Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Genetic Re-programming of Stem Cells to Fight Cancer

Funding Type: 
Disease Team Therapy Development - Research
Grant Number: 
DR2A-05309
ICOC Funds Committed: 
$19 999 563
Disease Focus: 
Melanoma
Cancer
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Science has made great progress in the treatment of certain cancers with targeted and combination therapies, yet prolonged remissions or cures are rare because most cancer therapies only inhibit cell growth and/or reduce such growth but do not stop the cancer. The study investigators propose to develop an Investigational New Drug (IND) and fully enroll a phase I clinical trial within the grant period to genetically redirect the patient’s immune response to specifically attack the cancer starting from hematopoietic (blood) stem cells (HSC) in patients with advanced forms of the aggressive skin cancer malignant melanoma. Evaluation of immune system reconstitution, effectiveness and immune response during treatment will use imaging with Positron Emission Tomography (PET) scans. The HSC treatment approach has been validated in extensive studies in the laboratory. The investigators of this grant have recently initiated a clinical trial where adult immune cells obtained from blood are genetically modified to become specific killer cells for melanoma. These cells are administered back to patients. The early data from this study is encouraging in terms of the ability to generate these cells, safely administer them to patients leading to beneficial early clinical effects. However, the adult immune cells genetically redirected to attack cancer slowly decrease over time and lose their killer activity, mainly because they do not have the ability to self-renew. The advantage of the proposed HSC method over adult blood cells is that the genetically modified HSC will continuously generate melanoma-targeted immune killer cells, hopefully providing prolonged protection against the cancer. The IND filing with the FDA will use the modified HSC in advanced stage melanoma patients. By the end of year 4, we will have fully accrued this phase 1 clinical trial and assessed the value of genetic modification of HSCs to provide a stable reconstitution of a cancer-fighting immune system. The therapeutic principles and procedures we develop will be applicable to a wide range of cancers and transferrable to other centers that perform bone marrow and HSC transplants. The aggressive milestone-driven IND timeline is based on our: 1) Research that led to the selection and development of a blood cell gene for clinical use in collaboration with the leading experts in the field, 2) Wealth of investigator-initiated cell-based clinical research and the Human Gene Medicine Program (largest in the world with 5% of all patients worldwide), 3) Experience filing a combined 15 investigator initiated INDs for research with 157 patients enrolled in phase I and II trials, and 4) Ability to have leveraged significant institutional resources of on-going HSC laboratory and clinical research contributed ~$2M of non-CIRM funds to pursue the proposed research goals, including the resulting clinical trial.
Statement of Benefit to California: 
Cancer is the leading cause of death in the US and melanoma incidence is increasing fastest (~69K new cases/year). Treatment of metastatic melanoma is an unmet local and national medical need (~9K deaths/year) striking adults in their prime (20-60 years old). Melanoma is the second greatest cancer cause of lost productive years given its incidence early in life and its high mortality once it metastasizes. The problem is severe in California, with large populations with skin types sensitive to the increased exposure to ultraviolet light. Most frequently seen in young urban Caucasians, melanoma also strikes other ethnicities, i.e., steady increases of acral melanoma in Latinos and African-Americans over the past decades. Although great progress has been made in the treatment of certain leukemias and lymphomas with targeted and combination therapies, few options exist for the definitive treatment of late stage solid tumors. When cancers like lung, breast, prostate, pancreas, and melanoma metastasize beyond surgical boundaries, prolonged remissions or cures are rare and most cancer therapies only inhibit cell growth and/or reduce such growth but do not stop the cancer. Our proposal, the filing of an IND and the conduct of a phase 1 clinical trial using genetically modified autologous hematopoietic stem cells (HSC) for the immunotherapy of advanced stage melanoma allowing sustained production of cancer-reactive immune cells, has the potential to address a significant and serious unmet clinical need for the treatment of melanoma and other cancers, increase patient survival and productivity, and decrease cancer-related health care costs. The advantage of the proposed HSC methodology over our current work with peripheral blood cells is that genetically modified stem cells will continuously generate melanoma-targeted immune cells in the patient’s body providing prolonged protection against the cancer. The therapeutic principles and procedures developed here will be applicable to a wide range of cancers. Good Manufacturing Practices (GMP) reagents and clinical protocols developed by our team will be transferable to other centers where bone marrow and peripheral blood stem cell transplantation procedures are done.

Genetic Re-programming of Stem Cells to Fight Cancer

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05309
ICOC Funds Committed: 
$110 000
Disease Focus: 
Melanoma
Cancer
oldStatus: 
Closed
Public Abstract: 
Science has made great progress in the treatment of certain cancers with targeted and combination therapies, yet prolonged remissions or cures are rare because most cancer therapies only inhibit cell growth and/or reduce such growth but do not stop the cancer. The study investigators propose to develop an Investigational New Drug (IND) and fully accrue a phase I clinical trial within the grant period to genetically redirect the patient’s immune response to specifically attack the cancer starting from hematopoietic (blood) stem cells (HSC) in patients with advanced forms of the aggressive skin cancer malignant melanoma. Evaluation of immune system reconstitution, effectiveness and immune response during treatment will use imaging with Positron Emission Tomography (PET) scans. The HSC treatment approach has been validated in extensive studies in the laboratory. The investigators of this grant have recently initiated a clinical trial where adult immune cells obtained from blood are genetically modified to become specific killer cells for melanoma. These cells are administered back to patients. The early data from this study is encouraging in terms of the ability to generate these cells, safely administer them to patients leading to beneficial early clinical effects. However, the adult immune cells genetically redirected to attack cancer slowly decrease over time and lose their killer activity, mainly because they do not have the ability to self-renew. The advantage of the proposed HSC method over adult blood cells is that the genetically modified HSC will continuously generate melanoma-targeted immune killer cells, hopefully providing prolonged protection against the cancer. The IND filing with the FDA will use the modified HSC in advanced stage melanoma patients. By the end of year 4, we will have fully accrued this phase 1 clinical trial and assessed the value of genetic modification of HSCs to provide a stable reconstitution of a cancer-fighting immune system. The therapeutic principles and procedures we develop will be applicable to a wide range of cancers and transferrable to other centers that perform bone marrow and HSC transplants. The aggressive milestone-driven IND timeline is based on our: 1) Research that led to the selection and development of a blood cell gene for clinical use in collaboration with the leading experts in the field, 2) Our wealth of investigator-initiated cell-based clinical research and the Human Gene Medicine Program (largest in the world with 5% of all patients worldwide), 3) Experience filing a combined 15 investigator initiated INDs for research with 157 patients enrolled in phase I and II trials, and 4) Ability to leverage significant institutional resources of on-going HSC laboratory and clinical research and contribute ~$1M of non-CIRM funds to pursue the proposed research goals, including the resulting clinical trial.
Statement of Benefit to California: 
Cancer is the leading cause of death in the US and melanoma incidence is increasing fastest (~69K new cases/year). Treatment of metastatic melanoma is an unmet local and national medical need (~9K deaths/year) striking adults in their prime (20-60 years old). Melanoma is the second greatest cancer cause of lost productive years given its incidence early in life and its high mortality once it metastasizes. The problem is severe in California, with large populations with skin types sensitive to the increased exposure to ultraviolet light. Most frequently seen in young urban Caucasians, melanoma also strikes other ethnicities, i.e., steady increases of acral melanoma in Latinos and African-Americans over the past decades. Although great progress has been made in the treatment of certain leukemias and lymphomas with targeted and combination therapies, few options exist for the definitive treatment of late stage solid tumors. When cancers like lung, breast, prostate, pancreas, and melanoma metastasize beyond surgical boundaries, prolonged remissions or cures are rare and most cancer therapies only inhibit cell growth and/or reduce such growth but do not stop the cancer. Our proposal, the filing of an IND and the conduct of a phase 1 clinical trial using genetically modified autologous hematopoietic stem cells (HSC) for the immunotherapy of advanced stage melanoma allowing sustained production of cancer-reactive immune cells, has the potential to address a significant and serious unmet clinical need for the treatment of melanoma and other cancers, increase patient survival and productivity, and decrease cancer-related health care costs. The advantage of the proposed HSC methodology over our current work with peripheral blood cells is that genetically modified stem cells will continuously generate melanoma-targeted immune cells in the patient’s body providing prolonged protection against the cancer. The therapeutic principles and procedures developed here will be applicable to a wide range of cancers. Good Manufacturing Practices (GMP) reagents and clinical protocols developed by our team will be transferable to other centers where bone marrow and peripheral blood stem cell transplantation procedures are done.
Progress Report: 
  • The planning award funds were entirely dedicated to the establishment of the disease team for the full award submission. This has included:
  • - Hiring the project leader, Dr. Phyllis Wu.
  • - Organization of the cell therapy manufacturing, quality assurance, and clinical groups.
  • - A meeting of the external advisory board.
  • - A site visit to the lentiviral vector manufacturing facility.
  • With these activities we were able to assemble and submit the full CIRM DT-2 application to pursue a translational research project based on the genetic programming of hematopoietic stem cells to become cancer-targeted by the insertion of T cell receptor (TCR) genes.

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

Funding Type: 
Early Translational II
Grant Number: 
TR2-01816-A
ICOC Funds Committed: 
$3 607 305
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Adult Stem Cell
Cancer Stem Cell
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.
Progress Report: 
  • 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 "expansion phase" and a "dormancy phase". The "expansion 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 "domancy 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.
  • Progress during Year 1: During the first year of this project, we discovered that the BCL6 factor is required to maintain leukemia stem cells in this well-protected safe haven. Our findings during year 1 demonstrate that the "dormancy 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 "dormancy phase" .
  • In year 1, we have performed screening procedures to identify novel therapeutic BCL6 inhibitors to eradicate leukemia stem cells: We have found that dual targeting of oncogenic tyrosine kinases ("expansion phase" ) and BCL6 ("dormancy phase") represents a powerful strategy to eradicate drug-resistant leukemia stem cells and prevent the acquisition of drug-resistance and recurrence of the disease.
  • Goal for years 2-3: 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.

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).
  • Our work during the fifth and last year of our CIRM New Faculty award has been essentially focused on understanding how deregulations in DNA repair mechanisms contribute to the aberrant functions of old hematopoietic stem cells (HSC) and the aging of the blood system.

Role of the tumor suppressor gene, p16INK4a, in regulating stem cell phenotypes in embryonic stem cells and human epithelial cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00444
ICOC Funds Committed: 
$639 150
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. In some cases these properties are not correctly regulated and cells with stem cell properties expand in number. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. This means that a cell can simultaneously acquire stem cell properties and cancer properties. In order to effectively use stem cells for therapeutic purposes we need to understand the link between these two programs and devise ways to access one program without turning on the other. In other words, we would like to expand stem cell populations without them turning into cancer. Recent work in our laboratory has found that the reduction of a specific tumor suppressor gene, p16, not only removes an important barrier to cancer but also confers stem cell properties within the cell. Cells that have reduced p16 activity can turn on a program that increases and reduces expression of specific genes that control differentiation. In this proposal we will test whether the continued reduction of this tumor suppressor gene creates human embryonic stem cells (hESC) that are unable to differentiate. We hypothesize that the lack of p16 represses multi-lineage potential by activating an epigenetic program and silencing genes that drive differentiation. To test this hypothesis we will first determine if lack of p16 activity is necessary for hESCs to develop into different cell types. Second, we will determine if continued lack of p16 activity is sufficient to inhibit differentiation of hESCs. Finally, we will determine if transient lack of p16 activity is sufficient for a non-stem cell to exhibit properties of a stem cell after propagation in a stem cell niche. Since these types of events are potentially reversible, targeting such events may become clinically useful. These new observations identify novel opportunities. They provide potential markers for determining if someone is susceptible to cancer, as well as, providing potential targets for prevention and therapy. We hypothesize that these properties are critically relevant to the formation of cancer and will provide insights into the role of epigenetic modifications in disease processes and stem cell characteristics.
Statement of Benefit to California: 
Stem cells hold great potential to help us in repairing injured body parts or replacing damaged organs. In order to realize this potential the rules that control stem cell behavior need to be understood. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. In the proposed study we hypothesize that we may learn about a fundamental switch that not only controls stem cells but also controls the formation of a cancer cell. In understanding how this switch works we may be able to identify biomarkers that indicate when a normal looking cell will become a cancer cell or identify a drug that will allow us to stop the potential cancer cell from increasing in number. Since cancer is a common disease in California, any insights we can gain to battle this disease will benefit the citizens of our State. There is also another side to the insights that may arise from the work in this proposal. Currently we believe the roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. We do not know how to encourage a stem cell to repair, for example, some heart tissue rather than some bone tissue. If we could understand the code that directs the stem cells to differentiate in the proper fashion into one tissue or another, we could use these cells for clinical benefit. The pathways we are studying in this proposal tell the stem cells which genes to silence and which to activate. This is the program that allows the one original cells of your body (the embryo) to diversify into the multitude of specialized cells that work together to make a functioning person (eye cells, skin cells, nerve cells, etc.). In order to effectively use stem cells for therapeutic purposes we need to understand how they code their decisions and whether they can be changed after they have been set. These insights would allow us to aid in maintaining the health of the citizens of California. Finally, if we do gain insight into the code that regulates the differences between cancer cells and stem cells, this information would be the basis of a new area of biotechnology. The generation of knowledge in this area would help in the development of companies, the recruitment of bright young minds and in the fiscal health of our State
Progress Report: 
  • Stem cells hold great potential to help us in repairing injured body parts or replacing damaged organs. In order to realize this potential the rules that control stem cell behavior need to be understood. Our laboratory has found that repression of the tumor suppressor p16 in human mammary epithelial cells (HMECs) endows them with specific properties that are only found in classical stem cells and tumor cells. Indeed, repression of p16INK4a in HMECs enables them to grow in culture for a long time, something that HMECs expressing p16INK4a cannot achieve. Importantly, we have previously shown that repression of p16INK4a is accompanied by the acquisition of pre-malignant features.
  • Thanks to the support of this CIRM grant, we have now established that a sub-population of these cells display stem cell properties. This means that these cells can self-renew but also differentiate in different breast cell types. Unexpectedly, these cells can also give rise to non-breast cells, such as brain cells, when grown in the appropriate cell culture conditions, making this unique cell model a powerful tool for cancer AND regenerative medicine research. Knowing that these cells can generate cells of different tissue types, we can now dissect the rules that dictate those different cell fates. We are also testing whether these exciting findings obtained in cell culture dishes (in vitro) can be confirmed in a mouse model (in vivo). In other words, can these cells generate a functional mammary gland? Other studies, beyond the scope of this application could also test whether these cells could rescue spinal injury.
  • So why do we bother using breast cells to generate brain cells (or other types of cells)? The answer is that we believe that the sub-population of cells we have identified in breast likely exists as a stem cell pool in any tissue (with some tissue-specific variations of course). If this hypothesis is confirmed, these cells could turn out to represent a major advancement in regenerative medicine. Another major advantage of these naturally occurring stem cells, compared to the widely used embryonic stem cell lines, is that they are directly isolated from fresh breast tissue without introducing artifacts that may result from establishment in long-term cell culture systems. Their properties are an accurate reflection of a fully functional stem cell pool actually existing physiologically in our body.
  • Understanding how stem cells code their decisions and whether cell fate can be changed after it has been set is key to the effective use of stem cells for therapeutic purposes. Gaining such insights will greatly improve our ability to manage wound repair and organ replacement. This should also help us characterize fundamental switches that control stem cells as well as control the formation of cancer cells since some of the genes that control stem cell properties are mutated in cancer. A mechanistic understanding of how these switches work may help us prevent adverse events that may result from the use of stem cells during regenerative medicine. Thus, we hope to contribute in improving the health of the citizens of California.
  • An important feature of adult stem cells is the ability to bypass negative growth signals and participate in wound healing. Based on this premise, we identified a small subpopulation of human breast epithelial cells that is capable of bypassing negative growth signals. We identified a differential expression of genes that allowed for the rapid isolation of this novel somatic cell population from fresh disease-free human breast tissue. Importantly, this cell population is characterized by the over-expression of Bmi-1, a protein that plays an essential role in the self-renewal of stem cells and represses the cell cycle inhibitor, p16. This population of cells is therefore poised to express pluripotency markers at a level similar to that measured in human embryonic stem cells. It has the ability to self-renew and can express phenotypes of any of the three mammary lineages in vitro using cell culture differentiation assays. Importantly, these cells are also functional in vivo as observed after implantation in mice. Indeed, these human cells can differentiate into functional mammary outgrowths of human origin in the host mouse as we could document secretion of human milk in mice transplanted with these human somatic cells. We are currently investigating whether these cells can also differentiate into other lineages (tissue types) when cultured in the appropriate conditions. Our preliminary studies support that these cells will hold great promise in regenerative medicine and cell replacement therapy and may help overcome some of the important ethical and technical roadblocks related to the use of human embryonic stem cells.

Sources of Genetic Instability in Human Embryonic Stem Cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00428
ICOC Funds Committed: 
$357 978
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The constant exposure of cells to endogenous and exogenous agents that inflict DNA damage requires active repair processes to eliminate potentially mutagenic events in stem cells leading to cancer. The same agents menace early human embryos with DNA damage that can ultimately lead to mutations, cancer, and birth defects. In vitro, human embryonic stem cells (HESCs) spontaneously undergo events leading to genetic instability and mutations. All these three types of genetic problems can have similar links to malfunctions in DNA repair systems, but little information now exists for HESCs. Therefore, the first step in understanding the causes of HESC genetic instability is to understand which DNA repair systems are defective. We will investigate the basis for this phenomenon in HESCs by evaluating their capacity to either repair DNA or form mutations. First, we will culture two HESC lines and compare HESC repair and mutation formation to that of control cells. We will use a new technique which simplifies the production and use of the feeder cells that support the growth of the HESCs. We will also test the genetic stability of HESCs grown on conventional feeder cells, as well as those grown in feeder free culture. We will use three types of DNA repair assays to monitor the genetic stability of the two HESC lines grown in these different ways. In the first of these assays, DNA molecules with different randomly-induced damage are transferred into HESCs, and DNA repair is followed by the re-establishment of the activity of a reporter protein that is coded for in the damaged DNA. A second assay will introduce specific DNA damage at a unique site in DNA that is transferred to HESCs and repair is determined using a polymerase chain reaction-based technique. Since aneuploidy is also known to be caused by double-strand DNA breaks, we will use two other assays to evaluate capacity of HESCs to repair that type of damage. These experiments will indicate if DNA repair pathways that eliminate DNA damage are dysfunctional and cause genetic instability. The final endpoint for these preliminary experiments is the formation of mutations. To study this, we have modified an assay system so that it will function in normal human cells to monitor mutations which arise spontaneously or those which are induced by various agents. In summary, these investigations will provide the basis for understanding genetic instability in HESCs that can direct cells to tumorgenic outcomes. The employment of HESCs clinically will require such knowledge. Moreover, these results will also yield information on susceptibility to mutations of cells early in development. The practical and basic science aspects of this seed grant proposal should lead to a complete proposal in the near future.
Statement of Benefit to California: 
Human embryonic stem cells (HESCs) hold the potential to cure or alleviate many chronic illnesses, including cancer, but an immense gap exists between the achievement of the goals of stem cell based medicine and the current state of the art. Several stages of development including the following are required: (1) Routine, standardized, simple protocols for the indefinite growth of HESC in the normal, undifferentiated state, in completely defined medium.(2) Control over differentiation of the cells in (1) to all adult cell types of interest. (3) Control over the maintenance of the differentiated state of derivatives of (1), in sufficient complexity to recreate normal functional histology. (4) Techniques, therapies, and protocols that allow immune tolerance of regenerated tissue, without rendering the human recipient immunodefficient. Researchers are still struggling with steps 1 and 2. Although claims of feeder cell and animal product-free, long-term, undifferentiated HESC culture have been made, this is not the current state of the art in laboratories. These claims may be fortuitous or true for only a few HESC lines. The public anticipates a quick success of human stem cell technology and application to human disease, but the promise of stem cell therapy requires basic scientific work that is critical, but may not make headlines. Imprudent claims of miraculous cures could dim public enthusiasm. Few if any data exist regarding DNA repair systems or mutation frequencies of HESCs. We propose to investigate mechanisms underlying genetic instability in cultured HESCs. This instability limits HESC research and therapeutic applications. The data generated by this research according to Proposition 71 will be of lasting value to the People of the State of California for the following reasons: (1) This proposal focuses on a serious, basic difficulty with respect to the growth of undifferentiated HESCs that is a barrier to their human therapeutic use.(2) In the future, if the focus of the stem cell field shifts to the as yet unavailable somatic nuclear transfer (SNT) methods, this proposed research, will provide a basis for the comparison of HESCs and SNT cell lines. (3) All humans begin as embryonic stem cells, therefore data generated by the proposed research will impact maternal health, well baby programs, early childhood development/learning, etc, because mutations are involved in birth defects as well as cancer. Therefore, understanding the causes of mutations in HESCs could assist in avoidance or reduction in birth defects that would aid both the families and the government of California.(5) All of the work described in this proposal will be conducted by individuals in California and most probably will result in the hiring of a graduate of a California institution of higher education, thus reducing unemployment and helping educate a new generation of California researchers in HESC use.
Progress Report: 
  • Human embryonic stem cells (hESCs) originate directly from human embryos, whereas induced pluripotent stem cells (iPSCs) originate from body (somatic) cells that are re-programmed by producing or introducing proteins that control the process making specific RNAs. Together, both these pluripotent cell types are referred to as human pluripotent stem cells (hPSCs). Several reports have observed that in hESCs grown for long times, their genetic material, DNA, is unstable. The stable maintenance of DNA is performed by groups of proteins functioning in different systems globally known as DNA repair pathways. Since the development of aneuploidy is closely linked to cancer and to deficiencies in DNA repair, we have studied the propensity of hPSCs to repair their DNA efficiently by 4 major known DNA repair pathways. In addition, we are also investigating if specific damage to DNA in either hPSCs or somatic cells is processed differently and could lead to deleterious mutations.
  • One major goal of the CIRM SEED grant mission is to bring new researchers into the hPSC field. The results we obtained during the funding period indicate that we have succeeded in that objective, since initially our laboratory had little experience with hESC culture. However, through courses and establishing critical collaborations with other hESC laboratories, we developed expertise in hPSC culture techniques. Most conditions for hPSCs growth require cells (feeder cells) that serve as a matrix and provide some factors needed for the pluripotent cells to divide. In accomplishing this aim, we perfected a method to generate reproducible feeder cells that significantly reduces the time and cost of feeder cell maintenance, and also developed a non-enzymatic and non-mechanical way to expand hPSCs. We now have experience with at least 5 hPSC lines and have methods to introduce foreign DNAs into hESCs and iPSCs to monitor DNA repair in hPSCs.
  • In Aim II of our grant, we used our accumulated knowledge of hPSCs and DNA repair to investigate 4 DNA repair mechanisms in hPSCs and in somatic cells. Depending on the DNA damage, there is often a preferred DNA repair pathway that cells use to alleviate potential harm. We initiated our investigation by treating hPSCs using different DNA damaging agents, including ultraviolet light and gamma radiation. However, we found that hPSCs exposed to these agents rapidly died compared to treatments that allowed somatic cells to continue growing. Therefore, we developed methods to study DNA repair in hPSCs without directly treating the cells with external agents. We treated closed, circular DNA (plasmids) with damaging agents separately, outside the hPSCs and then introduced them into the hPSCs. The plasmid DNA has a sequence that codes for a protein that is produced only when the damage is repaired. The length of time for repair both in hPSCs and in somatic cells was followed by determining the protein production. We have shown superior DNA repair ability and elevated protection against DNA damage in hPSCs compared to somatic cells for ultraviolet light and oxidative damage, two common sources of damage in cells. A major pathway for joining double-strand DNA breaks in mammalian cells, non-homologous end-joining (NHEJ) repair (error prone), is greater in H9 cells than in iPSCs. Another way to repair double-strand DNA breaks that uses similar (i.e., homologous) sequences is lower in iPSCs compared to hESCs and somatic cells. Further study of these repair pathways is warranted, since several methods can be used to form iPSCs. Therefore, the genomic stability for iPSCs could depend on the method used for their generation.
  • DNA repair analysis is critical to understanding how hPSCs protect against damage, but if left unrepaired, cells can turn damage into mutations when the damage is copied by enzymes (DNA polymerases) before repair occurs. Therefore, to monitor the mutations that ultimately lead to cancer or alter hPSC biology, we are using a plasmid that is damaged outside the cells and will make copies in hPSCs and somatic cells. That plasmid is introduced into cells and then the copies are recovered. The number of mutations found in the plasmid DNA indicates the likelihood of observing mutations in hPSCs compared to mutations in somatic cells. Together, these results will yield data on the stability of hPSCs and also a basis to monitor cells for stability which could serve as an indicator of safety for clinical use.

Using human embryonic stem cells to treat radiation-induced stem cell loss: Benefits vs cancer risk

Funding Type: 
SEED Grant
Grant Number: 
RS1-00413
ICOC Funds Committed: 
$625 617
Disease Focus: 
Cancer
Neurological Disorders
Skeletal Muscle
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
A variety of stem cells exist in humans throughout life and maintain their ability to divide and change into multiple cell types. Different types of adult derived stem cells occur throughout the body, and reside within specific tissues that serve as a reserve pool of cells that can replenish other cells lost due to aging, disease, trauma, chemotherapy or exposure to ionizing radiation. When conditions occur that lead to the depletion of these adult derived stem cells the recovery of normal tissue is impaired and a variety of complications result. For example, we have demonstrated that when neural stem cells are depleted after whole brain irradiation a subsequent deficit in cognition occurs, and that when muscle stem cells are depleted after leg irradiation an accelerated loss of muscle mass occurs. While an increase in stem cell numbers after depletion has been shown to lead to some functional recovery in the irradiated tissue, such recovery is usually very prolonged and generally suboptimal.Ionizing radiation is a physical agent that is effective at reducing the number of adult stem cells in nearly all tissues. Normally people are not exposed to doses of radiation that are cause for concern, however, many people are subjected to significant radiation exposures during the course of clinical radiotherapy. While radiotherapy is a front line treatment for many types of cancer, there are often unavoidable side effects associated with the irradiation of normal tissue that can be linked to the depletion of critical stem cell pools. In addition, many of these side effects pose particular threats to pediatric patients undergoing radiotherapy, since children contain more stem cells and suffer higher absolute losses of these cells after irradiation.Based on the foregoing, we will explore the potential utility and risks associated with using human embryonic stem cells (hESC) in the treatment of certain adverse effects associated with radiation-induced stem cell depletion. Our experiments will directly address whether hESCs can be used to replenish specific populations of stem cells in the brain and muscle depleted after irradiation in efforts to prevent subsequent declines in cognition and muscle mass respectively. In addition to using hESC to hasten the functional recovery of tissue after irradiation, we will also test whether implantation of such unique cells holds unforeseen risks for the development of cancer. Evidence suggests that certain types of stem cells may be prone to cancer, and since little is known regarding this issue with respect to hESC, we feel this critical issue must be addressed. Thus, we will investigate whether hESC implanted into animals develop into tumors over time. The studies proposed here comprise a first step in determining how useful hESCs will be in the treatment of humans exposed to ionizing radiation, as well as many other diseases where adult stem cell depletion might be a concern.
Statement of Benefit to California: 
Radiotherapy is a front line treatment used in California for many types of cancer, including brain, breast, prostate, bone and other cancer types presenting surgical complications. Treatment of these cancers through the use of radiation is however, often associated with side effects caused by the depletion of critical stem cell pools contained within non-cancerous normal tissue. While radiotherapy is clearly beneficial overall, many of these side effects have no viable treatment options. If we can demonstrate that human embryonic stem cells (hESC) hold promise as a safe therapeutic agent for the treatment of radiation-induced stem cell depletion, then cancer patients may have a new treatment for countering many of the debilitating side effects associated with radiotherapy. Once developed this new technology could position California to attract cancer patients throughout the United States, and the state would clearly benefit from the increased economic activity associated with a rise in patient numbers.
Progress Report: 
  • We have undertaken an extensive series of studies to delineate the radiation response of human embryonic stem cells (hESCs) and human neural stem cells (hNSCs) both in vitro and in vivo. These studies are important because radiotherapy is a frontline treatment for primary and secondary (metastatic) brain tumors. While radiotherapy is quite beneficial, it is limited by the tolerance of normal tissue to radiation injury. At clinically relevant exposures, patients often develop variable degrees of cognitive dysfunction that manifest as impaired learning and memory, and that have pronounced adverse effects on quality of life. Thus, our studies have been designed to address this serious complication of cranial irradiation.
  • We have now found that transplanted human embryonic stem cells (hESCs) can rescue radiation-induced cognitive impairment in athymic rats, providing the first evidence that such cells can ameliorate radiation-induced normal-tissue damage in the brain. Four months following head-only irradiation and hESC transplantation, the stem cells were found to have migrated toward specific regions of the brain known to support the development of new brain cells throughout life. Cells migrating toward these specialized neural regions were also found to develop into new brain cells. Cognitive analyses of these animals revealed that the rats who had received stem cells performed better in a standard test of brain function which measures the rats’ reactions to novelty. The data suggests that transplanted hESCs can rescue radiation-induced deficits in learning and memory. Additional work is underway to determine whether the rats’ improved cognitive function was due to the functional integration of transplanted stem cells or whether these cells supported and helped repair the rats’ existing brain cells.
  • The application of stem cell therapies to reduce radiation-induced normal tissue damage is still in its infancy. Our finding that transplanted hESCs can rescue radiation-induced cognitive impairment is significant in this regard, and provides evidence that similar types of approaches hold promise for ameliorating normal-tissue damage throughout other target tissues after irradiation.
  • A comprehensive series of studies was undertaken to determine if/how stem cell transplantation could ameliorate the adverse effects of cranial irradiation, both at the cellular and cognitive levels. These studies are important since radiotherapy to the head remains the only tenable option for the control of primary and metastatic brain tumors. Unfortunately, a devastating side-effect of this treatment involves cognitive decline in ~50% of those patients surviving ≥ 18 months. Pediatric patients treated for brain tumors can lose up to 3 IQ points per year, making the use of irradiation particularly problematic for this patient class. Thus, the purpose of these studies was to determine whether cranial transplantation of stem cells could afford some relief from the cognitive declines typical in patients afflicted with brain tumors, and subjected to cranial radiotherapy. Human embryonic (hESCs) and neural (hNSCs) stem cells were implanted into the brain of rats following head only irradiation. At 1 and 4 months later, rats were tested for cognitive performance using a series of specialized tests designed to determine the extent of radiation injury and the extent that transplanted cells ameliorated any radiation-induced cognitive deficits. These cognitive tasks take advantage of the innate tendency of rats to explore novelty. Successful performance of this task has been shown to rely on intact spatial memory function, a brain function known to be adversely impacted by irradiation. Our data shows that irradiation elicits significant deficits in learning and spatial task recognition 1 and 4-months following irradiation. We have now demonstrated conclusively, and for the first time, that irradiated animals receiving targeted transplantation of hESCs or hNSCs 2-days after, show significant recovery of these radiation induced cognitive decrements. In sum, our data shows the capability of 2 stem cell types (hESC and hNSC) to improve radiation-induced cognitive dysfunction at 1 and 4 months post-grafting, and demonstrates that stem cell based therapies can be used to effectively to reduce a serious complication of cranial irradiation.

Screening for Oncogenic Epigenetic Alterations in Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00408
ICOC Funds Committed: 
$685 000
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Embryonic stem cell-based therapies hold great promise for the treatment of many human diseases. These therapeutic strategies involve the culture and manipulation of embryonic stem cells grown outside the human body. Culture conditions outside the human body can encourage the development of changes to the cells that facilitate rapid and sustained cell growth. Some of these changes can resemble abnormal changes that occur in cancer cells. These include “epigenetic” changes, which are changes in the structure of the packaging of the DNA, as opposed to “genetic” changes, which are changes in the DNA sequence. Cancer cells frequently have abnormalities in one type of epigenetic change, called “DNA methylation”. We have found that cultured embryonic stem cells may be particularly prone to develop the type of DNA methylation abnormalities seen in cancer cells. A single rogue cell with DNA methylation abnormalities predisposing the cell to malignancy can jeopardize the life of the recipient of stem cell therapy. We have developed highly sensitive and accurate technology to detect DNA methylation abnormalities in a single cell hidden among 10,000 normal cells. In this seed grant, we propose to screen DNA methylation abnormalities at a large number of genes in different embryonic stem cells and compare their DNA methylation profiles to normal and cancer cells. This will allows us to identify the dangerous DNA methylation abnormalities most likely to occur in cultured embryonic stem cells. We will then develop highly sensitive assays to detect these DNA methylation abnormalities, using our technology. We will then use these assays to determine ES cell culture conditions and differentiation protocols most likely to cause these DNA methylation abnormalities to arise in cultured ES cells. The long-term benefits of this project include 1) an increased understanding of the epigenetics of human embryonic stem cells, 2) insight into culture conditions to avoid the occurrence of epigenetic abnormalities, and 3) a technology to monitor for epigenetic abnormalities in ES cells intended for introduction into stem cell therapy patients.
Statement of Benefit to California: 
The successful implementation of human embryonic stem cell therapy will require rigorous quality control measures to assure the safety of these therapies. Cells cultured outside the human body are known to be at risk of developing abnormalities similar to those found in cancer cells. Since a single rogue cell hidden among thousands of normal cells could cause cancer in an embryonic stem cell therapy recipient, it will be essential to have highly sensitive and accurate assays to detect these abnormalities in cultured embryonic stem cells before they are introduced into the patient. The goal of this proposal is to develop such sensitive and accurate assays. The citizens of the State of California will benefit from the availability of such assay technology to help assure the safety of human embryonic stem cell therapies.
Progress Report: 
  • Embryonic stem cell-based therapies hold great promise for the treatment of many human diseases. These therapeutic strategies involve the culture and manipulation of embryonic stem cells grown outside the human body. Culture conditions outside the human body can encourage the development of changes to the cells that facilitate rapid and sustained cell growth. Some of these changes can resemble abnormal changes that occur in cancer cells. These include epigenetic changes, which are changes in the structure of the packaging of the DNA, as opposed to genetic changes, which are changes in the DNA sequence. Cancer cells frequently have abnormalities in one type of epigenetic change, DNA methylation. In this grant, we screened for DNA methylation abnormalities at a large number of genes in different embryonic stem cells and compare their DNA methylation profiles to normal and cancer cells. This allowed us to identify potentially dangerous DNA methylation abnormalities, which occur in cultured embryonic stem cells. In the first year of this seed grant, we have developed a custom microarray to screen for DNA methylation changes at predisposed genes. In addition, we have analyzed DNA methylation in embryonic stem cells at more than 14,000 genes on a generic platform. This has allowed us to identify hundreds of genes that are abnormally methylated in various types of human cancers, and that show some evidence of this alteration in ES cells.
  • In the last phase of our study, we have screened the DNA methylation level of 1,536 genes in 142 different human embryonic stem cell pairs. Each member of the pair differed in the length of time it was in culture. Thus, our sample set was comprised of 284 paired specimens, one derived from an early passage and one derived from a late passage.
  • Our results indicate that the levels of DNA methylation varied considerably at a significant portion of the screened genes, some of which gained and some of which lost DNA methylation. These results indicate that DNA methylation in human embryonic stem cells seems to be susceptible to change over, at least in the genes examined in this study. Overall, our results suggest that the monitoring of DNA methylation changes in human embryonic stem cells may have to be incorporated as a routine protocol in stem cell manipulation.
  • During the past 12 months we have made significant progress on the data analysis of 141 paired (early passage-late passage) human embryonic stem cell lines (HESCs). The data in question was generated using a custom Illumina GoldenGate array of known Polycomb targets in HESCs, as described by Lee et al 2006. Briefly, we profiled the DNA methylation status of 1,536 loci on 282 specimens. This profiling was used to determine whether DNA methylation changes in HESCs arise as a result of time in culture at the examined loci. This determination was made by comparing the DNA methylation status of a sample of an early passage line with a late passage sample of the same line.
  • Interestingly, we found that DNA methylation in Polycomb target genes is highly affected by time in culture in a cell line-specific manner. That is, in some cell lines few DNA methylation changes were observed, while in the majority of them a large number of loci showed either an increase or decrease in DNA methylation. Via collaboration with the University of Sheffield, we were able to determine that DNA methylation instability seems to be independent of genetic instability. Furthermore, genetic instability seems to be a function of passage time in culture.

Derivation and Characterization of Cancer Stem Cells from Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$642 500
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Cancer is the leading cause of death for people younger than 85 (1). High cancer mortality rates underscore the need for more sensitive diagnostic techniques as well as therapies that selectively target cells responsible for cancer propagation (1) 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 therapeutic resistance (2-9). Although the majority of current 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 (2-9). We recently demonstrated that CSC are involved in progression of chronic phase chronic myelogenous leukemia (CML), a disease that has been the subject of many landmark discoveries in cancer research(19-30), to a more aggressive and therapeutically recalcitrant myeloid blast crisis (BC) phase. These CSC share the same cell surface markers as granulocyte-macrophage progenitors (GMP) but have aberrantly gained the capacity to self-renew as a result of activation of the Wnt/-catenin stem cell self-renewal pathway (4). 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 in vitro, they represent an ideal model system for generating and characterizing human CSC (10-18). 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 (2-18). To provide a robust model system for screening novel anti-CSC therapies, we propose to generate and characterize CSC from hESC (10-18). We will investigate the role of genes that are essential for initiation of CML such as BCR-ABL and additional mutations such as b-catenin implicated in CSC propagation (19-30). The efficacy of specific Wnt/b-catenin antagonists at inhibiting BCR-ABL+ human ES cell self-renewal, survival and proliferation alone and in combination with potent BCR-ABL antagonists will be assessed in sensitive in vitro and in vivo assays with the ultimate aim of developing highly active anti-CSC therapy that may halt cancer progression and obviate therapeutic resistance (4,31).
Statement of Benefit to California: 
The research outlined in this proposal represents a unique opportunity for collaborations between investigators from disparate disciplines to use human embryonic stem cells to challenge an existing paradigm namely that leukemic blasts are responsible for progression of chronic myelogenous leukemia (CML) rather than leukemic stem cells (LSC). Current clinical diagnostic tests are not sufficiently sensitive to predict timing of progression for all patients with CML nor are they adequate for determining the type of therapeutic intervention required. Moreover, the primary therapy for CML, Abl kinase inhibition, was shown to be cardiotoxic when given long-term at high doses. Furthermore, amplification of BCR-ABL is not the sole event that occurs during CML progression to blast crisis. Identification and inhibition of molecular mutations responsible for the generation of LSC in CML blood and/or marrow may prevent progression to blast crisis (BC) and would represent an innovative, effective form of CML therapy. Modeling of LSC responsible for CML progression in human embryonic stem cells could have a significant impact on our understanding of the pathophysiology of CML, provide novel diagnostic and therapeutic modalities and improve the quality and possibly quantity of life of patients with CML. By using BCR-ABL transduced human embryonic stem cells, we will rigorously evaluate the LSC hypothesis and as a consequence, the additional molecular events required for progression to blast crisis CML. The ultimate aims of this grant are to develop more sensitive methods to predict leukemic progression and to identify novel molecular therapeutic targets through the development of LSC models using human embryonic stem cells. We aim to provide a robust, reproducible system for testing novel anti-LSC compounds alone and in combination in order to expedite the development of novel therapeutic agents for anti-LSC clinical trials at {REDACTED}. Not only may the translational research performed in the context of this grant speed the delivery of innovative anti-LSC therapies for Californians with leukemia, it will help to train California’s 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 their research to cancer therapy and a better perspective on future career opportunities in California.
Progress Report: 
  • SEED Grant Research Summary
  • Compelling studies suggest that cancer stem cells (CSC) arise from primitive self-renewing progenitor cells. Although many cancer therapies target rapidly dividing cells, CSC may be quiescent i.e. asleep resulting in therapeutic resistance. Recently, we demonstrated that CSC drive progression of chronic phase (CP) chronic myeloid leukemia (CML), a subject of many landmark cancer research discoveries, to a therapeutically recalcitrant myeloid blast crisis (BC) phase. CML CSC share cell surface markers with granulocyte-macrophage progenitors (GMP) and have amplified expression of the CML fusion gene, BCR-ABL. In addition, they aberrantly gain self-renewal capacity, in part, as a result Wnt/β-catenin activation. Because human embryonic stem cells (hESC) have robust regenerative capacity and can provide a potentially limitless source of tissue specific progenitor cells in vitro, they represent an ideal model system for generating and characterizing human CSC. The main goals of this research were to generate CSC from hESC to provide an experimentally amenable platform to expedite the development of sensitive diagnostics that predict progression and combined modality anti-CSC therapy.
  • To this end, we tested whether BCR-ABL expression in hESC is sufficient to induce changes characteristic of CML stem cells. Unlike mouse ESC, introduction of a novel lentiviral BCR-ABL vector into hESC did not drive myeloid differentiation nor did it induce stromal independence in vitro underscoring key differences between mouse and human hESC and the importance of in vivo models. Notably, Hues16 cells had a higher propensity to differentiate into CD34+ cells than other hESC lines particularly in AGM co-cultures and thus, were used in subsequent in vivo experiments. Moreover, this SEED grant funded Yosuke Minami in Professor Jean Wang’s lab to create a unique CML blast crisis mouse model typified by GMP expansion and resistance to a BCR-ABL inhibitor, imatinib (Minami et al, PNAS 2008;105:17967-72). In addition, a bioluminescent humanized model of blast crisis CML was created based on transplantation of GMP from patient blood into immune deficient mice (RAG2-/-gc-/-). Cells were tagged with firefly luciferase that emits a bioluminescent signal so that leukemic transplantation efficiency could be tracked in vivo (IVIS). As few as 1,000 human blast crisis CML GMP could transplant leukemia in immune deficient mice thereby providing an important model for studying the molecular events that contribute to leukemic transformation (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the second aim, we hypothesized that BCR-ABL is sufficient for generating CML from self-renewing stem cells. In these studies, Hues16 cells differentiated into CD34+ cells were lentivirally transduced with BCR-ABL leading to sustained BCR-ABL engraftment in 50% of transplanted mice. Chronic phase CD34+ cells derived from CML blood were less efficient at sustaining CML engraftment (7%) suggesting that hESC derived CD34+ cells have higher self-renewal potential and are similar to advanced phase CML progenitors.
  • Thirdly, we hypothesized that BCR-ABL was necessary but not sufficient for progression to blast crisis. Introduction of lentiviral activated beta-catenin or shRNA to GSK3beta, together with BCR-ABL did not enhance BCR-ABL engraftment compared with BCR-ABL transduction of hESC alone. These studies suggested that hESC may already have sufficient self-renewal capacity to sustain the malignant CML clone and are molecularly comparable to advanced CML progenitors that behave like CSC. In addition, through extensive cDNA sequencing of human blast crisis CML progenitors, we found that 57% of samples harbored a misspliced form of GSK3beta that promoted tumor production and could serve as a novel prognostic marker in CML clinical trials (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the final aim, we hypothesized that CML CSC are not eliminated by BCR-ABL inhibitors alone and that combined modality therapy will be required. In collaborative research involving in vitro analysis of imatinib resistant CML progenitors and more recently in a humanized mouse model of blast crisis CML, we found that dasatinib, a potent BCR-ABL inhibitor, is necessary but not sufficient for CSC eradication. Discovery of a GSK3beta deregulation, a negative regulator of both beta-catenin and sonic hedgehog (Shh) pathways (Zhang et al, Nature 2009), led us to disover that Shh combined with BCR-ABL inhibition abrogated CSC driven tumor formation (manuscript in preparation) providing the impetus for an upcoming Pfizer sponsored Shh inhibitor clinical trial for refractory hematologic malignancies.

The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$777 467
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The successful use of human embryonic stem cells (hESCs) as novel regenerative therapies for a spectrum of currently incurable diseases critically depends upon the safety of such cell transfers. hESCs contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of their genetic material. While many of these retroelements have been permanently silenced during evolution by crippling mutations, many remain active and capable of moving to new chromosomal locations potentially producing disease-causing mutations or cancer. More mature differentiated cells control retroelement movement (retrotransposition) by methylating the DNA comprising these elements. Strikingly, such DNA methylation is largely absent in hESCs because these cells must be able to develop into a wide spectrum of different tissues and organs. Thus, in order to protect the integrity of their genomes, hESCs must deploy an additional defense to limit retroelement retrotransposition. Recent studies of HIV and other exogenous retroviruses have identified the APOBEC3 family of genes (A3A-A3H) as powerful anti-retroviral factors. These APOBEC3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also used by retroelements for their successful retrotransposition. We hypothesize that one or more of the APOBECs function as guardians of genome integrity in hESCs. We propose to compare and contrast which APOBEC3s are expressed in one federally approved and nine nonapproved hESC lines and to assess the natural level of retroelement RNA expression occurring in each of these lines. Next we will test whether the knockdown of expression of these APOBEC3s in the hESCS lines by RNA interference leads to a higher frequency of retrolement retrotransposition. Finally, if higher levels of retrotransposition are detected, we will examine whether these cells display an impaired ability to differentiate into specific tissue types corresponding to the three germ cell layers (ectoderm, mesoderm, and endoderm) and whether increased retrotransposition is associated with a higher frequency of malignant transformation within the hESC cultures. These studies promise to provide important new insights into how genomic stability in is maintained in hESCs and could lead to the identification of specific GMP culture conditions that minimize the chances of such unwanted retrotransposition events in cells destined for infusion into patients. These studies are directly responsive to the CIRM request for application. If funded, these studies would allow the entry of my laboratory with extensive APOBEC experience, into the exciting field of stem cell biology.
Statement of Benefit to California: 
Harnessing the exciting potential of embryonic stem cells as therapies for a wide range of diseases like diabetes, Alzheimer’s disease, myocardial infarction among others first requires ensuring that the infusion of these cells into patients can be performed safely. Of note, human embryonic stem cells contain up to 3 million “jumping genes” or mobile genetic retroelements that can potentially move from location to another in the genome. Great harm could occur if the movement of these retroelements in human embryonic stem cells results in the mutation of key genes or the inactivation of tumor suppressor genes, the latter could facilitate the development of cancer in recipients of these cells. The safety of stem cell therapy thus depends on the rigorous maintenance of genomic integrity and stability within the embryonic stem cell during its manipulation. Strikingly, the major cellular defense against the movement of the retroelements to new genetic locations, DNA methylation, is greatly reduced in human embryonic stem cells. A general state of hypomethylation is likely required to permit these pluripotent cells to differentiate into multiple cell types. With DNA methylation no longer able to constrain the activity of these retroelements, we believe a second natural defense springs into action to protect these stem cells. We proposeto identify and characterize this defensive network. These studies could lead to new approaches for maintaining or even enhancing this defense when embryotic stem cells are manipulated in culture, thereby helping to ensure the safety of embryonic stem cells destined for therapeutic transfer. Thus, the results of these studies will have both scientific and practical value. As such, we believe these studies will benefit the citizens of California certainly at a societal level and potentially at a personal level.
Progress Report: 
  • Human embryonic stem cells contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of human genome. While many of these retroelements have been silenced during evolution by crippling mutations, many remain active and capable of jumping to new chromosomal locations potentially producing disease-causing mutations or cancer. In tissues, mobility of these elements is suppressed by DNA methylation, which inactivates expression of the retroelement RNAs. In sharp contrast, embryonic stem cells exhibit very dynamic changes in DNA methylation, where the methylation patterns are gained and lost at high rates. During periods of low DNA methylation, retroelement RNA expression likely increases. Accordingly, hESCs must deploy other defensive strategies in order to maintain genomic integrity. Recent studies have identified the APOBEC3 family of genes (A3A-A3H) as powerful antiviral factors. These A3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also employed by retroelements for their successful retrotransposition. We hypothesized that one or more of the APOBECs function as guardians of genome integrity in hESCs. In the last two years we have found that six out of the seven human A3 genes located in a tandem array on chromosome 22 are expressed in hESCs. A3A, which in prior studies was suggested to exert the greatest anti-retroelement effects, surprisingly is not expressed in hESCs. Further, we find that the A3 proteins decrease when pluripotent cells differentiate into somatic cells suggesting an important function of these A3 proteins in pluripotent hESCs. We established a LINE1 retrotransposition assay in hESCs that allows us to visualize genetic jumping of this class of “marked” retroelements via flow cytometry. Using this assay we have found that LINE1 elements effectively jump in hESCs. To test our central hypothesis, namely that A3 proteins guard the genome in hESCs, we have established experimental conditions for RNAi knock-down of all expressed A3 genes. By combining the knock-down and the retrotransposition assay we demonstrated that the knock-down of one member of the A3 protein family leads to a 3.5-fold increase in LINE1 retrotranspositon. This finding highlights a protective role for the A3 family of cytidine deaminases that helps safeguard the genome integrity of hESCs.

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