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A HUMAN EMBRYONIC STEM CELL MODEL FOR THE DEVELOPMENT OF CD30-POSITIVE HODGKIN LYMPHOMA REED-STERNBERG CELLS (H-RS)

Funding Type: 
SEED Grant
Grant Number: 
RS1-00190
Funds requested: 
$642 497
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
A HUMAN EMBRYONIC STEM CELL MODEL FOR THE DEVELOPMENT OF CD30-POSITIVE HODGKIN LYMPHOMA REED-STERNBERG CELLS (H-RS) Chemotherapy and radiotherapy have improved the cure rates of classic Hodgkin lymphoma dramatically. However, 30-50% of patients with advanced disease will still die due to chemotherapy-resistant disease. New therapeutic approaches are needed. Hodgkin lymphoma is characterized by the presence of peculiar giant Hodgkin and Reed-Sternberg (HRS) cells. These are the “stem cells” of Hodgkin lymphoma, amounting to 0.1-1% of the total lymphoma mass. Significant advances have been made defining underlying molecular mechanisms. In about 50% of cases the HRS cells are infected with Ebstein Barr Virus (EBV). In addition, these cells have special molecular characteristics that are unique in their combination for this disease. They include CD30, an active NF-κB pathway, and overexpression of c-FLIP or XIAP. Only very few HRS cell lines exist and studies on primary patient material require single-cell laser capture micro-dissections. Here we propose to develop a novel Hodgkin lymphoma model system that will be very useful to understand the underlying molecular defects in this disease. Such a system would be invaluable for testing novel targeted therapies. We will use human embryonic stem (hES) cells and drive their development towards B-cells. In addition, we will introduce molecular defects into the hES cells that are typical for Hodgkin lymphoma. Our immediate goal is to test the “building blocks” of Hodgkin lymphoma in this system with the future goal to “re-engineer” this disease in the laboratory. Human embryonic stem (hES) cells can be driven towards B-cell development using special culture conditions. To introduce molecular defects, we will employ standard virus based expression systems. These systems will allow us to “turn on” the molecular defects at different stages of the B-cell development. In addition, we are developing a virus based therapy for Hodgkin lymphoma. As proof of principle we will target CD30 which is highly expressed on Hodgkin cells. We propose to use viruses (lentiviruses) encoding lethal genes (such as Pseudomonas exotoxin) to specifically target Hodgkin cells. These lentiviruses are engineered to express antibodies against a surface marker (CD30) that are present on the lymphoma cells, in order to direct the viruses to the target cells. We will test this approach in vitro and in mouse models growing human Hodgkin lymphoma cell lines. In this CIRM SEED grant proposal we aim to use hES cells to gain a greater understanding of and model HRS cells, the pathognomonic cell of HL. Such a model will allow the dissection and therapeutic targeting of aberrant pathways in HL. Grant support would allow us to produce sufficient data to submit for NIH funding to further expand this new program.
Statement of Benefit to California: 
A HUMAN EMBRYONIC STEM CELL MODEL FOR THE DEVELOPMENT OF CD30-POSITIVE HODGKIN LYMPHOMA REED-STERNBERG CELLS (H-RS) According to the United States National Cancer Institute, an estimated 7,300 new patients will be diagnosed with Hodgkin lymphoma this year. Older treatments for this disease were often associated with serious complications from therapy, including sterility and high incidence rates of secondary cancers. Given the young population of patients, these toxicities were not acceptable. Today, the standards of care, such as the chemotherapy combination followed by the selective use of low-dose radiation, have dramatically reduced treatment side effects and improved cure rates. However, 30-50% of patients with advanced disease will still die because of chemotherapy resistant disease, indicating the need for novel therapeutic approaches. Only very few H-RS cell lines exist and studies on primary patient material require single-cell laser capture micro-dissections. Here we propose to develop a novel Hodgkin lymphoma model system that will be very useful to understand the underlying molecular defects in this disease. We will use human embryonic stem (hES) cells and drive their development towards B-cells. In addition, we will introduce molecular defects into the hES cells that are typical for Hodgkin lymphoma. Our immediate goal is to test the “building blocks” of Hodgkin lymphoma in this system with the future goal to “re-engineer” this disease in the laboratory. In addition, we are developing a virus based therapy for Hodgkin lymphoma. As proof of principle we will target CD30 which is highly expressed on Hodgkin cells. We propose to use viruses (lentiviruses) encoding lethal genes (such as Pseudomonas exotoxin) to specifically target Hodgkin cells. These lentiviruses are engineered to express antibodies against a surface marker (CD30) that are present on the lymphoma cells, in order to direct the viruses to the target cells. We propose to develop the building blocks for a new Hodgkin lymphoma model system. The immediate goal of this study is to engineer human embryonic cell lines to harbor the hallmark molecular alterations of cHL and to describe their effects on the cell fate decisions and phenotype of these lines. The distant goal of this study is to recapitulate the cHL pathogenesis and engineer an in vitro model of cHL that will allow the testing of novel molecular targeted approaches. As an example, we will target CD30 on existing cHL cell lines using re-engineered lenti-viral vector systems delivering a potent cellular toxin. By testing the molecular mechanisms of cHL, new therapeutic targets may be identified that could give rise to a new generation of rationally designed therapies that may be even more effective and less toxic than current treatments.
Review Summary: 
SYNOPSIS: This project has 2 largely unrelated specific aims: Specific Aim 1: The effect of the introduction HRS pathogonomic molecular defects into human embryonic stem cells conditioned to develop towards B-cell lineage. The investigator purposed to differentiate human ES cells into CD34+, primitive hematopoietic cells on OP9 stromal cells. The CD34+ cells will be purified and then cultured on MS-5 stromal cell in the presence of various cytokines designed to induce B-lymphoid differentiation. The investigator will use this as a model system in an effort to generate Hodgkin Reed-Sternberg (HRS) cells by introducing genes that are over expressed in such cells in spontaneously occurring tumors. These genes include e-REL, CD30, and c-FLIP. A lentiviral vector will be used to generate polycistronic transcripts encoding these genes. Specific Aim 2: Cytotoxic lentiviral vector targeting of CD30-positive Hodgkin’s cells. The investigator purposes to use lentiviral vector mediated gene transfer to introduce the gene for a cellular toxin (Pseudomonas exotoxin A) into lymphoma cells. Targeting will be achieved by engineering a modified S sindbis envelope protein into which has been incorporated the coding sequences for monoclonal antibody with specificity for membrane bound CD30. The Pseudomonas exotoxin A will be expressed under the control of a NF-kB regulated promoter to increase tissue specific expression. SIGNIFICANCE AND INNOVATION: Generation of chronic Hodgkin Lymphoma (cHL) cells from hESC by differentiation along the B lineage pathway and transduction of genes associated with transformation. Lack of good model system for analysis of disease as primary cells are rare in the lymphoma mass. Proposal lacks innovation. STRENGTHS: Dr. de Vos has assembled a team of highly experienced collaborators both at UCLA and elsewhere that should be helpful in achieving the goals he has articulated. If strategy is successful, it would provide an excellent model system and insight into molecular pathway underlying the generation of these unusual transformed lymphoid cells. WEAKNESSES: -Complexity implicit in trying to recreate the genotype of a tumor cell and lack of specific biological endpoints to define success. - Lack of an published evidence that the investigator has used the methodology in the proposed experiments either in his own lab or in a training capacity - Rationale for various gene manipulations in hESC insufficiently developed. - The second aim not tightly related to first and does not require hESC derived cHL cells. DISCUSSION: Both reviewers predicted that the generation of Reed-Sternberg cells is likely to be more complex than the creation of CML or Bcr-Abl cells due to the complicated transformation pathway involved. Discussion also focused on whether the use of hESCs is appropriate given the studies outlined in the specific aims.
Conflicts: 

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