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RS1-00190-1: A HUMAN EMBRYONIC STEM CELL MODEL FOR THE DEVELOPMENT OF CD30-POSITIVE HODGKIN LYMPHOMA REED-STERNBERG CELLS (H-RS)

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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 (provided by applicant)

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

SYNOPSIS OF PROPOSAL: This project has two largely unrelated specific aims: Specific Aim 1 is to determine the effect of the introduction of “specific molecular defects” into human embryonic stem cells conditioned to develop towards B-cell lineage. The investigator will use this as a model system in an effort to generate and investigate spontaneously occurring tumors. Specific Aim 2 is to use a viral vector system to study lymphoma cells. Targeting will be achieved by employing a known method.

INNOVATION AND SIGNIFICANCE: This proposal lacks innovation.

STRENGTHS OF PROPOSAL:The Principal Investigator (PI) has assembled a team of highly experienced collaborators both at the home institution and elsewhere who should be helpful in achieving the goals he/she has articulated.

If the proposed strategy is successful, it would provide an excellent model system and provide insight into molecular pathway underlying the generation of these unusual cells.

WEAKNESSES OF PROPOSAL: There is complexity implicit in trying to recreate the genotype of a tumor cell and there is a lack of specific biological endpoints to define success. There is no published evidence that the investigator has used the methodology in the proposed experiments either in his own lab or in a training capacity.

The rationale for various gene manipulations in hESC was insufficiently developed. Furthermore, the second aim is not tightly related to first and does not require hESC derived cells.

DISCUSSION: Both reviewers predicted that the generation of “specific molecular defect” cells is likely to be more complex than the creation of other cells where the molecular defect is known; this is 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.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Lansing, Sherry