New Faculty I
$3 008 131
Cancer is the leading cause of death for people younger than 85. High cancer mortality rates underscore the need for more sensitive diagnostic techniques as well as therapies that selectively target cells responsible for cancer propagation. Compelling studies suggest that human cancer stem cells (CSC) arise from aberrantly self-renewing tissue specific stem or progenitor cells and are responsible for cancer propagation and resistance to therapy. Although the majority of cancer therapies eradicate rapidly dividing cells within the tumor, the rare CSC population may be quiescent and then reactivate resulting in disease progression and relapse. We recently demonstrated that CSC are generated in chronic myeloid leukemia by activation of catenin, a gene that allows cells to reproduce themselves extensively. However, relatively little is known about the sequence of events responsible for the 36-fold increased risk of leukemic transformation in more common myeloproliferative disorders (MPDs) that express an activating mutation in the JAK2 gene. Because human embryonic stem cells (hESC) have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells in vitro, they represent an ideal model system for generating and characterizing human JAK2+ MPD stem cells. Thus, hESC cell research harbors tremendous potential for developing life-saving therapy for patients with cancer by providing a platform to rapidly and rationally test new therapies that specifically target CSC. To provide a robust model system for screening novel anti-CSC therapies, we propose to generate and characterize JAK2+ MPD stem cells from hESC. We will investigate the role of genes that are essential for initiation of these MPDS such as JAK2 V617F and additional mutations such as b-catenin implicated in CSC propagation. The efficacy of a selective JAK2 inhibitor, TG101348, at inhibiting JAK2+ human ES cell self-renewal, survival and proliferation alone and in combination with potent and specific Wnt/b-catenin antagonists will be assessed in robust in vitro and in vivo assays with the ultimate aim of developing highly active anti-JAK2+ MPD stem cell therapy that may halt MPD progression and obviate therapeutic resistance.
Statement of Benefit to California:
Scientific Benefit Use of both federally approved and non-approved human embryonic stem cells (hESC) to engineer an inexhaustible supply of cancer stem cells (CSC) involved in leukemic transformation of myeloproliferative disorders (MPD) would establish California as a leader in cancer stem cell biology. Relatively little is known about the molecular pathogenesis of Philadelphia chromosome negative (Ph-) MPDs that confer a 1.4 fold increased rate of fatal thrombotic events as well as a striking 36-fold increased risk of death from transformation to acute leukemia compared with the general population. Recently, a point mutation, JAK2 V617F (JAK2+), resulting in constitutive activation of the JAK2 cytokine signaling pathway, was discovered in a large proportion of MPD patients. We found that JAK2 V617F is expressed at the hematopoietic stem cell level in these MPDs and that JAK2 skewed differentiation is normalized with a selective JAK2 inhibitor, TG101348, produced by TargeGen, a San Diego company. However, a detailed characterization of leukemic transforming events has been hampered by the paucity of stem and progenitor cells in JAK2 V617F+ MPD blood and marrow samples. Because hESC have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells in vitro, they represent an ideal model system for generating human JAK2+ MPD stem cells. Thus, California hESC research harbors tremendous potential for understanding the MPD initiating events that skew differentiation versus events that produce self-renewing CSC. Moreover, a more comprehensive understanding of primitive stem cell fate decisions may yield key insights into methods to expand blood cell production that may have major implications for blood banking. Clinical Benefit Generation of JAK2+ MPD stem cells from hESC would provide an experimentally amenable and relevant platform to expedite the development of sensitive diagnostic techniques to predict disease progression and to develop potentially curative therapies based on a small molecule JAK2 inhibitor, TG101348, developed by a California company, TargeGen for early phase disease and likely a combination of TG101348 and a self-renewal antagonist, such as dBHD, a -catenin inhibitor developed at UCSD. Economic Benefit The translational research performed in the context of this grant will help to train California’s future R&D workforce in addition to developing leaders in translational medicine. This grant will provide the personnel working on the project with a clear view of the importance of their research to cancer therapy and a better perspective on future career opportunities in California as well as direct revenue generated by innovative clinical trials aimed at eradicating JAK2+ MPDs that may be more broadly applicable to CSC in other malignancies.
SYNOPSIS: Philadelphia chromosome negative (Ph-) myeloproliferative disorders (MPD) include polycythemia vera (PV), essential thrombocytopenia (ET) and idiopathic myelofibrosis (MF). Recently, it has been discovered that a majority of these cases have an activating mutation in Jak2 (V617F), and this is associated with a 30 to 40 fold increased risk of leukemia compared to the general population. Because human embryonic stem (hES) cells can be differentiated in vitro into hematopoietic progenitors, they may serve as good test cells for introduction of mutant signaling molecules (such as JAK2 V617F) which are associated with MPD in humans. This is a project to define the role of Jak2 V617F in the initiation versus transformation to acute leukemia of hematopoietic stem cells (HSC) or progenitor cells in Ph- types of MPD (PV, ET, MF). The general idea is to use hESC tranduced with an activating JAK2 gene to generate a model of Ph- MPD and eventually to use this model to develop cancer stem cell (CSC)-targeted therapies for leukemia. The aims are organized around the creation of human stem cells with the appropriate genetic alterations to mimic human disease, an evaluation of whether these stem cells can recapitulate the progression to leukemia as is observed in human patients, and a test of whether these stem cells are refractory to targeted therapy as has been observed in the treatment of Philadelphia chromosome positive patients with Gleevac. All 3 aims are based on development of a model whereby CSCs are generated by transduction of hESCs with a Jak2+ retrovirus, followed by sorting GFP+ cells and either in vitro assays or transplant into a SCID mouse host. Aim 1 will characterize the human stem cells derived from the Jak2 transduction. Either hESC or hESCs differentiated to CD34+ cells will be transduced with Jak2+ or Jak2 WT. The effect of activated Jak2 on hematopoetic differentiation, proliferation and survival of human ES cells, in vitro and in vivo, will be determined. It has been found that progression of MPD to leukemia is associated with activation of the Wnt pathway. Aim 2 will test if Jak2+ is necessary but not sufficient for progression to AML. The PI will test whether expression of activated Jak2, either alone or in combination with activation of the Wnt pathway, is sufficient to cause leukemic transformation of human ES cells in a mouse model. The hypothesis is that the Jak2+ cells will require additional mutations to transform to a CSC. The hESC or CD34+ derived cells will be transduced with activated JAK2 alone or in combination with expression of an activated beta-catenin or notch1, and effects on colony replating will be quantified. Serial transplantation and implantation (with bioluminesence imaging) will test if the cells eventually derive an AML phenotype. As a preclinical model, Aim 3 will test if a specific Jak2 pharmacologic inhibitor(s) is able to reduce engraftment and clear the CSC phenotype, with or without a beta-catenin inhibitor or forced expression of axin. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is a focused, carefully designed proposal to create a human stem cell model of Philadelphia chromosome negative myeloproliferative disease. The PI is innovative in suggesting the use hES cells as a platform in which to perform the studies. This approach will obviate possible complications of using mouse cells. A major recognized problem in treating leukemia is the therapeutic resistance of CSCs. While the identification of Jak2+ cells in MPD is a major breakthrough, it is likely that this is not sufficient for transformation, and it is not at all clear why the various MPD diseases are so skewed for different lineages. The idea of generating Jak2+ HSC is a good one and could provide a key experimental model for testing drugs, looking for genetic interactions, etc. It would overcome the significant problem of obtaining enough of the Jak2+ CSCs to study the basic mechanisms and test therapeutics. Significance is therefore very high. Reviewers concurred that the PI has already some strong preliminary data, and a strong track record for studying CSCs. The current proposal is similar to a current SEED grant in that the PI is proposing the development of a novel model for leukemia. Preliminary data show that cord cells transduced with the JAK2 mutant generate increased progenitors, and she has also shown that cord blood cells can be established in the immune deficient mice. The PI has appropriate local expertise to assist, as needed, in the hES cell culture. Reviewers felt that this is a focused, carefully designed proposal; however, several problems with the approaches described in this proposal are noted, some more adequately addressed than others. One caveat that is not discussed relates to the stage at which the mutant signaling molecules act. It is stated that JAK2 increases the stem cell pool. If this is indeed the case and relevant to the disease, this aspect of the biology might be missed in the hES cell differentiation platform as it is unclear whether adult-type HSCs are generated in the in vitro differentiation protocols currently in use. The reviewer did not believe this was a fatal flaw in the proposed work, but considered it a significant and potentially limiting concern. Another reviewer questioned the biological relevance of studying the consequences of activated Jak2 expression in undifferentiated human ES cells. Certainly the biology of the Jak2 pathway will be very different in this setting than in the hematopoetic progenitors that give rise to MPD. In some of these experiments, the PI will test the effects of activated Jak2 on the differentiation of hES cells into hematopoetic cells types. This too is problematic, because the order of events is likely wrong. In this model, Jak2 activation is occurring at a very early stage of development, that precedes the differentiation of hES cells into hematopoetic stem cells. However, in human disease the activating mutation in Jak2 occurs much later, after the hematopoietic stem cells have formed. Therefore, its not clear what the biological meaning of these experiments will be. Finally, it is proposed that Jak2-expressing hES cells will be a virtually limitless source of hematopoietic stem cells for studying MPD. However, the effects of activated Jak2 on human ES cells has not been determined, even in a preliminary way. Indeed, it has not yet been shown that human ES cells expressing activated Jak2 are capable of differentiating into hematopoietic stem cells. These studies are planned in the first aim but would have been desirable as preliminary data for this proposal. In recognition of these problems, the PI also proposes to express activated Jak2 in CD34+ cells derived from human ES cells, which are the definitive hematopoetic progenitors. The idea is to directly create a stem cell that is representative of the cell of origin of MPD. However, hematopoietic stem cells produced in vitro from human ES cells do not have the self renewal capabilities that are typical of bona fide stem cells, and therefore lack the defining characteristic of a true cancer stem cell. Several groups have shown by FACS analysis that they can generate CD34+ cells as well as more mature hematopoietic cells, but no one has demonstrated the generation of long term repopulating hematopoietic stem cells. Even if they are shown to give rise to human hematopoiesis in immune-deficient mice (this can be done, but so far the level of engraftment is very poor) this xenograft assay does not prove self -renewing stem cells because the numbers produced during the assay time are easily accounted for by committed progenitors, because human committed progenitor cells can produce so much more compared to mouse. Self-renewal is likely to be an essential component of the disease, and its absence from this model is problematic. The ability to generate HSC from hESC cultures is an important element in the proposal, and while there has been some progress (such as the CD34+ phenotype), this will probably be the limiting step in progress. In this case, it is not clear why normal CD34+ cells would not just be a better starting place. The alternative plan is to use JAK V617F transduced cord blood cells, which is a good idea and at least at this point seems more likely to be successful. However the proposal in that case would not be an embryonic stem cell application, but is still relevant to CSC biology. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI has an outstanding training record as a physician scientist. Catriona Jamieson received her MD and PhD degrees from the University of British Columbia in 1995, followed by a bone marrow transplant fellowship and postdoc at Stanford until 2003, the latter with Irv Weissman. From 2003 through 2005 she was an instructor in Medicine at Stanford, and then in 2005 appointed as an Assistant Professor in Heme/Onc in the UCSD department of Medicine. In 2006 she became Director of the UCSD stem cell research program. The PI is truly a translational investigator, working in hematology/oncology. She has numerous high profile publications on leukemia and leukemia stem cells. During her postdoctoral work with Weissman she published two first author papers on leukemic CSCs, in PNAS and NEJM, describing activation of the Wnt pathway in CML, suggesting that it is a contributor to the leukemic stem cell phenotype. The candidate has secured independent funding, and the extent of her current support reflects her success. She has grants from Burroughs Wellcome, the Aplastic Amemia and MDS International Foundation, a cancer center collaborative translational research grant, a CIRM seed grant (to study Bcr-Abl CML stem cells in the hES model), a Mizrahi family foundation grant, and grants from TargeGen and Genentech. She has 2 CIRM-funded postdocs. Pending are grants from Celgene and Merck. The candidate’s career plans are excellent and well described. Dr. Jamieson has a clear focus on developing preclinical models for leukemia, using human cells in mouse xenotransplants, and testing therapeutic strategies. She has a well defined goal of translating basic studies toward leukemia CSC research. She is mentored at least in part by Dr. Larry Goldstein, who is the Director of the CIRM-funded UCSD Stem Cell facility. She also has recruited important assistance from several other strong investigators including Ken Kaushansky, who is Chair of her home Department of Medicine. There is no indication that her clinical responsibilities interfere with her research. Perhaps, on the contrary, they stimulate her research directions. A second reviewer who stated, “…She is clearly an ambitious and very talented young investigator” also commented that the PI is a new junior investigator who has taken on many diverse and ambitious research projects in collaboration with numerous biotechnology companies as well as the directorship of the stem cell program. In addition, the conditions of her appointment allow for the possibility that she spend up to 25% of her time outside her lab. The actual extent of those teaching responsibilities is not spelled out in the Institutional letter. Altogether this is an unusually busy, and already well-funded program for such a young investigator. This is especially true considering that her lab is only 825 square feet (1.5 bays). INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The commitment of the institution is excellent. There is strong important support from senior leadership at the Institution, in particular Drs. Goldstein and Kaushansky. Dr.Kaushanky , her department chair, is highly supportive. As he is an active investigator in the MPD field, the relationship will benefit this PI. She will be afforded 80% time to focus on research. The institution’s track record in training is also excellent. The facilities and collaborations available to the PI are outstanding. UCSD affords an outstanding research environment with a clear commitment to stem cell research. CIRM funding is already in place for facilities and training, as well as the funding of SEED grants, of which the candidate is a recipient. Excellent facilities, including excellent independent space in the Cancer Center with equipment including FACS and animal facilities are available, and this is a strength of the application. Finally, the candidate’s lab space is adequate, and her start-up package is average. DISCUSSION: Reviewers were consistent in their feeling that this was a very good candidate, with clinical training that complements the goals of the proposal. All reviewers felt that the applicant was very well trained, first in Canada, and then in Irv Weissman’s lab. One reviewer noted that the applicant is the recipient of a Wellcome award. The proposal was felt to be a natural extension of the applicant’s previous work in CML. However, reviewers noted that the proposal, although very solid, logical, connected to previous work and supported by preliminary data, was not as strong as the candidate. Discussion ensued regarding the choice of the platform in which to perform the experiments, that is to perform the studies in hESC-derived HSCs, rather than in HSCs themselves. The panel was divided in their opinions about this issue. Several members felt that deriving HSCs from hESCs was an uncertain strategy. However, others felt that this was important project to bring into a hESC platform, citing that a significant barrier in both human and mouse cells is to generate HSCs from ESCs. Overall, this concern did not dampen the panel’s enthusiasm to support this investigator.