Zebrafish as a Model for Identifying Cancer Genes, Year 3
Hemangioblastic Activity of Infant Leukemia.
Zebrafish as a Model for Identifying Cancer Genes.
A Non-Invasive Immune Therapy for Pediatric Cancers Resistant to Conventional Treatment.
Analysis of E2A-NMP4 and P120-E2A In Childhood Acute Lymphoblastic Leukemia.
Novel Immune-Type Genes: Role in Recognition of Cancer Cells.
Production of Viral Oncolysate Vaccine and Active Tumor-Specific Immunotherapy of Neuroblastoma.
Additional Funding Awarded by the Pediatric Cancer Foundation
Title of Project: The Kinomes of Pediatric ALL
Principal Investigative Researchers: William G. Kerr, Ph.D.
Research Facility: H. Lee Moffitt Cancer Center and Research Institute
Grant Information: Awarded $150,000
Grant Cycle: 2006
Gene profiling technology has enabled analysis of the transcriptome and proteome of tumor cells. In fact, massively parallel analysis of the Acute Lymphoblasic Leukemia (ALL) transcriptome has revealed a gene profile signature for this leukemia. This information has provided useful insights into molecular mechanisms that promote enhanced survival and proliferation in ALL. However, en equally, if not more important goal, is to define those proteins that participate in signaling pathways active in ALL and non-malignant cells present in the bone marrow that may have a trophic relationship with ALL cells. Enzymes that phosphorylate tyrosine, serine and threonine residues on other proteins play a major role in signaling cascades that control cell cycle entry, survival, angiogenesis and the immune response. Defining how these signaling pathways are altered in ALL and stromal cells present in these tumors will provide critical information for understanding how ALL survives, proliferates and interacts with other cells in the bone marrow microenvironment. We are applying to purified tumor cells a novel array strategy that allows the simultaneous detection of phosphorylation for 1176 different kinase substrates. Her we propose to apply this emerging technology to the analysis of phosphorylation-based cell signaling pathways in pediatric ALL. This proposal will be pursued in two aims. In Aim 1 we will use PepChip technology to identify kinome alteration sin primary pediatric ALL cells as compared to the normal counterparts of these cells. In Aim 2 we will compare the kinome of stroma in pediatric ALL to that of stroma in normal BM to identify signaling pathway alterations specific to tumor stroma in ALL.
Post-Doctoral
Fellowship: Andrew Phillips, PhD
Title of Project: Fission yeast as a model organism to investigate the
crucial interplay between apoptosis and mitosis.
Research Facility: University of Miami, School of Medicine
Programmed cell death plays a key role in maintaining the dynamic equilibrium of cells in fully grown organisms. Cells are born and die every minute, and this balance helps to maintain the overall shape and size of a tissue. In adult humans, typically 50-70 billion cells are eradicated via this cell suicide mechanism on a daily basis, thus making room for the equivalent number of cells produced daily through cell division. Homeostatic cell death is an important phenomenon and is often termed apoptosis. A disturbance in the subtle balance between mitosis and apoptosis can lead to the formation of a tumor as a result of cell proliferation exceeding cell death.
Although it is difficult to investigate such intricacy between the balances of cell growth and cell death in human cells, this interplay can easily be studied in the genetically amenable fission yeast, Schizosaccharomyces pombe. An apoptotic-like cell death event has recently been reported in S. pombe and a range of other yeasts including Saccharomyces cerevisiae, Aspergillus nidulans and Candida albicans. Dead or dying yeast cells display several key markers often associated with apoptotic mammalian cells, including chromatin condensation, exposure of phosphatidylserine, DNA fragmentation and delayed loss of plasma membrane integrity. It is not apparent that yeast cells contain the base cellular machinery to initiate and complete an active form of cell death, and the discovery in yeast of several functionally related components to the mammalian apoptotic, cascade, strengthens the notion that yeast undergoes programmed cell death. Furthermore, as with human cells, fission yeast cells have four distinct cell cycle phases and divide by medial fission. Moreover, most, if not all of the proteins involved in DNA replication and checkpoint controls in S. pombe are conserved in human cells. Taken together, this data suggests that S. pombe is a suitable model for studying the important interplay between cell proliferation and cell death.
Title
of Project: Zebrafish as a Model for Identifying Cancer Genes, Year 3
Principal Investigative Researchers: Gary W. Litman, Ph.D.
Research Facility: All Children’s Hospital/University of South Florida,
Molecular Genetics/Pediatrics
Grant Information: Awarded $75,000
Grant Cycle: 2005
Zebrafish offer many advantages as an animal model of development and disease. Their small size and transparent, rapidly developing embryo make them ideal for large scale studies involving multifactorial, complex diseases such as cancer. Zebrafish hematopoiesis closely resembles that observed in humans and models for human hematopoietic malignancies using transgenesis in zebrafish. Our primary focus is to generate transgenic fish representing models of leukemias/lymphomas that arise from the deregulated expression of hematopoietic-specific proto-oncogenes. We already have developed and currently are analyzing transgenic fish that express the mouse T-cell leukemia-1 cDNA under the control of the human vav and zebrafish rag promoters. We are using these promoters, as well as the zebrafish stem cell leukemia (scl) promoter, to drive expression of the zebrafish orthologs of scl, lymphoid enhancing factors-1 (Lef-1), acute myeloid leukemia-1 (AML1/Runx1), and B-Cell specific activator (Pax 5), which are associated with human hematopoietic malignancies. The transgenic fish will be analyzed at specific time points for developmental defects and the onset of malignant transformation. To demonstrate that the transgene is responsible for the tumor phenotype, the transgenic fish will be subjected to gene knockdown studies using the relevant antisense morpholino (a unique advantage of the zebrafish model) or shRNA and assayed for reversion of the tumor. These studies will develop new cancer models that will be amenable to genetic manipulations that can not be achieved readily in mouse or human.
Title
of Project: Post Transcriptional Silencing of Genes: A Novel Molecular
Approach to the Treatment of Pediatric Cancers of the Cerebellum, Year
2
Principal Investigative Researchers: Michael Lawman, Ph.D.
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Grant Information: Awarded $75,000
Grant Cycle: 2005
Cancer is the second most common disease and also one of the most feared. Cancer occurs when cells continue to divide and fail to die at the appropriate time. Under normal circumstances , the many types of cells that make up the body grow and divide to produce more cells as they are needed in order to maintain a healthy body. Tumors may form when this orderly process is disrupted by changes in genes that control normal cell growth and death whereby cellular growth becomes uncontrolled.
New and effective cancer treatments are constantly being pursued. While most malignant cells appear to be highly susceptible to current cancer treatments, there is some speculation that a certain subset of these cells are more resistant to drugs and radiation than normal, non-cancerous cells. Alternatively, tumor cells may simply develop resistance to chemical and radiation treatments, leading to recurrence of chemo and/or radio –resistant cancers.
The manipulation of the host immune system (immunotherapy) to identify cancer cells as non-self; i.e., methods to mobilize and strengthen the immune system so that it can selectively destroy and/or inhibit proliferation of cancerous cells, is gaining more attention. This is due to the recognition that the host itself may be able to generate the safest and most effective defense against cancer. Advantages of immunotherapy are considerable when compared to standard treatments in that little or no toxicity has been seen in clinical trials thus far, and vaccine-induced regression, when achieved., is usually durable, often lasting from months to years. However, past efforst to marshal host defenses by stimulating an immune response to specific cancers have generally failed, despite use of immunostimulatory and gene therapy methods. An effective cancer vaccine, for example, must elicit both humoral (antibody) and appropriate cellular (antigen-specific T cell) responses.
This will build upon the exciting preclinical results funded previously,
and further understanding of the nature of the immune response generated
by this novel cancer vaccine approach. It is anticipated that the research
proposed will give us a better insight as to how to develop effective non-invasive
therapies for many childhood cancer.
Title
of Project: Hemangioblastic Activity of Infant Leukemia
Principal Investigative Researchers: William B. Slayton, MD
Research Facility: University of Florida
Grant Information:
Grant Cycle: 2005
Infant leukemia is a particularly aggressive, frequently fatal form of childhood cancer. This proposal seeks to improve the understanding of the biology of this disease by studying the ability of infant leukemia cells to produce blood vessels. Solid tumors require constant production of blood vessel in order to receive enough oxygen to survive and grow. Traditionally, leukemia has not been thought to rely on vessel production for disease progression. However, recent studies suggest that certain forms of hematopoietic malignancies do produce their own blood vessels. A recent study has demonstrated that some infant leukemic blasts have gene expression profiles that are similar to hemangioblasts. Our laboratory is studying the menangioblastic activity of adult hematopoietic stem cells, and we have developed techniques that are well suited to understanding the hemangioblastic activity of infant leukemia. We hypothesize that infant leukemia cells are derived from malignant hemangioblasts and produce their own blood vessels in addition to leukemic blasts. Our specific aims are to determine whether infant leukemia cells express hemangioblastic proteins; how infant leukemia cells effect bone marrow vasculogenesis in xenogeneic (NOD/SCID) transfer experiments; and to determine the role of the transcription factor hypoxia inducible factor-1 in controlling leukemic blood vessel development. By understanding how leukemia develops it nutrient supply, we hope to identify more effective and less toxic ways to treat this aggressive form of leukemia.
Title of Project: Post Transcriptional Silencing of Genes: A Novel Molecular
Approach to the Treatment of Pediatric Cancers of the Cerebellum
Principal Investigative Researchers: Michael Lawman, Ph.D.
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Grant Information: Awarded $75,000
Grant Cycle: 2004
Cancer is the main cause of death, due to disease, in children and adolescents. Tumors of the central nervous system account for 22% of all childhood cancers. Medulloblastoma, the subject of this proposal, is a malignancy of the cerebellum and is the most common brain cancer. Despite significant advances made in the treatment of childhood cancers, brain tumors are still the leading cause of death among childhood cancers.
In recent years, much has been learned about gene regulation of the developing brain. Among these, the “sonic hedgehog” family of genes appears to have a pivotal role in the embryonic development in mammals including humans. Likewise, significant data has been accumulated regarding the biology and genetics of medulloblastoma. It is evident that certain genes and “signaling” pathways regulate the development of medulloblastoma. Previous data has shown that the dysregulation of sonic and Gli signaling pathways is a significant contributor to this process. Furthermore, the inability to down-regulate sonic hedgehog signaling in certain neuron brain cells underpins the development of tumors such as medulloblastoma.
Currently, the treatment of medulloblastoma involves surgery, radiation therapy and chemotherapy. While these treatments are successful, they can potentially cause significant neurological defects and can alter growth and development of young children as well as cause short and long-term psychosocial behavioral problems. Alternative therapies, based on the molecular biology of the tumor are needed.
The recent discovery of a molecular mechanism termed post-transcriptional gene silencing or RNA interference by which cells are able to shut down the expression of a targeted gene and alter a cellular pathway may offer a means to control such tumors without the side effects associated with conventional treatments. This study proposes to use these RNA interference molecules to “silence” genes within the hedgehog pathway and thereby alter the malignant nature of the cancer cell. This study requires the development of medulloblastoma cell lines, the construction of the RNA interference molecules, the development of methods to target the RNA interference molecules to tumor cells in the lab and to tumors in the transgenic ptch mouse model. If successful, this study would open a new avenue in the treatment of tumors in general and medulloblastoma in particular.
Title of Project: Zebrafish as a Model for Identifying Cancer Genes
Principal Investigative Researchers: Gary W. Litman, Ph.D.
Research Facility: All Children’s Hospital/University of South Florida,
Molecular Genetics/Pediatrics
Grant Information: Awarded $100,000
Grant Cycle: 2003, 2004
Environmental and genetic factors influence cancer susceptibility. Recent studies have provided evidence for the direct involvement of several different types of genes in the multistep process associated with the induction and progression of individual malignancies. Despite the significance of these findings, the current investigations of genetic factors that influence cancer in experimental animals are encumbered by high costs, technical complications and an inability to characterize malignancies at early stages of development without sacrificing the test animal. For these reasons, zebrafish are emerging as an important experimental animal. Forward genetic screens have been conducted in zebrafish, producing hundreds of mutations that emulate a wide variety of different human disorders. Notably, every mutational phenotype in zebrafish has a human disease equivalent, emphasizing the importance of this animal as a model of human disease. T cell acute lymphoblastic leukemia (T-ALL) has been induced in zebrafish by introducing a DNA construct consisting of a lymphoid lineage-specific promoter linked to a mammalian oncogene. The clinical presentation and progression of ALL in zebrafish closely resembles the human counterpart.
We propose to induce and further characterize zebrafish ALL by identifying genes that influence its development and progression. We will develop new models of leukemias in this species through transgenesis (introducing engineered recombinant DNA into zebrafish at the single cell stage) or protooncogenes associated with human leukemias that either affect earlier points in lymphoid differentiation or protect cells from apoptosis. The cytology and genetics of the malignancies will be characterized by a variety of approaches. We next will identify genes that influence the development of these leukemia(s) using DNA transposition to efficiently introduce mutations and will identify genes that affect survival. By developing this new model, we will have at our disposal a significant tool for identifying new therapeutic modalities that will provide alternative means to control and eliminate malignancies.
Title of Project: A Non-Invasive Immune Therapy for Pediatric Cancers
Resistant to Conventional Treatment
Principal Investigative Researchers: Michael Lawman, Ph.D.
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Grant Information: Awarded $50,000
Grant Cycle: 2003
Proposal: The object of this research project was to test the hypothesis that by introducing the emm55 gene from Streptococcus pyogenes into weakly immunogenic tumor cells, they will become more immunogenic. The experiments performed were with the intention of developing an immunotherapeutic cancer vaccine for the treatment of childhood cancers resistant to conventional treatment. It has been suggested that by modifying a tumor cell’s surface through the expression of a genetically engineered cell surface antigen, and anti-tumor response could be elicited, resulting in clearance of the malignancy. In order to accomplish this goal, several genetic vectors have been constructed and a murine neuroblastoma tumor model has been developed.
The Emm55 cancer vaccine is unique in several ways. First, Emm55 is highly antigenic. Second, it is one of few bacterial antigens that have been expressed on the surface of a mammalian cell. Third, while Emm55 shares the antigenicity characteristics of other M proteins, it is not believed to be rheumatogenic. Fourth, it is a common antigen and reintroduction might be expected to elicit a rapid and increased anamnestic response, which in turn could have a significant additional therapeutic effect. Fifth, in contrast to most true superantigens, which produce an overblown immune response in a non-immune fashion, the immune response to Emm55 should not result in clearance of the immune effectors before the therapeutic effect can be realized. Sixth, the determination and elucidation of immunogenicity of the Emm55 vaccine, a critical objective in the development of a cancer vaccine, will be more easily assessed than for other potential vaccines such as allo-MHC, heat shock proteins and superantigens.
Title of Project: Analysis of E2A-NMP4 and P120-E2A In Childhood Acute
Lymphoblastic Leukemia
Principal Investigative Researchers: Dr. Stephen Hunger
Research Facility: University of Florida, Department of Pediatrics
Grant Information: Awarded $300,000 over three years
Grant Cycle: 2002, 2003, 2004.
E2A alterations play a significant role in the pathogenesis of childhood acute lymphoblastic leukemia (ALL). The major E2A alteration is creation of an E2A fusion protein via chromosome translocation. The major known E2A fusion proteins are E2A-PBX1 (product of the 2nd most common translocation in ALL) and E2A-HLF, which have structural features and functional properties of chimeric transcription factors. Several hypothesis have been proposed to explain how E2A fusion proteins contribute to leukemia, including direct regulation of critical downstream target gene transcription and interference with normal E2A function. Using reagents developed in the laboratory, researchers have discovered two new E2A fusion proteins, E2A-NMP$ and p120-E2A, that result from a complicate translocation in childhood leukemia.
The goal of this research initiative is to identify acquired (somatic)
genetic abnormalities that occur in childhood ALL and understand how these
changes contribute to the development of leukemia. New treatment strategies
and drugs are needed to improve the outcome of patients not cured with
current therapies. Chromosome translocations occur in 70% of ALLs. Understanding
the changes that result from these translocations has led to major insights
into leukomegenesis and is now beginning to translate into new therapies
(e.g. Gleevec for CML). E2A is the 2nd most common translocation target
in childhood ALL. Improved understanding of the effects of E2A abnormalities
is critical to develop new therapies for this major subset of childhood
ALL.
Title of Project: Novel Immune-Type Genes: Role in Recognition of Cancer
Cells
Principal Investigative Researchers: Gary W. Litman, Ph.D.
Research Facility: All Children’s Hospital/University of South Florida,
Molecular Genetics/Pediatrics
Grant Information: Awarded $150,000 over two years
Grant Cycle: 2001, 2002
A family of novel immune-type receptor (NITR) genes that possess properties of immunoglobulin and T cell antigen receptors has been identified in pufferfish and zebrafish, two significant experimental models for gene investigation. Analysis of NITR structure in a third model system Ictalurus has shown NITRs to exhibit a wide range of form and presumably function. Comparisons of NITR structures in all three model systems has defined consensus features that will permit full scanning of the one million base pair NITR locus that we have defined in zebrafish. The sequence of this region will be resolved in the course of the zebrafish genome project; computational resources have been developed to facilitate the identification of NITR and NITR-line gene segments. In addition, a novel cDNA cloning vector/selection system has been developed that permits the rapid identification of molecules that share short regions of identity with prototypic NITR genes. Our intention is to use this cDNA selection system in conjunction with a SCID (severe combined immunodeficiency disease) mouse in which expression of immunoglobulin and T cell antigen receptor genes is suppressed and tumor killing function is markedly increased. Mouse genes sharing structural features with NITRs will be characterized by a number of immunological, biochemical and ultimately functional methods. Te identification of new genes that are involved in the recognition of cancer cells as well as in the control of normal cellular growth and development are critical to our understanding of how childhood cancer arises and how new therapies can be devised for its control.
Title of Project: A Novel Cancer Related Gene
Principal Investigative Researchers: Gary W. Litman, Ph.D.
Research Facility: All Children’s Hospital/University of South Florida,
Molecular Genetics/Pediatrics
Grant Information: Awarded $200,000 over two years
Grant Cycle: 2000, 2001
In the last ten years, it has become clear that cancers can be caused by disregulation of many different types of genes, and we have only begun to identify all genes involved in malignant transformation. The explosion of information emerging from Human Genome Project affords unique opportunities for identifying new genes that may function in malignancy. In the course of a gene discovery project, we identified a novel human gene that exhibits limited homology to genes that are involved in promoting cell growth and appears to be disregulated in cancer. Examination of its expression pattern suggests that it is ubiquitously expressed but is differentially expressed in several different cancers. This novel gene has been named cancer-related gene (CRG1). Additional studies have identified the mouse form of the gene (Crg1_ and further defined its expression patterns. Sequence analysis of the gene has identified a putative translational start site that should permit expression of the gene in mammalian cells and subsequent analysis of cellular, biochemical and biological function, especially pertaining to oncogenesis. Isolation of a mouse homolog of CRG1 has identified extended regions of sequence identity that can be used for deriving a CRG1-specific antibody that will recognize both human and mouse proteins. Identification of the mouse gene is a first step towards targeted disruption of the Crg1 gene which possibly will produce a recognizable phenotype associated with the null mutant (crg-1-) form. The GRG1 gene has been isolated in a human P1 artificial chromosome (PAC) and resolution of the genomic structure of the human form of the gene is a critical first step in examining its potential mutation patters in carious childhood cancer. Taken together, the studies will define the function of the CRG1 in the biological processes that give rise to malignancy.
Title of Project: Production of Viral Oncolysate Vaccine and Active Tumor-Specific
Immunotherapy of Neuroblastoma
Principal Investigative Researchers: Cameron K. Tebbi, MD
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Grant Information: Awarded $197,000
Grant Cycle: 2000, 2001
Proposal: Neuroblastoma is the most common extracranial tumor of children. However, the treatment of this disease has lagged behind a significant progress made in the treatment of childhood cancer. With the present therapeutic modalities, including chemotherapy and stem cell/bone marrow transplantation, less than 30% of patients with advanced stages survive their disease, thus a need for new modalities of treatment. The proposal is for the production of a neuroblastoma vaccine, modeled after successful melanoma vaccination and is designed to use the immunogenicity of this tumor to advantage for the treatment of the disease.
Key objectives of this study include:
- Production of a Newcastle Disease Virus (NDV) modified autologous or allogeneic viral onclysate neuroblastoma vaccine
- Determine the safety of NDV modified autologous or allogeneic viral oncolysate neuroblastoma vaccine
- Characterize the cellular response of patients with NB to the above vaccine
- Evaluate immunological modulation of tumor and marrow after the above vaccination (when marrow is involved).
Title of Project: Identification of Novel genes associated with undifferentiated
neuroblastoma by cDNA Library. Additional funds awarded in this three year
grant were redirectred to purchase a CDNA array machine.
Principal Investigative Researchers: Dr. Cheryl Johnson, Michael Lawman,
PhD
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Grant Information: Awarded $236,736
Grant Cycle: 1999, 2000, 2001
Funding Scope: To assist in the growth and development of the hematology/oncology
research laboratory at St. Joseph’s Children’s Hospital
Medical Director: Cameron K. Tebbi, MD
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Funding Information: Awarded $100,000
Year Awarded: 1997
Additional Funding Awarded by the Pediatric Cancer Foundation:
Funding Scope: Pediatric Oncology Laboratory
Medical Director: Gary Litman, PhD
Research Facility: All Children’s Research Institute
Funding Information: Awarded $100,000
Year Awarded: 1998
Funding Scope: Pediatric Oncology Laboratory
Medical Director: Dr Good, Dr. Day
Research Facility: All Children’s Research Institute
Funding Information: Awarded $100,000
Year Awarded: 1997
Funding Scope: To help the hospital enter into the area of gene research
through the purchase of a Polymerase Chain Reaction (PCR) system. This
process amplifies the nucleic acid reactions in genes and allows for investigators
to explore the implications at the gene level on oncology.
Medical Director: Cameron K. Tebbi, MD
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Funding Information: Awarded $43,500
Year Awarded: 1996
Funding Scope: Pediatric Oncology Laboratory
Medical Director: Cameron K. Tebbi, MD
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Funding Information: Awarded $150,000
Year Awarded: 1994
Funding Scope: Pediatric Oncology Laboratory
Medical Director: Cameron K. Tebbi, MD
Research Facility: St. Joseph’s Children’s Hospital of Tampa,
Hematology/Oncology
Funding Information: Awarded $78,000
Year Awarded: 1993, 1994, 1995