Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Clinical Development of an N-cadherin Antibody to Target Cancer Stem Cells

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06867
ICOC Funds Committed: 
$4 075 668
Disease Focus: 
Prostate Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Closed
Public Abstract: 
Metastatic disease and the castration resistance remain tremendous challenges in the treatment of prostate cancer. New targeted treatments, such as the ant-testosterone medication enzalutamide, have improved the survival of men with advanced disease, but a majority develops treatment resistance. The field of cancer stem cells hypothesizes that treatment resistance emerges because stem cells are inherently resistant to our current therapies and eventually repopulate tumors. One mechanism by which cancer stem cells resist therapy is through acquisition of an epithelial to mesenchymal transition (EMT), a phenomenon of normal development used by cancers to survive and metastasize. Our laboratory has shown that prostate cancers undergo an EMT that leads to invasion, metastasis and treatment resistance. N-cadherin, a critical regulator of EMT, is expressed in most castration resistant prostate cancers (CRPC) and is sufficient to promote treatment resistance. We therefore developed antibodies against N-cadherin, which are able to inhibit growth, metastasis and progression of prostate cancers in vivo. The goal of this translational application is to move this promising treatment from the laboratory to the clinic by making the antibody human, making it bind more strongly, and then testing it for toxicity, behavior and anti-tumor activity. At the completion of this project, we will be poised to manufacture this lead molecule and move expeditiously to Phase I clinical studies.
Statement of Benefit to California: 
Prostate cancer is the second leading cause of cancer-related death in Californian men. With an aging population, this problem is expected to continue to grow despite recent advances in treatment. The goal of this application is to develop a novel antibody targeting a cancer stem cell target in hormone and treatment refractory prostate cancer. The benefit to the California, if successful, will be the development of a novel therapy against this common disease.

Epigenetics in cancer stem cell initiation and clinical outcome prediction

Funding Type: 
New Faculty I
Grant Number: 
RN1-00550
ICOC Funds Committed: 
$3 238 450
Disease Focus: 
Solid Tumor
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Cancer is responsible for approximately 25% of all deaths in the US and other developed countries. For women, breast and lung cancers and for men, cancers of prostate and lung are the most prevalent and the most common cause of deaths from cancer. While a large number of treatment modalities such as surgery, chemotherapy, radiation therapy, etc. have been developed, we still are far from finding a cure for most cancers. So, more research is needed to understand the basic processes that are subverted by cancer cells to gain a proliferative advantage. In addition, cancer patients show a great deal of heterogeneity in the course and outcome of the disease. Therefore it is important to be able to predict the clinical outcome of the patients so that appropriate therapies can be administered. Clinical outcome prediction is based generally on tumor burden and degree of spread with additional information provided by histological type and patient demographics. However, patients with similar tumor characteristics still show heterogeneity in the course and outcome of disease. Thus, accurate sub-classification of patients with similar clinical outcomes is required for development of more efficacious therapies. One important molecular process that is altered in cancer is the epigenetic regulation of gene expression. In humans, DNA is tightly wrapped around a core of proteins called histones to form chromatin—the physiologically relevant form of the genome. The histones can be modified by small chemical molecules which can affect the structure of chromatin, allowing for a level of control on gene expression. The patterns of occurrences of the histone modifications throughout chromatin are highly regulated and affect all molecular processes that are based on DNA. This information which is heritable but not encoded in the sequence of DNA is referred to as ‘epigenetics.’ A challenge in biology is to understand how histone modifications which can number to more than 150, contribute to normal gene regulation and how their alterations contribute to development of cancer stem cells. These cells are thought to be responsible for maintain the bulk of the tumor and need to be completely eradicated if we were to cure a given cancer. By studying primary cancer tissues and viruses that cause tumor, we have found that one histone modification plays a critical role in transforming a normal call to a tumor cell, potentially generating a cancer stem cell. We have found that he same histone modifications can be used as a biomarker to predict clinical outcome of patients. We now propose to study this process in more depth, discover other important histone modifications that contribute to cancer development and progression and use this knowledge to develop standard, simple and robust assays for predicting clinical outcome of cancer patients. Our work may also lead to identification important molecules that can be targeted for cancer therapy.
Statement of Benefit to California: 
Cancer is a devastating disease that is becoming more prevalent as the population ages. While scientists have developed a general framework of how cancer initiates, there remains significant gaps in our knowledge about how cancer arises from a normal cell. One difficulty with studying cancer is the heterogeneity in the types of cells that exist within a given cancer tissue. Some of these cells have recently been shown to have stem cell-like properties and when isolated can reestablish the original tumor. These ‘cancer stem cells’ are thought to be responsible for maintaining the bulk of the tumor and need to be completely eradicated if we were to cure a given cancer. There is also a great deal of differences in the course and outcome of cancers with seemingly similar attributes, making application of appropriate therapies difficult. Our proposal aims to understand some of the basic processes that may contribute to development of cancer stem cells and to use this knowledge to develop proper clinical tests for prediction of cancer patients’ clinical outcome. This would be beneficial for people of California as it may lead to personalization of cancer therapy. Our work may also lead to identification of critical molecules that need to be therapeutically targeted to improve rates of cancer therapy. Identification of such molecules may lead to innovative discoveries and patents that may be exploited by the biotech industry in California, and thereby improve the economy of California as well.
Progress Report: 
  • Cancer is a genetic disease but epigenetic processes also contribute to cancer development and progression. Epigenetic processes include molecular pathways that modify the DNA itself or the proteins that are associated with DNA (i.e. histones), thereby affecting how the genetic information is used to maintain cellular states. Cancer cells exploit the normal epigenetic processes to their advantage to support uncontrolled growth and evade host defense mechanisms. Our proposal aims to understand the epigenetic requirements for cancer initiation and progression and how they can be used to develop prognostic assays that can predict cancer clinical outcome or response to therapeutics. We have made significant progress in all of our aims. We are discovering new basic principles governing epigenetic processes in human embryonic stem cells versus more differentiated cell types and understanding how these principles are implemented and regulated by the different types of cells. We have also shown that epigenetics can be used for cancer prognostic purposes as well as for prediction of response to specific cancer chemotherapeutics.
  • The goal of this proposal is to understand the dynamics of chromatin in various cellular differentiation states and how alteration of this dynamic may contribute to cancer development and progression. Our major findings are outlined as follows and further elaborated below.
  • 1) Among the various acetylation sites of histones, H3K18ac has a unique distribution in hESCs and is specifically affected during oncogenic transformation. As part of a screen to discover upstream regulators of this modification site (described in previous reports), we identified a non-coding RNA that is required for maintenance of H3K18ac, expression of SOX2 and its target genes, and growth of hESCs.
  • 2) We have discovered a highly novel and unanticipated role for histone acetylation. We have found that global histone acetylation and deacetylation coupled with flux of acetic acid in and out of the cells acts as a buffering system for regulation of intracellular pH. This phenomenon is a fundamental biological process and occurs in hESCs, cancer cells as well as normal differentiated human cells. (A paper reporting this finding is currently being reviewed at Nature.)
  • 3) We are continuing our efforts on the role of linker histone H1.5 in transcriptional regulation of terminally differentiated cells vs hESCs. This is a continuation project from a CIRM SEED grant. A manuscript on this project was submitted to Cell but was not accepted. We have performed additional experiments and preparing a new manuscript.
  • I. A non-coding RNA is required for hESC growth.
  • This aim was designed to understand how the global levels of histone modifications are regulated. As reported in previous progress reports, we carried out a kinase screen in which ~800 kinases were knocked down individually using siRNAs and the levels of two histone modifications were examined. We validated the top hits which were reported last year. The most significant effect on histone modifications, especially H3K18ac, was observed in knockdown of TPRXL (tetra-peptide repeat homeobox-like). We found that knockdown of TPRXL causes ~50-70% reductions in the global levels of H3K18ac specifically, suggesting that TPRXL is required for maintenance of a portion of H3K18ac throughout the genome. It turned out that the identification of TPRXL was a fortuitous finding. TPRXL is not a kinase but has been mis-annotated as a kinase in certain databases, hence its inclusion in the kinase siRNA library. TPRXL is a member of the TPRX homeobox gene family and is designated as a non-functional retrotransposed pseudogene (Booth and Holland, 2007). It is suggested that TPRXL was generated by reverse transcription of TPRX1 mRNA which was then integrated near an enhancer active in placenta. Consistently, TPRXL has a very high expression in placenta compared to other tissues. Subsequent to integration, TPRXL sequence has diverged from that of TPRX1 in an unusual way. In certain regions, such as over the homebox domain, TPRXL has retained 81% nucleotide identity but only 66% amino acid identity compared to TPRX1 (Booth and Holland, 2007). Despite its designation, TPRXL could possibly be a functional retrogene as it is transcribed and contains two potential open reading frames (ORFs). One ORF can code for a short protein (139 a.a.) that would contain the homeodomain and a polyglutamine stretch. Another ORF codes for a longer protein that would consist mostly of a long polyserine/proline stretch.
  • Epigenetic processes include molecular pathways that modify the DNA or the proteins that are associated with DNA (i.e. histones), thereby affecting how the genetic information is used to maintain cellular states. Thus epigenetics plays an important role in normal biology and disease. When deregulated, epigenetic processes could contribute to disease development and progression. Since embryonic stem cells (ESCs) and cancer cells share the capacity to divide indefinitely, our proposal aims to understand the epigenetic requirements for such capacity. We have found that a particular epigenetic process, which we previously linked to cancer progression, may contribute to regulation of DNA replication in human ESCs. We have also discovered how epigenetic processes could in novel ways exert control over metabolic state of the cell. Finally, we have discovered how chromatin – the complex of DNA and histones – at specific sets of gene families is differentially compacted in differentiated cell types vs. human ESCs. Altogether, we are providing novel insights into the functions of various epigenetic processes and how they may differ in stem cells vs. other normal and cancer cell types.
  • Epigenetic processes include molecular pathways that modify the DNA or the proteins that are associated with DNA (i.e. histones), thereby affecting how the genetic information is read. Epigenetics plays an important role in normal biology and disease because it can affect how genes are turned on and off. Deregulation of epigenetic processes indeed contributes to disease development and progression including cancer. Our proposal has aimed to understand how the epigenome exerts its control over gene regulation. We have found that in addition to gene regulation, on epigenetic process is unexpectedly linked to control of cellular physiology. We have shown that dynamic acetylation of histone proteins regulates intracellular level of acidity, providing an unprecedented function for the epigenome. Our data provides plausible explanations for why ESCs contain in general higher levels of histone acetylation than other cell types and why certain cancers with low levels of histone acetylation are more aggressive. In a separate study, we have found that replication of DNA in ESCs is associated with a unique epigenetic signature that is not found in differentiated cells or other rapidly dividing cell types such as cancer. We have proposed that this molecular property of replication in ESCs may be an important determinant of continual cell division without malignancy, fundamentally distinguishing ESC-specific from cancer-like cell division. Altogether, we are providing novel insights into the functions of various epigenetic processes and how they may be similar or differ in stem cells vs. other normal and cancer cell types.

Genetic Re-programming of Stem Cells to Fight Cancer

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

Combinatorial Chemistry Approaches to Develop LIgands against Leukemia Stem Cells

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

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

Funding Type: 
New Faculty II
Grant Number: 
RN2-00934
ICOC Funds Committed: 
$2 274 368
Disease Focus: 
Blood Cancer
Cancer
Trauma
oldStatus: 
Active
Public Abstract: 
Adult stem cells play an essential role in the maintenance of tissue homeostasis. Environmental and therapeutic insults leading to DNA damage dramatically impact stem cell functions and can lead to organ failure or cancer development. Yet little is known about the mechanisms by which adult stem cells respond to such insults by repairing their damaged DNA and resuming normal cellular functions. The blood (hematopoietic) system provides a unique experimental model to investigate the behaviors of specific cell populations. Our objective is to use defined subsets of mouse hematopoietic stem cells (HSCs) and myeloid progenitor cells to investigate how they respond to environmental and therapeutic insults by either repairing damaged DNA and restoring normal functions; accumulating DNA damage and developing cancer; or undergoing programmed cell death (apoptosis) and leading to organ failure. These findings will provide new insights into the fundamental mechanisms that regulate stem cell functions in normal tissues, and a better understanding of their deregulation during cancer development. Such information will identify molecular targets to prevent therapy-related organ damage or secondary cancers. These are severe complications associated with current cancer treatments and are among the leading causes of death worldwide. Originally discovered in blood cancers (leukemia), cancer stem cells (CSCs) have now been recognized in a variety of solid tumors. CSCs represent a subset of the tumor population that has stem cell-like characteristics and the capacity for self-renewal. CSCs result from the transformation of either stem or progenitor cells, which then generate the bulk of the cancer cells. Recent evidence indicates that CSCs are not efficiently killed by current therapies and that CSC persistence could be responsible for disease maintenance and cancer recurrence. Developing interventions that will specifically target CSCs is, therefore, an appealing strategy for improving cancer treatment, which is dependent on understanding how they escape normal regulatory mechanisms and become malignant. Few mouse models of human cancer are currently available in which the CSC population has been identified and purified. This is an essential prerequisite for identifying pathways and molecules amenable to interventional therapies in humans. We have previously developed a mouse model of human leukemia in which we have identified the CSC population as arising from the HSC compartment. We will use this model to understand how deregulations in apoptosis and DNA repair processes contribute to CSC formation and function during disease development. These results will provide new insights into the pathways that distinguish CSCs from normal stem cells and identify ways to prevent their transformation. Such information will be used to design novel and much-needed therapies that will specifically target CSCs while sparing normal stem cells.
Statement of Benefit to California: 
This application investigates how environmental and therapeutic insults leading to DNA damage impact stem cell functions and can lead to organ failure or cancer development. The approach is to study how specific population of blood (hematopoietic) stem, progenitor, and mature cells respond to DNA damaging agents and chose a specific cellular outcome. Such information could identify molecular pathways that are available for interventional therapies to prevent end-organ damage in patients who are treated for a primary cancer and reduce the risk of a subsequent therapy-induced cancer. These are severe complications associated with current mutagenic cancer treatments (radiation or chemotherapeutic agents) that comprise a substantial public health problem in California and in the rest of the developed world. The hematopoietic system is the first to fail following cancer treatment and the formation of therapy-related blood cancer (leukemia) is a common event. The development of novel approaches to prevent therapy-related leukemia will, therefore, directly benefit the health of the Californian population regardless of the type of primary cancer. This application also investigates a novel paradigm in cancer research, namely the role of cancer stem cells (CSCs) in the initiation, progression and maintenance of human cancer. The approach is to study how dysregulations in important cancer-associated pathways (apoptosis and DNA repair processes) contribute to CSC aberrant properties using one of the few established mouse model of human cancer where the CSC population has already been identified. Leukemia, the disease type investigated in this application, has been the subject of many landmark discoveries of basic principles in cancer research that have then been shown to be applicable to a broad range of other cancer types. Accordingly, this research should benefit the people of California in at least two ways. First, the information gained about the properties of CSCs should improve the ability of our physicians and scientists to design, develop and evaluate the efficacy of innovative therapies to target these rare disease-initiating cells for death. This would place Californian cancer research at the forefront of translational science. Second, an average of 11.55 out of 100,000 Californian inhabitants are diagnosed with primary leukemia each year. Thus, in California, leukemia occurs at approximately the same frequency as brain, liver and endocrine cancers. As is true for many types of cancer, most cases of leukemia occur in older adults. At this time, the only treatment that can cure leukemia is allogeneic stem cell transplantation, which is a high-risk and expensive procedure that is most successful in younger patients. The development of novel and safe curative therapies for leukemia would, therefore, particularly benefit the health of our senior population and the economy of the state of California by realizing savings in the healthcare sector.
Progress Report: 
  • Escape from apoptosis and increased genomic instability resulting from defective DNA repair processes are often associated with cancer development, aging and stem cell defects. Adult stem cells play an essential role in the maintenance of normal tissue. Removal of superfluous, damaged and/or dangerous cells is a critical process to maintain tissue homeostasis and protect against malignancy. Yet much remains to be learned about the mechanisms by which normal stem and progenitor cells respond to environmental and therapeutic genotoxic insults. Here, we have used the hematopoietic system as a model to investigate how cancer-associated mutations affect the behaviors of specific stem and progenitor cell populations. Our work during the first year of the CIRM New Faculty award has revealed the differential use of DNA double-strand break repair pathways in quiescent and proliferative hematopoietic stem cells (HSCs), which has clear implications for human health. Most adult stem cell populations, including HSCs, remain in a largely quiescent (G0), or resting, cell cycle state. This quiescent status is widely considered to be an essential protective mechanism stem cells use to minimize endogenous stress caused by cellular respiration and DNA replication. However, our studies demonstrate that quiescence may also have detrimental and mutagenic effects. We found both quiescent and proliferating HSCs to be similarly protected from DNA damaging genotoxic insults due to the expression and activation of cell type specific protective mechanisms. We demonstrate that both quiescent and proliferating HSCs resolve DNA damage with similar efficiencies but use different repair pathways. Quiescent HSCs preferentially utilize nonhomologous end joining (NHEJ) - an error-prone DNA repair mechanism - while proliferating HSCs essentially use homologous recombination (HR) - a high-fidelity DNA repair mechanism. Furthermore, we show that NHEJ-mediated repair in HSCs is associated with acquisition of genomic rearrangements. These findings suggest that the quiescent status of HSCs can, on one hand, be protective by limiting cell-intrinsic stresses but, on the other hand, be detrimental by forcing HSCs to repair damaged DNA with an error-prone mechanism that can generate mutations and eventually cause hematological malignancies. Our results have broad implications for cancer development and provide the beginning of a molecular understanding of why HSCs, despite being protected, are more likely than other cells in the hematopoietic system (i.e., myeloid progenitors) to become transformed. They also partially explain the loss of function occurring in HSCs with age, as it is likely that over a lifetime HSCs have acquired and accumulated numerous NHEJ-mediated mutations that hinder their cellular performance. Finally, our findings may have direct clinical applications for minimizing secondary cancer development. Many solid tumors and hematological malignancies are currently treated with DNA damaging agents, which may result in therapy-induced myeloid leukemia. Our results suggest that it might be beneficial to induce HSCs to cycle before initiating treatment, to avoid inadvertently mutating the patient's own HSCs by forcing them to undergo DNA repair using an error-prone mutagenic mechanism.
  • Our work during the second year of the CIRM New Faculty award has lead to the discovery of at least one key reason why blood-forming stem cells can be susceptible to developing genetic mutations leading to adult leukemia or bone marrow failures. Most adult stem cells, including hematopoietic stem cells (HSCs), are maintained in a quiescent or resting state in vivo. Quiescence is widely considered to be an essential protective mechanism for stem cells that minimizes endogenous stress associated with cellular division and DNA replication. However, we demonstrate that HSC quiescence can also have detrimental effects. We found that HSCs have unique cell-intrinsic mechanisms ensuring their survival in response to ionizing irradiation (IR), which include enhanced pro-survival gene expression and strong activation of a p53-mediated DNA damage response. We show that quiescent and proliferating HSCs are equally radioprotected but use different types of DNA repair mechanisms. We describe how nonhomologous end joining (NHEJ)-mediated DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. These results demonstrate that quiescence is a double-edged sword that, while mostly beneficial, can render HSCs intrinsically vulnerable to mutagenesis following DNA damage. Our findings have important implications for cancer biology. They indicate that quiescent stem cells, either normal or cancerous, are particularly prone to the acquisition of mutations, which overturns the current dogma that cancer development absolutely requires cell proliferation. They help explain why quiescent leukemic stem cells (LSC), which currently survive treatment in most leukemia, do in fact represent a dangerous reservoir for additional mutations that can contribute to disease relapse and/or evolution, and stress the urgent need to develop effective anti-LSC therapies. They also have direct clinical applications for minimizing the risk of therapy-related leukemia following treatment of solid tumors with cytotoxic agents. By showing that proliferating HSCs have significantly decreased mutation rates, with no associated change in radioresistance, they suggest that it would be beneficial to induce HSCs to enter the cycle prior to therapy with DNA-damaging agents in order to enhance DNA repair fidelity in HSCs and thus reduce the risk of leukemia development. While this possibility remains to be tested in the clinic using FDA approved agents such as G-CSF and prostaglandin, it offers exciting new directions for limiting the deleterious side effects of cancer treatment. Our findings also have broad biological implications for tissue function. While the DNA repair mechanism used by quiescent HSCs can indeed produce defective cells, it is likely not detrimental for the organism in evolutionary terms. The blood stem cell system is designed to support the body through its sexually reproductive years, so the genome can be passed along. The ability of quiescent HSCs to survive and quickly undergo DNA repair in response to genotoxic stress supports this goal, and the risk of acquiring enough damaging mutations in these years is minimal. The problem occurs with age, as these long-lived cells have spent a lifetime responding to naturally occurring insults as well as the effects of X-rays, medications and chemotherapies. In this context, the accumulation of NHEJ-mediated DNA misrepair and resultant genomic damages could be a major contributor to the loss of function occurring with age in HSCs, and the development of age-related hematological disorders. We are now using this work on normal HSCs as a platform to understand at the molecular level how the DNA damage response and the mechanisms of DNA repair become deregulated in leukemic HSCs during the development of hematological malignancies.
  • Our work during the third year of the CIRM New Faculty award has extended and broaden up our investigations in two novel directions that are still within the scope of our initial Aims: 1) identifying novel stress-response mechanisms that preserve hematopoietic stem cells (HSC) fitness during periods of metabolic stress; and 2) understanding how deregulations in DNA repair mechanisms contribute to the aberrant functions of old and transformed HSCs. Blood development is organized hierarchically, starting with a rare but well-defined population of HSCs that give rise to a series of committed progenitors and mature cells with exclusive functional and immunophenotypic properties. HSCs are the only cells within the hematopoietic system that self-renew for life, whereas other hematopoietic cells are short-lived and committed to the transient production of mature blood cells. Under steady-state conditions, HSCs are a largely quiescent, slowly cycling cell population, which, in response to environmental cues, are capable of dramatic expansion and contraction to ensure proper homeostatic replacement of all blood cells. While considerable work has deciphered the molecular networks controlling HSC activity, still little is known about how these mechanisms are integrated at the cellular level to ensure life-long maintenance of a functional HSC compartment. HSCs reside in hypoxic niches in the bone marrow microenvironment, and are mostly kept quiescent in order to minimize stress and the potential for damage associated with cellular respiration and cell division. Last year, we showed that HSCs can also engage specialized response mechanisms that protect them from the killing effect of environmental stresses such as ionizing radiation (IR) (Mohrin et al., Cell Stem Cell, 2010). We demonstrated that long-lived HSCs, in contrast to short-lived myeloid progenitors, have enhanced expression of pro-survival members of the bcl2 gene family and robust induction of p53-mediated DNA damage response, which ensures their specific survival and repair following IR exposure. We reasoned that HSCs have other unique protective features, which allow them to contend with a variety of cellular insults and damaged cellular components while maintaining their life-long functionality and genomic integrity. Now, we show that HSCs use the self-catabolic process of autophagy as an essential survival mechanism in response to metabolic stress in vitro or nutriment deprivation in vivo. Last year, we also reported that although HSCs largely survive genotoxic stress their DNA repair mechanisms make them intrinsically vulnerable to mutagenesis (Mohrin et al., Cell Stem Cell, 2010). We showed that their unique quiescent cell cycle status restricts them to the use of the error-prone non-homologous end joining (NHEJ) DNA repair mechanism, which renders them susceptible to genomic instability and transformation. These findings provide the beginning of an understanding of why HSCs, despite being protected at the cellular level, are more likely than other hematopoietic cells to initiate blood disorders (Blanpain et al., Cell Stem Cell, review, 2011). Such hematological diseases increase with age and include immunosenescence (a decline in the adaptive immune system) as well as the development of myeloproliferative neoplasms, leukemia, lymphoma and bone marrow failure syndromes. Many of these features of aging have been linked to changes in the biological functions of old HSCs. Gene expression studies and analysis of genetically modified mice have suggested that errors in DNA repair and loss of genomic stability in HSCs are driving forces for aging and cancer development. However, what causes such failures in maintaining HSC functionality over time remains to be established. We therefore asked whether the constant utilization of error-prone NHEJ repair mechanism and resulting misrepair of DNA damage over a lifetime could contribute to the loss of function and susceptibility to transformation observed in old HSCs. Similarly, we started investigating how mutagenic DNA repair could contribute to the genomic instability of HSC-derived leukemic stem cells (LSC).
  • Our work during the fourth year of the CIRM New Faculty award has been focused on achieving the goals set forth last year for the two first aims of the grant: 1) identifying the stress-response mechanisms that preserve hematopoietic stem cells (HSC) fitness during periods of metabolic stress; and 2) understanding how deregulations in DNA repair mechanisms contribute to the aberrant functions of old HSCs and the aging of the blood system.
  • Blood development is organized hierarchically, starting with a rare but well-defined population of HSCs that give rise to a series of committed progenitors and mature cells with exclusive functional and immunophenotypic properties. HSCs are the only cells within the hematopoietic system that self-renew for life, whereas other hematopoietic cells are short-lived and committed to the transient production of mature blood cells. Under steady-state conditions, HSCs are a largely quiescent, slowly cycling cell population, which, in response to environmental cues, are capable of dramatic expansion and contraction to ensure proper homeostatic replacement of all needed blood cells. While considerable work has deciphered the molecular networks controlling HSC activity, still little is known about how these mechanisms are integrated at the cellular level to ensure life-long maintenance of a functional HSC compartment.
  • HSCs reside in hypoxic niches in the bone marrow microenvironment, and are mostly kept quiescent in order to minimize stress and the potential for damage associated with cellular respiration and cell division. Previously, we found that HSCs also have a unique pro-survival wiring of their apoptotic machinery, which contribute to their enhanced resistance to genotoxic stress (Mohrin et al., Cell Stem Cell, 2010). Now, we identified autophagy as an essential mechanism protecting HSCs from metabolic stress (Warr et al., Nature, in press). We show that HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy following ex vivo cytokine withdrawal and in vivo caloric restriction. We demonstrate that FoxO3a is critical to maintain a gene expression program that poise HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FoxO3a-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system.
  • Previous studies have also suggested that increased DNA damage could contribute to the functional decline of old HSCs. Therefore, we set up to investigate whether the reliance on the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism we previously identified in young HSCs (Mohrin et al., Cell Stem Cell, 2010) could render old HSCs vulnerable to genomic instability. We confirm that old HSCs have increased numbers of γH2AX DNA foci but find no evidence of associated DNA damage. Instead, we show that γH2AX staining in old HSCs entirely co-localized with nucleolar markers and correlated with a significant decrease in ribosome biogenesis. Moreover, we observe high levels of replication stress in proliferating old HSCs leading to severe functional impairment in condition requiring proliferation expansion such as transplantation assays. Collectively, our results illuminate new features of the aging HSC compartment, which are likely to contribute to several facets of age-related blood defects (Flach et al, manuscript in preparation).

Trop2 dependent and independent mechanisms of self-renewal in human cancer stem cells

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06209
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Cancer
Prostate Cancer
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Progress from our group and others has led to the identification of normal prostate tissue stem cells and the definition of important signaling pathways that regulate their growth and maintenance. Human cancers utilize these same pathways to promote malignancy and drive tumor progression. Our recent studies have uncovered an important regulatory molecule (Trop2) that is expressed on a subset of prostate cancer cells capable of regenerating tumors. Trop2 expression is selected for in advanced disease and predicts poor prognosis for many tumors including prostate, ovarian, pancreatic, breast, gastric and colorectal cancer. We predict that blocking Trop2 and other regulatory signaling pathways will be an effective strategy to prevent disease progression in prostate and other human cancers.
Statement of Benefit to California: 
In 2012 alone in the state of California, an estimated 29,000 men will be diagnosed with prostate cancer and almost 3,400 men will die from the disease. The advanced stages of prostate cancer are treated with hormonal therapy which causes significant changes in mood, body weight and composition, impotence and gynecomastia in addition to the pain and suffering from the disease. Our proposed experiments will define new therapeutic targets and combinatorial therapies with the potential to significantly extend life and minimize suffering of men with advanced prostate cancer. Many of the molecules that we are investigating are implicated in a range of tumors, suggesting that our findings may provide benefit to patients suffering from numerous cancers.
Progress Report: 
  • Stem cells are characterized by longevity, self-renewal throughout the lifetime of a tissue or organism and the ability to generate all lineages of a tissue. Pathways involved in stem cell function are commonly dysregulated in cancer. Emerging evidence in leukemias and epithelial cancers suggests that tumors can be maintained by self-renewing cancer stem cells (CSCs), defined functionally by their ability to regenerate tumors. Delineating mechanisms that regulate self-renewal in human CSCs are essential to design new therapeutic strategies to combat cancer.
  • We have developed an in vivo tissue-regeneration model of primary human prostate cancer and identified two distinct populations of CSCs that can self-renew and serially propagate tumors. Both CSC subsets express the transmembrane protein Trop2. We have previously shown that Trop2 is a marker and a new regulator of stem/progenitor activity in the prostate. Trop2 controls self-renewal, proliferation and tissue hyperplasia through two cleavage products—intracellular domain (ICD) and extracellular domain (ECD) generated by regulated intramembrane proteolysis (RIP). RIP of Trop2 is carried out by TACE metalloprotease and gamma-secretase complex.
  • We have also demonstrated that cleaved Trop2 ICD is found in human prostate cancer but not in the cancer-adjacent benign tissue, suggesting a role for Trop2 cleavage in tumorigenesis. Now we are generating antibodies that will block Trop2 cleavage and activation. Blocking Trop2 signaling will be an effective strategy to prevent disease progression not only in the prostate but also in other epithelial cancers.

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

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

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

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

Stem cell-based carriers for RCR vector delivery to glioblastoma

Funding Type: 
Early Translational II
Grant Number: 
TR2-01791
ICOC Funds Committed: 
$3 370 607
Disease Focus: 
Brain Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Modified viruses can be used to infect tumor cells and alter the tumor cell to make anti-tumor proteins. Most researchers use virus that can infect and modify the tumor cell it enters, but can not make more of itself to infect additional cells surrounding the original infected cell. This type of virus is called replication-incompetent virus. Use of replication-incompetent virus is considered safe because no additional virus, which potentially could get out of control, is generated inside of the tumor. However such therapies have been shown to have only limited beneficial effects, presumably because too many tumor cells never get infected. Newer approaches investigate the use of replication-competent viruses to achieve highly efficient gene transfer to tumors. A successfully transduced tumor cell itself becomes a virus-producing cell, sustaining further transduction events even after initial administration. We propose here to use a type of replication-competent virus that only infects dividing cells and therefore will infect the rapidly dividing cancer cells but not normal brain cells. The use of replication-competent virus is potentially more risky but is well justified in clinical scenarios involving highly aggressive and rapidly progressing metastatic tumor growth in the brain. To administer therapeutic virus into the brain, the virus is injected right into the center of the tumor. Yet, human brain tumors are often found as diffusely spreading foci in the brain and may be difficult to eliminate by locally-administered replication-competent retrovirus (RCR) vectors alone. In this study we propose to use a type of adult stem cell called a "mesenchymal stem cell" (MSC) as a delivery system for the RCR vectors. Mesenchymal stem cells (MSCs) have been shown to have natural tumor-homing abilities, and can migrate to tumor foci and penetrate through into the interior of tumor masses. We propose to engineer them into "aircraft carriers" that release tumor-selective viruses, which can then efficiently spread suicide genes from one cancer cell to another in multiple tumor foci in the brain.
Statement of Benefit to California: 
This research is based on a solid foundation that combines two innovative technologies for the treatment of primary brain tumors, particularly glioblastoma multiforme (GBM) the most malignant form of brain tumor, which afflicts men, women, and children in California and elsewhere. Each of these technologies has been approved separately by FDA for clinical testing in humans: human mesenchymal stem cells (MSCs), and replication-competent retrovirus (RCR) vectors. MSCs have been reported to exhibit a natural ability to migrate to solid tumors and penetrate into the tissue mass. Once inside a tumor, RCR vectors can spread selectively in the cancer cells and their replication can keep up with their uncontrolled proliferation, and their ability to integrate themselves into the cancer cell genome allows them to permanently "seed" tumor cells with therapeutic genes. Here we propose to utilize the natural tumor homing ability of MSCs to deliver RCR vectors into brain tumors. This "virus vs. cancer" strategy takes advantage of the amplification process inherent in the spread of virus from cell to cell, and by using MSCs to initiate the virus infection efficiently in brain tumors, represents an approach that will have the potential to effectively treat this poor prognosis disease. If successful, clinical application of this strategy can be implemented by an "off-the-shelf" mesenchymal stem cell (MSC) primary cell lines that have been pre-characterized for their tumor homing ability and virus production capability, and can be offered to patients without requiring an invasive procedure to harvest their own stem cells. Furthermore, this represents a treatment that could potentially be administered through a needle, thus making it unnecessary for patients to undergo major neurosurgical procedures entailing craniotomy at an advanced medical center. Hence this research could lead to a novel treatment approach that would particularly address the needs of brain tumor patients in California who are underserved due to socioeconomic and geographic constraints, as well as the elderly who are poor-risk for surgical interventions.
Progress Report: 
  • The goal of this project is to develop clinically translatable methods for engineering human mesenchymal stem cells (hMSC) to serve as tumor-homing cellular carriers that will deliver a replication-competent retrovirus (RCR) vector throughout primary brain tumors (gliomas). RCR vectors expressing a prodrug activator (also known as a "suicide gene"), which converts a non-toxic "pro-drug" compound into a potent chemotherapy drug directly generated within the infected tumor cells, have recently initiated testing in Phase I/II clinical trials for suicide gene therapy of recurrent high-grade gliomas. We are examining whether MSCs can serve as producer cells for this RCR vector, and whether the tumor transduction efficiency and therapeutic efficacy of this vector can be significantly enhanced, without compromising its safety profile, hMSC-based RCR producer cells (MSC-RCR) are used as a tumor-homing mobile carrier system that releases the virus as the cells migrate toward and within tumor masses in the brain. In particular, we are comparing this MSC-RCR cell-based carrier method against conventional delivery methods by direct intratumoral injection of 'naked' virus, in subcutaneous and intracranial brain tumor models.
  • To date, we have accomplished our milestone tasks for Year 1, by:
  • - successfully developing efficient methods to transduce hMSCs with RCR vectors and thereby convert them into vector producer cells
  • - developing and comparing in vitro and in vivo assays to evaluate the tumor-homing migratory activity of hMSCs
  • - applying these assays to screen and evaluate commercially available hMSC isolates
  • - demonstrating that the MSC-RCR delivery system can achieve significantly more efficient transduction of subcutaneous glioma models as compared to virus by itself
  • - confirming that enhanced transduction efficiency by MSC-RCR achieves more rapid tumor growth inhibition, as compared to 'naked' RCR alone, when applied to suicide gene therapy in subcutaneous tumor models of human glioma
  • - confirming that hMSC-mediated RCR delivery does not increase vector biodistribution to normal tissues, nor incur any increased risk of secondary leukemogenesis
  • Interestingly, through these studies we have found considerable variability in tumor-homing migration activity and intratumoral migration activity between hMSC isolates from different sources, a finding that may have significant implications for the development of hMSC-based clinical products. We are continuing to characterize additional hMSC isolates from various tissue sources, and are preparing a manuscript to publish these results.
  • Furthermore, based on our favorable results as described above, indicating the enhanced efficiency of tumor transduction and growth inhibitory effects when suicide gene therapy is delivered by MSC-RCR, as compared to RCR alone, we have fulfilled the success criteria for each of our milestone tasks in Year 1, and are currently proceeding with Year 2 studies.
  • Modified viruses can be used to infect tumor cells and alter the tumor cell to make anti-tumor proteins. We have developed a type of replication-competent virus that efficiently infects rapidly dividing cancer cells, but not normal brain cells. This virus is currently being tested clinically in patients with malignant brain tumors. However, to administer therapeutic virus into the brain, the virus is injected right into the center of the tumor, or in around the margins of the cavity after surgical removal of most of the tumor. Yet, human brain tumors are often found as diffusely spreading foci in the brain and may be difficult to eliminate by locally-administered replication-competent retrovirus (RCR) vectors alone. In this project, we propose to use a type of adult stem cell, called a "mesenchymal stem cell" (MSC), as a delivery system for the RCR vectors. Human mesenchymal stem cells (hMSCs) have been shown to have natural tumor-homing abilities, and can migrate to tumor foci and penetrate through into the interior of tumor masses.
  • During this project period, we have established and optimized manufacturing methods to engineer hMSCs into "aircraft carriers" that release our tumor-selective RCR vectors, which we then confirmed can efficiently spread a non-therapeutic marker gene to brain tumor cells. We have further confirmed that the use of hMSCs as a cellular delivery system for RCR vectors achieves more rapid spread of the vectors through the tumor mass, as compared to injecting the virus by itself, both in tumor models implanted under the skin as well as implanted in the brain. We have also obtained initial results demonstrating that hMSC delivery of RCR vectors does not result in unwanted spread of virus to normal tissues outside the brain. This stem cell-based RCR vector delivery system, which we have so far tested and validated using a marker gene, in our current studies is now being applied to delivery of a therapeutic anti-tumor 'suicide' gene. We have also initiated discussions with the UC Davis Stem Cell Institute to develop clinical grade manufacturing processes for hMSC-based RCR vector producer cells, and with a San Diego-based biotech partner, Tocagen Inc., toward the initiation of a clinical trial to test this strategy in brain tumor patients in the near future.

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

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

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