Mechanisms Underlying the Responses of Normal and Cancer Stem Cells to Environmental and Therapeutic Insults
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) and myeloid progenitor cells to investigate how they respond to 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 deregulation during cancer development. Such information will 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 deregulations 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 will be used to design novel and much-needed therapies that will specifically target CSCs while sparing normal stem cells.
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.
Escape from apoptosis and increased genomic instability resulting from defective DNA repair processes are often associated with cancer development, aging and stem cell defects. Adult stem cells play an essential role in the maintenance of normal tissue. Removal of superfluous, damaged and/or dangerous cells is a critical process to maintain tissue homeostasis and protect against malignancy. Yet much remains to be learned about the mechanisms by which normal stem and progenitor cells respond to environmental and therapeutic genotoxic insults. Here, we have used the hematopoietic system as a model to investigate how cancer-associated mutations affect the behaviors of specific stem and progenitor cell populations. Our work during the first year of the CIRM New Faculty award has revealed the differential use of DNA double-strand break repair pathways in quiescent and proliferative hematopoietic stem cells (HSCs), which has clear implications for human health. Most adult stem cell populations, including HSCs, remain in a largely quiescent (G0), or resting, cell cycle state. This quiescent status is widely considered to be an essential protective mechanism stem cells use to minimize endogenous stress caused by cellular respiration and DNA replication. However, our studies demonstrate that quiescence may also have detrimental and mutagenic effects. We found both quiescent and proliferating HSCs to be similarly protected from DNA damaging genotoxic insults due to the expression and activation of cell type specific protective mechanisms. We demonstrate that both quiescent and proliferating HSCs resolve DNA damage with similar efficiencies but use different repair pathways. Quiescent HSCs preferentially utilize nonhomologous end joining (NHEJ) - an error-prone DNA repair mechanism - while proliferating HSCs essentially use homologous recombination (HR) - a high-fidelity DNA repair mechanism. Furthermore, we show that NHEJ-mediated repair in HSCs is associated with acquisition of genomic rearrangements. These findings suggest that the quiescent status of HSCs can, on one hand, be protective by limiting cell-intrinsic stresses but, on the other hand, be detrimental by forcing HSCs to repair damaged DNA with an error-prone mechanism that can generate mutations and eventually cause hematological malignancies. Our results have broad implications for cancer development and provide the beginning of a molecular understanding of why HSCs, despite being protected, are more likely than other cells in the hematopoietic system (i.e., myeloid progenitors) to become transformed. They also partially explain the loss of function occurring in HSCs with age, as it is likely that over a lifetime HSCs have acquired and accumulated numerous NHEJ-mediated mutations that hinder their cellular performance. Finally, our findings may have direct clinical applications for minimizing secondary cancer development. Many solid tumors and hematological malignancies are currently treated with DNA damaging agents, which may result in therapy-induced myeloid leukemia. Our results suggest that it might be beneficial to induce HSCs to cycle before initiating treatment, to avoid inadvertently mutating the patient's own HSCs by forcing them to undergo DNA repair using an error-prone mutagenic mechanism.
Our work during the second year of the CIRM New Faculty award has lead to the discovery of at least one key reason why blood-forming stem cells can be susceptible to developing genetic mutations leading to adult leukemia or bone marrow failures. Most adult stem cells, including hematopoietic stem cells (HSCs), are maintained in a quiescent or resting state in vivo. Quiescence is widely considered to be an essential protective mechanism for stem cells that minimizes endogenous stress associated with cellular division and DNA replication. However, we demonstrate that HSC quiescence can also have detrimental effects. We found that HSCs have unique cell-intrinsic mechanisms ensuring their survival in response to ionizing irradiation (IR), which include enhanced pro-survival gene expression and strong activation of a p53-mediated DNA damage response. We show that quiescent and proliferating HSCs are equally radioprotected but use different types of DNA repair mechanisms. We describe how nonhomologous end joining (NHEJ)-mediated DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. These results demonstrate that quiescence is a double-edged sword that, while mostly beneficial, can render HSCs intrinsically vulnerable to mutagenesis following DNA damage. Our findings have important implications for cancer biology. They indicate that quiescent stem cells, either normal or cancerous, are particularly prone to the acquisition of mutations, which overturns the current dogma that cancer development absolutely requires cell proliferation. They help explain why quiescent leukemic stem cells (LSC), which currently survive treatment in most leukemia, do in fact represent a dangerous reservoir for additional mutations that can contribute to disease relapse and/or evolution, and stress the urgent need to develop effective anti-LSC therapies. They also have direct clinical applications for minimizing the risk of therapy-related leukemia following treatment of solid tumors with cytotoxic agents. By showing that proliferating HSCs have significantly decreased mutation rates, with no associated change in radioresistance, they suggest that it would be beneficial to induce HSCs to enter the cycle prior to therapy with DNA-damaging agents in order to enhance DNA repair fidelity in HSCs and thus reduce the risk of leukemia development. While this possibility remains to be tested in the clinic using FDA approved agents such as G-CSF and prostaglandin, it offers exciting new directions for limiting the deleterious side effects of cancer treatment. Our findings also have broad biological implications for tissue function. While the DNA repair mechanism used by quiescent HSCs can indeed produce defective cells, it is likely not detrimental for the organism in evolutionary terms. The blood stem cell system is designed to support the body through its sexually reproductive years, so the genome can be passed along. The ability of quiescent HSCs to survive and quickly undergo DNA repair in response to genotoxic stress supports this goal, and the risk of acquiring enough damaging mutations in these years is minimal. The problem occurs with age, as these long-lived cells have spent a lifetime responding to naturally occurring insults as well as the effects of X-rays, medications and chemotherapies. In this context, the accumulation of NHEJ-mediated DNA misrepair and resultant genomic damages could be a major contributor to the loss of function occurring with age in HSCs, and the development of age-related hematological disorders. We are now using this work on normal HSCs as a platform to understand at the molecular level how the DNA damage response and the mechanisms of DNA repair become deregulated in leukemic HSCs during the development of hematological malignancies.
Our work during the third year of the CIRM New Faculty award has extended and broaden up our investigations in two novel directions that are still within the scope of our initial Aims: 1) identifying novel stress-response mechanisms that preserve hematopoietic stem cells (HSC) fitness during periods of metabolic stress; and 2) understanding how deregulations in DNA repair mechanisms contribute to the aberrant functions of old and transformed HSCs. Blood development is organized hierarchically, starting with a rare but well-defined population of HSCs that give rise to a series of committed progenitors and mature cells with exclusive functional and immunophenotypic properties. HSCs are the only cells within the hematopoietic system that self-renew for life, whereas other hematopoietic cells are short-lived and committed to the transient production of mature blood cells. Under steady-state conditions, HSCs are a largely quiescent, slowly cycling cell population, which, in response to environmental cues, are capable of dramatic expansion and contraction to ensure proper homeostatic replacement of all blood cells. While considerable work has deciphered the molecular networks controlling HSC activity, still little is known about how these mechanisms are integrated at the cellular level to ensure life-long maintenance of a functional HSC compartment. HSCs reside in hypoxic niches in the bone marrow microenvironment, and are mostly kept quiescent in order to minimize stress and the potential for damage associated with cellular respiration and cell division. Last year, we showed that HSCs can also engage specialized response mechanisms that protect them from the killing effect of environmental stresses such as ionizing radiation (IR) (Mohrin et al., Cell Stem Cell, 2010). We demonstrated that long-lived HSCs, in contrast to short-lived myeloid progenitors, have enhanced expression of pro-survival members of the bcl2 gene family and robust induction of p53-mediated DNA damage response, which ensures their specific survival and repair following IR exposure. We reasoned that HSCs have other unique protective features, which allow them to contend with a variety of cellular insults and damaged cellular components while maintaining their life-long functionality and genomic integrity. Now, we show that HSCs use the self-catabolic process of autophagy as an essential survival mechanism in response to metabolic stress in vitro or nutriment deprivation in vivo. Last year, we also reported that although HSCs largely survive genotoxic stress their DNA repair mechanisms make them intrinsically vulnerable to mutagenesis (Mohrin et al., Cell Stem Cell, 2010). We showed that their unique quiescent cell cycle status restricts them to the use of the error-prone non-homologous end joining (NHEJ) DNA repair mechanism, which renders them susceptible to genomic instability and transformation. These findings provide the beginning of an understanding of why HSCs, despite being protected at the cellular level, are more likely than other hematopoietic cells to initiate blood disorders (Blanpain et al., Cell Stem Cell, review, 2011). Such hematological diseases increase with age and include immunosenescence (a decline in the adaptive immune system) as well as the development of myeloproliferative neoplasms, leukemia, lymphoma and bone marrow failure syndromes. Many of these features of aging have been linked to changes in the biological functions of old HSCs. Gene expression studies and analysis of genetically modified mice have suggested that errors in DNA repair and loss of genomic stability in HSCs are driving forces for aging and cancer development. However, what causes such failures in maintaining HSC functionality over time remains to be established. We therefore asked whether the constant utilization of error-prone NHEJ repair mechanism and resulting misrepair of DNA damage over a lifetime could contribute to the loss of function and susceptibility to transformation observed in old HSCs. Similarly, we started investigating how mutagenic DNA repair could contribute to the genomic instability of HSC-derived leukemic stem cells (LSC).
Our work during the fourth year of the CIRM New Faculty award has been focused on achieving the goals set forth last year for the two first aims of the grant: 1) identifying the stress-response mechanisms that preserve hematopoietic stem cells (HSC) fitness during periods of metabolic stress; and 2) understanding how deregulations in DNA repair mechanisms contribute to the aberrant functions of old HSCs and the aging of the blood system.
Blood development is organized hierarchically, starting with a rare but well-defined population of HSCs that give rise to a series of committed progenitors and mature cells with exclusive functional and immunophenotypic properties. HSCs are the only cells within the hematopoietic system that self-renew for life, whereas other hematopoietic cells are short-lived and committed to the transient production of mature blood cells. Under steady-state conditions, HSCs are a largely quiescent, slowly cycling cell population, which, in response to environmental cues, are capable of dramatic expansion and contraction to ensure proper homeostatic replacement of all needed blood cells. While considerable work has deciphered the molecular networks controlling HSC activity, still little is known about how these mechanisms are integrated at the cellular level to ensure life-long maintenance of a functional HSC compartment.
HSCs reside in hypoxic niches in the bone marrow microenvironment, and are mostly kept quiescent in order to minimize stress and the potential for damage associated with cellular respiration and cell division. Previously, we found that HSCs also have a unique pro-survival wiring of their apoptotic machinery, which contribute to their enhanced resistance to genotoxic stress (Mohrin et al., Cell Stem Cell, 2010). Now, we identified autophagy as an essential mechanism protecting HSCs from metabolic stress (Warr et al., Nature, in press). We show that HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy following ex vivo cytokine withdrawal and in vivo caloric restriction. We demonstrate that FoxO3a is critical to maintain a gene expression program that poise HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FoxO3a-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system.
Previous studies have also suggested that increased DNA damage could contribute to the functional decline of old HSCs. Therefore, we set up to investigate whether the reliance on the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism we previously identified in young HSCs (Mohrin et al., Cell Stem Cell, 2010) could render old HSCs vulnerable to genomic instability. We confirm that old HSCs have increased numbers of γH2AX DNA foci but find no evidence of associated DNA damage. Instead, we show that γH2AX staining in old HSCs entirely co-localized with nucleolar markers and correlated with a significant decrease in ribosome biogenesis. Moreover, we observe high levels of replication stress in proliferating old HSCs leading to severe functional impairment in condition requiring proliferation expansion such as transplantation assays. Collectively, our results illuminate new features of the aging HSC compartment, which are likely to contribute to several facets of age-related blood defects (Flach et al, manuscript in preparation).
Our work during the fifth and last year of our CIRM New Faculty award has been essentially focused on understanding how deregulations in DNA repair mechanisms contribute to the aberrant functions of old hematopoietic stem cells (HSC) and the aging of the blood system.
- Nature (2014) Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. (PubMed: 25079315)
- Nature (2013) FOXO3A directs a protective autophagy program in haematopoietic stem cells. (PubMed: 23389440)
- Cell Stem Cell (2013) Myeloproliferative Neoplasia Remodels the Endosteal Bone Marrow Niche into a Self-Reinforcing Leukemic Niche. (PubMed: 23850243)
- Cell Stem Cell (2011) DNA-damage response in tissue-specific and cancer stem cells. (PubMed: 21211780)
- Cell Stem Cell (2010) Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis. (PubMed: 20619762)