell growth. Selected inhibitors of the PTN-stimulated ALK kinase will be tested for their ability to block PTN-induced cell proliferation, anti-apoptosis and soft agar colony formation in comparison to their blockade of other growth factors. In summary, we propose the design and study of structure-based inhibitors of the PTN/ALK signaling as a close collaboration between a biology and a molecular modeling/chemistry laboratory with the purpose of discovering novel anticancer drugs. [unreadable] [unreadable]

pancreas neoplasms; health science research;

DESCRIPTION (provided by applicant): The Division of Clinical Pharmacology Postdoctoral Research Training Program at Georgetown University was established in July, 1988. It received support as an NIGMS sponsored training program in 1990. During the last 15 years the goals and objectives of the training program have remained unchanged, that is to provide a clinical and basic research experience in order to prepare the trainee to become an independent investigator. During the time period growth in the Training Program has been exceptional, due in part to the strong institutional commitment to Clinical Pharmacology and in part to continuing recruitment to research oriented faculty who participate with enthusiasm as faculty for the Research Training Program. The applicant pool for Clinical Pharmacology training at Georgetown is now proven and national in scope. Presently the Program consists of 7 Core and 21 Participating Faculty and 6 postdoctoral fellows in training. Candidates for the program are physicians who have completed training in a clinical specialty. Highly qualified Ph.D. (or equivalent degree) applicants with clearly defined career goals in Clinical Pharmacology will also be considered for training. Training is for 2-3 years, with fellows strongly encouraged to complete 3 years unless they have substantial prior research experience. The research training is carried out under the supervision of a mentor from the training faculty, monitored on an ongoing basis by the Program Director. Research training is complemented by formal instruction in clinical pharmacology, clinical investigation, bioethics, pharmacometrics, other graduate courses as appropriate, training in the responsible conduct of research, as well as weekly research conferences, and clinical consulting rounds. A broad range of research opportunities are available to trainees including cardiovascular, molecular, immuno, neuropharmacology, population pharmacokinetics, and drug metabolism programs in the laboratories of the faculty. Graduates of the program accept positions in academics, the Food and Drug Administration, and industry.

The goal of this Specialized Program of Research Excellence (SPORE) in pancreas cancer is to translate outstanding hypothesis-driven basic research into clinical treatment that saves lives. Pancreatic cancer is a devastating cancer that kills patients not just because it may be extremely aggressive but also because it is diagnosed late. This SPORE is a unique collaboration between Georgetown University, Geisinger Health System and INDIVUMED a German Research Institute with a German hospital consortium that brings together an outstanding academic institution, a large regional healthcare system and a German basic and clinical research system to address the biology of pancreatic cancer and clinical care. Project 1 focuses on inhibition of pleiotrophin, a novel heparin-binding growth factor that promotes invasion and metastasis in pancreatic cancer. Project 2 focuses on the role of neuregulins and pleiotrophin in an established model of neuron:tumor cell interaction with the goal of developing methods to block neural invasion by malignant pancreas cells that may ultimately aid in disease palliation. Project 3 focuses on recent findings that inhibition of the nuclear receptor coactivator AIB1 within pancreas cancer cells and adjacent endothelium may block signaling from multiple growth factors to enhance apoptosis. The activation of the ras/MAP kinase pathway that occurs in up to 90% of pancreas cancer is the focus for novel approaches to radiation sensitization in Proiect 4. Investigators at Georgetown have developed a rafl antisense inhibitor that is already in Phase I trial and have developed other molecular targets in the pathway that are complementary. In addition, this SPORE is developing the infrastructure and resources to acquire tissues and bodily fluids from patients (Pancreatic Tumor Bank and Serum Detection & Molecular Identification Cores) with an expected 120 curative resections a year. The SPORE will utilize a biostatistical core to insure that all projects have appropriate designs, methods of analysis, and an integrated database for reporting patient outcomes. Further, the SPORE will support developmental projects designed to generate new knowledge and approaches to early detection and promote the development of career tracks dedicated to translational research in pancreatic cancer. This SPORE is also establishing interactions with SPOREs in other institutions as well as with other NCI- and NIH-funded programs to promote the aggressive development of novel therapies for the control of pancreas cancer

Fibroblast growth factors (FGF-1 or -2) are present at significant concentrations in most normal tissues in[unreadable] the adult. However, these FGFs are immobilized in an inactive state on the extracellular matrix and it is only[unreadable] poorly understood how they are solubilized and activated to reach their extracellular receptors. One[unreadable] mechanism through which FGFs can be mobilized is by binding to secreted binding proteins (BPs) and we[unreadable] showed that BP1 can enhance the activity of locally stored, immobilized FGFs. BP1 expression is controlled[unreadable] by stress pathways in cultured cells and found upregulated after wounding, toxic or infectious injury of the[unreadable] skin or kidneys. BP1 expression in mice carrying an inducible BP1 transgene caused a significant rise in[unreadable] mean arterial blood pressure (MAP) by +30 mm Hg within two days of transgene induction and analysis of[unreadable] vascular contractility showed a sensitization to angiotensin II. The rise of MAP after BP1 transgene[unreadable] expression was inhibited by systemic administration of the superoxide dismutase mimetic Tempol[unreadable] suggesting an essential role of oxidative stress. We hypothesize that BP1/FGF signaling modulates the[unreadable] sensitivity of blood vessels towards contractile signaling and propose to study this under the following aims:[unreadable] Aim 1. To evaluate the contribution of FGF-2 or other FGFs to the BP1-induced hypertensive effect. We will[unreadable] study blood pressure, vessel contractility and renal tubular function in FGF-2(-/-) mice that are crossed with[unreadable] mice carrying an inducible BP1 transgene. Systemic administration of BP1 and FGF-2 will complement this.[unreadable] Aim 2. To study the contribution of kidney expression of BP1 to blood pressure regulation, vessel[unreadable] contractility and renal tubular function we will use mice harboring a HoxB7-controlled, tetracycline inducible[unreadable] BP1 transgene. To evaluate the role of endogenous BP1 to oxidative stress-regulated blood pressure, we[unreadable] will generate mice that are null for BP1 expression.[unreadable] Aim 3. To study the intracellular cross-talk between BP1 / FGF signaling and G-protein coupled receptor[unreadable] pathways we will monitor signal transduction and phenotypic effects in preglomular smooth muscle cells[unreadable] from experimental animals. Biochemical signaling via known integrators of the pathways (i.e. MAPKs) and[unreadable] proliferation/cell survival and superoxide generation will be used as read-outs. Mass spectrometry to identify[unreadable] new signaling proteins in the cross-talk will complement this.[unreadable]

The project is administered by the PI, the Program Administrator, Ms. Tolise Miles with the assistance of the Program Coordinator, Ms. Wing Kam Emily Chan. They provide the routine and strategic management for the administration of the Core. They oversee the finances of the project. The Core provides the interface between the scientific investigators and the purchasing, regulatory, and administrative requirements. The Core allocates the financial resources to individual subprojects and cores. It provides a monthly financial review for project and core leaders. It maintains a log of purchase order requests and receipt of supplies and equipment and for the tracking of expenditure, changes in personnel, and service contracts for the project leaders. It provides expert editorial assistance for preparation of manuscripts and word processing. It coordinates the convening of the internal and external scientific review groups. It handles the travel arrangements for the external review committee members and remuneration for their expenses. It coordinates the weekly research seminar, the monthly meeting of subproject and core leaders and faculty where data are presented and discussed and administrative plans are made and the visits of invited professors. The Core coordinates a biannual meeting on New insights into Cardiovascular Kidney Disease. This is a 3 day, small group program for opinion leaders in the US and overseas where selected topics from the program project are discussed in depth. The subproject and core leaders present their results and the external review group is invited to participate. This provides the reviewers with an opportunity to become deeply acquainted with the progress of the program and to provide their own comments on detail and substance. The meeting is financed by Division Funds. The Core prepares the reports and summaries which are required by the NIH for administrative review. The Core will support Xin (James) Li, Ph.D. as a biostatistician. He is skilled and experienced in the analysis of gene array data. He will provide statistical guidance and data interpretation to all investigators.

Core B will manage the supply of animals to all projects in the program, by initiating and maintaining breeding colonies for multiple strains of genetically engineered mice. In addition, the Core will evaluate cardiovascular and renal function for cohorts of all mice proposed in the program. Consious blood pressure will be measured by radiotelemetryJn mice with reduced renal mass (RRM) to mimic chronic kidney disease and in separate hypertensive mice infused with angiotensin II (Ang II). Renal function and more specifically, components of renal autoregulation will me measured in anesthetized mice. Renal autoregulaiton will be assessed by two methods that will assess both the myogenic response (MR) and tubuloglomerular feedback (TGF) response that constitutes renal autoregulation. The roles of several genes in blood pressure control and the regulation of renal function in these models will be tested in mice with selected gene deletions, gene overexpression or tissue specific deletions. Also, the roles of genes specifically in the kidney will be tested in mice with gene knockdown in the kidney by the delivery of small interfering RNA (siRNA) constructs. Aim 1 will breed and manage colonies to produce knockout mice for EC-SOD, IC-SOD, p47phox,CD38 eNOS, dopamine-2 receptor (D2-R), paraoxonase-2 (PON-2), and fibroblast growth factor-binding protein-1 (FGF-BP1) for use in 3 projects. In addition, two tissue-specific transgenic strains, p22phox and catalase in vascular smooth muscle cells (VSMCp22phox tg; VSMCcat tg) will be bred from stock . Aim 2 will develop mouse models of CKD and hypertension in each of the mouse strains. CKD will be mimicked by surgical reduction of 5/6ths of the kidney at 8-10 weeks of age. Hypertension will be achieved by placement of osmotic minipumps that infuse Ang II at a low pressor dose for 4 weeks. Aim 3 will measure conscious blood pressure, renal function and renal autoregulation in cohorts of each group of mice. Conscious MAP, heart rate and renal blood flow and GFR, RBF, 0 0 2 under anesthesia will be measured in these mice for the following projects. RELEVANCE (See instructions): The ability of the kidney to adjust to long-term disease and to hypertension predicts the survival of these patients. How the kidney makes those adjustments will help these investigators identify pathways that can be targeted in the treatment of kidney disease and injury. Understanding renal autoregulation is an important component to how the kidney adjusts and protects the patient.

The Bioassay Core (Core C) will provide services to all three projects in this program project grant (PPG) and will be directed by Dr. Kathryn Sandberg. The overall goal of this core is to provide analytical support for each project of the PPG by aiding in gene silencing and gene expression through si_RNA and lentiviral strategies and to ensure quality control of employed strategies by genotyping knockout and transgenic (tg) animals and by measuring mRNA and protein expression of targeted genes of interest. Genotyping will be performed on single-gene deleted mice and at least two strains of transgenic mice bred in Core B including: gene deletions of extracellular superoxide dismutase (SOD-3); NADPH oxidase-4 (NOX-4); p47'^¿'', a critical subunit of NADPH oxidase-2 (p47''¿''); ADP ribosyl cyclase 38 (CD38); fibroblast growth factor binding protein-1 (FGF-BP1) and -3 (FGF-BP3); dopamine-2 receptor (D2R); paraoxonase-2 (PON-2); overexpr-ession in vascular smooth muscle cells of p22''^¿'*, a membrane subunit of NOX (VSMCP^^'^'' tg) and catalase (VSMC^^' tg). This core will also determine levels of oxidative stress by measuring nitrates and nitrites, superoxide and the redox status of thiols by enzyme assays and capillary electrophoresis. Furthermore, this core is poised to develop new methods for gene manipulation for each project as gene manipulation technologies advance in the field. Aim 1 will construct and develop gene silencing techniques including siRNA and lentiviral approaches to study the role of genes of interest both in vivo and in cell culture. Aim 2 will assess gene expression at the DNA, mRNA and/or protein levels to ensure quality control of animal breeding performed in Core B and the efficacy of gene manipulation strategies in each of the three projects including via gene knockout, siRNA and shRNA-lentiviral approaches. Aim 3 will determine the degree of oxidative stress by measuring nitrates, nitrites, reactive oxygen species (ROS) and the redox status of thiols in each of the three projects. RELEVANCE (See instructions): Hypertension is a global health concern. Fifty million Americans have hypertension that requires treatment and over 1 billion people woridwide have hypertension. In fact, suboptimal blood pressure is the number one risk factor for death throughout the worid. Though much research has been conducted on hypertension, the mechanisms underiying the main cause of hypertension, i.e., essential hypertension, remain unknown. This Core serves a crucial resource for this program project that is devoted to understanding the role of oxidative stress in hypertension.

DESCRIPTION (provided by applicant): Our recent discovery of conditional reprogramming (CR) to generate cell cultures from human tissues offers new and exciting opportunities for biospecimen repositories. This cell technology makes it possible to rapidly generate cell cultures from surgical specimens and small biopsies, thereby providing an unlimited amount of patient material for genetic and proteomic analysis. However, this technology goes further: it will allow the functional analysis of tumor cells and comparison with the patients' normal cells from the same tissue. In this proposal we will extend and validate aspects of the CR technology and optimize its usage for biobanking. It is important to verify that the genotype and functional responses of CR cells mimics that of the primary tumor and experiments will address these issues using exome sequencing and TruSeq analysis. We will also examine whether the CR cells can predict patient responses to therapies, as we have recently described for a single case in the New England Journal of Medicine. Indeed, we believe that this technology will alter how Pathology departments and tissue repositories freeze patient specimens. Rather than simply quick freezing samples for future molecular analysis, tumor samples will be frozen in cryopreservative, which will additionally permit the generation of cell cultures for diagnostic and therapeutic evaluation.

The D2 dopamine receptor (D2R) is important in the pathogenesis of human essential hypertension. D2R, rs6276 is associated with salt-sensitive hypertension in Caucasians. Depending on the genetic background, deletion of Drd2 (D2-/-) in mice causes salt-sensitive hypertension. In addition, as with the D5-/-mice, D2-/- mice are in a state of oxidative stress caused by increased expression and activity of NADPH oxidase and decreased expression and activity of HO-2 rather than HO-1, as is the case in D5-/- mice. Although the D2R and DSR can co-regulate certain enzymes (e.g., phospholipase C), there is no evidence for such a direct interaction in renal tubules. We hypothesize that NADPH oxidase isoforms are targets of both DSR and D2R but are regulated differentially by these two dopamine receptor genes. One such D2R target may be paraoxonase (PON). The PON family comprises three members: P0N1, P0N2, and PONS. P0N1 and PONS are circulating in serum associated with the high-density lipoprotein fraction. In contrast, P0N2 is cell- associated and protects against cellular oxidative stress. Renal P0N2 expression is decreased in D2-/- mice. Silencing P0N2 in RPT cells increases the production of ROS that is not affected by D2R/D3R agonist, indicating a response downstream to D2R. Other genes involved in the negative regulation of oxidative stress and regulated by D2R include sestrin-2 and DJ1. Sestrins accumulate in cells in response to oxidative stress and DJ1 may function as an atypical peroxiredoxin-like peroxidase. The deletion or silencing of Drd2 in mice decreases the renal expression of DJ1 and silencing DJ1 in RPT cells increases ROS production, reminiscent of P0N2. We will test the overall hypothesis that the inhibition of ROS production by D2R is caused by a positive regulation of P0N2, interacting with sestrin-2 and DJ1. Specific aim 1 will test the hypothesis that P0N2 mediates the inhibitory effect of D2R on the production of ROS. Specific Aim 2 will test the hypothesis that D2R directly regulates P0N2 and other interacting proteins that negatively regulate ROS production, e.g., sestrin-2 and DJ1. Abnormal D2R expression or function may result in salt-dependent hypertension in humans, caused by abnormal regulation of PON, sestrin 2, and DJ1 function. RELEVANCE (See instructions): Recent large association studies identified genes that cause 12% of blood pressure variability. A D2R gene variant, rs6276, is associated with salt-sensitive hypertension in a Caucasian population and DJ1 locus is linked to hypertension. Abnormal D2R expression or function may result in salt-dependent hypertension iri humans, caused by abnormal regulation of PON, sestrin 2, and DJ1 function.

Fibroblast growth factors (FGF) function during development and adult tissue homeostasis. Activity of these FGFs is modulated by three known secreted binding proteins (FGFBP or BP). Genome-wide association studies (GWAS) and familial hypertension studies have shown significant associations between the risk of hypertension and single nucleotide polymorphisms (SNPs) in different genes in the FGF pathway including BP1, FGF1 and FGFS. BP1 and FGF1 expression were also found elevated in kidney epithella and macrophages in subjects with hypertension. Whilst most of the known FGFs act locally, FGF19, 21 and 23 function as circulating factors in a hormone-like fashion and utilize the extracellular glucuronidase klotho as a co-receptor. Loss of klotho has been associated with defective FGF signaling and oxidative stress. We observed that conditional expression of the BP1 in mice caused a significant rise in blood pressure and a sensitization of resistance vessels to angiotensin II. Reversal by the superoxide dismutase mimetic Tempol suggests an essential role of oxidative stress. We also observed that BPS can function as a co- receptor for the circulating FGF19, 21 and 23 and impact their signaling. Thus, we hypothesize that BP1 and BPS interactions with FGF signaling modulates oxidative stress, blood pressure control and the sensitivity of glomerular afferent arterioles. To evaluate the contribution of endogenous BP1 expression, we have generated mice with a floxed BP1 gene for conditional deletion. Under Aim 1 we plan to study the role endogenous BP1 expression during normal development and its contribution to the initiation and maintenance of disease caused by oxidative stress. BP1 will be conditionally deleted in the germline (Aim la), before induction of oxidative stress (Aim lb) and after chronic induction of oxidative stress (Aim 1c). Under Aim 2 we will evaluate the contribution of BP1 expression to oxidative stress signaling by tissue specific deletion from kidney epithella (Aim 2a) and macrophages (Aim 2b). Under Aim S we will evaluate the effect of BPS as a co-receptor for endocrine FGFs (FGF19, 21 and 23) and the impact of BPS on oxidative stress signaling in vivo and in vitro. RELEVANCE (See instructions): Alterations in, the FGF pathway appear to be significant drivers of chronic kidney disease as well as hypertension. FGF pathway activity is complex due to the high number of FGF ligands, receptors, binding proteins and co-receptors. Understanding the role of different contributors in the FGF pathway can reveal novel therapeutic targets in the treatment of chronic kidney disease as well as hypertension.

The tone of the renal afferent arteriole (Aff) increases with perfusion pressure (PP) to protect the kidneys from damage (barotrauma). The normal myogenic response (MR) of the Aff was accompanied by generation of superoxide anion (02')- MRs and ROS signals were reduced by deletion of p47''*'¿'' or addition of pegalated (PEG) superoxide dismutase (SOD) but were unaffected by PEG-catalase or knockout of nitric oxide synthase (NOS-3) relating normal Aff MRs to stretch-induced O2 from NADPH oxidase rather than to H2O2. However, incubation of Affs with H2O2 (from 1 to 30 IJM) caused dose-dependent blunting of MRs. Thus ROS in Affs can have opposite MR effects. Normotensive high salt (HS) mice with surgical reduction in renal mass (RRM, 5/6 nephrectomy) will model chronic kidney disease (CKD) and angiotensin II slow pressor infusion (Ang) will model hypertension. The MRs from these two were severely impaired, despite increased ROS in the kidneys and the Affs. However, the culprit ROS was apparently H2O2 since defective MRs were corrected by PEG-catalase but were unaffected by PEG-SOD, and were preserved in RRM mice drinking tempol for 3 months which normalized the increased excretion of H2O2 and Aff ROS.This proposal will investigate the hypothesis that ROS have opposite effects on MRs: short-term 02'enhances normal MRs by increasing VSMC Ca^^ entry mechanisms and Ca^^ sensitivity while prolonged H2O2 in models of oxidative stress impair MRs by downregulation of voltage operated calcium channels, PKC^ and transient receptor potential canonical 6 channels, whose mRNAs were downregulated in a ROS-dependent manner in Affs from RPM mice. It will study short- vs. long- term effects of 02vs. H2O2 on normal MRs (Aim 1) and blunted MRs in mice with RRM or Ang infusion (Aim 2). Gene analysis of individual Affs by gene array, confirmed by RNAseq and RT-PCR will select ROS-dependent candidate pathways. MRs will be recorded directly from perfused Affs during step increases in PD. Fluorescence methods will quantitate ROS subtypes, intracellular Ca^^ and membrane potential. Use of knockout or VSMC transgenic models combined with transfection or silencing of candidate genes will test directly their roles in PP-induced MRs, ROS generation and Ca^^ entry and signaling. This project is focused on the roles of ROS in regulating the MR. Results should provide novel treatment targeted for patients with hypertension or CKD. It is integrated with the animal core where whole kidney MRs will be studied and the biomarkers core where genes and protein responses will be assessed. RELEVANCE (See Instructions): An increase in the tone of the afferent arteriole of the kidney resets the blood pressure that the kidney regulates and thereby can lead to hypertension, whereas a breakdown in tone allows transmission of hypertensive pressure into the kidney and worsens chronic kidney disease. This project will study how oxidative stress regulates afferent arteriolar tone in animal models of hypertension or chronic kidney diseases which are major causes of cardiovascular disease.

Tumor cells escape recognition by the immune system by multiple mechanisms that include activation of inhibitory immune checkpoint pathways. Therapeutic inhibition of these immune checkpoints has demonstrated striking efficacy in a number of cancers and is a promising new approach to cancer treatment in combination with other anti-cancer regimens. Antibodies against the PD-1 pathway have in particular revolutionized the treatment of patients with advanced melanoma and produce tumor responses in >40% of patients. Combinations of anti-PD-1 with anti-CTLA-4 produces tumor responses in approximately 60% of patients. However, immune checkpoint blockade frequently causes inflammatory and immune-related Adverse Events (irAEs) due to the disruption of self-tolerance protection of normal tissues. These irAEs can be severe, lead to discontinuation of immune checkpoint inhibitor therapy and can require immunosuppressive treatment. Any tissue can be injured with the most frequent occurrences in the skin, gastro-intestinal tract, endocrine glands, liver, and lungs. Combined anti-PD-1 and CTLA-4 has significantly higher toxicity than monotherapy and requires more frequent and aggressive management. Although treatment with steroids and other immune modulators can reverse these irAEs, immunosuppression may compromise the anti-tumor activity of the checkpoint blockade. Thus, there is an unmet need for availability of clinically validated, non-invasive biomarkers for real time monitoring and prediction of on- or post-treatment irAEs and therapeutic efficacy to allow for proper management of cancer patients exposed to immune checkpoint inhibitors. Here we propose to monitor anti-tumor efficacy (Aim 1) as well as organ-specific irAEs (Aim 2) using cell-free DNA analysis from serial blood samples obtained before and at regular intervals during and after treatment. Under Aim 1 we propose to monitor changes in circulating cell-free mutant tumor DNA (ctDNA) patterns as a readout of anti- tumor treatment efficacy. Under Aim 2 we propose to assess autoimmune organ damage by monitoring changes in the abundance of circulating cell-free, tissue-specific methylated DNA (cmeDNA). We are currently leading a national cooperative group trial (EA6134) that involves combination anti-CTLA-4 and anti-PD-1 treatment of patients with BRAF mutant melanoma. Serially collected blood samples from patients on this trial will be analyzed as they respond to treatment, develop irAEs, require immunosuppressive therapy or discontinuation of treatment. We will compare the ctDNA and cmeDNA biomarker readouts with clinical observations of efficacy and adverse events in the trial to establish their utility.