Funding opportunities

Identification and functional analysis of stem cells in acute lymphoblastic leukemia

Funding Type: 
New Faculty I
Grant Number: 
RN1-00560
Funds requested: 
$3 237 448
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Acute lymphoblastic leukemia (ALL), a malignancy derived from lymphocyte precursors in the bone marrow, is by far the most frequent type of cancer in children and is seen throughout adulthood as well. In 2006, 3,930 patients were newly diagnosed with leukemia in the United States, the majority of them are children and teenagers. Therapeutic advances over the past 40 years have made ALL a curable disease in many cases. In the majority of patients, the initial therapy is successful in eradicating the leukemia cells entirely. However, in some patients, a small number of leukemia cells survive, become resistant to the treatment and the disease recurs. When this happens, it is termed a “relapse” of the leukemia and very often, relapse leukemia cannot be treated successfully. Cutting edge research now focuses on the understanding of the few drug-resistant leukemia cells that initiate the leukemia and that may eventually cause the relapse. These cells are termed “leukemia stem cells” because they share their capacity of self-renewal with normal stem cells. In addition, leukemia stem cells (like normal stem cells) can give rise to various distinct progeny populations of the leukemia. While as few as 20 leukemia stem cells are enough to induce full-blown leukemia in mice, more than 50,000 “regular” leukemia cells are otherwise needed to induce the disease. The overall objective of this proposal is the identification of the “Achilles heel” of these leukemia stem cells. Our proposal focuses on the concept to force the leukemia stem cells to mature so that they eventually lose their stem cell features and become more sensitive to drug-treatment. Depending on the outcome of the studies we are proposing in leukemia animal models, we plan to develop a novel therapeutic approach for the treatment of ALL that will focus on leukemia stem cells. Our proposal brings together a team of investigators with expertise in patient care, drug development, basic science and structural chemistry. This multidisciplinary approach allows us to translate the identification of stem cell-like signaling pathways in ALL cells into a novel therapeutic agent to specifically target these pathways in patients towards a cure for otherwise drug-resistant ALL.
Statement of Benefit to California: 
Acute lymphoblastic leukemia (ALL) represents the malignant outgrowth of a transformed lymphocyte progenitor within the bone marrow. For yet unknown reasons, ALL is particularly frequent in children and teenagers and represents by far the most frequent type of cancer in childhood and adolescence. According to a statewide survey of the California Cancer registry and California Department of Health Services, every year 680 children and teenagers under the age of 20 are newly diagnosed with ALL (ICCCIa) in California. This corresponds to an annual age-corrected incidence of 6.2 new patients with ALL among 100,000 Californians. This almost corresponds to the half of the total annual incidence for all types of childhood cancer in California, which is at 14.5 per 100,000 inhabitants. Over the past four decades, constant improvement of cytotoxic drug treatment has made ALL a curable disease in many cases. In fact, about 60 percent of children and about 35% adults with ALL can be cured during the initial course of chemotherapy. However, in many patients, a small subset of particularly drug resistant cells persist and initiate the recurrence of leukemia. Relapse leukemia is typically even more drug resistant than the primary tumor, which makes it particularly difficult to treat patients with ALL relapse successfully. In this case, there are only few treatment options including highly aggressive chemotherapy often followed by allogeneic bone marrow stem cell transplantation. However, aggressive chemotherapy has severe side-effects. In addition, consequences of allogeneic bone marrow transplantation may include the requirement of life-long immunosuppression to prevent life-threatening graft versus host disease. For this reasons, California State legislation recently initiated a state-wide bank for umbilical cord blood. Assembly bill 34, introduced by assembly member Anthony J. Portantino became effective on July 1st 2007. The California Cord Blood Bank expands the pool of stem cell preparations that are available for transplantation and even allows for transplantation of healthy autologous stem cells. In an approach to even prevent the development of relapse of ALL and to avoid extensive side effects of aggressive chemotherapy, we propose a strategy that will target leukemia stem cells in ALL. We hypothesize that complete eradication of leukemia stem cells will drastically reduce the frequency of ALL relapse, improve overall survival of ALL patients, increase the quality of life of the cancer survivors, reduce the burden of chemotherapy-related side-effects and shorten the duration of therapy. Our proposal aims to lay the foundation for the development of a new generation of stem cell-targeted agents that will help to achieve these goals.
Review Summary: 
SYNOPSIS: This is a cancer stem cell (CSC) project to identify and characterize acute lymphoblastic leukemia stem cells (ALLSC) from human tumor samples using a murine xenograft model. While the LSC is fairly well characterized in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), this is not the case for acute lymphoblastic leukemia (ALL). The goals of this proposal are to identify and isolate a stem cell population that maintains tumor growth in ALL, to determine the phenotype and cell of origin of ALL, and to assess the importance of 3 distinct signaling pathways (IGFII, Wnt, and Cripto) in maintaining leukemia growth, in order to develop strategies to target the self-renewal properties. Based on preliminary RT-PCR and reporter gene analyses, the PI proposes that normal hematopoietic stem cell (HSC) and ALL samples contain a distinct population of cells which express the pluripotency-associated factor OCT4. He also proposes that these cells are CD133+, and express IGF1R, beta-catenin activity, and TDGF1. These candidate ALL stem cell markers were identified based on analysis of publically available gene array data, which showed enrichment of these genes and their partners in embryonic stem (ES) cells over a compilation of differentiated tissues, in 34+38- cord blood HSC over B cell precursors, and were expressed in bulk ALL samples of 4 different subtypes. Three specific Aims are described in the proposal. First the PI will use the information derived from the gene expression analysis to try and sort out sub-populations that could be ALLSC. In specific Aim 1, to test whether OCT4+, CD133+, IGF1R+, and Wnt signaling cells are in fact leukemia stem cells for human ALL, the PI will obtain samples of bone marrow (BM) from ALL patients, and engraft them into NOD/SCID/gammachain-mice. He will then reisolate these cells from engrafted mice and look for co-expression of the putative ALL markers he has identified, using antibody staining and reporter gene analysis. Finally, he will transduce primary samples with LV-encoded Oct4-GFP reporter, sort CD133+ GFP+ cells and look for enrichment of SCID reconstituting potential. An alternative approach is to include a bcr-abl/Oct4:eGFP double transgene, in case an extra transforming event is needed. In specific Aim 2, the issue will be addressed whether the LSC are derived from HSC or from more committed B cell progenitors that regain self-renewal capacity. He will sort BM LSK (Lin- sca-1+ c-kit+) cells and CD19+c-kit+B220low B cell progenitor cells and splenic CD19+IgD+IgK+ B cells from pre-leukemic BCR-ABL transgenic mice, and transfer these 3 populations to wild type congenic recipients. He will then determine which groups of mice develop ALL. Finally, in specific Aim 3, the applicant proposes to test the importance of a variety of signaling pathways (identified in the preliminary data from published transcriptional profiling signatures) in the self-renewal of normal and leukemic stem cells. For a loss of function screen, he will use inducible barcoded shRNA constructs in combination with an Oct4-GFP reporter mouse. Oct4+ HSC will be compared directly with Oct4+ putative ALLSC by transducing each with lentiviruses expressing the shRNA constructs. One week post transplantion, the shRNA lentivectors are induced for one week, cells are sorted Oct4+ vs Oct4-, and then compared using a microarray to measure the barcode and determine if there was enrichment or depletion of cells targeted for down regulation of certain genes. Currently 16 potential targets are planned, with up to 50 considered. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Determining the cell of origin and tumor-propagating cell for hematopoietic malignancies is important for understanding the molecular events that lead to emergence of these cancers and for developing more effective treatments. Similar approaches to cancer stem cell isolation have been successful in identifying enriched populations of tumor forming cells in AML, CML, and several solid tumors. While LSC are at least somewhat characterized in myeloid leukemias, this is not the case in ALL and this project has the potential to clarify this important issue. The source of the tumor is also important since it would affect subsequent strategies. The project has the potential to identify novel therapeutic targets and the significance is very high. The proposal, rationale, and strategies are very well described. The experiments in the first two aims are fairly straightforward, based on interesting preliminary data, and the PI is clearly capable of carrying them out. Experiments in the third aim are clearly more risky and untested. It is not obvious why the one week time frame was chosen or if this would provide enough time to reveal changes in phenotype. How long do the lentiviruses remain active? What percent knockdown is anticipated? This does not appear to be a simple assay for high throughput screening. It also relies on the assumption that an Oct4-GFP transgene will faithfully serve as a surrogate marker for ALLSC self-renewal. However, the experiments are feasible to try and identification of a single new potential target would be exciting. Specific concerns about the proposal’s aims are discussed below. Much of the design of this study is based on gene profiling comparisons, which showed co-enrichment of expression of a subset of genes in ES cells, HSC, and ALL samples. Based on this co-enrichment, the PI proposes that this subset of genes will mark ALL stem cells and regulate their self-renewal. However, there is a conceptual difficulty with this rationale. First, comparisons of ES cells to differentiated tissues, and cord blood HSC to BM B cell precursors could also identify genes that are altered simply by the process of development. How does this subset compare with subsets of genes previously identified by the Melton and Lemischka groups as "stemness" genes, or by the Armstrong group as a leukemia "self-renewal" signature. Preliminary data compared ESC, HSC, and primitive ALL cells to their more differentiated counterparts, and revealed coordinate enhanced expression for components of IGFII, beta-catenin, and Cripto signaling pathways. 0.1-3% of ALL cells were found to express an Oct4 reporter and expression also correlates with PROM1/CD133; these are hypothesized to represent the LSC. The enrichment of these genes in bulk ALL is not really consistent with ALL stem cells representing a rare subset (0.1-3% as estimated by the PI's Oct4 reporter) of total ALL cells. This is a key issue, that the putative ALLSC might be a heterogeneous population, and this would complicate the analysis. The PI is well aware of this caveat and so plans to focus first on defined subsets of ALL, and this should at least help to reduce the heterogeneity. Finally, the levels of expression of these genes (Oct4, CTGF, IMP-3) in ALL cells as detected in RT-PCR validation assays are in most cases 10-100 times lower than in ES cells, calling into question their biological significance. Likewise, while the PI states that "FZD7 and Cripto correlate with Oct4 expression", based on the data in Fig. 3, more than half of the FZD7+ cells and most TDGF1+ cells are Oct4-. Most troublingly, there are no functional data provided to indicate that Oct4+ cells in normal or leukemic BM are enriched in stem cell activity. The expression of the Oct4 reporter shown in Fig. 4 is very low, but is not correlated with purified HSC or leukemic cells (Sca1 alone is not sufficient). Moreover, recent data from the Jaenisch group (published recently in Cell Stem Cell) demonstrates genetically (by conditional deletion in the hematopoietic compartment) that Oct4 expression is not important for HSC function in maintaining hematopoiesis or in reconstituting hematopoiesis after transplantation. Alternative approaches to identify ALL stem cells if these markers do not enrich for this activity are not adequately discussed. Regarding Aim 2, the proposed experiments would likely identify an ALL stem cell if it were present in one of the 3 populations that will be analyzed, but such cells could be in any population in the marrow. A more systematic analysis including cells expressing a given phenotype AND cells not expressing that phenotype, would be better able to comprehensively identify ALL transplanting populations. Also, the PI's assertion that re-acquisition of self-renewal activity in BCR-ABL transduced B cell progenitors will be associated with phenotypic changes in cell surface markers is not necessarily true, and is not supported by data from the Armstrong group, which indicates that leukemic stem cells (which re-activate a "self-renewal signature") retain the phenotypic profile of differentiated granulocyte-monocyte progenitors. Under Specific Aim 3, the PI will use an shRNA based screen to assay for the role of IGF, Wnt and TDGF1 signaling pathways in maintaining self-renewal in leukemic stem cells. These studies are problematic because they will employ a screening method based on Oct4 expression. Oct4+ cells will be sorted from wild type mice, or mice transgenically expressing BCR-ABL, and then transduced with a mixture of DOX-inducible bar-coded shRNAs (to down regulate expression of genes hand selected from the literature and based on expression analysis). After transplant, shRNA expression will be induced and Oct4+ and Oct4- cells (which the PI presumes will represent a subset that has lost self-renewal activity) will be isolated and assayed for enrichment of certain shRNAs. The problems are: 1) that the PI doesn't know if Oct4 expression will in fact enrich for ALL stem cells, so it is not clear this will be the population of interest, 2) it's not clear that Oct4 downregulation in ALL stem cells will affect their self-renewal (the data provided on ES cells is not relevant to this issue, and actually shows that FZD7 shRNA reduces expression of FZD7 more effectively in Oct4+ over Oct4- cells), 3) from the PI's own preliminary data (Fig. 3), a majority of Oct4+ cells normally down-regulate Oct4(GFP)after sorting for GFP+ cells, so the background in this assay will likely be quite high. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Muschen is a trained Hematology / Oncology physician scientist. Dr. Muschen received his MD from Heinrich-Heine University in Dusseldorf, and completed postdoctoral training with Klaus Rajewsky and for 1 year with Janet Rowley in Chicago. He became a Professor of Immunology and started his laboratory in Germany at Cologne in 2003, and then became a Professor of Molecular Stem Cell Biology at Dusseldorf in 2004. In 2006, he moved to USC where he was appointed as an Associate Professor of Pediatrics and currently serves as Director of the Leukemia Research Program at Childrens Hospital LA. Thus, he is a bit more senior than most candidates. He is well qualified to carry out this work. The applicant has a strong CV, a strong publication record, both as a postdoc and as an independent investigator, indicating 39 publications, including many in excellent journals, with 9 senior author publications since 2003. He is well supported and has received several substantial grants to fund his research by: remaining German funds, the Cancer Center, an endowment, and a grant from the V foundation. The candidate's plan for career development is strong. He outlines a basic and translational research program that takes advantage of the facilities, resources and expertise available in his own laboratory and his collaborators'. Dr. Muschen describes a good plan to grow his group with a focus on LSC and the development of novel therapeutic strategies. He is well positioned with collaborators from CHLA and USC. He has sought out and interacts regularly with senior colleagues, who provide mentorship regarding his role as PI and as Program Director. He has strong mentors in Stuart Siegel and Gay Crooks. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Childrens Hospital, LA and USC have clearly made a significant committment to this investigator. They have committed a large amount of laboratory space and provided an endowed chair to support his research budget. There is strong support and commitment demonstrated for Dr. Muschen, by placing him in a leadership position, as Director of the Leukemia Research Program and Program Director of the Leukemia and Lymphoma Program. This places him in a position to support his own salary + 4 additional positions over the next 5 years. In addtion, he is directly involved in ongoing recruitment of additional junior scientists, who will expand the local stem cell program. They have also committed to protect his research time. He has 65% effort for research and no patient care requirements. The track record at Childrens Hospital LA is strong with respect to stem cell biology. The institution provides ES cell and cell sorting core facilities that will support the investigations. They are committed to recruiting new faculty in the stem cell area and participate actively with other institutions in the area to foster cross-institutional interaction and collaboration. There is a clear commitment to integrate efforts with the stem cell initiatives at USC. Other investigators at CHLA have obtained CIRM funding including Drs. Kohn (facilities) and Lawlor (SEED). They integrate well with the stem cell facility directed (with CIRM funding) by Gay Crooks. Although not relevant to this proposal, the USC ES cell facility (Pera) is also available. DISCUSSION: Reviewers were in general agreement that the applicant is an outstanding candidate. However, all reviewers expressed concern that the proposal hinges on Oct4 for identification and isolation of the desired cell population. Because the entire proposal depends on Oct4, the proposal was judged to be fatally flawed. Several problems with respect to this marker were identified in the scientific approach. Oct4 is not proven to be a functional marker of BM stem cells, e.g. its expression is not needed for maintaining hematopoiesis or reconstituting hematopoiesis after HSC transplantation. Oct 4 pseudogenes can confound RT-PCR results. Finally in Aim 3, the background in the experiments will be huge, since the PI is looking for the disappearance of Oct4-GFP expression as a read-out, yet 80% of cells naturally lose Oct 4 expression. Another major scientific concern was expressed, that while the proposed experiments would likely identify an ALL stem cell if it were present in one of the 3 populations that will be analyzed, such cells could be in any population in the marrow, and the investigator does not address this possibility. PROGRAMMATIC DISCUSSION: A motion was made to recommend that this application be moved to Tier 2 - Recommended if Funds Are Available. The discussant cited that this is a strong cancer and immunobiology candidate, who is a physician scientist. Scientific concerns regarding the proposal were sufficient to defeat the motion. The motion to move this application to Tier 2 failed.
Conflicts: 

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