Normal hematopoietic stem cells generate all the different mature blood cell types. Similarly leukemia stem cells represent a small subpopulation that generates the other cells that represent the bulk of the leukemic clone. Effective targeting and elimination of the leukemia stem cell population is likely to be required in order to achieve long term remission and cure from leukemia. Leukemia stem cells are often resistant to treatment and may contribute to disease persistence and relapse. Here we plan to use human embryonic stem cells to derive leukemia stem cells to identify the mechanisms underlying malignant transformation of hematopoietic stem cells in leukemia and to develop improved treatments directed against these cells. Currently investigations of leukemia stem cells are hampered by the very small numbers of such cells that are available for study. Since human embryonic stem cells possess almost infinite proliferative capacity and can differentiate into hematopoietic stem cells, we reason that expression of leukemia-related oncogenes in human embryonic stem cells or their progeny can lead to the development of a novel, innovative model system to study leukemia stem cells. Chronic myeloid leukemia (CML) is a lethal hematological disorder resulting from hematopoietic stem cell transformation by the BCR/ABL oncogene. Here we propose to model leukemia stem cell transformation in CML through expression of the BCR/ABL oncogene in hematopoietic stem cells derived from human embryonic stem cells. These studies are highly significant since successful establishment of an efficient and representative human stem cell model of CML will allow detailed investigation of altered mechanisms of stem cell regulation in this disease, and will allow identification of molecular targets for anti-leukemia therapy that can effectively target leukemia stem cells. Development of human leukemia stem cell model will also facilitate screening of libraries of candidate therapeutic agents for activity against leukemia stem cells. Successful completion of these studies will support the use of similar technologies to model other leukemia such as acute myeloid leukemia. Finally, expression of BCR/ABL may enhance expansion and in vivo engraftment of hematopoietic stem cells derived from human embryonic stem cell, and may thereby provide improved insights into the process of hematopoietic stem cell differentiation from embryonic stem cells which could be applied towards improved methods to generate hematopoietic stem cells for transplantation and gene therapy purposes in the future.
Statement of Benefit to California:
Each year 35,000 new cases of leukemia are diagnosed in the US, with 3500 new patients being diagnosed in California last year. About 32 percent of cancers in children ages 0-14 years are leukemia. Hispanic children of all races under the age of 20 have the highest rates of leukemia. Although the relative five-year survival rate has more than tripled in the past 46 years for patients with leukemia, and in 1996-2002 was nearly 49 percent, approximately 22,280 deaths in the United States will be attributed to leukemia in 2006. Therefore, despite advances in treatment leukemia continues to be a devastating, life-threatening and often incurable illness which imposes a huge burden on the individual, society and the state. The leukemia stem cell subpopulation generates all other cells that represent the bulk of the leukemic clone and is often resistant to treatment and may contribute to disease persistence and relapse. More effective targeting and elimination of the leukemia stem cells is needed to improve long term remission and cure from leukemia. Here we propose to express a leukemia oncogene in hematopoietic stem cells derived from human embryonic stem cells to model human leukemia stem cells. Successful completion of these studies have the potential to significantly benefit the State of California and its residents in several ways: (1) Establishment of an efficient and representative human stem cell model of CML will allow improved understanding of the differences between leukemia stem cells and normal hematopoietic stem cells and could lead to the identification of novel, new molecular targets for therapy directed against leukemia stem cells; (2) Development of human leukemia stem cell model will facilitate screening of libraries of candidate therapeutic agents for activity against leukemia stem cells.; Development of candidate compounds would be pursued within our institution and in partnership with collaborators from industry. (3) Successful completion of these studies will support the use of similar technologies to model other leukemia such as AML; (4) Finally, these studies may provide improved insights into the process of hematopoietic stem cell differentiation from human embryonic stem cells which could be applied towards improved methods to generate hematopoietic stem cells for therapeutic transplantation and gene therapy purposes in the future. The intermediate and long-term aim of the proposed research is therefore to improve the outcomes for treatment of leukemia and hematological malignancies. Successful therapeutic and diagnostic strategies to emerge from these studies could immensely benefit patients and families within and outside California.
SYNOPSIS: In this proposal the investigators will establish a new model for studying human CML. Based on the idea that leukemias arise from small populations of malignant stem cells, the applicants will attempt to recreate and characterize this “cell of origin” by transducing human ES cells, and ES cells that have differentiated into hematopoeitic progenitors, with a lentivirus expressing BCR/ABL. In Aim 1 the applicants will study the abilities of these cells to differentiate in vitro into cells in the hematopoeitic lineages in response to specific extracellular signals. In Aim 2 the applicants will inject these cells, and their differentiated derivatives, into immunocompromised mice to study their engraftment and progression into leukemias. It is proposed that this model will be useful in the development of new therapeutic agents that are most effective against leukemic stem cells. SIGNIFICANCE AND INNOVATION: The applicant accurately points out that the clinical failures of Gleevec therapy show persistence of primitive progenitor or stem-like CML cells. If this new system for the generation of leukemic stem cells from hESC using BCR/ABL replicates that in the mouse, then it could certainly be useful in the development of new compounds. Thus, the proposed experiments offer the opportunity of some immediate practical benefit, in the sense of providing a new and better model system for chronic myeloid leukemia for testing an important class of therapeutic agents. STRENGTHS: The experimental approach is clear and should not present any unusual technical problems. The applicants are highly qualified to perform the proposed experiments. They have already developed a human model of CML by transduction of BCR/ABL in cord blood HSC, and the laboratory has considerable expertise in the in vivo as well as in vitro analysis of leukemic cells. WEAKNESSES: This proposal is descriptive in design without a clear, overriding hypothesis for what would be pursued with the new model system. It lacks innovation in part because it will use existing protocols for inducing hESC to differentiate into hematopoietic progenitors. Also, it is unclear why the hESC derived leukemic cells will be a better model than those derived from cord blood HSC. Previous work transducing human umbilical cord stem cells with BCR/Abl successfully generated progenitor cell lines with the properties of CML cells in vitro, however these failed to cause CML in mouse models in vivo. No insight into this failure is offered, and no good reason to anticipate a more successful outcome with human ES cell lines is provided. The applicant cites previous work describing expression of BCR/Abl in mouse ES cells and injection of these cells into mice. Indeed, BCR/Abl transduced murine ES cells have been available since 2001, and it is possible to induce them to differentiate along the myeloid lineage. However, again, there have been no reports of these cells being used successfully to create a leukemia model in vivo. This would have constituted strong support for extending this approach to human ES cells, while conversely the apparent difficulties with this approach using murine ES cells dampens enthusiasm for this application. One additional point requires clarification, and may potentially represent a significant weakness of the proposal. Reviewers were not certain that "definitive" adult HSCs can be derived from ES cells "in vitro". Certainly, embryonic (yolk sac) progenitors are easily formed, but whether definitive, multilineage hematopoiesis can be established in this manner is unknown. DISCUSSION: Reviewers suggested during discussion that extending the cord blood work to hESCs is very sound, but the applicant should have better articulated the rationale for the hypothesis that BCR/Abl transduced into hESC was likely to result in CML in vivo when so far it has not been done with cord blood or mESC.