Blood Cancer

Coding Dimension ID: 
287
Coding Dimension path name: 
Cancer / Blood

Combinatorial Chemistry Approaches to Develop LIgands against Leukemia Stem Cells

Funding Type: 
New Faculty I
Grant Number: 
RN1-00561
ICOC Funds Committed: 
$2 567 397
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Cancer Stem Cell
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Various cells and organs in the human body originate from a small group of primitive cells called stem cells. Human cancer cells were also recently found to arise from a group of special stem cells, called cancer stem cells (CSCs). At present, cancer that has spread throughout the body (metastasized) is difficult to treat, and survival rates are low. One major reason for therapeutic failure is that CSCs are relatively resistant to current cancer treatments. Although most mature cancer cells are killed by treatment, resistant CSCs will survive to regenerate additional cancer cells and cause a recurrence of cancer. As opposed to other human stem cells, CSCs have their own unique molecules on their cell surface. This project aims to develop agents that specifically target the unique cell surface molecules of CSCs. These agents will have the potential to eradicate cancer from the very root, i.e., from the stem cells (CSCs) that produce mature cancer cells. In this project, we will develop agents that specifically target leukemia stem cells to determine the feasibility of our approach. Leukemia is the fourth most common cause of cancer death in males and the fifth in females. If our approach is successful, we can use the same approach for other cancer types. To develop these specific agents, we will screen a library of billions of molecules to identify those that specifically target the unique cell surface molecules of leukemia stem cells (LSCs). After we identify these specific molecules, we will optimize their structure to increase their specific binding to LSCs. Specific binding to LSCs is crucial, as the optimized molecules will be able to uniquely kill LSCs and spare normal blood cells. Many leukemia patients need stem cell transplantation during treatment. There are two approaches to harvesting stem cells for transplantation: those harvested from patients themselves and those harvested from healthy donors. Stem cells harvested from healthy donors need to genetically match patients’ cells. Otherwise, these transplanted cells from the donor recognize the recipient’s (host or patient) cells as non-self cells and attack these cells. This response leads to a serious disease called graft-versus-host disease (GVHD). It is often difficult to find matched donors. Stem cells harvested from patients are usually not used for the treatment of acute leukemia because they are contaminated with LSCs that will lead to recurrence of leukemia after transplantation. If this project is successful, the targeting agents developed in this project can be used to eliminate the contaminating LSCs and decrease the leukemia recurrence after transplantation.
Statement of Benefit to California: 
Acute leukemia is the sixth most common cause of cancer death in males and females in California. The outcome for acute leukemia is poor and over 70% of patients will die from this disease. This project aims to develop therapeutic agents that specifically target leukemia stem cells and therefore eradicate leukemia from its root. These agents can also be used for stem cell transplantation. Many leukemia patients need stem cell transplantation during treatment. There are two approaches to harvesting stem cells for transplantation: those harvested from patients themselves and those harvested from healthy donors. Stem cells harvested from healthy donors need to genetically match patients’ cells. Otherwise, these transplanted cells from the donor recognize the recipient’s (host or patient) cells as non-self cells and attack these cells. This response leads to a serious disease called graft-versus-host disease (GVHD). It is often difficult to find matched donors. This is especially true in California because of the genetically diversified population. Stem cells harvested from patients are usually not used because they are contaminated with leukemia stem cells that will lead to recurrence of leukemia after transplantation. If this project is successful, the targeting agents developed in this project can be used to eliminate the contaminated leukemia cells and decrease the likelihood of leukemia recurrence after transplantation. The ligands developed in this project can be used for targeted therapy for leukemia. Since no such ligands have been identified so far that specifically target leukemia stem cells, these ligands can be patented and eventually commercialized. This may have huge financial benefits to California. If this project is successful, the same approach can be used to treat other cancers and for the development of more commercialized drugs. If this grant is funded, it will secure my career as a physician-scientist in stem cell and cancer research. The physician-scientist is a diminishing breed in that it is difficult for physicians to do research while meeting the huge demands of the clinic. However, there is a huge gap between basic research and clinical applications. This gap is in part traced to the fact that it is difficult to find researchers who know and can integrate clinical needs with basic research. I consider myself a promising physician-scientist who has received extensive, rigorous and systematic training in medical science and basic research ([REDACTED]). If this grant is funded, I will not only carry out this important research, but this will also give me protected time for this research.
Progress Report: 
  • Human cancer cells were recently found to arise from a group of special stem cells, called cancer stem cells (CSCs). At present, cancer that has spread throughout the body (metastasized) is difficult to treat, and survival rates are low. One major reason for therapeutic failure is that CSCs are relatively resistant to current cancer treatments. Although most cancer cells are killed by treatment, resistant CSCs will survive to regenerate additional cancer cells and cause a recurrence of cancer. As opposed to other human stem cells, CSCs may have some unique molecules that can be targeted for cancer treatment. This project is to use such technologies as our patented one-bead one-compound technology (OBOC) to develop small molecules that can specifically target cancer stem cells. With OBOC, trillions copies of small molecules are synthesized in tiny beads around 90 microns. During development, millions of molecules can be screened against cancer stem cells with hours to days. So far, we have identified six molecules that target CSC. Currently, we are optimizing these molecules to increase their efficiency of these molecules on CSC. Once fully developed, these molecules will have the potential to eradicate cancer from the very root, i.e., from the stem cells (CSCs) that produce mature cancer cells.
  • Acute myeloid leukemia is a group of serious blood malignant diseases. The treatment outcome is poor, in large part, to the fact that a small group of cells named leukemia stem cells can survive treatment, regenerate more leukemic cells and cause recurrence. This project aims to improve the treatment outcomes of acute leukemia by eradicating leukemia stem cells. During the previous two years, we identified several small molecules that can specifically bind to leukemia stem cells. Over the last one year, we determined that one of these small molecules has the potential to work like a “smart missile” to guide the delivery of chemotherapeutic drugs to leukemia stem cells. More specifically, we linked this small molecule on the surface of nanoparticles that are small particles with the size of about 1/100th of one micron (much smaller than the width of a human hair). Inside of these nanoparticles, we can load chemotherapeutic drugs. We found that our small molecules can specifically attach the nanoparticles to leukemia stem cells, and deliver the drug load to the inside of the cells. Therefore, these “smart” nanoparticles can potentially target leukemia stem cells, and eradicate leukemia from the very root. Furthermore, chemotherapeutic drugs formulated in these nanoparticles are less toxic, suggesting that high-dose chemotherapeutic drugs can be given to patients to treat leukemia without increasing the horrendous toxicity associated with regular chemotherapy.
  • Acute myeloid leukemia is a group of serious blood malignant diseases. The treatment outcome is poor, in large part, due to the fact that a small group of cells named leukemia stem cells can survive treatment, regenerate more leukemic cells and cause recurrence. This project aims to improve the treatment outcomes of acute leukemia by eradicating leukemia stem cells. We identified one molecule that can specifically bind to leukemia stem cells. We also developed nanoparticles that are small particles with the size of about 1/100th of one micron (much smaller than the width of a human hair). Inside of these nanoparticles, we can load chemotherapeutic drugs, such as daunorubicin that is one of the two drugs used for the upfront treatment of acute leukemia. When we attached the stem cell-targeting molecules on the surface of nanoparticles, these nanoparticles work like “small missiles” that can seek and delivery daunorubicin into leukemia stem cells. We have shown that these “smart” nanoparticle can delivery chemotherapeutic drug daunorubicin to leukemia cells directly isolated from clinical patient specimens, and kills these cells more efficient that the regular nanoparticles. Therefore, these “smart” nanoparticles can potentially target leukemia stem cells, and eradicate leukemia from the very root. Furthermore, chemotherapeutic drugs formulated in these nanoparticles are less toxic, suggesting that high-dose chemotherapeutic drugs can be given to patients to treat leukemia without increasing the horrendous toxicity associated with regular chemotherapy.
  • Acute myeloid leukemia (AML) is the most common acute leukemia in adults and a very serious disease. Most AML cells arise from a group of special stem cells, named leukemia stem cells (LSCs). One major reason for treatment failure is that LSCs are relatively resistant to current treatments. Although most leukemia cells are killed by treatment, resistant LSCs will survive to regenerate additional leukemia cells and cause a recurrence of leukemia. Recently, we have developed a small molecule that can recognize and bind to AML LSCs. We have also developed tiny particles named nanomicelles. These nanomicelles have a size of about 1-2/100th of one micron (one millionth of a meter), and can be loaded with chemotherapy drug called daunorubicin that can kill LSCs. In this project, we will coat the drug-loaded nanomicelles with small molecules that specifically bind and kill LSCs. In patient’s body, these drug-loaded nanomicelles will work like “smart bombs”, and deliver a high concentration of daunorubicin to kill LSCs. Over the last one year, we found that these LSC-targeting nanomicelles could target and kill LSC more efficiently that free daunorubicin or nanomicelles that do not target LSC. We also found that, compared to free daunorubicin commonly used in the treatment of AML now, daunorubicin in nanomicelles could raise the blood daunorubicin concentration by more than 20 times. This is clinically significant as leukemia cells and LSC are located inside blood vessels and bone, and have direct contact with blood. Therefore, increase in blood daunorubicin concentration may represent more efficiency in killing leukemia and LSC.
  • Acute myeloid leukemia (AML) is the most common acute leukemia in adults and a very serious disease. Most AML cells arise from a group of special stem cells, named leukemia stem cells (LSCs). One major reason for treatment failure is that LSCs are relatively resistant to current treatments. Although most leukemia cells are killed by treatment, resistant LSCs will survive to regenerate additional leukemia cells and cause a recurrence of leukemia. Recently, we have developed a small molecule that can recognize and bind to AML LSCs. We have also developed tiny particles named nanomicelles. These nanomicelles have a size of about 1-2/100th of one micron (one millionth of a meter), and can be loaded with chemotherapy drug called daunorubicin that can kill LSCs. In this project, we will coat the drug-loaded nanomicelles with small molecules that specifically bind and kill LSCs. In patient’s body, these drug-loaded nanomicelles will work like “smart bombs”, and deliver a high concentration of daunorubicin to kill LSCs. Over the last one year, we found that daunorubicin-loaded nanomicelles could significantly increase the blood daunorubicin concentration by 20-35 times after intravenous administration. This is clinically significant as leukemia cells and leukemia stem cells are mainly located inside blood vessels. Therefore, increase in blood daunorubicin concentration by nanomicelles means leukemia and leukemia stem cells are exposed to 20-35 times more daunorubicin than regular chemotherapy. one of the major toxicity of daunorubicin is toxicity to the heart. As acute myeloid leukemia usually occurs in elderly patients, many of them already have heart diseases that prevent them from receiving the most effective chemotherapeutic drug daunorubicin. We found that, when compared to the standard daunorubicin, daunorubicin in nanomicelle has 3-5 folds less toxicity to the heart. In addition, the toxicity to other vital organs, such as liver and spleen, is significantly decreased. Compared to the standard daunorubicin, daunorubicin in nanomicelles dramatically increases the drug efficacy in killing cancer cells and prolonging the survival in animal models.

Mechanisms Underlying the Responses of Normal and Cancer Stem Cells to Environmental and Therapeutic Insults

Funding Type: 
New Faculty II
Grant Number: 
RN2-00934
ICOC Funds Committed: 
$2 274 368
Disease Focus: 
Blood Cancer
Cancer
Trauma
oldStatus: 
Active
Public Abstract: 
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.
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.
Progress Report: 
  • 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).

Prostaglandin pathway regulation of self-renwal in hematopoietic and leukemia stem cells

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06036
ICOC Funds Committed: 
$1 244 455
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Leukemias are cancers of the blood cells that result from corruption of the normal controls that regulate blood-forming stem cells. They are serious causes of illness and death, and are particularly devastating in children and the elderly. Despite substantial advances in treatment of leukemia, a significant proportion of cases are unresponsive to current therapy. Since more aggressive chemotherapy regimens provide only marginal improvements in therapeutic efficacy, we have reached a point of diminishing returns using currently available drugs. Thus, there is an urgent need for more targeted, less toxic, and more effective treatments. To this end, our studies focus on defining the defects that corrupt the normal growth controls on blood stem cells. The proposed studies build on our discovery of a key enzyme with an unexpected causative role in leukemia. We propose to further characterize its function using various proteomic approaches, and employ a cross-species comparative approach to identify additional pathways unique to cancer stem cell function. The proposed characterization of crucial growth controls that go awry in blood stem cells to cause leukemia will identify new drug targets for more effective and less toxic treatments against these devastating, life-threatening diseases.
Statement of Benefit to California: 
Leukemias are cancers of the blood cells that cause serious illness and death in children and adults. They result from corruption of the normal controls that regulate blood-forming stem cells. Despite many attempts to improve treatments with new drug combinations, this approach has reached a point of diminishing returns since intensified chemotherapies contribute only marginal improvement in outcome and are associated with increasing toxicity. The proposed characterization of crucial growth controls that go awry in blood stem cells to cause leukemia will identify new drug targets for more effective and less toxic treatments against these devastating, life-threatening diseases.
Progress Report: 
  • Leukemias are cancers of the blood cells that cause serious illness and death in children and adults. Even patients who are successfully cured of their disease often suffer from long-term deleterious health effects of their curative treatment. Thus, there is a need for more targeted, less toxic, and more effective treatments. Our studies focus on the defects and mechanisms that induce leukemia by disrupting the normal growth controls that regulate blood-forming stem cells. Using a comparative genomics approach we have identified genes that are differentially expressed in leukemia stem cells. These genes have been the focus of our studies to establish better biomarkers and treatment targets. One candidate gene codes for an enzyme with a previously unknown, non-canonical causal role in a specific genetic subtype of leukemia caused by abnormalities of the MLL oncogene. To characterize its molecular contributions, we are identifying and characterizing protein partners that may assist and interact with the enzyme in its oncogenic role. Candidate interaction partners have been identified using proteomic techniques, and are being investigated for their possible mechanistic roles in leukemia stem cell functions. Another promising candidate that we identified in the comparative gene expression approach encodes a cell surface protein that is preferentially expressed on leukemia stem cells. We have exploited this cell surface protein as a marker to isolate the rare population of cells in human leukemias with stem cell properties. This technical approach has resulted in the isolation of leukemia stem cell populations that are more highly enriched than those obtained using previous techniques. The highly enriched sub-population of leukemia stem cells has been used for comparative gene expression profiling to define a dataset of genes that are differentially expressed between highly matched populations of leukemia cells that are enriched or depleted of leukemia stem cells. Bioinformatics analysis of the dataset has further suggested specific cellular processes and transcriptional regulatory factors that distinguish human leukemia stem cells caused by abnormalities of the MLL oncogene. These newly identified factors will be studied using in vitro and in vivo assays for their specific contributions to leukemia stem cell function and leukemia pathogenesis. Continued characterization of crucial growth controls that go awry in blood stem cells to cause leukemia will identify new drug targets for more effective and less toxic treatments against these devastating, life-threatening diseases.

Dual targeting of tyrosine kinase and BCL6 signaling for leukemia stem cell eradication

Funding Type: 
Early Translational II
Grant Number: 
TR2-01816
ICOC Funds Committed: 
$3 607 305
Disease Focus: 
Blood Cancer
Cancer
Collaborative Funder: 
Germany
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Leukemia is the most frequent form of cancer in children and teenagers, but is also common in adults. Chemotherapy has vastly improved the outcome of leukemia over the past four decades. However, many patients still die because of recurrence of the disease and development of drug-resistance in leukemia cells. In preliminary studies for this proposal we discovered that in most if not all leukemia subtypes, the malignant cells can switch between an “proliferation phase” and a “quiescence phase”. The “proliferation phase” is often driven by oncogenic tyrosine kinases (e. g. FLT3, JAK2, PDGFR, BCR-ABL1, SRC kinases) and is characterized by vigorous proliferation of leukemia cells. In this phase, leukemia cells not only rapidly divide, they are also highly susceptible to undergo programmed cell death and to age prematurely. In contrast, leukemia cells in “quiescence phase” divide only rarely. At the same time, however, leukemia cells in "quiescence phase" are highly drug-resistant. These cells are also called 'leukemia stem cells' because they exhibit a high degree of self-renewal capacity and hence, the ability to initiate leukemia. We discovered that the BCL6 factor is required to maintain leukemia stem cells in this well-protected safe haven. Our findings demonstrate that the "quiescence phase" is strictly dependent on BCL6, which allows them to evade cell death during chemotherapy treatment. Once chemotherapy treatment has ceased, persisting leukemia stem cells give rise to leukemia clones that reenter "proliferation phase" and hence initiate recurrence of the disease. Pharmacological inhibition of BCL6 using inhibitory peptides or blocking molecules leads to selective loss of leukemia stem cells, which can no longer persist in a "quiescence phase". In this proposal, we test a novel therapeutic concept eradicate leukemia stem cells: We propose that dual targeting of oncogenic tyrosine kinases (“proliferation”) and BCL6 (“quiescence”) represents a powerful strategy to eradicate drug-resistant leukemia stem cells and prevent the acquisition of drug-resistance and recurrence of the disease. Targeting of BCL6-dependent leukemia stem cells may reduce the risk of leukemia relapse and may limit the duration of tyrosine kinase inhibitor treatment in some leukemias, which is currently life-long.
Statement of Benefit to California: 
Leukemia represents the most frequent malignancy in children and teenagers and is common in adults as well. Over the past four decades, the development of therapeutic options has greatly improved the prognosis of patients with leukemia reaching 5 year disease-free survival rates of ~70% for children and ~45% for adults. Despite its relatively favorable overall prognosis, leukemia remains one of the leading causes of person-years of life lost in the US (362,000 years in 2006; National Center of Health Statistics), which is attributed to the high incidence of leukemia in children. In 2008, the California Cancer Registry expected 3,655 patients with newly diagnosed leukemia and at total of 2,185 death resulting from fatal leukemia. In addition, ~23,300 Californians lived with leukemia in 2008, which highlights that leukemia remains a frequent and life-threatening disease in the State of California despite substantial clinical progress. Here we propose the development of a fundamentally novel treatment approach for leukemia that is directed at leukemia stem cells. While current treatment approaches effectively diminish the bulk of proliferating leukemia cells, they fail to eradicate the rare leukemia stem cells, which give rise to drug-resistance and recurrence of the disease. We propose a dual targeting approach which combines targeted therapy of the leukemia-causing oncogene and the newly discovered leukemia stem cell survival factor BCL6. The power of this new therapy approach will be tested in clinical trials to be started in the State of California.

Preclinical development of a pan Bcl2 inhibitor for cancer stem cell directed therapy

Funding Type: 
Early Translational II
Grant Number: 
TR2-01789
ICOC Funds Committed: 
$3 341 758
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Cancer is the leading cause of death for individuals under 85. Relapse and metastatic disease are the leading causes of cancer related mortality. Anti-apoptotic BCL2 family member overexpression has been shown to promote disease progression in both chronic myeloid leukemia (CML) and prostate cancer. Andr., the emergence of cancer stem cells (CSC) promotes apoptosis resistance in the bone marrow metastatic microenvironment. While targeted therapy with BCR-ABL inhibitors has improved survival of patients with chronic phase CML, the prevalence has doubled since 2001 with over 22,000 people living with CML in the US in 2009. Unfortunately, a growing proportion of patients become intolerant or simply cannot afford full dose BCR-ABL inhibitor therapy and thus, progress to advanced phase disease with a 5 year survival rate of less than 30%. Although prostate cancer prevalence was high at 2.26 million in 2007, distant disease was relatively rare at 5%. However, like blast crisis CML, metastatic prostate cancer survival was only 30% over 5 years. Overexpression of B-cell lymphoma/leukemia-2 (BCL2) family genes has been observed in human blast crisis CML and advanced prostate cancer and may fuel CSC survival. Recent RNA sequencing data demonstrate that human CSC express a panoply of anti-apoptotic Bcl-2 isoforms in response to extrinsic signals in vivo, indicating that a pan BCL2 inhibitor will be required to abrogate CSC survival. Through binding and anti-tumor studies, a potent inhibitor of BCL2 pro-survival family proteins, BI-97C1, has been identified which inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl1-1 with nanomolar IC50 values. Notably, BI-97C1 potently inhibits growth of human prostate cancer in a xenograft model as well as blast crisis CML CSC engrafted in RAG2-/-c-/- mice while exerting minimal cytotoxicity toward bax-/-bak-/- cells. Because BI-97C1 inhibits all six anti-apoptotic Bcl-2 family members including Bcl-2, Mcl-1 (myeloid cell leukemia 1), Bcl-XL (BCL2L1), Bfl-1 (BCL-2A1), Bcl-W (BCL2L2) and Bcl-B (BCL2L10) proteins, with improved chemical, plasma and microsomal stability relative to apogossypol, we anticipate that it will have clinical utility for targeting apoptosis resistant human CSC in two malignancies with proven reliance on BCL2 signaling – blast crisis CML and advanced prostate cancer. Thus, anti-apoptotic BCL2 family member inhibition with BI-97C1 could represent a vital component of a potentially curative strategy for advanced malignancies that may obviate the need for costly continuous tyrosine kinase inhibitor therapy by increasing sensitivity to therapy. Elimination of CSC contributing to therapeutic resistance, the primary cause of cancer death, is of high clinical importance and thus, development of a small molecule pan-BCL2 inhibitor would fulfill a vital unmet medical need, fuel California biotechnology stem cell R&D efforts and decrease health care costs for patients with cancer.
Statement of Benefit to California: 
Cancer is the leading cause of death for individuals under 85 and usually results from metastatic disease in the setting of therapeutic recalcitrance. Anti-apoptotic BCL2 family member overexpression has been shown to promote disease progression in both chronic myeloid leukemia and prostate cancer. Moreover, the emergence of quiescent cancer stem cells promotes apoptosis resistance in the bone marrow niche for. While targeted BCR-ABL inhibition has resulted in improved survival of patients with chronic phase CML, the prevalence has doubled since 2001 with over 22,000 people living with CML in the US in 2009 (http://www.leukemia-lymphoma.org). Unfortunately, a growing proportion of patients become intolerant or simply cannot afford full dose BCR-ABL inhibitor therapy as a result of spiraling annual costs and thus, progress to advanced phase disease with a 5 year survival rate of less than 30%. Although prostate cancer prevalence was high at 2.26 million in 2007, distant disease was relatively rare at 5%. Like CML, metastatic prostate cancer survival was only 30% over 5 years (http://seer.cancer.gov/statfacts/html/prost.html#prevalence <http://seer.cancer.gov/statfacts/html/prost.html#prevalence> ). Like blast crisis CML, prostate cancer progression and metastasis is associated with BCL2 overexpression. Thus, anti-apoptotic BCL2 family member inhibition with BI-97C1 could represent a vital component of a potentially curative strategy for advanced malignancies that may obviate the need for costly continuous tyrosine kinase inhibitor therapy by increasing sensitivity to therapy. Elimination of CSC contributing to therapeutic resistance, the primary cause of cancer death, is of high clinical importance and thus, development of a small molecule pan-BCL2 inhibitor would fulfill a vital unmet medical need, fuel California biotechnology stem cell R&D efforts and decrease health care costs for patients with cancer.
Progress Report: 
  • Overexpression of Bcl-2 family genes may fuel CSC survival. Recent RNA sequencing data demonstrate that human CSC express a panoply of antiapoptotic Bcl-2 isoforms in response to extrinsic signals in vivo, indicating that a pan Bcl-2 inhibitor will be required to abrogate CSC survival. Sabutoclax inhibits growth of blast crisis CML CSC engrafted in RAG2-/-c-/- mice with minimal cytotoxicity toward bax-/-bak-/- cells. Because sabutoclax inhibits all six antiapoptotic Bcl-2 family members including Bcl-2, Mcl-1, Bcl-XL, Bfl-1, Bcl-W and Bcl-B proteins, with good chemical, plasma and microsomal stability, we anticipate that it will have clinical utility for targeting apoptosis resistant human CSC in malignancies
  • Significant progress against milestones in the first year was accomplished and we have made early progress on several milestones projected for Year 2. During this 6 month reporting period, sabutoclax was licensed by a biotech company, Oncothyreon. The license was previously held by Coronado Biosciences. Dr. Pellecchia (SBMRI ) continues to provide sabutoclax to Dr. Jamieson for use in cellular and in vivo studies. SBMRI conducted QC analyses (integrity and purity) on samples’ used in preclinical studies and provided comparative analyses of compound produced by the CMO produced by different methods of synthesis. Importantly, the sabutoclax manufacturing process was optimized allowing scale-up of drug. In formulation studies, a method was developed and qualified that separates impurities and degradation compounds from sabutoclax for quantitation of the drug. Additional solubility and stability studies were performed by Oncothyreon to identify an IV formulation that could be used for both nonclinical studies and the clinic. Several pilot PK studies in mice, rats and dogs, planned for Year 2, were also conducted by Oncothyreon. Through whole transcriptome RNA sequencing Dr. Jamieson showed that Bcl-W was up-regulated in CP and BC progenitors compared to normal CB progenitors. Previous qRT-PCR results for Mcl-1 were confirmed, showing that the long isoform was preferentially expressed in BC CML. Results for Bcl-2 and Mcl-1 were also confirmed at the protein level by FACS analysis and immunohistochemistry of bone marrow (BM) from mice engrafted with human CML CD34+ LSC.
  • Sabutoclax treatment ablated BC CML progenitor cells in vivo and in vitro. Colony formation of BC CML (vs normal progenitor cells) was decreased by sabutoclax in a dose dependent manner. When CML cells were co-cultured with stromal cells or in stroma conditioned media, BCL-2 mRNA expression was increased and colony formation was improved. Knockdown of endogenous BCL2 in BC CML cells by shRNA resulted in decreased colony formation. Preliminary results suggest that BM is a protective niche for BC CML CSC and that sabutoclax may target these niche protected cells.
  • In BC CML engrafted mice, dasatinib increased quiescent BC CML cell engraftment in mouse BM measured by FACS for cell cycle markers. Sabutoclax decreased BCL-2 and MCL1 protein expression by immunohistochemistry staining and decreased quiescent BC CML CSC in BM however sabutoclax increased TUNEL staining in BM suggesting that while dasatinib may increase the number of quiescent BC CML CSC, sabutoclax may do the reverse.
  • High doses of sabutoclax administered in combination with dasatinib resulted in a significant decrease in human cell engraftment in BM versus dasatinib alone. Mice serially transplanted with tissues from combination treated mice had increased survival compared to serial transplants of single agent treated tissues. Human CD34+ cells from the BM of combination treated mice had more cells in cycle than CD34+ cells compared to the BM of mice treated with dasatinib alone. The frequency of CD34+BCL2+ and CD34+MCL1+ BC LSC were significantly lower in BM treated with a combination of sabutoclax and dasatinib suggesting that sabutoclax and dasatinib may act synergistically to increase survival of BC CML engrafted mice.
  • Dormant cancer stem cells (CSC) contribute to therapeutic resistance and relapse in chronic myeloid leukemia (CML) and other recalcitrant malignancies. Cumulative data demonstrate that overexpression of BCL2 family pro-survival splice isoforms fuels quiescent CSC survival in human blast crisis (BC) CML. Whole transcriptome RNA sequencing data, apoptosis PCR array and splice isoform specific qRT-PCR demonstrate that human CSC express anti-apoptotic long BCL2 isoforms in response to extrinsic signals in the marrow niche, indicating that a pan BCL2 inhibitor will be required to abrogate CSC survival. Sabutoclax, a novel pan BCL2 inhibitor, prevents survival of BC CSC engrafted in RAG2-/-c-/- mice, commensurate with downregulation of pro-survival BCL2 splice isoforms and proteins, and sensitizes CSC to a BCR-ABL inhibitor, dasatinib, while exerting minimal cytotoxicity toward normal hematopoietic stem cells. Because sabutoclax inhibits all six anti-apoptotic BCL2 family members, with good chemical, plasma and microsomal stability, in addition to a scaleable production process, we anticipate that it will have broad clinical utility for targeting apoptosis resistant quiescent human CSC in a number of recalcitrant malignancies as featured in our recent lead article (Goff D et al, Cell Stem Cell. 2013 Mar 7;12(3):316-28).
  • Significant progress against milestones in the second year was accomplished and we have made early progress on several milestones projected for Year 3. Whole transcriptome RNA sequencing, qRT-PCR array and splice isoform specific qRT-PCR analysis performed on FACS purified progenitors derived from 8 CP, 8 BC and 6 normal samples demonstrated splice isoform switching favoring pro-survival long isoform expression during progression from CP to blast BC CML and in CSC engrafted in the bone marrow (BM) niche. Both human BCL2 and MCL1 protein expression co-localized with engrafted human leukemic CD34+ cells in the bone marrow epiphysis and served as important biomarkers of response to sabutoclax. Importantly, intravenous treatment with sabutoclax reduced BC CML CSC survival in both marrow and splenic niches at doses that spared normal hematopoietic stem cells in RAG2-/-gamma c-/- xenograft models established with cord blood CD34+ cells.
  • While dasatinib treatment alone increased serially transplantable quiescent BC CML CSC in BM, sabutoclax decreased CSC survival commensurate with upregulation of short pro-apoptotic and downregulation of long anti-apopoptotic BCL2 family isoforms. While previous studies involved intraperitoneal administration, in the last 12 months we have focused on a more clinically relevant intravenous (IV) administration schedule with IV sabutoclax administered alone or in combination with oral dasatinib. In these studies, sabutoclax sensitized quiescent CSC to dasatinib resulting in a significant decrease in CSC survival versus dasatinib alone. Moreover, mice serially transplanted with human cells from combination treated mice had increased survival compared to serial transplants of single agent treated tissues. Human CD34+ cells from the BM of combination treated mice had more cells in cycle than CD34+ cells compared to the BM of mice treated with dasatinib alone. The frequency of CD34+BCL2+ and CD34+MCL1+ BC CSC were significantly lower in BM treated with a combination of sabutoclax and dasatinib suggesting that the combination acts synergistically to decrease CSC survival and increase the lifespan of CSC engrafted mice.
  • During this 12-month reporting period, sabutoclax production was successfully scaled up by two separate CMOs, Syncom and Norac. Dr. Pellecchia (SBMRI) provided flash chromatography purified sabutoclax to Dr. Jamieson for use in cellular and in vivo studies in addition to conducting QC analyses (integrity and purity) on scaled up sabutoclax formulations produced by Norac (4g) and Syncom (30g) in different vehicles. In formulation studies, a flash chromatography method was developed and qualified that separates impurities and degradation compounds from sabutoclax. Additional solubility and stability studies were performed to identify an IV Solutol formulation, compared with the previous IP DMSO/PBS Tween formulation, which could be used for both pre-clinical studies and in future clinical trials. Pilot PK studies in mice and rats were conducted with the Solutol formulated sabutoclax and showed weight loss associated with impurities that could be readily removed by standard flash chromatography. As a result, ssabutoclax production will include flash chromatography to enhance purity and stability and this material will be used for further PK and PD studies. In conclusion, we are on track to accomplish our milestones as set forth in the grant and anticipate that sabutoclax will form the basis of combination clinical studies aimed at eradicating quiescent CSC in a broad array of refractory malignancies.

Human endothelial reprogramming for hematopoietic stem cell therapy.

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06479
ICOC Funds Committed: 
$3 259 000
Disease Focus: 
Blood Disorders
Blood Cancer
Cancer
Stem Cell Use: 
Directly Reprogrammed Cell
Cell Line Generation: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
The current roadblocks to hematopoietic stem cell (HSC) therapies include the rarity of matched donors for bone marrow transplant, engraftment failures, common shortages of donated blood, and the inability to expand HSCs ex vivo in large numbers. These major obstacles would cease to exist if an extensive, bankable, inexhaustible, and patient-matched supply of blood were available. The recent validation of hemogenic endothelium (blood vessel cells lining the vessel wall give rise to blood stem cells) has introduced new possibilities in hematopoietic stem cell therapy. As the phenomenon of hemogenic endothelium only occurs during embryonic development, we aim to understand the requirements for the process and to re-engineer mature human endothelium (blood vessels) into once again producing blood stem cells (HSCs). The approach of re-engineering tissue specific de-differentiation will accelerate the pace of discovery and translation to human disease. Engineering endothelium into large-scale hematopoietic factories can provide substantial numbers of pure hematopoietic stem cells for clinical use. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the ability to program mature lineage restricted cells into more primitive versions of the same cell lineage will capitalize on cell renewal properties while minimizing malignancy risk.
Statement of Benefit to California: 
Bone marrow transplantation saves the lives of millions with leukemia and other diseases including genetic or immunologic blood disorders. California has over 15 centers serving the population for bone marrow transplantation. While bone marrow transplantation can be seen as a standard to which all stem cell therapies should aspire, there still remains the difficulty of finding matched donors, complications such as graft versus host disease, and the recurrence of malignancy. While cord blood has provided another donor source of stem cells and improved engraftment, it still requires pooling from multiple donors for sufficient cell numbers to be transplanted, which may increase transplant risk. By understanding how to reprogram blood vessels (such as those in the umbilical cord) for production of blood stem cells (as it once did during human development), it could eventually be possible to bank umbilical cord vessels to provide a patient matched reproducible supply of pure blood stem cells for the entire life of the patient. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the proposed work will introduce a new approach to engineering human cells. The ability to turn back the clock to near mature cell specific stages without going all the way back to early embryonic stem cell stages will reduce the risk of malignancy.

RUNNING TITLE: Stem Cell Gene Therapy for HIV in AIDS Lymphoma Patients

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05327
ICOC Funds Committed: 
$74 195
Disease Focus: 
Blood Cancer
Cancer
HIV/AIDS
oldStatus: 
Closed
Public Abstract: 
The Human Immunodeficiency Virus (HIV) is still a major health problem. In both developed and underdeveloped nations, millions of people are infected with this virus. HIV infects cells of the immune system, becomes part of the cell’s genetic information, stays there for the rest of the life of these cells, and uses these cells as a factory to make more HIV. In this process, the immune cells get destroyed. Soon a condition called AIDS, the Acquired Immunodeficiency Syndrome sets in where the immune system cannot fight common infections. If left untreated, death from severe infections occurs within 8 to 10 years. Although advances in treatment using small molecule drugs have extended the life span of HIV infected individuals, neither a cure for HIV infection nor a well working vaccine could be developed. Drug treatment is currently the only option to keep HIV infected individuals alive. Patients have to take a combination of drugs daily and reliably for the rest of their lives. If not taken regularly, HIV becomes resistant to the drugs and continues to destroy immune cells. What makes this situation even more complicated is the fact that many patients cannot take these drugs due to severe side effects. Stem cell gene therapy for HIV may offer an alternative treatment. Blood forming stem cells, also called bone marrow stem cells make all blood cells of the body, including immune system cells such as T cells and macrophages that HIV destroys. If “anti-HIV genes” were inserted into the genetic information of bone marrow stem cells, these genes would be passed on to all new immune cells and make them resistant to HIV. Anti-HIV gene containing immune cells can now multiply in the presence of HIV and fight the virus. In previous and current stem cell gene therapy clinical trials for HIV, only one anti-HIV gene has been used. Our approach, however, will use a combination of three anti-HIV genes which are much more potent. They will not only prevent HIV from entering an immune cell but will also prevent HIV from mutating, since it would have to escape the anti-HIV effect of three genes, similar to triple combination anti-HIV drug therapy. To demonstrate safety and effectiveness of our treatment, we will perform a clinical trial in HIV lymphoma patients. In such patients, the destruction of the immune system by HIV led to the development of a cancer of the lymph nodes called B cell lymphoma. High dose chemotherapy together with the transplantation of the patient’s own bone marrow stem cells cures B cell lymphoma. We will insert anti-HIV genes in the patient’s bone marrow stem cells and then transplant these gene containing cells into the HIV infected lymphoma patient. The gene containing bone marrow stem cells will produce a new immune system and newly arising immune cells will be resistant to HIV. In this case, we have not only cured the patient's cancer but have also given the patient an HIV resistant immune system which will be able to fight HIV.
Statement of Benefit to California: 
As of September 30, 2010, over 198,883 cumulative HIV/AIDS cases were reported in California. Another 40,000 un-named cases of HIV were also reported before 2006 although some of them may be duplicates of the named HIV cases. Patients living with HIV/AIDS totaled 108,986 at the end of September 2010. These numbers continue to grow since new cases of HIV and AIDS are being reported on a daily basis and patients now live much longer. In fact, after New York, California has the second highest number of HIV cases in the nation. Although the current and improved anti-retroviral small molecule drugs have prolonged the life of these patients, they still have to deal with the emotional, financial, and medical consequences of this disease. The fear of side effects and the potential generation of drug resistant strains of HIV is a constant struggle that these patients have to live with for the rest of their lives. Furthermore, not every patient with HIV responds to treatment and not every complication of HIV dissipates upon starting a drug regimen. In fact, the risk of some AIDS-related cancers still remains high despite the ongoing drug therapy. Additionally, in the current economic crisis, the financial burden of the long term treatment of these patients on California taxpayers is even more obvious. In 2006, the lifetime cost of taking care of an HIV patient was calculated to be about $618,900. Most of this was related to the medication cost. With the introduction of new HIV medications that have a substantially higher price and with the increase in the survival of HIV/AIDS patients, the cost of taking care of these patients can be estimated to be very high. The proposed budget cuts and projected shortfall in the California AIDS assistant programs such as ADAP will make the situation worse and could result in catastrophic consequences for patients who desperately need this of kind of support. Consequently, improved therapeutic approaches and the focus on developing a cure for HIV infected patients are issues of great importance to the people of California. Our proposed anti-HIV stem cell gene therapy strategy comprises the modification of autologous hematopoietic blood forming stem cells with a triple combination of potent anti-HIV genes delivered by a single lentiviral vector construct. This approach would engineer a patient’s immune cells in a way to make them completely resistant to HIV infection. By transplanting these anti-HIV gene expressing stem cells back into an HIV infected patient, the ability of HIV to further replicate and ravage the patient’s immune system would be diminished. The prospect of such a stem cell based therapy which may require only a single treatment to cure an HIV infected patient and which would last for the life of the individual would be especially compelling to the HIV community and the people of California.
Progress Report: 
  • HIV is still a major health problem. In both developed and underdeveloped nations, millions of people are infected with this virus. If left untreated, death from severe infections occurs within 8 to 10 years. Although advances in treatment using small molecule drugs have extended the life span of HIV infected individuals, neither a cure for HIV infection nor a well working vaccine could be developed. Drug treatment is currently the only option to keep HIV infected individuals alive. Patients have to take a combination of drugs daily and reliably for the rest of their lives. If not taken regularly, HIV becomes active again and may even become resistant to the drugs and continues to destroy immune cells. What makes this situation even more complicated is the fact that many patients cannot take these drugs due to severe side effects. Stem cell gene therapy for HIV may offer an alternative treatment. If “anti-HIV genes” were inserted into the genetic information of bone marrow stem cells, these genes would be passed on to all new immune cells and make them resistant to HIV. Anti-HIV gene containing immune cells can now multiply in the presence of HIV and fight the virus. In our approach, we are planning to use a combination of three anti-HIV genes which are much more potent. They will not only prevent HIV from entering an immune cell but will also prevent HIV from mutating, since it would have to escape the anti-HIV effect of three genes, similar to triple combination anti-HIV drug therapy. To demonstrate safety and effectiveness of our treatment, we have proposed a clinical trial in HIV lymphoma patients with stem cell gene therapy incorporated into their routine treatment with high dose chemotherapy together with the transplantation. The fund provided by CIRM (California Institute for Regenerative Medicine) gave us the opportunity to put together a panel of experts within the University of California at Davis and another panel of international experts in the area of gene therapy (an external advisory board). Intense discussion in multiple meeting with members of these two panels as well as many other meetings with individual researches within our institution resulted in the design of a clinical trial for treating patients with HIV disease using our gene therapy approach. It further helped us to identify the necessary means needed to support such a regulatory intensive gene therapy trial. To be able to recruit enough patients for such a trial, we used the funds from this planning grant for several presentations to our colleagues in other institutions for a multi-institutional clinical trial approach. The funds provided to us through this grant helped to calculate the budget required to 1) finish our application with Federal Drug Administration (FDA) to obtain the appropriate license for starting such a trial and 2) to manufacture the target drug and 3) to run the actual clinical trial. Finally, with the help of this grant, we have put together a CIRM disease grant proposal and have applied for necessary funds based on the above calculation.
  • The original progress report was submitted to the CIRM on March 1st 2012. The no cost extension was requested to perform the necessary work related to further development of our clinical trial before submission to RAC. During this period, in multiple meetings we rewrote our clinical trial based on the comments of our external advisory board and other consultants. We submitted our clinical trial protocol and Appendix M to RAC committee and after receiving their preliminary comments, we formulated our response. As the last step, we presented our clinical trial to the members of RAC committee and received a unanimous approval to move forward with the IND application to FDA.

Forming the Hematopoietic Niche from Human Pluripotent Stem Cells

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05217
ICOC Funds Committed: 
$1 375 983
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop. To approach this question we will use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation. Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells. The findings from these studies will have broad applicability to the production of other types of tissues from pluripotent stem cells, all of which have stem cells that require interaction with a specialized niche. In addition to the biological questions explored in this proposal, our focus on the blood system has direct clinical relevance to the field of bone marrow and cord blood transplantation. The development of a human hematopoietic niche from pluripotent stem cells could potentially be used to expand hematopoietic stem cells from adult tissues like cord blood. Most importantly, the ability to control differentiation from pluripotent stem cells into the blood lineage could provide an unlimited source of matched cells for transplantation for patients with leukemia and other diseases of the bone marrow and the immune system who currently lack suitable donors.
Statement of Benefit to California: 
The unique combination of pluripotentiality and unlimited capacity for proliferation has raised the hope that pluripotent stem cells will one day provide an inexhaustible source of tissue for transplantation and regeneration. Diseases that might be treated from such tissues affect millions of Californians and their families. However, much is still to be learned about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The clinical potential of pluripotent stem cells for regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and better controlled than is currently the case. The research proposed in this application has broad potential benefits for Californians both through the biological questions it will answer and the relevance of these studies for clinical translation. Our goal is to understand the way the microenvironment influences tissue production from pluripotent stem cells, a critical issue for the field of stem cell biology. Specifically we will explore the question- Do the cell types produced during differentiation of pluripotent stem cells produce an adequate microenvironment for the differentiation of tissue or are some cells inhibitory to tissue production? Our approach to these questions will be to use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation and offers a range of unique technical tools with which to study these questions rigorously. However, the fundamental concepts formed from these studies will have great relevance for the clinical production of other types of tissues from pluripotent stem cells, such as islets, neural cells and cardiac muscle. In addition to the broad biological questions explored in this proposal, our focus on the blood system has direct clinical relevance to the field of bone marrow and cord blood transplantation. One goal in the proposal is to generate a cellular platform from pluripotent stem cells that will create an environment in which adult blood stem cells can grow and be expanded. Cell numbers collected from cord blood at birth are often insufficient for transplantation in adult patients and older children. The development of a human cell culture system that could expand the number of cord blood stem cells would provide new opportunities for transplantation for patients with leukemia and other diseases of the bone marrow and the immune system who currently lack suitable donors. All scientific findings and technical tools developed in this proposal will be made available to researchers throughout California, under the guidelines from the California Institute of Regenerative Medicine.
Progress Report: 
  • The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop.
  • To approach this question we use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation.
  • Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells.
  • During the first year of support, we have made significant progress in the first two specific aims. We have developed a method that allows us to track the common origin of the blood forming cells and their microenvironment. We also have identified subsets of cells generated from pluripotent cells that have distinct functions in blood formation. Our plan during the next year is to fully characterize these subsets to understand how they function, and to improve our methods to expand them in culture.
  • The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop.
  • To approach this question we use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells (HSC) survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation.
  • Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells.
  • During the second year of support, we have made significant progress in all three specific aims. We continue to refine our method that allows us to track the common origin of the blood forming cells and their microenvironment during development. We have identified subsets of cells generated from pluripotent cells that can support cord blood HSC and now we are determining the mechanisms by which these cells act and how they can be best used to support HSC that develop from PSC.

Derivation and Characterization of Myeloproliferative Disorder Stem Cells from Human ES Cells

Funding Type: 
New Faculty II
Grant Number: 
RN2-00910
ICOC Funds Committed: 
$3 240 572
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Cancer is the leading cause of death for people younger than 85. High cancer mortality rates related to resistance to therapy and malignant progression underscore the need for more sensitive diagnostic techniques as well as therapies that selectively target cells responsible for cancer propagation. Compelling studies suggest that human cancer stem cells (CSC) arise from aberrantly self-renewing tissue specific stem or progenitor cells and are responsible for cancer propagation and resistance to therapy. Although the majority of cancer therapies eradicate rapidly dividing cells within the tumor, the rare CSC population may be quiescent and then reactivate resulting in disease progression and relapse. We recently demonstrated that CSC are generated in chronic myeloid leukemia by activation of beta-catenin, a gene that allows cells to reproduce themselves extensively. However, relatively little is known about the sequence of events responsible for leukemic transformation in more common myeloproliferative disorders (MPDs) that express an activating mutation in the JAK2 gene. Because human embryonic stem cells (hESC) have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells, they represent an ideal model system for generating and characterizing human MPD stem cells. Thus, hESC cell research harbors tremendous potential for developing life-saving therapy for patients with cancer by providing a platform to rapidly and rationally test new therapies that specifically target CSC. To provide a robust model system for screening novel anti-CSC therapies, we propose to generate and characterize BCR-ABL+ and JAK2+ MPD stem cells from hESC. We will investigate the role of genes that are essential for initiation of these MPDs such as BCR-ABL and JAK2 V617F as well as additional mutations in beta-catenin or GSK3betaï€ implicated in CSC propagation. The efficacy of a selective BCR-ABL and JAK2 inhibitors at blocking BCR-ABL+ and JAK2+ human ES cell self-renewal, survival and proliferation alone and in combination with a potent and specific beta-catenin antagonist will be assessed in robust in vitro and in vivo assays with the ultimate aim of developing highly active anti-MPD stem cell therapy that may halt progression to acute leukemia and obviate therapeutic resistance.
Statement of Benefit to California: 
Although much is known about the genetic and epigenetic events involved in CSC production in a Philadelphia chromosome positive MPD like chronic myeloid leukemia (CML), comparatively little is known about the molecular pathogenesis of the five-fold more common Philadelphia chromosome negative (Ph-) MPDs. MPD patients have a moderately increased risk of fatal thrombotic events as well as a striking 36-fold increased risk of death from transformation to acute leukemia. Recently, a point mutation, JAK2 V617F(JAK2+), resulting in constitutive activation of the JAK2 cytokine signaling pathway was discovered in a large proportion of MPD patients. A critical barrier to developing potentially curative therapies for both BCR-ABL+ and JAK2+ MPDs is a comprehensive understanding of relative contribution of BCR-ABL and JAK2 V617F to disease initiation versus transformation to acute leukemia. We recently discovered that JAK2 V617F is expressed at the hematopoietic stem cell level in PV, ET and MF and that JAK2 skewed ifferentiation in PV is normalized with a selective JAK2 inhibitor, TG101348. However, a detailed molecular pathogenetic characterization has been hampered by the paucity of stem and progenitor cells in MPD derived blood and marrow samples. Because hESC have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells in vitro, they represent an ideal model system for generating human MPD stem cells. Thus, California hESC research harbors tremendus potential for understanding the MPD initiating events that skew differentiation versus events that promote self-renewal and thus, leukemic transformation. Moreover, a more comprehensive understanding of primitive stem cell fate decisions may yield key insights into methods to expand blood cell production that may have major implications for blood banking. Clinical Benefit Generation of MPD stem cells from hESC would provide an experimentally amenable and relevant platform to expedite the development ofsensitive diagnostic techniques to predict disease progression and to develop potentially curative anti-CSC therapies. Economic Benefit The translational research performed in the context of this grant will not only speed the delivery of innovative MPD targeted therapies for Californians, it will help to train Californiaís future R&D workforce in addition to developing leaders in translational medicine. This grant will provide the personnel working on the project with a clear view of the importance of thir research to cancer therapy and a better perspective on future career opportunities in California as well as directly generate revenue through development and implementation of innovative therapies aimed at eradicating MPD stem cells that may be more broadly applicable to CSC in other malignances.
Progress Report: 
  • Summary of Overall Progress
  • This grant focuses on generation of MPN stem cells from hESC or CB and correlates leukemic potential with MPN patient samples. In the first year of this grant, we have demonstrated that 1) hESC differentiate on AGM stroma to the CD34+ stage, which is associated with increased GATA-1, Flk2, GATA-2 and ADAR1 expression; 2) hESC CD34+ differentiation is enhanced in vitro and in vivo in the presence of a genetically engineered mouse stroma, which produces human stem cell factor, IL-3 and G-CSF; 3) hESC CD34+ cells can be transduced with our novel lentiviral BCR-ABL vector, which, unlike retroviral BCR-ABL, can transduce quiescent stem cells; 4) BCR-ABL expression by CP CML progenitors does not sustain engraftment but rather leukemic transformation is predicated, in part, on bcl-2 overexpression; 5) JAK2V617F expression in hES or CB stem cells is insufficient to induce leukemic transformation; 6) BCR-ABL transduced hESC CD34+ cells have significantly higher BCR-ABL transplantation potential than CP CML progenitors suggesting that they have higher survival capacity; 7) lentiviral -catenin transduction of BCR-ABL hESC CD34+ cells leads to serial transplantation indicative of LSC formation; 8) CML BC LSC persist in vivo despite potent BCR-ABL inhibition with dasatinib therapy and will likely require combined inhibitor therapy to eradicate. Currently, HEEBO arrays and phospho-flow studies are underway to detect bcl-2 family members and self-renewal protein expression in BCR-ABL and JAK2 V617F transduced hESC and CB CD34+ cells compared with MPN patient derived progenitors. This will aid in development of combined MPN stem cell inhibitor strategies in this grant.
  • This grant focuses on generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent (hESC) or multipotent (CB) stem cells and seeks to correlate their leukemic potential with that of MPN patient sample-derived stem cells. To provide a platform for testing induction of stem cell differentiation, survival and self-renewal by BCR-ABL versus JAK2, hESC were utilized in the first year and as more patient samples and cord blood became available these were utilized.
  • In the first year of this grant, we found that hESC undergo hematopoietic differentiation on AGM stroma to the CD34+ stage resulting in increased GATA-1, Flk2, ADAR1 and GATA-2 expression. Moreover, CD34+ differentiation was enhanced on a genetically engineered mouse stroma (SL/M2) secreting human SCF, IL-3 and G-CSF. Lentiviral BCR-ABL transduced hESC-derived CD34+ cells had higher BCR-ABL+ cellular transplantation potential than chronic phase (CP) CML progenitors, indicative of a higher survival capacity. However, they sustained self-renewal only when co-transduced with lentiviral -catenin (Rusert et al, manuscript in preparation) suggesting that blast crisis evolution requires acquisition of both enhanced survival and self-renewal potential. Similarly, lentiviral mouse mutant JAK2 expression in hESC or CB stem cells was insufficient to produce self-renewing MPN stem cells, indicating that the cellular context, nature of the genetic driver and responses to extrinsic cues from the microenvironment play seminal roles in regulating therapeutically resistant MPN stem cell properties such as aberrant survival, differentiation, self-renewal and dormancy.
  • In the second year of this five year grant, we have focused on human cord blood (CB) stem cells compared with a large number of MPN patient samples propagated on SL/M2 stroma or in RAG2-/-c-/- mice to more adequately recapitulate the human MPN stem cell niche. Also, to more faithfully recapitulate human (rather than the previously published lentiviral mouse JAK2 vectors, Cancer Cell 2008) JAK2 driven MPNs, we cloned human wild-type JAK2 and human JAK2 V617F from MPN patient samples into lentiviral-GFP vectors (Court Recart A*, Geron I* et al, manuscript in preparation). We also incorporated full transcriptome RNA (ABI SOLiD 4.0) sequencing, PCR array and nanofluidic phosphoproteomics technology to better gauge the impact of JAK2 versus BCR-ABL on stem cell fate, survival, self-renewal and dormancy in the context of specific malignant microenvironments and the relative susceptibility of MPN stem cells in these niches to single agent molecularly targeted inhibitors.
  • This grant focuses on generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent human embryonic stem cells (hESC) or multipotent cord blood (CB) stem cells, and seeks to correlate their leukemic potential with that of disease progression in MPN patient sample-derived stem cells. In the first and second years of this grant, we found that lentiviral BCR-ABL transduced hESC-derived CD34+ cells had higher leukemic transplantation potential than chronic phase (CP) chronic myeloid leukemia (CML) progenitors. However, they sustained self-renewal only when co-transduced with lentiviral beta-catenin suggesting that blast crisis (BC) evolution requires acquisition of both enhanced survival and self-renewal potential. Similarly, we have shown using lentiviral vectors that mouse and human mutant JAK2 were insufficient to produce self-renewing MPN stem cells. New results in Year 3 demonstrate that BCR-ABL and JAK2 activation drive differentiation of hematopoietic progenitors towards an erthyroid/myeloid lineage bias. We have used full transcriptome RNA-Sequencing (RNA-Seq) technology to evaluate the genetic and epigenetic status of BCR-ABL and JAK2-transduced normal progenitor cells as well as patient-derived MPN progenitors. This has allowed us to probe the mechanisms of aberrant differentiation and self-renewal of MPN progenitors and identify unique gene expression signatures of disease progression.
  • We previously found that overexpression and splice isoform switching of a key RNA editing enzyme – adenosine deaminase acting on dsRNA (ADAR), and splice isoform changes in pro-survival BCL2 family members, correspond with disease progression in CML. In the current reporting period, RNA-Seq analyses revealed that ADAR1-driven activation of RNA editing contributed to malignant progenitor reprogramming, promoting aberrant differentiation and self-renewal of MPN stem cells. Knocking down ADAR1 using lentiviral shRNA vectors reduced the self-renewal potential of CML progenitors. This work has culminated in a manuscript that has now been submitted to PNAS (Jiang et al.). Recent results also show that ADAR1 is activated in progenitors from patients with JAK2-driven MPNs. Thus, ADAR1 may be an important factor that works in concert with BCR-ABL or JAK2 to facilitate disease progression in MPNs.
  • Our results show that another self-renewal factor that may drive BCR-ABL or JAK2-mediated propagation of disease from quiescent MPN progenitors is Sonic hedgehog (Shh). We have examined the expression patterns of this pathway in MPN progenitors using qRT-PCR and RNA-Seq, and have tested a pharmacological inhibitor of this pathway in a robust stromal co-culture model of MPN progression to Acute Myeloid Leukemia (AML).
  • In sum, we have utilized full transcriptome RNA-Seq and qRT-PCR coupled with hematopoietic progenitor assays and in vivo studies to evaluate the impact of JAK2 versus BCR-ABL on stem cell fate, survival, self-renewal and dormancy. These techniques have allowed us to investigate in more detail the role of genetic and epigenetic alterations that drive disease progression in the context of specific malignant microenvironments, and the relative susceptibility of MPN stem cells in these niches to single agent molecularly targeted inhibitors.
  • The main objectives of this project are generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent human embryonic stem cells (hESC) or multipotent stem cells, and identification of crucial leukemia stem cell (LSC) survival and self-renewal factors that contribute to the development and progression of BCR-ABL and JAK2-driven hematopoietic disorders. A key finding of our work thus far is that in addition to activation of BCR-ABL or JAK2 oncogenes, generation of self-renewing MPN LSC requires stimulation of other pro-survival and self-renewal factors such as β-catenin, Sonic hedgehog (SHH), BCL2, and in particular the RNA editing enzyme ADAR1, which we identified as a novel regulator of LSC differentiation and self-renewal.
  • We have now completed comprehensive gene expression analyses from next-generation RNA-sequencing studies performed on normal and leukemic human hematopoietic progenitor cells from primary cord blood samples and adult normal peripheral blood samples, along with normal cord blood transduced with BCR-ABL or JAK2 oncogenes, and primary samples from patients with BCR-ABL+ chronic phase and blast crisis chronic myeloid leukemia (CML). These studies revealed that gene expression patterns in survival and self-renewal pathways (SHH, JAK2, ADAR1) clearly distinguish normal and leukemic progenitor cells as well as MPN disease stages. These data provide a vast resource for identification of LSC-specific biomarkers with diagnostic and prognostic clinical applications, as well as providing new potential therapeutic targets to prevent disease progression.
  • New results from RNA-sequencing studies reveal high levels of expression of inflammatory mediators in human blast crisis CML progenitors and in BCR-ABL transduced normal cord blood stem cells. Moreover, expression of the inflammation-responsive form of ADAR1 correlated with generation of an abnormally spliced GSK3β gene product that has been previously linked to LSC self-renewal. These results have now been published in the journal PNAS (Jiang et al.). Together, we have demonstrated that ADAR1 drives hematopoietic cell fate by skewing cell differentiation – a trend which occurs during normal bone marrow aging – and promotes LSC self-renewal through alternative splicing of critical survival and self-renewal factors. Notably, inhibition of ADAR1 through genetic knockdown strategies reduced self-renewal capacity of CML LSC, and may have important applications in treatment of other disorders that transform to acute leukemia. Thus, these results suggest that RNA editing (ADAR1) and splicing represent key therapeutic targets for preventing LSC self-renewal – a primary driver of leukemic progression.
  • Whole transcriptome profiling studies coupled with qRT-PCR, hematopoietic progenitor assays and in vivo studies have shown that combined inhibition of BCR-ABL and JAK2 is another effective method to reduce LSC self-renewal in pre-clinical models. New results show that lentivirus-enforced BCR-ABL or JAK2 expression in normal cord blood stem cells drives generation of distinct splice isoforms of STAT5a. While inhibition of JAK2/STAT5a signaling or BCR-ABL tyrosine kinase activity alone did not eradicate self-renewing LSC, combined JAK2 and BCR-ABL inhibition dramatically impaired LSC survival and self-renewal in the protective bone marrow niche, and increased the lifespan of serial transplant recipients. These effects were associated with reduction in STAT5a isoform expression – which represents a novel molecular marker of response to combined BCR-ABL/JAK2 inhibition – and altered expression of cell cycle genes in human progenitor cells harvested from the bone marrow of transplanted mice. These results are the subject of a new manuscript currently under review (Court et al.). Moreover, this work has led to the development of new experimental tools that will facilitate study of LSC maintenance and cell cycle status in the context of normal versus diseased bone marrow microenvironments. In sum, studies completed thus far have uncovered a role for RNA editing and splicing alterations in leukemic progression, particularly in specific microenvironments. Using specific inhibitors targeting BCR-ABL and JAK2, along with strategies to block RNA editing and aberrant splicing activities, we have been able to establish the relative susceptibility of MPN stem cells to molecular inhibitors with activity against LSC residing in select hematopoietic niches that are difficult to treat with conventional chemotherapeutic agents.
  • In the final year of this project, we focused on elucidating the mechanisms of leukemia stem cell (LSC) generation in JAK2 compared with BCR-ABL1 initiated myeloproliferative neoplasms (MPN, previously called myeloproliferative disorders). To this end, we investigated the MPN stem cell propagating effects of BCR-ABL1 or JAK2 alone or in combination with activation of the human embryonic stem cell RNA editase, ADAR1. Recently, we discovered that ADAR1, which edits adenosine to inosine bases in the context of primate specific Alu sequences, leads to GSK3β missplicing and β-catenin activation in chronic phase (CP) CML progenitors leading to blast crisis (BC) transformation and LSC generation. In addition, variant isoform expression of a Wnt/β-catenin target gene, CD44, was also characteristic of LSC. In a previous report (Jiang et al., PNAS 2013), identification of ADAR1 as a malignant reprogramming factor represented the first description of RNA editing as a regulator of reprogramming. When lentivirally overexpressed, ADAR1 endows committed CP myeloid progenitors with self-renewal capacity. Further studies revealed that JAK2/STAT5a activates ADAR1 leading to deregulation of cell cycle progression and global down-regulation of microRNA expression thereby uncovering two additional key mechanisms of LSC generation in MPNs. This is consistent with our findings from gene expression profiling studies performed in the previous year, along with functional classification and network analysis using Ingenuity Pathway Analysis (IPA), showing that cell cycle-related genes were significantly altered in human progenitors from xenografted mice treated with combination JAK2 and BCR-ABL inhibitor therapy compared with single agent therapies alone. Together these data suggest that combined BCR-ABL and JAK2 inhibition impairs LSC survival and self-renewal via cell cycle modulation. ADAR1 and other stem cell regulatory pathways such as CD44 represent novel targets to detect and eradicate the self-renewing LSC. We also performed new studies that elucidate the stem cell-intrinsic genetic changes that occur during human bone marrow aging, which may contribute to BCR-ABL or JAK2-dependent functional alterations.
  • This work has led to discovery of a novel role for embryonic stem cell genes and splice isoforms, including ADAR1 p150 and a transcript variant of CD44, in the maintenance of LSC that promote MPN progression. In addition, through the course of this research we have 1) developed novel lentiviral tools for investigating normal hematopoietic stem and progenitor (HSPC) and malignant LSC survival, differentiation, self-renewal, and cell cycle regulation, and 2) devised innovative LSC diagnostic strategies and 3) tested therapeutic strategies targeting LSC-associated RNA editing and splice isoform generation that selectively inhibit LSC self-renewal.

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