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RN1-00560-1: Identification and functional analysis of stem cells in acute lymphoblastic leukemia
Recommendation: Not recommended for funding
Public Abstract (provided by applicant)
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 (provided by applicant)
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
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 model. While the leukemic stem cell (LSC) is fairly well characterized in both acute and chronic myeloid leukemias, 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 signaling pathways in maintaining leukemic growth, in order to develop strategies to target the self-renewal properties.
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 myeloid leukemias 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 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 proposed time frame to achieve down regulation of genes in the proposed screen was chosen or if this would provide enough time to reveal changes in phenotype. This does not appear to be a simple assay for high throughput screening. It also relies on the assumption that the proposed readout strategy 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 among these cell types could also identify genes that are altered simply by the process of development. An important question is how this subset compares with subsets of genes previously identified by other groups as “stemness” genes, or as a leukemia “self-renewal” signature. Additionally, the enrichment of these genes in bulk ALL is not really consistent with ALL stem cells representing a rare subset (as estimated from the PI’s data) 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.
The levels of expression of certain marker genes in ALL cells as detected in validation assays are in most cases much lower than in ES cells, calling into question their biological significance. Most troublingly, there are no functional data provided to indicate that cells expressing the pluripotency-associated factor are enriched in stem cell activity. Recently published data demonstrates that expression of this factor 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 stem cell activity are not adequately discussed.
The proposed experiments in the next aim 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 assertion that re-acquisition of self-renewal activity in genetically-altered progenitors will be associated with phenotypic changes in cell surface markers is not necessarily true, and is not supported by data from another group, which indicates that leukemic stem cells (which re-activate a “self-renewal signature”) retain the phenotypic profile of a different population of differentiated progenitors.
In the last aim, the PI will use a screening method to test the role of multiple signaling pathways in maintaining self-renewal in leukemic stem cells. These studies are problematic because they will employ a screening method based on expression of a specific factor. The problems are: 1) that the PI does not know if the expression of this factor will in fact enrich for ALL stem cells, so it is not clear this will be the population of interest, 2) it is not clear that altering the expression of this factor in ALL stem cells will affect their self-renewal, and 3) from the PI’s own preliminary data, a majority of cells normally down-regulate this factor after sorting, so the background in this assay will likely be quite high.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI is a trained Hematology / Oncology physician scientist. The PI received an MD outside of the U.S., and completed postdoctoral training in the U.S. The PI became a Professor of Immunology and started a laboratory in 2003, and then became a Professor of Molecular Stem Cell Biology in 2004. In 2006, the PI moved to California where s/he was appointed as an Associate Professor and currently directs the leukemia research program at the applicant institution. Thus, the PI is a bit more senior than most candidates. The PI 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 nearly 40 publications, including many in excellent journals, with nearly 10 senior author publications since 2003. The PI is well supported and has received several substantial grants to fund his/her research
The candidate’s plan for career development is strong. The PI outlines a basic and translational research program that takes advantage of the facilities, resources and expertise available in his/her own laboratory and his/her collaborators’. The PI describes a good plan to grow his/her group with a focus on LSC and the development of novel therapeutic strategies. The PI is well positioned with collaborators from both the applicant and neighboring institutions. The PI has sought out and interacts regularly with senior colleagues, who provide mentorship regarding his/her role as PI and as a program director.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution and a neighboring university have clearly made a significant commitment to this investigator. They have committed a large amount of laboratory space and provided an endowed chair to support this PI’s research budget. There is strong support and commitment demonstrated for the applicant, by placing him/her in a leadership position as director of leukemia programs. This places the PI in a position to support his/her own salary + 4 additional positions over the next 5 years. In addition, the PI is directly involved in ongoing recruitment of additional junior scientists, who will expand the local stem cell program. The applicant institution has also committed to protect the PI’s research time — s/he has 65% effort for research and no patient care requirements.
The track record at the applicant institution 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 a neighboring institution. Other investigators at the applicant institution have obtained CIRM funding, and they integrate well with the stem cell facility.
DISCUSSION: Reviewers were in general agreement that the applicant is an outstanding candidate. However, all reviewers expressed concern that the proposal hinges on the pluripotency-associated factor for identification and isolation of the desired cell population. Because the entire proposal depends on this factor, the proposal was judged to be fatally flawed. Several problems with respect to this marker were identified in the scientific approach. The factor 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. Pseudogenes can confound the gene expression results. Furthermore, in the proposed screen, the background in the experiments will be very high, since the PI is looking for the disappearance of expression as a read-out, yet a large proportion of cells naturally lose 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.
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:
- None