Mechanisms Underlying the Responses of Normal and Cancer Stem Cells to Environmental and Therapeutic Insults
New Faculty I
$2 257 484
Adult stem cells play an essential role in the maintenance of tissue homeostasis. Environmental and therapeutic insults leading to DNA damage dramatically impact stem cell functions and can lead to organ failure or cancer development. Yet little is known about the mechanisms by which adult stem cells respond to such insults by repairing their damaged DNA and resuming normal cellular functions. The blood (hematopoietic) system provides a unique experimental model to investigate the behaviors of specific cell populations. Our objective is to use defined subsets of mouse hematopoietic stem cells (HSCs), progenitors and mature cells to investigate how they respond to a range of environmental and therapeutic insults by either repairing damaged DNA and restoring normal functions; accumulating DNA damage and developing cancer; or undergoing programmed cell death (apoptosis) and leading to organ failure. These findings will provide new insights into the fundamental mechanisms that regulate stem cell functions in normal tissues, and a better understanding of their dysregulation during cancer development. Such information could identify molecular targets to prevent therapy-related organ damage or secondary cancers. These are severe complications associated with current cancer treatments and are among the leading causes of death worldwide. Originally discovered in blood cancers (leukemia), cancer stem cells (CSCs) have now been recognized in a variety of solid tumors. CSCs represent a subset of the tumor population that has stem cell-like characteristics and the capacity for self-renewal. CSCs result from the transformation of either stem or progenitor cells, which then generate the bulk of the cancer cells. Recent evidence indicates that CSCs are not efficiently killed by current therapies and that CSC persistence could be responsible for disease maintenance and cancer recurrence. Developing interventions that will specifically target CSCs is, therefore, an appealing strategy for improving cancer treatment, which is dependent on understanding how they escape normal regulatory mechanisms and become malignant. Few mouse models of human cancer are currently available in which the CSC population has been identified and purified. This is an essential prerequisite for identifying pathways and molecules amenable to interventional therapies in humans. We have previously developed a mouse model of human leukemia in which we have identified the CSC population as arising from the HSC compartment. We will use this model to understand how dysregulations in apoptosis and DNA repair processes contribute to CSC formation and function during disease development. These results will provide new insights into the pathways that distinguish CSCs from normal stem cells and identify ways to prevent their transformation. Such information could be used to design novel and much-needed therapies that will specifically target CSCs while sparing normal stem cells.
Statement of Benefit to California:
This application investigates how environmental and therapeutic insults leading to DNA damage impact stem cell functions and can lead to organ failure or cancer development. The approach is to study how specific population of blood (hematopoietic) stem, progenitor, and mature cells respond to DNA damaging agents and chose a specific cellular outcome. Such information could identify molecular pathways that are available for interventional therapies to prevent end-organ damage in patients who are treated for a primary cancer and reduce the risk of a subsequent therapy-induced cancer. These are severe complications associated with current mutagenic cancer treatments (radiation or chemotherapeutic agents) that comprise a substantial public health problem in California and in the rest of the developed world. The hematopoietic system is the first to fail following cancer treatment and the formation of therapy-related blood cancer (leukemia) is a common event. The development of novel approaches to prevent therapy-related leukemia will, therefore, directly benefit the health of the Californian population regardless of the type of primary cancer. This application also investigates a novel paradigm in cancer research, namely the role of cancer stem cells (CSCs) in the initiation, progression and maintenance of human cancer. The approach is to study how dysregulations in important cancer-associated pathways (apoptosis and DNA repair processes) contribute to CSC aberrant properties using one of the few established mouse model of human cancer where the CSC population has already been identified. Leukemia, the disease type investigated in this application, has been the subject of many landmark discoveries of basic principles in cancer research that have then been shown to be applicable to a broad range of other cancer types. Accordingly, this research should benefit the people of California in at least two ways. First, the information gained about the properties of CSCs should improve the ability of our physicians and scientists to design, develop and evaluate the efficacy of innovative therapies to target these rare disease-initiating cells for death. This would place Californian cancer research at the forefront of translational science. Second, an average of 11.55 out of 100,000 Californian inhabitants are diagnosed with primary leukemia each year. Thus, in California, leukemia occurs at approximately the same frequency as brain, liver and endocrine cancers. As is true for many types of cancer, most cases of leukemia occur in older adults. At this time, the only treatment that can cure leukemia is allogeneic stem cell transplantation, which is a high-risk and expensive procedure that is most successful in younger patients. The development of novel and safe curative therapies for leukemia would, therefore, particularly benefit the health of our senior population and the economy of the state of California by realizing savings in the healthcare sector.
SYNOPSIS: The goal of the proposal is to study apoptosis and DNA repair pathways in response to insult in normal hematopoetic progenitor cells, and to compare this with the integrity of the same pathways in a mouse leukemia model. In this model, the PI has identified a cancer stem cell (CSC) cohort that is present in the pre-cancerous myeloproliferative disorder phase that eventually progresses to blast crisis and to acute myeloid leukemia (AML). In Aim 1 the applicant will study the apoptosis during normal hematopoiesis. Defined populations of stem, progentior, and mature hematopoietic cells will be isolated, and the responses of these to specific death signals will be studied. At the same time, the expression of various mediators of apoptosis will be measured in order to address whether any differences in the frequency of apoptosis might be corrleated with differences in the regulation of the apoptotic machinery. These correlations will be pursued at the functional level by overexpressing or inhibiting the expression of specific proteins in apoptosis pathways, and determining whether this alters the frequency of apoptosis in vivo and in vitro. In Aim 2 a similar strategy will be used to study DNA repair pathways in the same subsets of hemotopoietic progenitors. The DNA damage response will be determined in response to various insults to evaluate how specific cell types recognize and respond to damage, and which cellular choices they preferentially follow (resume normal function, apoptosis, accumulate mutations). Aim 3 will focus on a junB-null mouse model of leukemia developed by the applicant. The hypothesis is that JunB target genes are deregulated in this model leading to defects in apoptosis and/or DNA repair. In this aim the applicant will study apoptosis and DNA repair in the junB-null hematopoeitic progenitor cells, and compare this to what was observed in the normal progenitor cells in Aims 1 and 2. The focus will be on hematopoietic stem cells (HSC) compared to a more commited progenitors in order to identify the more susceptible target cell to bast crisis. Any differences will be functionally evaluated by knock-down and overexpression experiments. In a second part of this Aim, the applicant will follow-up the observation that transition to blast crisis in the junB leukemia model is associated with emergence of variant stem cells containing a (1,17) chromosome translocation. The goal will be to clone the fusion gene and study its oncogenic properties as well as to determine additional translocations that arise during blast crisis progression. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The overall research plan is clearly organized, and addresses significant problems in stem cell biology and tumorigenesis. The preliminary data indicate that the applicant has the tools necessary to successfully describe the molecular and physiological aspects of apoptosis and DNA repair in discrete hematopoietic progenitor cell compartments. The studies in the first two aims are largely descriptive but are still important to carry out and catalog. The major concern is whether the studies can proceed beyond the descriptive to more mechanistic insights. Its not clear that manipulation of single proteins will be able to explain, even in part, any significant differences in apoptosis or DNA repair among different progenitor cell compartments. Usually these processes are regulated by multiple overlapping or redundant pathways. Also, the proposal does not adequately address how these observations will be used to understand homeostasis of progenitor cell compartments in vivo, either during normal development or in responses to specific stresses. The studies in the first two aims on apoptosis and DNA repair in all the subsets are extremely ambitious and somewhat unfocused, although at this stage it is an advantage to seek widely. Characterizing multiple progenitor cell compartments in response to multiple apoptotic signals and in response to multiple DNA damaging agents is already a large amount of work. The focus in subsequent aim 2 on HSC and granulocyte/macrophage progenitors (GMP) is wise .It was not made clear how the PI will introduce genotoxic stress, does the method matter? In addition, the applicant proposes to genetically modify each progenitor compartment in multiple ways in an effort to understand any functional differences. Certainly, not all of this could be accomplished and the application fails to prioritize the goals. If mutant mice must be used instead of overexpression and knock-down vectors, then the timeline may become unreasonable. The applicant has recruited experts in the respective fields as co-PIs (Oates and Morrison). The third aim is quite interesting, albeit much more risky, and promises to provide new insight into the emergence and evolution of a cancer stem cell. The junB-null model has been developed by the applicant, and the experiments proposed here are a logical extension of that work. The question as to whether the junB deletion might alter apoptotic and/or DNA repair pathways is reasonable and the experiments proposed here should provide answers. The proposal to clone the putative 1,17 fusion gene is risky, but probably the right thing to try at this point. The alternative strategy briefly mentioned here, a retroviral insertion screen to identify genes that cause blast crisis in this model, is a major undertaking and probably will not be possible within a reasonable time frame. Another reviewer considered the screen for breakpoints promising and may provide early targets to consider. The proposal tackles a fundamental aspect of cell biology that has important and broad implications for human therapy and has been an area of intensive investigation in cancer research.The identification of the cancer stem cell is important and exciting yet still somewhat controversial. The PI has developed an outstanding model and has strong preliminary data that the cancer stem can be studied and potential targets identified. The proposed studies in this model could shed insight into how leuckemic stem cells are derived from HSC, and how the balance of normal repair/apoptosis is deregulated in cancer. Understanding how cells escape from apoptosis is relevant to cancer and ageing. It is clear that defective DNA repair leads to genomic instability and cancer. Many of the players are identified and extensively studied, but this study has the potential to identify mechanisms that occur during the natural development of a cancer in vivo, and to develop model therapeutics. The proposed research thus has high significance. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Passegue is a highly productive expert in hematopoiesis with a long-term commitment to these studies. She received her Ph.D. in 1990 at the Paris University, and did postdoctoral work with Ernst Wagner at the Research Institute of Molecular Pathology in Vienna and with Irving Weissman at Stanford. She developed the junB null mouse model of leukemia while in the Wagner lab, and demonstrated in the Weissman lab that this involved a misregulation of a stem cell population. There she also participated in studies on the effect of the MLL fusion oncogene in hematopoetic stem cells. These studies resulted in multiple first author publications in top journals, including Nature Genetics, two in Cell, and PNAS. More recently she has had a senior author paper in Cell, multiple middle author papers in other top journals, and numerous high profile review articles. Active international collaboration is already an important element of the work and the PI is a regular invited speaker at international cancer meetings. She was appointed Assistant Professor in the Department of Medicine at USCF in 2005. In recognition of her accomplishments and promise, she has received a Jose Carreras Leukemia Foundation Fellowship, and a career development award from the American Society of Hematology. Dr. Passegue describes a coherent, focused, and ambitious plan to develop a leading research program on leukemia stem cells. However, this is not expressed in the form of measurable objectives and targets and there is also no indication of how progress would be measured and reviewed formally. She has already developed a strong mentoring committee. She has demonstrated a long-term commitment to this field and is clearly in the vanguard. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Institutional commitment to the PI is quite high. She was recruited to USCF to lead their effort in hematopoetic stem cell biology with a tenure track appointment in the Department of Medicine, with concurrent appointments in the Microbiology Department, the Institute for Regenerative Medicine (IRM), and the Comprehensive Cancer Center. She received a generous start-up package and occupies 1600 square feet of personal lab space. She has access to facilities including tissue culture, FACS, microscopes, a vivarium, etc. for her studies. A CIRM-funded facilities grant will provide an additional FACS machine. She has also been provided with a very strong mentoring committee and has outstanding colleagues available. The intellectual environment is outstanding, as are the other institutional resources. UCSF provides a terrific environment and has a long track record of supporting junior faculty and stem cell research. An excellent letter of support is provided by the Dean (Kessler) and IRM Director (Kriegstein), who assure us that Dr. Passegue will not lack resources or opportunities. The IRM has also ensured low teaching commitments to permit personal research and development. The Institute for Regenerative Medicine at UCSF is a growing, vibrant center that should provide an ideal setting for her career development.. DISCUSSION: This proposal addresses the role of apoptosis and the response to DNA damage during hematopoiesis in a mouse model of leukemia. These are important questions in stem cell biology and tumorigenesis. The PI is uniquely able to perform these experiments because she developed a leukemia animal model, one of the best animal models available. One reviewer considered the first two aims to be descriptive but noted that the aims were interesting in the context of one of the best animal models in the field. Reviewers commented that the applicant is an accomplished and productive researcher, well-integrated in the field. The criticism was made that apoptosis and DNA damage are complex pathways with redundancies and that it may be naïve to think that the PI will see causality from this analysis. One panel member questioned the significance of the research given that many (especially childhood) leukemias are curable, but the reviewers noted that this model was one of myeloid, not lymphoid, leukemia which is not easily curable. One reviewer noted that one could take issue that the model is generated by inactivating junB, which doesn’t necessarily show up in myeloid tumors. However, it was noted that the value to this study is that leukemia is a model system where one can purify cells, study them, and define leukemic stem cells in order to develop concepts and conduct proof-of- principle studies. The junB mutation model may not be perfect, it does enable one to address the specific mechanisms of stem cell dysregulation in tumorigenesis. One reviewer identified the career development plan as an area of weakness, but all reviewers commented on the high level of institutional commitment to the applicant. The letters of support for this applicant from the Dean and from the Head of the Program were characterized by one reviewer as “beautiful” and “moving”. The proposal was regarded as enabling the further development of the applicant to be a leader in the field.