Tannishtha Reya - US grants

Hematopoietic stem cells (HSCs) have the capacity to generate all the cells of the blood. A defining feature of HSCs is their ability to perpetuate themselves through self-renewal. While the phenotypic and functional properties of HSCs have been extensively characterized a fundamental question that remains unanswered is how self-renewal is regulated. Our work has revealed that I_-catenin,a key mediator of Wnt signaling, can promote self-renewal of HSC in vitro. Moreover, Axin, which enhances _-catenin degradation, causes inhibition of growth and reconstitution by HSC. These studies reveal an important role for 13-cateninin the regulation of HSC growth and self-renewal. To further elucidate the role of Wnt and {3-cateninsignaling in HSC, we propose to 1) Determine whether Wnt signaling regulates HSC function in vivo, 2) Examine whether the effects of I_-catenin and Axin on HSC reflect the activity of Wnt proteins and 3) Identify the molecular mechanisms by which I_-catenin exerts its effects on HSC self-renewal. These studies will not only advance our understanding of self-renewal, a key characteristic of all stem cells, but will also help develop strategies for in vitro expansion of stem cells, and thereby contribute to improving transplantation therapies for hematopoietic and degenerative disorders.

A large body of work exists on the efficacy of hematopoietic stem cells (HSCs) to rescue patients from lethal irradiation such as in the case of a nuclear disaster. These observations form the basis for the field of bone marrow transplantation and its successes in treating malignancies. This suggests that an important avenue of therapy for radiation-induced damage may be the delivery of hematopoietic stem cells, or the stimulation of accelerated repair of endogenous stem cells. The recent identification of signaling modulators that can control and enhance hematopoietic stem cell growth, renewal and regeneration raise the possibility that the same modulators may be utilized to induce rapid regeneration during time of need. Our own experiments have identified the Wnt signaling pathway as a modulator of homeostatic hematopoietic stem cell growth, and as a signal that is upregulated during regeneration of HSCs after damage. Thus, we propose to test 1) Whether Wnt signaling can stimulate proliferation of human hematopoietic stem cells such that they may be expanded and stored for delivery as a cellular therapy following radiation exposure and 2) Whether direct in vivo delivery of activators of Wnt signaling will allow enhanced regeneration of HSCs following exposure. Cumulatively aims would allow the identification of both cellular and molecular deliverables that can be utilized rapidly in the aftermath of a nuclear disaster.

During aging, hematopoietic stem cells (HSCs) are able to function normally under homeostatic conditions but are unable to mount effective regenerative responses under conditions of acute damage. Thus, in response to bleeding, infection or chemotherapy, elderly people often fail to replenish their blood effectively,and have an increased risk of hematopoietic disorders such as anemia and neutropenia that can often be fatal. To understand why this hematopoietic dysfunction occurs during aging, it is important to identify the molecular mechanisms that allow young HSCs to respond to acute damage and determine how these mechanisms fail in the aging hematopoietic system. To understand the molecular signals that normally allow the effective regeneration of the blood, we have studied animals treated with the chemotherapeutic drug Cyclophosphamide (Cy) and the growth factor G-CSF which causes an acute loss of proliferating progenitors in the bone marrow followed by expansion of HSCs to regenerate the progenitor pool. Using this as a damage model, we have found that that the Wnt signaling is sharply upregulated in a large fraction of HSCs responding to Cy/G-CSF suggesting that Wnt signaling may be a critical mediator of HSC regeneration after damage. In order to test this hypothesis, we now propose to define the significance of Wnt activation in regenerating HSCs and determine if this activity changes as HSCs age. Identifying the mechanisms that underlie the regenerative response to Cyclophosphamide/ G-CSF treatment will enhance our ability to harvest and expand stem cells for transplantation therapy. Furthermore, understanding the basis of impaired regeneration in the aging hematopoietic system may allow us to design novel means to improve the health and quality of life of aging patients.

DESCRIPTION Abstract Hematopoietic stem cells (HSCs) have the unique capacity to regenerate themselves, as well as to generate all the lineages of the blood. The fine balance between renewal and commitment is effortlessly achieved in vivo. Yet how this balance is regulated is one of the most fundamental unsolved problems in biology. Although in the last four decades we have learned a lot about HSCs, ultimately we have remained incapable of recapitulating what they do in vivo. What's missing is a true view of the signals and the interactions that these cells normally experience in their native microenvironment. Here we propose to study how HSC renewal is controlled by the stem cell niche by imaging HSC interactions with the stromal cell microenvironment, and imaging the signals they receive in vivo, under both homeostatic conditions as well as conditions of regeneration and transformation. To this end, we propose to develop a transgenic system in which hematopoietic stem cells are specifically fluorescently labeled and can be detected in vivo. These cells can then be visualized using live imaging technology to visualize cells within the bone marrow niche in living animals. This would allow a dynamic view of the cell-cell interactions, signaling pathways and cell movements that regulate normal HSC function and transformation. Understanding the in vivo signals and interactions that occur during these processes is key to utilizing these signals for expansion of human hematopoietic stem cells for transplantation. In addition, defining how mechanisms of normal growth and division are properly controlled in stem cells may allow us to find ways to correct their dysregulation in cancer. Delineating the basis of stem cell and cancer cell self-renewal in vivo will in the long term, contribute to novel approaches to regenerative medicine and cancer therapy. Public Health Relevance The research proposed here will allow for in vivo monitoring of the signals and niche components that contribute

DESCRIPTION (provided by applicant): Stem cells have a tremendous capacity for self-renewal and regeneration of damaged tissues. Although we have learned a great deal about how these processes are regulated from studies of stem cells in culture, we have remained largely in the dark about the dynamic behavior of stem cells in their native microenvironment. To begin to view and understand the mechanisms and interactions that make stem cells renew, differentiate, and regenerate after injury it is essential to develop strategies that allow imaging of these cells in the context of a living animal. We have recently utilized a combination of transgenic approaches and live animal confocal microscopy to develop a novel imaging strategy that allows us to visualize in real time how individual hematopoietic stem cells (HSCs) behave in vivo over extended periods of time. Using this strategy we now propose to define 1) the influence of the niche on asymmetric and symmetric division of HSCs in vivo 2) how the niche and the interaction of a stem cell with the niche changes during regeneration and 3) how this changes during oncogenic transformation. Dynamic high resolution viewing of living stem cells in their native environment will be a powerful new and exciting avenue to understand endogenous regulation of stem cell function and how this is altered in regeneration and cancer. PUBLIC HEALTH RELEVANCE: The research proposed here will allow for in vivo monitoring of the signals and niche components that contribute to stem cell self renewal, regeneration and transformation. Insights gained from these studies will allow a better understanding of the signals influencing stem cells and aid in the development of new stem cell based therapeutic approaches enhancing human hematopoietic repair and may uncover new targets for treating leukemias.

DESCRIPTION (provided by applicant): The unique ability of hematopoietic stem cells to balance self-renewal with differentiation is crucial for development and homeostasis as well as for regeneration and repair. One fundamental way in which HSCs may be able to balance both self-renewal and differentiation is by utilizing a combination of asymmetric or symmetric division, differing modes of division mediated by the differential inheritance of cell fate determinants. In previous studies we have used live cell imaging to show that hematopoietic stem/progenitor cells can undergo both symmetric and asymmetric divisions. But how these types of divisions are controlled, and whether the loss of these mechanisms can affect cell fate and maintenance of the stem cell state are specific questions that remain to be answered. To address these issues, we have focused on Lis1, a dynein-binding protein that anchors the spindle to the cellular cortex and thereby regulates spindle orientation and correct inheritance of fate determinants in the nervous system. Using conditional knockout mice, we have found that loss of Lis1 leads to severe defects in the establishment of the hematopoietic system and a failure of self-renewal. Using real time imaging approaches that we have developed, we now propose to define whether the defects in hematopoietic stem cell fate in Lis1-deficient mice are linked to defects in asymmetric division as well as elucidate the downstream mechanisms important for Lis1 function. A better understanding of the mechanisms that regulate the balance between self-renewal and differentiation may enable development of new strategies to control hematopoietic stem cells and accelerate hematopoietic regeneration in times of need.

?R35 Abstract *REVISED* Tannishtha Reya, Ph.D Grant # 1R35CA197699-01 Over the last few decades our remarkable understanding of the molecular basis of oncogenesis has begun to influence cancer therapy at many levels. In the recent past this has led us away from simply relying on blunt tools like chemotherapy and radiation and towards including and designing more molecularly-targeted therapies. Despite these advances, cancer continues to claim millions of lives worldwide. In some very significant part this extraordinary toll is due to our inability to detect the disease early. Early detection of cancer vastly increases the likelihood of effective and durable responses to therapy, and can make the difference between life and death. Considering the potential impact of effective early detection, research in this area lags markedly behind the development of therapeutics. While the field of cancer research has largely focused on developing new therapeutics, there is a critical need to combine this effort with designing new and sensitive strategies that will allow effective early detection. The work we describe here focuses on bridging this gap and providing a more balanced approach to controlling cancer, by combining molecular strategies for design and development of early detection tools coupled with understanding of control points of the transition from benign hyperproliferative phase to a more malignant phase, allowing development of new therapies that can be used for more effective interventions.

DESCRIPTION (provided by applicant): Pancreatic cancer is now the 4th leading cause of cancer death in the United States. Despite some recent advances in systemic therapy, survival remains dismal in large part due to the aggressive nature of this disease and its propensity for early metastasis. Thus, there is a critical need to define new therapeutic targets that can more effectively block tumor growth and spread. To define new ways to approach cancer therapy we have focused on stem cell signals that are hijacked to drive cancer initiation, propagation, and recurrence. Using this approach we previously identified the stem cell fate determinant Musashi (Msi) as critically required for progression of hematologic malignancies. Importantly, we have recently found that Msi is highly expressed in pancreatic cancer cells in both mouse models of the disease and in primary patient samples. Further, our preliminary studies indicate that Msi inhibition functionally blocks the growth of pancreatic cancer cell lines as well as patient samples in vitro and in xenografts. These data have led us to hypothesize that pancreatic cancer growth and propagation is critically dependent on Msi. We will test this by determining 1) whether Msi expression marks cancer stem cells, cells with preferential capacity for pancreatic cancer cell propagation, 2) whether Msi is necessary for propagation of pancreatic cancer in genetically engineered mouse models, and 3) whether Msi is necessary for human pancreatic cancer propagation in primary patient-derived xenografts. These studies will help define whether Msi is a key regulator of pancreatic cancer and if it may be an effective target for therapy.

DESCRIPTION (provided by applicant): The unique ability of hematopoietic stem cells to balance self-renewal with differentiation is crucial for development and homeostasis as well as for regeneration and repair. One fundamental way in which HSCs may be able to balance both self-renewal and differentiation is by utilizing a combination of asymmetric or symmetric division, differing modes of division mediated by the differential inheritance of cell fate determinants. In previous studies we have used live cell imaging to show that hematopoietic stem/progenitor cells can undergo both symmetric and asymmetric divisions. But how these types of divisions are controlled, and whether the loss of these mechanisms can affect cell fate and maintenance of the stem cell state are specific questions that remain to be answered. To address these issues, we have focused on Lis1, a dynein-binding protein that anchors the spindle to the cellular cortex and thereby regulates spindle orientation and correct inheritance of fate determinants in the nervous system. Using conditional knockout mice, we have found that loss of Lis1 leads to severe defects in the establishment of the hematopoietic system and a failure of self-renewal. Using real time imaging approaches that we have developed, we now propose to define whether the defects in hematopoietic stem cell fate in Lis1-deficient mice are linked to defects in asymmetric division as well as elucidate the downstream mechanisms important for Lis1 function. A better understanding of the mechanisms that regulate the balance between self-renewal and differentiation may enable development of new strategies to control hematopoietic stem cells and accelerate hematopoietic regeneration in times of need.