DESCRIPTION (provided by applicant)*3
COLLABORATING INSTITUTION (S)*3
OVERALL CRITIQUE*3
PROGRAM AS AN INTEGRATED EFFORT*5
PRINCIPAL INVESTIGATOR*5
BUDGETARY OVERLAP*6
HUMAN SUBJECTS RESUME*6
VERTEBRATE ANIMAL RESUME*6
INDIVIDUAL PROJECTS AND CORES
Project 1:*KSHV Latency and Reactivation in DMVEC Spindle Cells
**(Gary Hayward, Ph.D.)*7
Project 2:*The Oncogenic Functions of KSHV/HHV-8 Encoded LANA-2
**(Paula Pitha-Rowe, Ph.D.)*15
Project 3:*Viral Chemokine Signaling in HHV-8 Infection
**(John Nicholas, Ph.D.)*21
Project 4:*LANA-1 Mediated Negative Regulation of Gene Expression
**(Diane Hayward, Ph.D.)*26
Core A:*Administrative Core
**(Gary Hayward, Ph.D.)*33
Core B:*KSHV Genetics and DNA
**(Prashant Desai, Ph.D.)*34
Core C:*Gene Delivery and Gene Expression Analysis
**(John Nicholas, Ph.D.)*36
COMMITTEE BUDGET RECOMMENDATIONS
REVIEW PANEL ROSTER
DESCRIPTION (provided by applicant): The AIDS epidemic was recognized in part because of a 20,000-fold increase in the occurrence of unusual malignancies such as Kaposi's Sarcoma (KS) in gay men. Despite success in controlling HIV levels in Western countries with HAART, as well as empiric advances in the treatment of AIDS malignancies, affected patients are rarely cured of their malignancies. Furthermore, because of the spread of HTV, Kaposi's Sarcoma in particular has now become the most prevalent tumor encountered in Southern Africa. The presence of Kaposi's Sarcoma-Associated Herpesvirus (KSHV or HHV8) in KS, Epstein-Barr virus (EBV) in a large subset of AIDS-associated lymphomas, and both viruses in primary effusion lymphomas (PELs) provide an important target for the investigation of the pathogenesis and ultimately the specific treatment of these neoplasms. The overall goal of this Program Project is to investigate how KSHV takes control of vascular endothelial cells and B-lymphocytes, with a mechanistic focus on viral and cellular genes expected to be elevant to tumorigenesis, prevention, and treatment of KS and PEL. Project 1 (Gary Hayward) will focus on control of latency, reactivation and spindle cell conversion in KSHV-infected dermal microvascular endothelial cells in culture, including the role of the tyrosine kinase signaling proteins VIP (Kl) and TMP (K15). Project 2 (Paula Pitha-Rowe) will focus on the mechanism of action of a KSHV latency protein vIRFS (or LANA2) that is homologous to cellular interferon response factors (IRF). Project 3 (John Nicholas) will focus on KSHV homologues of cellular beta-chemokines, which induce VEGF and include autocrine interactions with vGPCR that may play a role in supporting neoplastic proliferation. Project 4 (S. Diane Hayward and Richard Ambinder) will focus on repression of cellular gene expression by the key latent state LANAI protein involved in KSHV genome maintenance and in driving cell proliferation. Current models of KSHV pathogenesis envisage roles for the combined activities of both constitutively expressed KSHV latency gene products plus the occasional expression of abortive lytic cycle gene products These four Projects all address several different and overlapping aspects of the role of viral and cellular genes in maintenance of latency, the role of the early lytic cycle chemokines and the processes that controls switching between the two states. Collecting them together as a PPG centered around extensive utilization of the expertise and reagents generated by the skilled personnel in the two Laboratory Cores involving KSHV genetics and DNA and Viral vector driven gene expression and analysis, provides synergy, reagent sharing and overall beneficial interactions that would not otherwise occur.
COLLABORATING INSTITUTION (S): None.
OVERALL CRITIQUE: This is a new program from the applicant group at the Johns Hopkins University School of Medicine to investigate how KSHV takes control of vascular endothelial cells and B-lymphocytes, with a mechanistic focus on viral and cellular genes expected to be relevant to tumorigenesis, prevention, and treatment of KS and PEL. The four projects focus on the role of viral and cellular genes in maintenance of latency, the role of the early lytic cycle, the role of chemokines, and the processes that control switching between the latent and lytic states. Given the concerns about KS as prevalent form of cancer, particularly among immunocompromised individuals, understanding the mechanism of latency, lytic infection, and transformation is important. This program has broad implications in several fields such as tumorigenesis, and therapeutics.
The merit of the projects is uneven, with variable levels of innovation. In Project 1, even though a large amount of data may be generated, the merit is reduced due to inadequate consideration to the key experimental details that may not address issues directly reflecting the KS pathogenesis. Project 2 has a great potential despite a lack of clear hypothesis and some concerns related to biological relevance. Projects 3 and 4 are strong with minor deficiencies within the experimental design and approach. The interactions between projects were evident from the interactions of the investigators over a period of several years resulting in joint publications. It is clear demonstration of the synergistic flow of ideas with collaborative planning of experiments among the projects. The Principal investigator is internationally recognized molecular virologist whose work in the field of KSHV is excellent. He is eminently qualified to lead this program. Support for five years is appropriate for this integrated program.
In Project 1, "KSHV Latency and Reactivation in DMVEC Spindle Cells," (Gary S. Hayward, Ph.D.) the goal is to define which viral and cellular proteins are principally responsible for both establishing and maintaining the viral latent state and the spindle cell phenotype in KSHV-infected vascular endothelial cells. Strengths are the excellent expertise and experience of the Project Leader; the state-of-the-art technology that will be used for the proposed labor intensive studies; and the potential for the generation of important data with the studies of relevant genes in the endothelium cell system. However, the overall merit of the project is reduced due to weakness in the consideration of key experiments that will address issues directly reflecting the KS pathogenesis in the KSHV-infected DMVECs. There are insufficient experiments to confirm that down-regulated genes are low in vivo as compared to uninfected cells, and in fact some of the molecules that are down regulated in vitro are expressed in KS lesions (like CD31 and VWF). There is inadequate consideration given to the potential weaknesses of this model system. Even though the model provides a good tool to study viral infection, concerns with regards to its use in the study of pathogenesis remains. Additionally, there is a lack of focus in numerous experiments that are described in insufficient detail.
Furthermore, there were questions about the ability to correlate latency with spindle cell formation and cellular gene down-regulation, and to exclude a lytic component (for example paracrine effects of inhibitory cytokines) that acts in conjunction with latent gene expression. The Project Leader partially clarified this question during the phone review by stating that a better model is now available based on GFP expressing viruses, where latency and lytic replication may be better distinguished. This project received an average merit rating of 1.9.
In Project 2, "The Oncogenic Functions of KSHV/HHV-8 Encoded LANA-2," (Paula Pitha-Rowe, Ph.D.) the overall goal of this project is to identify the oncogenicity of KSHV and to determine how this virus is regulated by cellular immune systems. The proposed study therefore focuses on the pro-oncogenic properties of KSHV-encoded LANA-2, both in vitro and in vivo. Strengths of the project include the straightforward and focused approach, and the significance of studying LANA-2 due to its oncogenic potential. The expertise of the Project Leader and the environment are both outstanding. The weaknesses include the deficiency of a central hypothesis, the biological relevance of the luciferase reporter data, and the discrepancies between the reporter data and the microarray results. This project received an average merit rating of 1.9.
In Project 3, "Viral Chemokine Signaling in HHV-8 Infection," (John Nicholas, Ph.D.) the investigators plan to characterize the roles of the v-chemokines in virus replication to provide the basis for the development of anti-viral vCCL-2-based vGPCR antagonists. Specifically, the goal is to investigate details of vCCL-mediated anti-apoptosis mechanism and its role in KSHV replication to address several important unresolved issues concerning the replication and pathogenesis of this human cancer virus. This project is based on published results on PEL cells and new results showing that the vMIPs CCL-1 and CCL-2 can promote cell survival and VEGF secretion in PEL cells, and that vCCL-2 a reverse-agonist to vGPCR binds to different residues than Gro-a. Strengths include the proposed focused study; sound approaches that can lead to development of novel inhibitory molecules, and new insights on the role of viral chemokines in KSHV replication and pathogenesis; expertise and experience of the investigators; and the excellent environment. The weaknesses include the descriptive nature of the specific aims. Furthermore, focus on functional differences between the two v-chemokines that may determine important differences in their biological function is not adequately addressed. This project received an average merit rating of 1.5.
In Project 4 "LANA-1 Mediated Negative Regulation of Gene Expression" (Diane Hayward, Ph.D.), the investigator addresses an important clinical and scientific problem relevant to AIDS-associated malignancies; how does KSHV regulate cell gene expression during transformation and latency. The significance is that if the aims of this project are achieved, new information regarding the cellular pathways that are important in transformation in both endothelial and B lymphocyte cell types will be obtained. The studies have the potential to identify new mechanisms of transcriptional control, oncogenesis and targets for therapeutic intervention. The experimental approaches are, for the most part, well described, logical, well justified, and complementary. The project incorporates and integrates several novel ideas, such as the use of Rett's syndrome patient-derived cells and several ingenious methodologies to dissect LANA effects. The expertise of investigator and the environment at the Johns Hopkins University are excellent. However, some of the approaches planned are based on modest preliminary data. In addition, some of the proposed experiments may not yield clearly interpretable data or conclusions. This project received an average merit rating of 1.5.
Core A, "Administrative Core, (Gary Hayward, Ph.D.), is to provide secretarial, budget management, publication, and organizational support for the investigators, and laboratory cores. Arrangements regarding visiting speakers, the external Advisory Board and intramural meetings and retreats will be made through this core. The experience of the support personnel appear to be appropriate for the functions they will be asked to perform, as are the roles for the external visitors and advisors. It would have been helpful to have details on the structure of the evaluation by the external Advisory Committee and how their recommendations will be implemented. This core is rated satisfactory.
Core B, "KSHV Genetics and DNA," (Prashant Desai, Ph.D.) is to generate mutant derivatives of KSHV; to generate and culture dermal microvascular endothelial cells (DMVEC) and their immortalized derivatives; to generate and culture KSHV-infected DMVEC, and to maintain and distribute KSHV DNA isolates. The investigators of the core are developing the expertise in mutagenesis techniques and under the leadership of Dr. Desai will use alternative approaches to construct desired derivatives of KSHV. A second major effort will be to propagate and distribute uninfected and infected DMVEC. The core is utilized by Projects 1,3, and 4. However, specific details regarding Bac technology are inadequately addressed. Nevertheless, the core has adequate expertise and experience to provide the reagents to various projects. This core is rated satisfactory.
Core C, "Gene Delivery and Gene Expression Analysis," (John Nicholas, Ph.D.) is to provide services particularly vectors and engineered cells to all the projects of the program project. There are preliminary results in the application and the supplementary information indicating that many of the vectors and cells designed by the core are already working. The leadership includes outstanding scientists with proven experience in vector design, gene transfer and cell engineering. However, the core has investigators with limited experience in IHC and in situ. Issues of overlap with Core B were resolved during the telephone conference. The core is rated satisfactory.
PROGRAM AS AN INTEGRATED EFFORT: The Principal Investigator, Dr. Gary Hayward, leads this program with four projects. The four Project Leaders share interests in KSHV and have adjacent space on one floor of the new Oncology Center Research Building. They describe one administrative and two scientific cores that are headed by experts. One measure of the potential integration of this program is that Drs. Gary and Diane Hayward clearly have collaborated recently, having co-authored 10 papers since 2000. Dr. Gary Hayward has also previously collaborated with Drs. Nicholas and Pitha-Rowe. A second measure of the integration of this program is the commitment of Dr. Gary Hayward to refine the use of DMVEC as a model for infection with KSHV and to have members of the program use this model. He has acknowledged that this model is not a complete reflection of KS lesions in vivo but it is now as good a model as any available and his fostering its use in the program will both accelerate the understanding of its strengths and weaknesses and bring the program together in this effort. It was also clear during the phone conference that Dr. Gary Hayward, by answering several of the questions directed at other project leaders, not only is technically immersed in all of the projects but also contributes to their design. His interactions with the project leaders and core directors therefore constitute a third measure of the integration of the program.
RECOMMENDED RATING: Integrated.
PRINCIPAL INVESTIGATOR: Dr. Gary S. Hayward received his Ph.D. in Molecular Biology in 1972 from the University of Otago, in Dunedin, New Zealand. He completed his postdoctoral studies in Molecular Genetics and Virology at several institutions and in 1976accepted an Assistant Professorship in the Department of Pharmacology at the Johns Hopkins University School of Medicine (JHUSOM). He is presently a Professor in the Departments of Pharmacology, Molecular Sciences, and Pathology, as well as the Program Leader in Molecular Virology in the Oncology Center at JHUSOM. Dr. Hayward has received a number of honors, awards, and served on a number of committees. He is an internationally recognized leader in molecular virology and his work on KSHV is outstanding. Dr. Hayward is well qualified to lead this program project.
BUDGETARY OVERLAP: None
HUMAN SUBJECTS RESUME:
THE FOLLOWING RESUME SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW ADMINISTRATOR TO SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE ON THE FOLLOWING ISSUES:
PROTECTION OF HUMAN SUBJECTS (Resume):
Protection of human subjects against research risk: No concerns. Blood and biopsy specimens will be obtained from patients at the John Hopkins Hospital under a human investigation approved protocol under Dr. Richard Ambinder who is a co-investigator on Project 4 of the program. HS-30.
INCLUSION OF WOMEN PLAN (Resume): Acceptable. Both male and female patients' samples will be used for the study. G1A.
INCLUSION OF MINORITIES PLAN (Resume): Acceptable. Minorities are included in the study. M1A.
INCLUSION OF CHILDREN PLAN (Resume): Acceptable. Children are appropriately excluded from the study. C3A.
VERTEBRATE ANIMALS (Resume): No concerns. The applicants plan to generate mouse monoclonal antibodies against the same VIP and TMP synthetic polypeptides used successfully already in rabbits. This will be carried out for a service fee within an established Johns Hopkins Monoclonal Core, and which has local IUCAC approval under the leadership of Dr. James Hildreth. A-30.
INDIVIDUAL PROJECTS AND CORES**
PROJECT 1: **KSHV Latency and Reactivation in DMVEC Spindle Cells
(Gary Hayward, Ph.D., 20 percent effort)
DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) in the causative agent of angiogenic proliferative skin lesions in vascular endothelial cells (KS), as well as several rare B-cell lymphomas (PEL and MCD). Following the spread of HIV, the rates of KS increased 20,000 fold in homosexual males in the United States and KS has now become the most prevalent of all tumors in Southern Africa. We have established a biological assay system for both latent and lytic cycle infection by KSHV in primary adult dermal microvascular endothelial cells (DMVEC) in culture, which includes a proliferative aligned spindle cell conversion phenotype associated with viral latency and stable episome maintenance that closely resembles the key feature of nodular KS lesions. Previous gene array and real-time RT-PCR analyses of LANA1-positive KSHV-infected DMVEC spindle cell cultures compared to uninfected DMVEC cobblestone monolayers led to the identification and confirmation of 22 of the most highly up and down-regulated cellular genes. We have also identified and characterized two highly variable KSHV-encoded tyrosine kinase membrane signaling proteins known as VIP (or Kl) and IMP (or K15) that have distinct subtypes in different human populations and are likely to contribute to the control of latency and reactivation. Our goals in this project are to define which viral and cellular proteins are principally responsible for both establishing and maintaining the viral latent state and the spindle cell phenotype in KSHV-infected vascular endothelial cells. The three Specific Aims include: (I) More extensive analysis of altered cellular gene regulation at the mRNA and protein levels to define the contributions of true latent state effects compared to possible virion surface contact signaling and occasional spontaneous lytic cycle functions. Evaluation of specific selected up-or down-regulated cellular genes, exemplified by BMP4, a4-Integrin and cKIT to the latent state and spindle phenotype; (II) Using genetic knock-out and interference approaches to identify which specific sets of cellular genes and pathways are regulated by the major LANA1, vCYC or vFLIP latent state proteins; and (III) Define the biological roles and functions of VIP and TMP in the vascular endothelial cell background, both as isolated genes and by interference or inactivation including mutation of their IT AM and SH2 domains within the viral genome. Assays will include measurements of the efficiency of establishing latency, maintaining latency and of suppressing reactivating from latency. |