Blood Cancer

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

Mechanisms of Hematopoietic stem cell Specification and Self-Renewal

Funding Type: 
New Faculty I
Grant Number: 
RN1-00557
ICOC Funds Committed: 
$2 286 900
Disease Focus: 
Blood Cancer
Cancer
Anemia
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • The goal of this grant is to investigate the cell intrinsic mechanisms that govern hematopoietic stem cell specification and self-renewal. During the second year of this award, we have further elucidated the regulatory mechanisms that dictate hematopoietic fate specification by validating the target genes that Scl/tal1 activates and represses in vivo (Aim 1). We have also shown that loss of Scl results not only results in loss of all blood cells, but also causes defective arterio-venous identity that precludes generation of hemogenic endothelium and hematopoietic stem cells. We have defined the phenotype of hemogenic endothelium and emerging HSCs in both mouse and human embryos (Aim 2), and identified novel markers that can be used to isolate developing HSCs at distinct stages, as well as to purify functional HSCs further (Aim 3). We have also established an inducible lentiviral based expression system that will now be used to test functionally candidate HSC regulators that were identified by comparing gene expression profiles between freshly isolated HSCs and dysfunctional HSCs that were expanded in culture or generated from human ES cells. We hope that these studies will provide better understanding of the key regulatory mechanisms that govern HSC properties, and ultimately lead to development of improved methods for generation of functional HSCs in culture.
  • Our work has focused on defining mechanisms that govern the specification and self-renewal of hematopoietic stem cells during mouse and human development. Using gene targeted mouse ES cells and mouse embryos, we defined the transcriptional programs that are regulated by Scl, the master regulator for blood formation. We discovered that Scl not only establishes the transcriptional programs that are critical for specifying hemogenic endothelium and hematopoietic stem cells, but it also represses heart development. Strikingly, in the absence of Scl, hemogenic endothelium in embryonic hematopoietic tissues becomes converted to cardiogenic fate, and gives rise to fully functional, beating cardiomyocytes.
  • In order to define the key programs that distinguish self-renewing HSCs from their downstream progenitors or the compromised HSPCs (hematopoietic stem/progenitor cells) that were generated in vitro, we performed microarray analysis for human phenotypic HSCs from various sources. We identified novel markers for human HSCs that can be used to purify transplantable HSCs to a higher purity. We have identified key molecular defects in HSCs that are expanded in culture, or generated from human ES cells. We have further validated that dysregulation of certain Hox genes is a major bottleneck for generating functional HSCs from human ES cells. Future studies are focused on establishing methods that would allow correction of the compromised HSC regulatory networks in cultured HSCs.
  • We have defined key regulatory mechanisms that are required for generation and maintenance of blood forming stem cells. We showed that transcription factor Scl is critical for specifying hemogenic endothelium from where blood stem cells emerge, and moreover, we discovered and unexpected repressive function for Scl to suppress cardiomyogenesis; in the absence of Scl, the blood vessels in start to generate beating cardiomyocytes. We have also identified factors that are critical for blood stem cells to maintain the unique properties: to self-renew (make more of themselves) and engraft (interact with the niche cells that support them). We will now continue to define how these key regulators act so that we can design better strategies to generate blood stem cells as well as heart muscle precursors for therapeutic applications.
  • The goal of this grant was to define mechanisms that govern blood stem cell specification and self-renewal. We have completed the studies on hematopoietic fate specification by defining how Scl/tal1 establishes hemogenic endothelium. We documented that, in addition to Scl’s critical function in activating blood cell regulators, Scl also has to repress heart factors to prevent the misspecification of blood precursors to heart muscle. We documented that Scl controls blood and heart regulators through enhancers that have been primed for activation prior to Scl action (Aim 1). We identified a new surface marker that is expressed in hemogenic endothelium and blood forming cells in the yolk sac (Lyve1), which provides new tools to investigate the origin of blood stem and progenitor cells during development (Aim 2). We identified GPI-80 as a novel marker for transplantable blood stem cells during human fetal development (Aim 2, 3). Taking advantage of this new marker for blood stem cells, we narrowed down the critical defects in the dysfunctional blood precursors that are generated from human ES cells, or expanded in culture from fetal liver blood stem cells (Aim 3). We showed that the inability to induce HOXA cluster genes and other novel blood stem cell regulators that cannot be sustained in culture hinder the generation of blood stem cells from pluripotent cells, and further validated these novel regulators using lentiviral knockdown and overexpression. These findings will now be used to develop novel strategies to generate blood stem cells in culture.

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: 
Statement of Benefit to California: 
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.

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

Funding Type: 
New Faculty II
Grant Number: 
RN2-00910
ICOC Funds Committed: 
$3 065 572
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • Summary of Overall Progress
  • This grant focuses on generation of MPN stem cells from hESC or CB and correlates leukemic potential with MPN patient samples. In the first year of this grant, we have demonstrated that 1) hESC differentiate on AGM stroma to the CD34+ stage, which is associated with increased GATA-1, Flk2, GATA-2 and ADAR1 expression; 2) hESC CD34+ differentiation is enhanced in vitro and in vivo in the presence of a genetically engineered mouse stroma, which produces human stem cell factor, IL-3 and G-CSF; 3) hESC CD34+ cells can be transduced with our novel lentiviral BCR-ABL vector, which, unlike retroviral BCR-ABL, can transduce quiescent stem cells; 4) BCR-ABL expression by CP CML progenitors does not sustain engraftment but rather leukemic transformation is predicated, in part, on bcl-2 overexpression; 5) JAK2V617F expression in hES or CB stem cells is insufficient to induce leukemic transformation; 6) BCR-ABL transduced hESC CD34+ cells have significantly higher BCR-ABL transplantation potential than CP CML progenitors suggesting that they have higher survival capacity; 7) lentiviral -catenin transduction of BCR-ABL hESC CD34+ cells leads to serial transplantation indicative of LSC formation; 8) CML BC LSC persist in vivo despite potent BCR-ABL inhibition with dasatinib therapy and will likely require combined inhibitor therapy to eradicate. Currently, HEEBO arrays and phospho-flow studies are underway to detect bcl-2 family members and self-renewal protein expression in BCR-ABL and JAK2 V617F transduced hESC and CB CD34+ cells compared with MPN patient derived progenitors. This will aid in development of combined MPN stem cell inhibitor strategies in this grant.
  • This grant focuses on generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent (hESC) or multipotent (CB) stem cells and seeks to correlate their leukemic potential with that of MPN patient sample-derived stem cells. To provide a platform for testing induction of stem cell differentiation, survival and self-renewal by BCR-ABL versus JAK2, hESC were utilized in the first year and as more patient samples and cord blood became available these were utilized.
  • In the first year of this grant, we found that hESC undergo hematopoietic differentiation on AGM stroma to the CD34+ stage resulting in increased GATA-1, Flk2, ADAR1 and GATA-2 expression. Moreover, CD34+ differentiation was enhanced on a genetically engineered mouse stroma (SL/M2) secreting human SCF, IL-3 and G-CSF. Lentiviral BCR-ABL transduced hESC-derived CD34+ cells had higher BCR-ABL+ cellular transplantation potential than chronic phase (CP) CML progenitors, indicative of a higher survival capacity. However, they sustained self-renewal only when co-transduced with lentiviral -catenin (Rusert et al, manuscript in preparation) suggesting that blast crisis evolution requires acquisition of both enhanced survival and self-renewal potential. Similarly, lentiviral mouse mutant JAK2 expression in hESC or CB stem cells was insufficient to produce self-renewing MPN stem cells, indicating that the cellular context, nature of the genetic driver and responses to extrinsic cues from the microenvironment play seminal roles in regulating therapeutically resistant MPN stem cell properties such as aberrant survival, differentiation, self-renewal and dormancy.
  • In the second year of this five year grant, we have focused on human cord blood (CB) stem cells compared with a large number of MPN patient samples propagated on SL/M2 stroma or in RAG2-/-c-/- mice to more adequately recapitulate the human MPN stem cell niche. Also, to more faithfully recapitulate human (rather than the previously published lentiviral mouse JAK2 vectors, Cancer Cell 2008) JAK2 driven MPNs, we cloned human wild-type JAK2 and human JAK2 V617F from MPN patient samples into lentiviral-GFP vectors (Court Recart A*, Geron I* et al, manuscript in preparation). We also incorporated full transcriptome RNA (ABI SOLiD 4.0) sequencing, PCR array and nanofluidic phosphoproteomics technology to better gauge the impact of JAK2 versus BCR-ABL on stem cell fate, survival, self-renewal and dormancy in the context of specific malignant microenvironments and the relative susceptibility of MPN stem cells in these niches to single agent molecularly targeted inhibitors.
  • This grant focuses on generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent human embryonic stem cells (hESC) or multipotent cord blood (CB) stem cells, and seeks to correlate their leukemic potential with that of disease progression in MPN patient sample-derived stem cells. In the first and second years of this grant, we found that lentiviral BCR-ABL transduced hESC-derived CD34+ cells had higher leukemic transplantation potential than chronic phase (CP) chronic myeloid leukemia (CML) progenitors. However, they sustained self-renewal only when co-transduced with lentiviral beta-catenin suggesting that blast crisis (BC) evolution requires acquisition of both enhanced survival and self-renewal potential. Similarly, we have shown using lentiviral vectors that mouse and human mutant JAK2 were insufficient to produce self-renewing MPN stem cells. New results in Year 3 demonstrate that BCR-ABL and JAK2 activation drive differentiation of hematopoietic progenitors towards an erthyroid/myeloid lineage bias. We have used full transcriptome RNA-Sequencing (RNA-Seq) technology to evaluate the genetic and epigenetic status of BCR-ABL and JAK2-transduced normal progenitor cells as well as patient-derived MPN progenitors. This has allowed us to probe the mechanisms of aberrant differentiation and self-renewal of MPN progenitors and identify unique gene expression signatures of disease progression.
  • We previously found that overexpression and splice isoform switching of a key RNA editing enzyme – adenosine deaminase acting on dsRNA (ADAR), and splice isoform changes in pro-survival BCL2 family members, correspond with disease progression in CML. In the current reporting period, RNA-Seq analyses revealed that ADAR1-driven activation of RNA editing contributed to malignant progenitor reprogramming, promoting aberrant differentiation and self-renewal of MPN stem cells. Knocking down ADAR1 using lentiviral shRNA vectors reduced the self-renewal potential of CML progenitors. This work has culminated in a manuscript that has now been submitted to PNAS (Jiang et al.). Recent results also show that ADAR1 is activated in progenitors from patients with JAK2-driven MPNs. Thus, ADAR1 may be an important factor that works in concert with BCR-ABL or JAK2 to facilitate disease progression in MPNs.
  • Our results show that another self-renewal factor that may drive BCR-ABL or JAK2-mediated propagation of disease from quiescent MPN progenitors is Sonic hedgehog (Shh). We have examined the expression patterns of this pathway in MPN progenitors using qRT-PCR and RNA-Seq, and have tested a pharmacological inhibitor of this pathway in a robust stromal co-culture model of MPN progression to Acute Myeloid Leukemia (AML).
  • In sum, we have utilized full transcriptome RNA-Seq and qRT-PCR coupled with hematopoietic progenitor assays and in vivo studies to evaluate the impact of JAK2 versus BCR-ABL on stem cell fate, survival, self-renewal and dormancy. These techniques have allowed us to investigate in more detail the role of genetic and epigenetic alterations that drive disease progression in the context of specific malignant microenvironments, and the relative susceptibility of MPN stem cells in these niches to single agent molecularly targeted inhibitors.
  • The main objectives of this project are generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent human embryonic stem cells (hESC) or multipotent stem cells, and identification of crucial leukemia stem cell (LSC) survival and self-renewal factors that contribute to the development and progression of BCR-ABL and JAK2-driven hematopoietic disorders. A key finding of our work thus far is that in addition to activation of BCR-ABL or JAK2 oncogenes, generation of self-renewing MPN LSC requires stimulation of other pro-survival and self-renewal factors such as β-catenin, Sonic hedgehog (SHH), BCL2, and in particular the RNA editing enzyme ADAR1, which we identified as a novel regulator of LSC differentiation and self-renewal.
  • We have now completed comprehensive gene expression analyses from next-generation RNA-sequencing studies performed on normal and leukemic human hematopoietic progenitor cells from primary cord blood samples and adult normal peripheral blood samples, along with normal cord blood transduced with BCR-ABL or JAK2 oncogenes, and primary samples from patients with BCR-ABL+ chronic phase and blast crisis chronic myeloid leukemia (CML). These studies revealed that gene expression patterns in survival and self-renewal pathways (SHH, JAK2, ADAR1) clearly distinguish normal and leukemic progenitor cells as well as MPN disease stages. These data provide a vast resource for identification of LSC-specific biomarkers with diagnostic and prognostic clinical applications, as well as providing new potential therapeutic targets to prevent disease progression.
  • New results from RNA-sequencing studies reveal high levels of expression of inflammatory mediators in human blast crisis CML progenitors and in BCR-ABL transduced normal cord blood stem cells. Moreover, expression of the inflammation-responsive form of ADAR1 correlated with generation of an abnormally spliced GSK3β gene product that has been previously linked to LSC self-renewal. These results have now been published in the journal PNAS (Jiang et al.). Together, we have demonstrated that ADAR1 drives hematopoietic cell fate by skewing cell differentiation – a trend which occurs during normal bone marrow aging – and promotes LSC self-renewal through alternative splicing of critical survival and self-renewal factors. Notably, inhibition of ADAR1 through genetic knockdown strategies reduced self-renewal capacity of CML LSC, and may have important applications in treatment of other disorders that transform to acute leukemia. Thus, these results suggest that RNA editing (ADAR1) and splicing represent key therapeutic targets for preventing LSC self-renewal – a primary driver of leukemic progression.
  • Whole transcriptome profiling studies coupled with qRT-PCR, hematopoietic progenitor assays and in vivo studies have shown that combined inhibition of BCR-ABL and JAK2 is another effective method to reduce LSC self-renewal in pre-clinical models. New results show that lentivirus-enforced BCR-ABL or JAK2 expression in normal cord blood stem cells drives generation of distinct splice isoforms of STAT5a. While inhibition of JAK2/STAT5a signaling or BCR-ABL tyrosine kinase activity alone did not eradicate self-renewing LSC, combined JAK2 and BCR-ABL inhibition dramatically impaired LSC survival and self-renewal in the protective bone marrow niche, and increased the lifespan of serial transplant recipients. These effects were associated with reduction in STAT5a isoform expression – which represents a novel molecular marker of response to combined BCR-ABL/JAK2 inhibition – and altered expression of cell cycle genes in human progenitor cells harvested from the bone marrow of transplanted mice. These results are the subject of a new manuscript currently under review (Court et al.). Moreover, this work has led to the development of new experimental tools that will facilitate study of LSC maintenance and cell cycle status in the context of normal versus diseased bone marrow microenvironments. In sum, studies completed thus far have uncovered a role for RNA editing and splicing alterations in leukemic progression, particularly in specific microenvironments. Using specific inhibitors targeting BCR-ABL and JAK2, along with strategies to block RNA editing and aberrant splicing activities, we have been able to establish the relative susceptibility of MPN stem cells to molecular inhibitors with activity against LSC residing in select hematopoietic niches that are difficult to treat with conventional chemotherapeutic agents.
  • In the final year of this project, we focused on elucidating the mechanisms of leukemia stem cell (LSC) generation in JAK2 compared with BCR-ABL1 initiated myeloproliferative neoplasms (MPN, previously called myeloproliferative disorders). To this end, we investigated the MPN stem cell propagating effects of BCR-ABL1 or JAK2 alone or in combination with activation of the human embryonic stem cell RNA editase, ADAR1. Recently, we discovered that ADAR1, which edits adenosine to inosine bases in the context of primate specific Alu sequences, leads to GSK3β missplicing and β-catenin activation in chronic phase (CP) CML progenitors leading to blast crisis (BC) transformation and LSC generation. In addition, variant isoform expression of a Wnt/β-catenin target gene, CD44, was also characteristic of LSC. In a previous report (Jiang et al., PNAS 2013), identification of ADAR1 as a malignant reprogramming factor represented the first description of RNA editing as a regulator of reprogramming. When lentivirally overexpressed, ADAR1 endows committed CP myeloid progenitors with self-renewal capacity. Further studies revealed that JAK2/STAT5a activates ADAR1 leading to deregulation of cell cycle progression and global down-regulation of microRNA expression thereby uncovering two additional key mechanisms of LSC generation in MPNs. This is consistent with our findings from gene expression profiling studies performed in the previous year, along with functional classification and network analysis using Ingenuity Pathway Analysis (IPA), showing that cell cycle-related genes were significantly altered in human progenitors from xenografted mice treated with combination JAK2 and BCR-ABL inhibitor therapy compared with single agent therapies alone. Together these data suggest that combined BCR-ABL and JAK2 inhibition impairs LSC survival and self-renewal via cell cycle modulation. ADAR1 and other stem cell regulatory pathways such as CD44 represent novel targets to detect and eradicate the self-renewing LSC. We also performed new studies that elucidate the stem cell-intrinsic genetic changes that occur during human bone marrow aging, which may contribute to BCR-ABL or JAK2-dependent functional alterations.
  • This work has led to discovery of a novel role for embryonic stem cell genes and splice isoforms, including ADAR1 p150 and a transcript variant of CD44, in the maintenance of LSC that promote MPN progression. In addition, through the course of this research we have 1) developed novel lentiviral tools for investigating normal hematopoietic stem and progenitor (HSPC) and malignant LSC survival, differentiation, self-renewal, and cell cycle regulation, and 2) devised innovative LSC diagnostic strategies and 3) tested therapeutic strategies targeting LSC-associated RNA editing and splice isoform generation that selectively inhibit LSC self-renewal.

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
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to 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.

Development of Therapeutic Antibodies Targeting Human Acute Myeloid Leukemia Stem Cells

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01485
ICOC Funds Committed: 
$19 999 996
Disease Focus: 
Blood Cancer
Cancer
Collaborative Funder: 
UK
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow that is rapidly fatal within months if untreated. Even with aggressive treatment, including chemotherapy and bone marrow transplantation, five-year overall survival rates range between 30-40%. Evidence indicates that not all cells in this cancer are the same, and that there is a rare population of leukemia stem cells (LSC) that are responsible for maintaining the disease. Thus, in order to cure this cancer, all LSC must be eliminated, while at the same time sparing the normal blood forming stem cells in the bone marrow. We propose to develop therapeutic antibodies directed against surface markers present in much larger amounts on LSC than on the surface of normal blood forming stem cells. We recently identified and validated several such protein markers including CD47, which we determined contributes to leukemia development by blocking the ingestion and removal of leukemia cells by immune system cells called macrophages. In this way, CD47 acts as a “don’t eat me” signal on LSC. Moreover, we determined that monoclonal antibodies (mAbs) directed against CD47, able to block its interaction with macrophages, mask the “don’t eat me” signal resulting in ingestion and elimination of leukemia in mouse pre-clinical models. We propose a combination of clinical studies, basic research, and pre-clinical development to prepare a therapeutic antibody directed against CD47 and/or other LSC-specific proteins for Initial New Drug (IND) filing with the FDA, and then a Phase I clinical trial to be conducted at {REDACTED} and in the Collaborative Funding Partner country. In collaboration with the pioneering Collaborative Funding Partner country AML Working Group, we will track expression of the LSC proteins in patient samples and correlate with clinical outcomes. This will allow us to identify particular LSC proteins that must be targeted to achieve cure, thereby prioritizing candidate therapeutic antibodies for clinical development. Concurrently, we will conduct basic research and pre-clinical development to prepare these candidates. Basic research during years 1 and 2 will focus on the characterization of anti-CD47 mAb efficacy, investigation of mAb targeting of additional LSC molecules, and determination of efficacy in combinations with anti-CD47. Pre-clinical development during years 1 and 2 will focus on blocking anti-CD47 mAbs, including antibody humanization and large animal model pharmacologic and toxicity studies. Similar studies will be conducted with the most promising antibodies resulting from our basic research. During years 3-4, we will proceed with GMP grade production of the best candidate, followed by efficacy testing in mouse models and large animal models. Finally, in year 4, we will prepare an IND filing with the FDA/MHRA and develop a Phase I clinical trial with this antibody for the treatment of AML. Ultimately, therapeutic antibodies specifically targeting AML LSC offer the possibility of less toxicity with the potential for cure.
Statement of Benefit to California: 
Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with nearly 13,000 new diagnoses annually in the US and 2,200 in the Collaborative Funding Partner country. Current standard of care for medically fit patients consists of several cycles of high dose chemotherapy, and often includes allogeneic hematopoietic cell transplantation. Even with these aggressive treatments, which cause significant morbidity and mortality, relapse is common and the five-year overall survival is 30-40%, but <10% in patients with relapsed or refractory disease or in the majority of AML patients who are over age 65. The goal of this research proposal is to prepare therapeutic antibodies directed against AML stem cell-specific antigens for IND filing with the FDA and a Phase I clinical trial. There are several potential benefits of this research for California: (1) most importantly, this research has the potential to revolutionize current clinical practice and provide a targeted therapy for AML that offers the possibility of less toxicity with the potential for cure; (2) this research will directly contribute to the California economy by funding a contract manufacturing organization to generate and produce GMP-grade clinical antibody, by employing several individuals who will be essential for the conduct of these studies, and through the purchase of equipment and reagents from California vendors; (3) additional clinical and economic benefits for California will derive from the potential application of clinical agents developed here to a number of other human cancers and cancer stem cells; (4) our animal models indicate that a significant fraction of patients with fatal AML can be cured, resulting in savings on their clinical care plus their return as productive contributors to the California economy; (5) if our therapeutic antibodies show clinical benefit in AML, they will be commercialized, and under CIRM policy, profits derived from treating insured patients and lower cost therapies for uninsured patients, would enrich the state and the lives of its citizens; (6) finally, this research has the potential to maintain California as the national and world-wide leader in stem cell technology.
Progress Report: 
  • Our program is focused on producing new therapeutic candidates to prolong remission and potentially cure highly lethal cancers where patients have few alternative treatment options. We have selected Acute Myelogenous Leukemia (AML) as the initial clinical indication for evaluating our novel therapeutics, but anticipate a full development program encompassing many other types of solid tumor cancers.
  • Our strategy is to develop an antibody that binds to and eliminates the cancer-forming stem cells in leukemia and other solid tumors. While current cancer treatments (e.g. surgery, chemotherapy, radiation) will frequently get rid of the bulk of the tumor, they rarely touch the tiny number of cancer stem cells that actually re-generate the masses of cancer cells that have been eliminated. When the latter occurs, the patient is described as having a relapse, leading to a disease recurrence with poor prognosis. Our strategy is to eliminate the small number of cancer-regenerating stem cells by targeting cell membrane proteins expressed by these cells.
  • We have discovered that many cancer cells coat themselves with a protein called CD47 that prevents them from being eaten and disposed of by the patient’s blood cells. In this context, CD47 can be considered a ‘don’t eat me’ signal that protects the cancer cells from being phagocytosed i.e. ‘eaten’. The antibody we are developing binds to and covers the ‘don’t eat me’ CD47 protein, so that the patient’s blood cells are now able to ‘eat’ the cancer cells by standard physiological responses, and eliminate them from the body.
  • Developing an antibody such as this for use in humans requires many steps to evaluate it is safe, while at the same ensuring it targets and eliminates the cancer forming stem cells. The antibody must also ‘look’ like a human antibody, or else the patient will ‘see’ it as a foreign protein and reject it. To achieve these criteria, we have made humanized antibodies that bind to human CD47. We have shown that the antibodies eliminate cancer cells in two ways: (i) blood cells from healthy humans rapidly “ate” and killed leukemia cells collected from separate cancer patients when the anti-human CD47 antibody was added to a mixture of both cell types in a research laboratory test tube; (ii) the anti-human CD47 antibody eliminates human leukemia cells collected from patients, then transferred into special immunodeficient mice which are unable to eliminate the human tumor cells themselves. In these experiments, the treated mice remained free of the human leukemia cells for many weeks post-treatment, and could be regarded as being cured of malignancy.
  • To show the antibodies were safe, we administered to regular mice large amounts of a comparable anti-mouse CD47 antibody on a daily basis for a period of many months. No adverse effects were noted. Unfortunately our antibody to human CD47 did not bind to mouse CD47, so it’s safety could not be evaluated directly in mice. Since the anti-human CD47 antibody does bind to non-human primate CD47, safety studies for our candidate therapeutic need to be conducted in non-human primates. These studies have been initiated and are in progress. Following administration of the anti-human CD47 antibodies, the non-human primates will be monitored for clinical blood pathology, which, as in humans, provides information about major organ function as well as blood cell function in these animals.
  • The next step after identifying an antibody with strong anti-cancer activity, but one that can be safely administered to non-human primates without causing any toxic effects, is to make large amounts of the antibody for use in humans. Any therapeutic candidate that will be administered to humans must be made according to highly regulated procedures that produce an agent that is extremely “clean”, meaning free of viruses, other infectious agents, bacterial products, and other contaminating proteins. This type of production work can only be performed in special facilities that have the equipment and experience for this type of clinical manufacturing. We have contracted such an organization to manufacture clinical grade anti-human CD47 antibodies. This organization has commenced the lengthy process of making anti-CD47 antibody that can be administered to humans with cancer. It will take another 18 months to complete the process of manufacturing clinical grade material in sufficient quantities to run a Phase I clinical trial in patients with Acute Myelogenous Leukemia.
  • Our program is focused on producing new therapeutic candidates to prolong remission and potentially cure highly lethal cancers where patients have few alternative treatment options. Our strategy is to develop an antibody that will eliminate the cancer stem cells which are the source of the disease, and responsible for the disease recurrence that can occur months-to-years following the remission achieved with initial clinical treatment. The cancer stem cells are a small proportion of the total cancer cell burden, and they appear to be resistant to the standard treatments of chemotherapy and radiation therapy. Therefore new therapeutic approaches are needed to eliminate them.
  • In year 2 of the CIRM award, we have continued to develop a clinical-grade antibody that will eliminate the cancer stem cells in Acute Myelogenous Leukemia (AML). We have identified several antibodies that cause human leukemia cells to be eaten and destroyed by healthy human white blood cells when tested in cell culture experiments. These antibodies bind to a protein called CD47 that is present on the outer surface of human leukemia cells. The anti-CD47 antibodies can eliminate leukemia growing in mice injected with AML cells obtained from patients. We have now extensively characterized the properties of our panel of anti-CD47 antibodies, and have identified the lead candidate to progress though the process of drug development. There are several steps in this process, which takes 18-24 months to fully execute. In the last 12 months, we have focused on the following steps:
  • (i) ‘Humanization’ of the antibody: The antibody needs to be optimized so that it looks like a normal human protein that the patient’s immune system will not eliminate because it appears ‘foreign’ to them.
  • (ii) Large scale production of the antibody: To make sufficient quantities of the antibody to complete the culture and animal model experiments required to progress to clinical safety trials with patients, we have contracted with a highly experienced manufacturing facility capable of such large-scale production. We have successfully transferred our antibody to them, and they have inserted it into a proprietary expression cell that will produce large amounts of the protein. This process is managed through weekly interactions with this contract lab. They send us small amounts of the material from each step of their manufacturing process and we test it in our models to ensure the antibody they are preparing retains its anti-cancer properties throughout production.
  • (iii) Pre-clinical safety studies: The antibody must be tested extensively in animals to ensure it does not cause serious limiting damage to any of the normal healthy tissues in the recipient. We have spent much of the last 12 months performing these types of safety experiments. The antibody has been administered to both mice and non-human primates and we have evaluated their overall health status, as well as analyzing their blood cells, blood enzyme levels, and urine, for up to 28 days. We have also collected samples of their organs and tissues to evaluate for abnormalities. Thus far, these assessments have appeared normal except for the development of a mild anemia a few days after the initial antibody injection. Subsequent experiments indicate that this anemia can be managed with existing approved clinical strategies
  • (iv) Determination of optimal dose: We have used mice injected with human cancer cells from AML patients, and determined how much antibody must be injected into these mice to produce a blood level that destroys the leukemia cells. This relationship between antibody dose and anti-cancer activity in the mouse cancer model enables us to estimate the dose to administer to patients.
  • Hematologic tumors and many solid tumors are propagated by a subset of cells called cancer stem cells. These cells appear to be resistant to the standard cancer treatments of chemotherapy and radiation therapy, and therefore new therapeutic approaches are needed to eliminate them. We have developed a monoclonal antibody (anti-CD47 antibody) that recognizes and causes elimination of these cancer stem cells and other cells in the cancer, but not normal blood-forming stem cells or blood cells. Cancer stem cells regularly produce a cell surface ‘invisibility cloak’ called CD47, a ‘don’t eat me signal’ for cells of the native immune system. Anti-CD47 antibody counters the ‘cloak, allowing the patient’s natural immune system eating cells, called macrophages, to eliminate the cancer stem cells.
  • As discussed in our two-year report, we optimized our anti-CD47 antibody so that it looks like a normal human protein that the patient’s immune system will not eliminate because it appears ‘foreign’. In this third year of the grant, we initiated the pre-clinical development of this humanized antibody, and assigned the antibody the development name of Hu5F9. Our major accomplishments in the third year of our grant are as follows:
  • (i) In addition to the hematological malignancies we have studied in previous years, we have now demonstrated the Hu5F9 is effective at inhibiting the growth and spread throughout the body [metastasis] of a large panel of human solid tumors, including breast, bladder, colon, ovarian, glioblastoma [a very aggressive brain cancer], leiomyosarcoma, head & neck squamous cell carcinoma, and multiple myeloma.
  • (ii) We have performed extensive studies optimizing the production and purification of Hu5F9 to standards compatible with use in humans, including that it is sterile, free of contaminating viruses, microorganisms, and bacterial products. We will commence manufacturing of Hu5F under highly regulated sterile conditions to produce what is known as GMP material, suitable for use in humans.
  • (iii) Another step to show Hu5F9 is safe to administer to humans is to administer it to experimental animals and observe its effects. We have demonstrated that Hu5F9 is safe and well tolerated when administered to experimental animals. Notably, no major abnormalities are detected when blood levels of the drug are maintained in the potentially therapeutic range for an extended duration of time.
  • (iv) We have initiated discussions with the FDA regarding the readiness of our program for initiating clinical trials, which we anticipate to start in the first quarter of 2014. To prepare for these trials we have established a collaboration between the Stanford Cancer Institute and the University of Oxford in the United Kingdom, currently our partners in this CIRM-funded program.
  • To our knowledge, CD47 is the first common target in all human cancers, one which has a known function that enables cancers to grow and spread, and one which we have successfully targeted for cancer therapy. Our studies show that Hu5F9 is a first-in-class therapeutic candidate that offers cancer treatment a totally new mechanism of enabling the patient’s immune system to remove cancer stem cells and their metastases.

Development of Highly Active Anti-Leukemia Stem Cell Therapy (HALT)

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01430
ICOC Funds Committed: 
$19 999 826
Disease Focus: 
Blood Cancer
Cancer
Collaborative Funder: 
Canada
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Leukemias are cancers of the blood forming cells that afflict both children and adults. Many drugs have been developed to treat leukemias and related diseases. These drugs are often effective when first given, but in many cases of adult leukemia, the disease returns in a form that is not curable, causing disability and eventual death. During the last few years, scientists have discovered that some leukemia cells possess stem cell properties that make them more potent in promoting leukemia growth and resistance to common types of treatment. These are called leukemia stem cells (LSC). More than in other cancers, scientists also understand the exact molecular changes in the blood forming cells that cause leukemias, but it has been very difficult to translate the scientific results into new and effective treatments. The main difficulty has been the failure of existing drugs to eliminate the small numbers of LSC that persist in patients, despite therapy, and that continue to grow, spread, invade and kill normal cells. In fact, the models used for drug development in the pharmaceutical industry have not been designed to detect drugs or drug combinations capable of destroying the LSC. Drugs against LSC may already exist, or could be simple to make, but there has not been an easy way to identify these drugs. Recently, physicians and scientists at universities and research institutes have developed tools to isolate and to analyze LSC donated by patients. By studying the LSC, the physicians and scientists have identified the molecules that these cells need to survive. The experimental results strongly suggest that it will eventually be possible to destroy LSC with drugs or drug combinations, with minimal damage to most normal cells. Now we need to translate the new knowledge into practical treatments. The CIRM Leukemia Team is composed of highly experienced scientists and physicians who first discovered LSC for many types of leukemia and who have developed the LSC systems to test drugs. The investigators in the Team have identified drug candidates from the vigorous California pharmaceutical industry, who have already performed expensive pharmacology and toxicology studies, but who lack the cells and model systems to assess a drug’s ability to eliminate leukemia stem cells. This Team includes experts in drug development, who have previously been successful in quickly bringing a new leukemia drug to clinical trials. The supported interactive group of physicians and scientists in California and the Collaborative Funding Partner country has the resources to introduce into the clinic, within four years, new drugs for leukemias that may also represent more effective therapies for other cancers for the benefit of our citizens.
Statement of Benefit to California: 
Thousands of adults and children in California are afflicted with leukemia and related diseases. Although tremendous gains have been made in the treatment of childhood leukemia, 50% of adults diagnosed with leukemia will die of their disease. Current therapies can cost tens of thousands of dollars per year per patient, and do not cure the disease. For the health of the citizens of California, both physical and financial, we need to find a cure for these devastating illnesses. What has held up progress toward a cure? Compelling evidence indicates that the leukemias are not curable because available drugs do not destroy small numbers of multi-drug resistant leukemia stem cells. A team approach is necessary to find a cure for leukemia, which leverages the expertise in academia and industry. Pharmaceutical and biotech companies have developed drugs that inhibit pathways known to be involved in leukemia stem cell survival and growth, but are using them for unrelated indications. In addition, they do not have the expertise to determine whether the inhibitors will kill leukemia stem cells. The Leukemia Team possesses stem cell expertise and has developed state of the art systems to determine whether drugs will eradicate leukemia stem cells. They have also have access to technologies that may allow them to identify patients who will respond to the treatment. The development plan established by the Leukemia Disease Team will also serve as a model for the clinical development of drugs against solid tumor stem cells, which are not as well understood. In summary, the benefits to the citizens of California from the CIRM disease specific grant in leukemia are: (1) direct benefit to the thousands of leukemia patients (2) financial savings due to definitive treatments that eliminate the need for costly maintenance therapies
Progress Report: 
  • Development of Highly Active Leukemia Therapy (HALT)
  • Leukemias are cancers of the blood forming cells that affect both children and adults. Although major advances have been made in the treatment of leukemias, many patients still succumb to the disease. In these patients, the leukemias may progress despite therapy because they harbor primitive malignant stem-like cells that are resistant to most drugs. This CIRM disease specific grant aims to develop a combination of highly active anti-leukemic therapy (HALT) that can destroy the drug-resistant cancer stem-like cells, without severely harming normal cells.
  • During the current year of support, substantial progress has been made in achieving this goal. The CIRM investigators have shown that two different drugs that inhibit different proteins in leukemia stem cells can sensitize them to chemotherapeutic agents, and block their ability to self-renew. The CIRM investigators have also demonstrated that two different antibodies against molecules on the surface of the leukemia cells can inhibit their survival in both test tube experiments and in mouse models.
  • Extensive experiments are underway to confirm these promising results. The results will enable the planning and implementation of potentially transforming clinical trials in leukemia patients, during the period of CIRM grant support.
  • During the past 12 months, our disease team has made further progress in
  • the development of stem cell targeted treatment for chronic lymphocytic
  • leukemias and other leukemias. Stem cells express some molecules on the
  • surface that are different from the corresponding molecules on adult
  • cells. The ROR1 molecule is highly expressed by malignant cells from
  • patients with chronic lymphocytic leukemia, as well as by progenitor cells
  • from other forms of leukemia and lymphoma. It is not expressed by normal
  • adult cells. With the support of the CIRM Disease Team grant, the
  • cooperating investigators have prepared monoclonal antibodies against the
  • ROR1 molecule, that are potent and specific. In animal models, the
  • antibodies can retard leukemia growth and spread. Unlike other anti-cancer
  • drugs, the new antibodies are not toxic for normal bone marrow cells.
  • Thus, they can potentiate the action of other agents used for the
  • treatment of leukemia.
  • The disease team is now focused on the pre-clinical development, safety
  • testing, and scale-up manufacturing of our new, promising agents, in
  • preparation for their introduction into the clinic.
  • During the past 12 months, our disease team has made further progress in
  • the development of stem cell targeted treatment for chronic lymphocytic
  • leukemias and other leukemias. Stem cells express some molecules on the
  • surface that are different from the corresponding molecules on adult
  • cells. The ROR1 molecule is highly expressed by malignant cells from
  • patients with chronic lymphocytic leukemia, as well as by progenitor cells
  • from other forms of leukemia and lymphoma. It is not expressed by normal
  • adult cells. With the support of the CIRM Disease Team grant, the
  • cooperating investigators have prepared a humanized monoclonal antibody against the
  • ROR1 molecule, that is potent and specific. In animal models, the
  • antibodies can retard leukemia growth and spread. Unlike other anti-cancer
  • drugs, the new antibodies are not toxic for normal bone marrow cells.
  • Thus, they can potentiate the action of other agents used for the
  • treatment of leukemia.
  • The disease team is now focused on the pre-clinical development, safety
  • testing, and scale-up manufacturing of our new, promising agents, in
  • preparation for their introduction into the clinic.
  • During the past 12 months, our disease team has made further progress in
  • the development of stem cell targeted treatment for chronic lymphocytic
  • leukemias and other leukemias. Stem cells express some molecules on the
  • surface that are different from the corresponding molecules on adult
  • cells. The ROR1 molecule is highly expressed by malignant cells from
  • patients with chronic lymphocytic leukemia, as well as by progenitor cells
  • from other forms of leukemia and lymphoma. It is not expressed by normal
  • adult cells. With the support of the CIRM Disease Team grant, the
  • cooperating investigators have prepared a humanized monoclonal antibody against the
  • ROR1 molecule, that is potent and specific. In animal models, the
  • antibodies can retard leukemia growth and spread.
  • The disease team has now finalized the pre-clinical development, safety
  • testing, and scale-up manufacturing of our new, promising agent, in
  • preparation for their introduction into the clinic.

Therapeutic potential of genetically modified human ES cells in an Alzheimer's disease model: Contribution of IGF-1

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Alzheimer’s disease (AD) is a progressive and irreversible disease of the brain leading to deterioration of mental function and eventual morbidity and death. The major defining characteristic of AD brains is the excessive accumulation of amyloid plaques (composed of clumps of Abeta) outside of nerve cells and tangles (composed of clumps of tau) inside nerve cells. These lesions are toxic to nerve cells and likely explain the progressive degeneration seen in AD brains. Currently available treatments for AD provide only limited symptomatic relief and are unable to prevent, stop, or cure the disease. Even if next generation drugs prove to be more effective, they are unlikely to reverse the disease progression. Thus, it may be necessary to replace dead or dying nerve cells in order to reverse the course of the disease in many AD patients. The long-term objective of this proposal is to use genetically modified human embryonic stem cells (ESCs) as an inexhaustible source for replacing lost or damaged nerve cells, supplying the host brain with protection from further damage, and working against the underlying factors that promote amyloid and tangle lesions. Such objective ultimately may lead to a strategy for therapeutic intervention in AD patients who do not respond to available pharmacological treatments. It is well known that mouse embryonic stem cells exhibit the remarkable ability to respond to damaged nerve cells and home in on these degenerative environments in brain. At present, the capacity of human embryonic stem cells (ESCs) to integrate into the diseased brain such as those with amyloid and tangle lesions is unknown. In this proposal, we will use a mouse model of AD that develops both amyloid plaques and tangles to test the idea that transplantation of ESCs might be beneficial in treating AD. Our hypothesis is that human ESCs possess the inherent capacity to home in and integrate into sites surrounding plaques and tangles, where nerve cell damage is occurring. In addition, we hypothesize that human ESCs genetically modified to produce a protective factor called IGF-1 will further enhance this capability, help host nerve cells from further damage, and block the accumulation of plaques and tangles. It is known that IGF-1 promotes ESCs to become nerve cells, protects nerve cells from damage by Abeta, and decreases the levels of Abeta in brain. Furthermore, IGF-1 levels are reduced in AD, and loss of IGF-1 promotes tangle-like lesions in mice. If the above hypotheses can be even partially demonstrated, the current proposal is expected significantly advance our long-term objective of applying genetically modified human ESCs as a therapeutic technology for AD patients who are refractory to available pharmacological treatments.
Statement of Benefit to California: 
Alzheimer’s disease (AD) is an age-related debilitating disease of the brain characterized by progressive deterioration of mental function and accounts for more than 70% of all dementias of the brain. AD inflicts more than 465,000 residents in California alone and places substantial medical, social, psychological, and financial burden on the patients, their families, and social/medical institutions. The per capita cost of caring for an AD patient in California was estimated to be more than $65,000 per year in 1998. It was also projected at the time that the cost of caring for AD patients in California (in 1998 dollars) will be ~$25.9 billion in 2000, ~$47.5 billion in 2020, and ~$75.4 billion in 2040. During the same time period, the number of AD patients in California is projected to rise from ~395,000 in 2000 to ~1.2 million in 2040. At present, no effective treatment is available for AD. First generation drugs can temporarily mask symptoms of the disease but rapidly lose effectiveness during the progression of AD. Even if next generation drugs prove to be more effective, they will only help to slow down the progression of AD but not reverse it. As such, it may be necessary use an alternate therapeutic strategy to replace dead or dying nerve cells, especially in patients that do not respond to available drugs. Human embryonic stem cells have emerged in recent years to hold enormous potential for cell replacement therapy for wide variety of neurological disorders, including AD. As California continues to be at the forefront of new and innovative technologies, the passage of Proposition 71 to fund stem cell research further extends this spirit of innovation. The research proposed in this application attempts to generate genetically modified human embryonic stem cells capable of not only replacing lost nerve cells but also delivering protective factors that prevent further degeneration of existing nerve cells in an animal model of AD. Such kind of technological coupling between stem cell therapy and gene therapy poses therapeutic potential for application in AD where irreversible nerve cell damage cannot be treated with even the best of next generation drugs. If successful, this will also help to offset the enormous social and financial burden of caring for AD patients in California. Technologies and therapeutics derived from stem cell research funded by the California Institute for Regenerative Medicine (CIRM) are in part the contractual property of the state of California, and hence its residents. In the event that such intellectual property leads to commercialization or licensing down the line, a portion of the proceeds are contracted to enter the California state general fund, ensuring that all California residents benefit from potential successes of this research.
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.

Epigenetic regulation of AAVS1

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Development and differentiation is regulated by spatial and temporal regulation of genes. Genes in the nucleus are found associated with proteins and this is called chromatin, which regulates genes. Genes in stem cells are also regulated by chromatin and the structure of chromatin undergoes changes during differentiation. Understanding the sequence of events that occur in specific chromatin domains during stem cell self-renewal and differentiation becomes vital before we can begin to use these in regenerative medicine. Genetically modifying stem cells may be necessary prior to their use in therapy. The non-pathogenic virus AAV is employed as a vector in numerous gene therapy trials and holds promise for use in modifying stem cells. This virus establishes a latent infection by integrating into a specific region of the human genome called AAVS1. This is in contrast to other viruses used in gene therapy that randomly insert into the genome and thus can be mutagenic. We propose to investigate the chromatin structure at AAVS1 so that AAV based vectors can be used optimally in regenerative medicine. This proposal will improve our toolkit for modifying stem cells using gene therapy. One way to reverse the effects of dysfunctional genes is to deliver a corrected copy to the affected individual. By virtue of their ability to propagate indefinitely, stem cells offer an unlimited supply of healthy genes but undifferentiated stem cells transplanted into patients give rise to problems. These problems can potentially be circumvented by genetically manipulating stem cells in vitro to direct their differentiation into the lineage of choice prior to transplantation but will necessitate integrating transgenes into these cells. The proposed experiments will allow us to better genetically modify stem cells. The experiments outlined in this proposal will characterize the chromatin domains around the AAVS1 region in depth. We will determine how the AAVS1 genomic locus changes with respect to its chromatin structure as stem cells undergo differentiation into specific lineages. Furthermore, we will establish the chromatin determinants that (i) promote the stable integration of AAV into a specific region of the genome and (ii) allow stable expression of transgenes in stem cells. As our long-term goal we will study the changes that occur in the chromatin structure of the AAVS1 region in stem cells expressing an AAV-mediated transgene that induces these cells to differentiate along a specific lineage. These studies will enable the development of vectors for the expression of specific transgenes in stem cells that will direct their differentiation into specific cell types. Such a system could then be exploited to generate large cell banks with diverse histocompatibilities for use in patients with hereditary disorders.
Statement of Benefit to California: 
This proposal seeks to combine the potential of two of the most promising approaches in modern medicine: stem cell and gene therapy. Over 1800 genes have been determined to cause hereditary disorders and the most obvious way to reverse the effects of such dysfunctional genes is to deliver a corrected copy to the affected individual. By virtue of their ability to propagate indefinitely, stem cells offer an unlimited supply of healthy genes. However, when undifferentiated embryonic stem cells are transplanted into the patient they have the potential to form teratomas while adult stem cells can potentially give rise to tissues that are not desirable at the site of transplantation. These problems can potentially be circumvented by genetically manipulating stem cells in vitro to direct and control their differentiation into the lineage of choice prior to transplantation. In the future one can envision CA-based large therapeutic cell bank repositories of different lineages and immune characteristics that would enable physicians to find immunologically compatible cells for corrective cell therapy. Results from experiments in this proposal will allow the stable expression of proteins and growth factors that can direct stem cell differentiation without being subjected to position effects resulting from random integrations and can therefore be utilized for generating cell banks. A second application for the proposed research is in gene transfer therapy where stem cells derived from the patient are corrected for the defective gene, expanded, characterized and allowed to differentiate prior to re-transplantation into that patient thus avoiding immune rejection. Although this approach requires heavy logistics and might be limited to small numbers of patients, therapies such as these could be developed from the proposed research and will have the advantage that the integrated genes will not be subject to variations in expression by gene silencing and additionally will avoid the problems of histocompatibility mismatches and immune rejection. Knowledge from this research will also spur growth in new biotechnology firms to develop gene delivery vectors in stem cells thus offering a direct advantage to the state in terms of revenue and employment opportunities. This research will also put the state of California at the forefront of stem cell technology along with other nations.
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.

Role of Notch signaling in human embryonic stem cell differentiation to neuronal cell fates

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Human embryonic stem cells (HESCs) are capable of giving rise to a variety of differentiated human cell types that in principle could be used therapeutically to treat tissue damage that arises in human disease. The promise of HESCs is still quite limited because of technical limitations in our ability to propagate these cells in culture, while retaining their potency to become many different types of cells, and to guide them to become the right type of cell needed for clinical use. The proposed work will develop the tools to address these issues, by focusing on the Notch signaling pathway. Studies of the Notch pathway in model organisms like mice has shown that it plays a pivotal role in regulating the development of embryonic cells, by activating critical target genes that maintain cells in a proliferative, undifferentiated state. The proposed experiments will examine the activity of the Notch pathway in HESCs, as they are experimentally induced to form the precursors to nerve cells. The long-term goal of this work is to develop the information and tools needed to manipulate HESCs in culture via the Notch pathway, allowing one to better control their proliferation and differentiation into defined cell types.
Statement of Benefit to California: 
The goal of the proposed research is to develop tools that can be used to manipulate human embryonic stem cells, thus allowing them to be more effectively used as therapeutic agents. The process we are studying will help define optimal procedures to encourage human embryonic stem cells to produce homogeneous populations of specific neural cell types that are needed to replace damaged neural tissues for patients with Parkinson’s and other neural diseases.
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.

Brain Aging and hESC-derived Neural Stem Cell Transplantation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Aging is an important risk factor for human diseases. In addition to cognitive decline in the elderly, brain aging also increases the risk for neurological diseases like Alzheimer’s disease (AD), Parkinson's disease (PD), and stroke, which are major causes of disability and mortality in the elderly. Current therapeutic approaches typically fail to treat the underlying cause of these disorders. The potential capacity of human neural stem cells to replace cells and tissues damaged due to aging and age-realted diseases is, therefore, of great importance. Activation or transplantation of these cells has already shown promise in animal models of these disorders. However, little is known about how aging affects host receptivity to hESC-derived cell transplants, or about the proliferation, survival, differentiation, migration and functionality of the donor cells. This is partly because in vivo studies of aged-related neurological diseases have relied almost universally on experimental models using young adult animals.We hypothesize that the ability of hESC-derived transplants to proliferate, survive, differentiate, migrate and function may be compromised in the aged brain, requiring that new strategies be devised to overcome this deficiency. The experiments we propose are designed to increase our understanding of how the age of the recipient alters the ability of transplanted neural stem cells to differentiate into mature, functional neurons and integrate into local neuronal circuits. Our first Specific Aim will examine these issues using immunocytochemistry and electrophysiological methods, after transplanting neural stem cells into young adult, middle aged and aged rats. In addition, because brain aging produces progressive changes in learning and memory, a critical area to examine is whether neural stem cell therapy can produce functional improvement in learning and memory deficits in aged rats. Our second Specific Aim will address this question by using a battery of behavioral tests to assess the ability of transplanted neural stem cells to reverse age-related losses of cognitive function. The use of neural stem cells to repair age-related neurological diseases will require an increased understanding of stem cell biology, the environment of the aged tissue, and the interaction between the two, which this proposed work will focus on. If the aims of the application are achieved, significant advances will be made in establishing a new paradigm of brain aging and the biological behaviors of transplanted neural stem cells, and substantial progress will be made in developing basic science knowledge that can be translated fairly rapidly into clinical treatment of aged-related neurological diseases.
Statement of Benefit to California: 
Aging is destined to become a serious public health problem for California over the next several decades. California’s elderly population is expected to grow more than twice as fast as the total population from 1990 to 2020, and will reach 12.5 million by 2040. One in five Californians will be 60 years of age or older beginning in 2010. Consequently, age-related diseases, including neurological diseases, will dramatically increased in parallel, presenting enormous social and economic challenges. Determining whether transplanted human neural stem cells can differentiate into functional neurons in the aging brain, and thereby slow or prevent cognitive decline in the aged, could have great social and economic impact in our state.
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.

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