Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Therapeutic Eradication of Cancer Stem Cells

Funding Type: 
Disease Team Therapy Development III
Grant Number: 
DR3-06924
ICOC Funds Committed: 
$4 179 600
Disease Focus: 
Blood Cancer
Cancer
oldStatus: 
Closed
Public Abstract: 
Cancer is a leading cause of death in California. Research has found that many cancers can spread throughout the body and resist current anti-cancer therapies because of cancer stem cells, or CSC. CSC can be considered the seeds of cancer; they can resist being killed by anti-cancer drugs and can lay dormant, sometimes for long periods, before growing into active cancers at the original tumor site, or at distant sites throughout the body. Required are therapies that can kill CSC while not harming normal stem cells, which are needed for making blood and other cells that must be replenished. We have discovered a protein on the surface of CSC that is not present on normal cells of healthy adults. This protein, called ROR1, ordinarily is found only on cells during early development in the embryo. CSC have co-opted the use of ROR1 to promote their survival, proliferation, and spread throughout the body. We have developed a monoclonal antibody that is specific for ROR1 and that can inhibit these functions, which are vital for CSC. Because this antibody does not bind to normal cells, it can serve as the “magic bullet” to deliver a specific hit to CSC. We will conduct clinical trials with the antibody, first in patients with chronic lymphocytic leukemia to define the safety and best dose to use. Then we plan to conduct clinical trials involving patients with other types of cancer. To prepare for such clinical trials, we will use our state-of-the-art model systems to investigate the best way to eradicate CSC of other intractable leukemias and solid tumors. Finally, we will investigate the potential for using this antibody to deliver toxins selectively to CSC. This selective delivery could be very active in killing CSC without harming normal cells in the body because they lack expression of ROR1. With this antibody we can develop curative stem-cell-directed therapy for patients with any one of many different types of currently intractable cancers.
Statement of Benefit to California: 
The proposal aims to develop a novel anti-cancer-stem-cell (CSC) targeted therapy for patients with intractable malignancies. This therapy involves use of a fully humanized monoclonal antibody specific for a newly identified, CSC antigen called ROR1. This antibody was developed under the auspices of a CIRM disease team I award and is being readied for phase I clinical testing involving patients with chronic lymphocytic leukemia (CLL). Our research has revealed that the antibody specifically reacts with CSC of other leukemias and many solid-tumor cancers, but does not bind to normal adult tissues. Moreover, it has functional activity in blocking the growth and survival of CSC, making it ideal for directing therapy intended to eradicate CSC of many different cancer types, without affecting normal adult stem cells or other normal tissues. As such, treatment could avoid the devastating physical and financial adverse effects associated with many standard anti-cancer therapies. Also, because this therapy attacks the CSC, it might prove to be a curative treatment for California patients with any one of a variety different types of currently intractable cancers. Beyond the significant benefit to the patients and families that are dealing with cancer, this project will also strengthen the position of the California Institute of Regenerative Medicine as a leader in cancer stem cell biology, and will deliver intellectual property to the state of California that may then be licensed to pharmaceutical companies. In summary, the benefits to the citizens of California from the CIRM disease team 3 grant are: (1) Direct benefit to the thousands of patients with cancer (2) Financial savings through definitive treatment that obviates costly maintenance or salvage therapies for patients with intractable cancers (3) Potential for an anti-cancer therapy with a high therapeutic index (4) Intellectual property of a broadly active uniquely targeted anti-CSC therapeutic agent.

Human endothelial reprogramming for hematopoietic stem cell therapy.

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06479
ICOC Funds Committed: 
$3 084 000
Disease Focus: 
Blood Disorders
Blood Cancer
Cancer
Stem Cell Use: 
Directly Reprogrammed Cell
Cell Line Generation: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
The current roadblocks to hematopoietic stem cell (HSC) therapies include the rarity of matched donors for bone marrow transplant, engraftment failures, common shortages of donated blood, and the inability to expand HSCs ex vivo in large numbers. These major obstacles would cease to exist if an extensive, bankable, inexhaustible, and patient-matched supply of blood were available. The recent validation of hemogenic endothelium (blood vessel cells lining the vessel wall give rise to blood stem cells) has introduced new possibilities in hematopoietic stem cell therapy. As the phenomenon of hemogenic endothelium only occurs during embryonic development, we aim to understand the requirements for the process and to re-engineer mature human endothelium (blood vessels) into once again producing blood stem cells (HSCs). The approach of re-engineering tissue specific de-differentiation will accelerate the pace of discovery and translation to human disease. Engineering endothelium into large-scale hematopoietic factories can provide substantial numbers of pure hematopoietic stem cells for clinical use. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the ability to program mature lineage restricted cells into more primitive versions of the same cell lineage will capitalize on cell renewal properties while minimizing malignancy risk.
Statement of Benefit to California: 
Bone marrow transplantation saves the lives of millions with leukemia and other diseases including genetic or immunologic blood disorders. California has over 15 centers serving the population for bone marrow transplantation. While bone marrow transplantation can be seen as a standard to which all stem cell therapies should aspire, there still remains the difficulty of finding matched donors, complications such as graft versus host disease, and the recurrence of malignancy. While cord blood has provided another donor source of stem cells and improved engraftment, it still requires pooling from multiple donors for sufficient cell numbers to be transplanted, which may increase transplant risk. By understanding how to reprogram blood vessels (such as those in the umbilical cord) for production of blood stem cells (as it once did during human development), it could eventually be possible to bank umbilical cord vessels to provide a patient matched reproducible supply of pure blood stem cells for the entire life of the patient. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the proposed work will introduce a new approach to engineering human cells. The ability to turn back the clock to near mature cell specific stages without going all the way back to early embryonic stem cell stages will reduce the risk of malignancy.
Progress Report: 
  • We aim to understand how blood stem cells develop from blood vessels during development. We are also interested in learning whether the blood-making program can be turned back on in blood vessel cells for blood production outside the human body. During the past year we have been able to extract and culture blood vessel cells that once had blood making capacity. We have also started experiments that will help uncover the regulation of the blood making program. In addition, we have developed tools to help the process of understanding whether iPS technology can "turn back time" in mature blood vessels and turn on the blood making program.

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.

Combinatorial Chemistry Approaches to Develop LIgands against Leukemia Stem Cells

Funding Type: 
New Faculty I
Grant Number: 
RN1-00561
ICOC Funds Committed: 
$2 392 397
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.

Epigenetics in cancer stem cell initiation and clinical outcome prediction

Funding Type: 
New Faculty I
Grant Number: 
RN1-00550
ICOC Funds Committed: 
$3 063 450
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.

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

Funding Type: 
New Faculty II
Grant Number: 
RN2-00910
ICOC Funds Committed: 
$3 065 572
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Cancer is the leading cause of death for people younger than 85. High cancer mortality rates related to resistance to therapy and malignant progression underscore the need for more sensitive diagnostic techniques as well as therapies that selectively target cells responsible for cancer propagation. Compelling studies suggest that human cancer stem cells (CSC) arise from aberrantly self-renewing tissue specific stem or progenitor cells and are responsible for cancer propagation and resistance to therapy. Although the majority of cancer therapies eradicate rapidly dividing cells within the tumor, the rare CSC population may be quiescent and then reactivate resulting in disease progression and relapse. We recently demonstrated that CSC are generated in chronic myeloid leukemia by activation of beta-catenin, a gene that allows cells to reproduce themselves extensively. However, relatively little is known about the sequence of events responsible for leukemic transformation in more common myeloproliferative disorders (MPDs) that express an activating mutation in the JAK2 gene. Because human embryonic stem cells (hESC) have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells, they represent an ideal model system for generating and characterizing human MPD stem cells. Thus, hESC cell research harbors tremendous potential for developing life-saving therapy for patients with cancer by providing a platform to rapidly and rationally test new therapies that specifically target CSC. To provide a robust model system for screening novel anti-CSC therapies, we propose to generate and characterize BCR-ABL+ and JAK2+ MPD stem cells from hESC. We will investigate the role of genes that are essential for initiation of these MPDs such as BCR-ABL and JAK2 V617F as well as additional mutations in beta-catenin or GSK3betaï€ implicated in CSC propagation. The efficacy of a selective BCR-ABL and JAK2 inhibitors at blocking BCR-ABL+ and JAK2+ human ES cell self-renewal, survival and proliferation alone and in combination with a potent and specific beta-catenin antagonist will be assessed in robust in vitro and in vivo assays with the ultimate aim of developing highly active anti-MPD stem cell therapy that may halt progression to acute leukemia and obviate therapeutic resistance.
Statement of Benefit to California: 
Although much is known about the genetic and epigenetic events involved in CSC production in a Philadelphia chromosome positive MPD like chronic myeloid leukemia (CML), comparatively little is known about the molecular pathogenesis of the five-fold more common Philadelphia chromosome negative (Ph-) MPDs. MPD patients have a moderately increased risk of fatal thrombotic events as well as a striking 36-fold increased risk of death from transformation to acute leukemia. Recently, a point mutation, JAK2 V617F(JAK2+), resulting in constitutive activation of the JAK2 cytokine signaling pathway was discovered in a large proportion of MPD patients. A critical barrier to developing potentially curative therapies for both BCR-ABL+ and JAK2+ MPDs is a comprehensive understanding of relative contribution of BCR-ABL and JAK2 V617F to disease initiation versus transformation to acute leukemia. We recently discovered that JAK2 V617F is expressed at the hematopoietic stem cell level in PV, ET and MF and that JAK2 skewed ifferentiation in PV is normalized with a selective JAK2 inhibitor, TG101348. However, a detailed molecular pathogenetic characterization has been hampered by the paucity of stem and progenitor cells in MPD derived blood and marrow samples. Because hESC have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells in vitro, they represent an ideal model system for generating human MPD stem cells. Thus, California hESC research harbors tremendus potential for understanding the MPD initiating events that skew differentiation versus events that promote self-renewal and thus, leukemic transformation. Moreover, a more comprehensive understanding of primitive stem cell fate decisions may yield key insights into methods to expand blood cell production that may have major implications for blood banking. Clinical Benefit Generation of MPD stem cells from hESC would provide an experimentally amenable and relevant platform to expedite the development ofsensitive diagnostic techniques to predict disease progression and to develop potentially curative anti-CSC therapies. Economic Benefit The translational research performed in the context of this grant will not only speed the delivery of innovative MPD targeted therapies for Californians, it will help to train Californiaís future R&D workforce in addition to developing leaders in translational medicine. This grant will provide the personnel working on the project with a clear view of the importance of thir research to cancer therapy and a better perspective on future career opportunities in California as well as directly generate revenue through development and implementation of innovative therapies aimed at eradicating MPD stem cells that may be more broadly applicable to CSC in other malignances.
Progress Report: 
  • Summary of Overall Progress
  • This grant focuses on generation of MPN stem cells from hESC or CB and correlates leukemic potential with MPN patient samples. In the first year of this grant, we have demonstrated that 1) hESC differentiate on AGM stroma to the CD34+ stage, which is associated with increased GATA-1, Flk2, GATA-2 and ADAR1 expression; 2) hESC CD34+ differentiation is enhanced in vitro and in vivo in the presence of a genetically engineered mouse stroma, which produces human stem cell factor, IL-3 and G-CSF; 3) hESC CD34+ cells can be transduced with our novel lentiviral BCR-ABL vector, which, unlike retroviral BCR-ABL, can transduce quiescent stem cells; 4) BCR-ABL expression by CP CML progenitors does not sustain engraftment but rather leukemic transformation is predicated, in part, on bcl-2 overexpression; 5) JAK2V617F expression in hES or CB stem cells is insufficient to induce leukemic transformation; 6) BCR-ABL transduced hESC CD34+ cells have significantly higher BCR-ABL transplantation potential than CP CML progenitors suggesting that they have higher survival capacity; 7) lentiviral -catenin transduction of BCR-ABL hESC CD34+ cells leads to serial transplantation indicative of LSC formation; 8) CML BC LSC persist in vivo despite potent BCR-ABL inhibition with dasatinib therapy and will likely require combined inhibitor therapy to eradicate. Currently, HEEBO arrays and phospho-flow studies are underway to detect bcl-2 family members and self-renewal protein expression in BCR-ABL and JAK2 V617F transduced hESC and CB CD34+ cells compared with MPN patient derived progenitors. This will aid in development of combined MPN stem cell inhibitor strategies in this grant.
  • This grant focuses on generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent (hESC) or multipotent (CB) stem cells and seeks to correlate their leukemic potential with that of MPN patient sample-derived stem cells. To provide a platform for testing induction of stem cell differentiation, survival and self-renewal by BCR-ABL versus JAK2, hESC were utilized in the first year and as more patient samples and cord blood became available these were utilized.
  • In the first year of this grant, we found that hESC undergo hematopoietic differentiation on AGM stroma to the CD34+ stage resulting in increased GATA-1, Flk2, ADAR1 and GATA-2 expression. Moreover, CD34+ differentiation was enhanced on a genetically engineered mouse stroma (SL/M2) secreting human SCF, IL-3 and G-CSF. Lentiviral BCR-ABL transduced hESC-derived CD34+ cells had higher BCR-ABL+ cellular transplantation potential than chronic phase (CP) CML progenitors, indicative of a higher survival capacity. However, they sustained self-renewal only when co-transduced with lentiviral -catenin (Rusert et al, manuscript in preparation) suggesting that blast crisis evolution requires acquisition of both enhanced survival and self-renewal potential. Similarly, lentiviral mouse mutant JAK2 expression in hESC or CB stem cells was insufficient to produce self-renewing MPN stem cells, indicating that the cellular context, nature of the genetic driver and responses to extrinsic cues from the microenvironment play seminal roles in regulating therapeutically resistant MPN stem cell properties such as aberrant survival, differentiation, self-renewal and dormancy.
  • In the second year of this five year grant, we have focused on human cord blood (CB) stem cells compared with a large number of MPN patient samples propagated on SL/M2 stroma or in RAG2-/-c-/- mice to more adequately recapitulate the human MPN stem cell niche. Also, to more faithfully recapitulate human (rather than the previously published lentiviral mouse JAK2 vectors, Cancer Cell 2008) JAK2 driven MPNs, we cloned human wild-type JAK2 and human JAK2 V617F from MPN patient samples into lentiviral-GFP vectors (Court Recart A*, Geron I* et al, manuscript in preparation). We also incorporated full transcriptome RNA (ABI SOLiD 4.0) sequencing, PCR array and nanofluidic phosphoproteomics technology to better gauge the impact of JAK2 versus BCR-ABL on stem cell fate, survival, self-renewal and dormancy in the context of specific malignant microenvironments and the relative susceptibility of MPN stem cells in these niches to single agent molecularly targeted inhibitors.
  • This grant focuses on generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent human embryonic stem cells (hESC) or multipotent cord blood (CB) stem cells, and seeks to correlate their leukemic potential with that of disease progression in MPN patient sample-derived stem cells. In the first and second years of this grant, we found that lentiviral BCR-ABL transduced hESC-derived CD34+ cells had higher leukemic transplantation potential than chronic phase (CP) chronic myeloid leukemia (CML) progenitors. However, they sustained self-renewal only when co-transduced with lentiviral beta-catenin suggesting that blast crisis (BC) evolution requires acquisition of both enhanced survival and self-renewal potential. Similarly, we have shown using lentiviral vectors that mouse and human mutant JAK2 were insufficient to produce self-renewing MPN stem cells. New results in Year 3 demonstrate that BCR-ABL and JAK2 activation drive differentiation of hematopoietic progenitors towards an erthyroid/myeloid lineage bias. We have used full transcriptome RNA-Sequencing (RNA-Seq) technology to evaluate the genetic and epigenetic status of BCR-ABL and JAK2-transduced normal progenitor cells as well as patient-derived MPN progenitors. This has allowed us to probe the mechanisms of aberrant differentiation and self-renewal of MPN progenitors and identify unique gene expression signatures of disease progression.
  • We previously found that overexpression and splice isoform switching of a key RNA editing enzyme – adenosine deaminase acting on dsRNA (ADAR), and splice isoform changes in pro-survival BCL2 family members, correspond with disease progression in CML. In the current reporting period, RNA-Seq analyses revealed that ADAR1-driven activation of RNA editing contributed to malignant progenitor reprogramming, promoting aberrant differentiation and self-renewal of MPN stem cells. Knocking down ADAR1 using lentiviral shRNA vectors reduced the self-renewal potential of CML progenitors. This work has culminated in a manuscript that has now been submitted to PNAS (Jiang et al.). Recent results also show that ADAR1 is activated in progenitors from patients with JAK2-driven MPNs. Thus, ADAR1 may be an important factor that works in concert with BCR-ABL or JAK2 to facilitate disease progression in MPNs.
  • Our results show that another self-renewal factor that may drive BCR-ABL or JAK2-mediated propagation of disease from quiescent MPN progenitors is Sonic hedgehog (Shh). We have examined the expression patterns of this pathway in MPN progenitors using qRT-PCR and RNA-Seq, and have tested a pharmacological inhibitor of this pathway in a robust stromal co-culture model of MPN progression to Acute Myeloid Leukemia (AML).
  • In sum, we have utilized full transcriptome RNA-Seq and qRT-PCR coupled with hematopoietic progenitor assays and in vivo studies to evaluate the impact of JAK2 versus BCR-ABL on stem cell fate, survival, self-renewal and dormancy. These techniques have allowed us to investigate in more detail the role of genetic and epigenetic alterations that drive disease progression in the context of specific malignant microenvironments, and the relative susceptibility of MPN stem cells in these niches to single agent molecularly targeted inhibitors.
  • The main objectives of this project are generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent human embryonic stem cells (hESC) or multipotent stem cells, and identification of crucial leukemia stem cell (LSC) survival and self-renewal factors that contribute to the development and progression of BCR-ABL and JAK2-driven hematopoietic disorders. A key finding of our work thus far is that in addition to activation of BCR-ABL or JAK2 oncogenes, generation of self-renewing MPN LSC requires stimulation of other pro-survival and self-renewal factors such as β-catenin, Sonic hedgehog (SHH), BCL2, and in particular the RNA editing enzyme ADAR1, which we identified as a novel regulator of LSC differentiation and self-renewal.
  • We have now completed comprehensive gene expression analyses from next-generation RNA-sequencing studies performed on normal and leukemic human hematopoietic progenitor cells from primary cord blood samples and adult normal peripheral blood samples, along with normal cord blood transduced with BCR-ABL or JAK2 oncogenes, and primary samples from patients with BCR-ABL+ chronic phase and blast crisis chronic myeloid leukemia (CML). These studies revealed that gene expression patterns in survival and self-renewal pathways (SHH, JAK2, ADAR1) clearly distinguish normal and leukemic progenitor cells as well as MPN disease stages. These data provide a vast resource for identification of LSC-specific biomarkers with diagnostic and prognostic clinical applications, as well as providing new potential therapeutic targets to prevent disease progression.
  • New results from RNA-sequencing studies reveal high levels of expression of inflammatory mediators in human blast crisis CML progenitors and in BCR-ABL transduced normal cord blood stem cells. Moreover, expression of the inflammation-responsive form of ADAR1 correlated with generation of an abnormally spliced GSK3β gene product that has been previously linked to LSC self-renewal. These results have now been published in the journal PNAS (Jiang et al.). Together, we have demonstrated that ADAR1 drives hematopoietic cell fate by skewing cell differentiation – a trend which occurs during normal bone marrow aging – and promotes LSC self-renewal through alternative splicing of critical survival and self-renewal factors. Notably, inhibition of ADAR1 through genetic knockdown strategies reduced self-renewal capacity of CML LSC, and may have important applications in treatment of other disorders that transform to acute leukemia. Thus, these results suggest that RNA editing (ADAR1) and splicing represent key therapeutic targets for preventing LSC self-renewal – a primary driver of leukemic progression.
  • Whole transcriptome profiling studies coupled with qRT-PCR, hematopoietic progenitor assays and in vivo studies have shown that combined inhibition of BCR-ABL and JAK2 is another effective method to reduce LSC self-renewal in pre-clinical models. New results show that lentivirus-enforced BCR-ABL or JAK2 expression in normal cord blood stem cells drives generation of distinct splice isoforms of STAT5a. While inhibition of JAK2/STAT5a signaling or BCR-ABL tyrosine kinase activity alone did not eradicate self-renewing LSC, combined JAK2 and BCR-ABL inhibition dramatically impaired LSC survival and self-renewal in the protective bone marrow niche, and increased the lifespan of serial transplant recipients. These effects were associated with reduction in STAT5a isoform expression – which represents a novel molecular marker of response to combined BCR-ABL/JAK2 inhibition – and altered expression of cell cycle genes in human progenitor cells harvested from the bone marrow of transplanted mice. These results are the subject of a new manuscript currently under review (Court et al.). Moreover, this work has led to the development of new experimental tools that will facilitate study of LSC maintenance and cell cycle status in the context of normal versus diseased bone marrow microenvironments. In sum, studies completed thus far have uncovered a role for RNA editing and splicing alterations in leukemic progression, particularly in specific microenvironments. Using specific inhibitors targeting BCR-ABL and JAK2, along with strategies to block RNA editing and aberrant splicing activities, we have been able to establish the relative susceptibility of MPN stem cells to molecular inhibitors with activity against LSC residing in select hematopoietic niches that are difficult to treat with conventional chemotherapeutic agents.
  • In the final year of this project, we focused on elucidating the mechanisms of leukemia stem cell (LSC) generation in JAK2 compared with BCR-ABL1 initiated myeloproliferative neoplasms (MPN, previously called myeloproliferative disorders). To this end, we investigated the MPN stem cell propagating effects of BCR-ABL1 or JAK2 alone or in combination with activation of the human embryonic stem cell RNA editase, ADAR1. Recently, we discovered that ADAR1, which edits adenosine to inosine bases in the context of primate specific Alu sequences, leads to GSK3β missplicing and β-catenin activation in chronic phase (CP) CML progenitors leading to blast crisis (BC) transformation and LSC generation. In addition, variant isoform expression of a Wnt/β-catenin target gene, CD44, was also characteristic of LSC. In a previous report (Jiang et al., PNAS 2013), identification of ADAR1 as a malignant reprogramming factor represented the first description of RNA editing as a regulator of reprogramming. When lentivirally overexpressed, ADAR1 endows committed CP myeloid progenitors with self-renewal capacity. Further studies revealed that JAK2/STAT5a activates ADAR1 leading to deregulation of cell cycle progression and global down-regulation of microRNA expression thereby uncovering two additional key mechanisms of LSC generation in MPNs. This is consistent with our findings from gene expression profiling studies performed in the previous year, along with functional classification and network analysis using Ingenuity Pathway Analysis (IPA), showing that cell cycle-related genes were significantly altered in human progenitors from xenografted mice treated with combination JAK2 and BCR-ABL inhibitor therapy compared with single agent therapies alone. Together these data suggest that combined BCR-ABL and JAK2 inhibition impairs LSC survival and self-renewal via cell cycle modulation. ADAR1 and other stem cell regulatory pathways such as CD44 represent novel targets to detect and eradicate the self-renewing LSC. We also performed new studies that elucidate the stem cell-intrinsic genetic changes that occur during human bone marrow aging, which may contribute to BCR-ABL or JAK2-dependent functional alterations.
  • This work has led to discovery of a novel role for embryonic stem cell genes and splice isoforms, including ADAR1 p150 and a transcript variant of CD44, in the maintenance of LSC that promote MPN progression. In addition, through the course of this research we have 1) developed novel lentiviral tools for investigating normal hematopoietic stem and progenitor (HSPC) and malignant LSC survival, differentiation, self-renewal, and cell cycle regulation, and 2) devised innovative LSC diagnostic strategies and 3) tested therapeutic strategies targeting LSC-associated RNA editing and splice isoform generation that selectively inhibit LSC self-renewal.

Stem Cells in Lung Cancer

Funding Type: 
New Faculty II
Grant Number: 
RN2-00904
ICOC Funds Committed: 
$2 381 572
Disease Focus: 
Lung Cancer
Cancer
Respiratory Disorders
Stem Cell Use: 
Adult Stem Cell
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Lung cancer is the most deadly cancer worldwide and accounts for more deaths than prostate cancer, breast cancer and colon cancer combined. Non small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. The current 5-year survival rate for all stages of NSCLC is only 15%. Although early stage lung cancer has a much better survival rate. Current therapeutic strategies of chemotherapy, radiation therapy and trials with new targeted therapies have only demonstrated, at best, extension in survival by a few months. Clearly, a novel approach is required to develop new therapies for this devastating disease and to detect the disease at an early stage. Cancer stem cells have been identified as the initial cell in the formation of carcinomas. Chemotherapy, radiation and even targeted therapies are all designed to eliminate dividing cells. However, cancer stem cells “hide out” in the quiescent phase of growth. This provides an explanation as to why our cancer therapies may produce an initial response but are often unsuccessful in curing patients. Lung cancer develops through a series of step wise changes that result in the progression of pre-malignant lesions to invasive lung cancer. The mechanisms of how lung cancer develops are not known and if we can prevent the formation of pre-malignant lesions, we will likely be able to prevent lung cancer. We have discovered a subpopulation of stem cells that circulates in the blood and is essential for normal lung repair. Blocking these cells from entering the lung results in a pre-malignant condition in the lungs. We have also identified a subpopulation of stem cells in the lung that is responsible for generating pre-malignant lung cancer lesions. We hypothesize that the interaction between the stem cells in the blood and the stem cells in the lung are critical to prevent lung cancer. We plan to use cutting edge technologies to characterize these different stem cell populations in the lung, and determine how they form pre-malignant lung cancer lesions. We also plan to use preclinical models to try to prevent lung cancer by giving additional stem cells derived from the blood as a therapy. Lastly, we plan to determine whether levels of stem cells in the blood in patients may be used as a blood test to measure the chance of recurrence of lung cancer after therapy. The long term goals of our work are to develop a screening test for lung cancer stem cells that can predict which patients are at high risk for developing lung cancer in order to diagnose lung cancer at an early stage, and to potentially develop a new stem cell based therapy for preventing and treating lung cancer.
Statement of Benefit to California: 
According to the Center for Health Statistics, California Department of Health Services, 13,427 people died of lung cancer in the state of California in 2005. This is more than the deaths attributed to breast, prostate and colon cancers combined. The devastating effects of this disease on the citizens of California and the health care costs involved are enormous. Most cases of lung cancer occur in smokers, but non smokers, people exposed to second hand smoke and ex-smokers are also at risk. In addition, of special concern to California residents, is that exposure to air pollution is associated with an increased risk of lung cancer. Current therapeutic strategies for lung cancer are in general only able to prolong survival by a few months, especially for late stage disease. One reason for this may be that the cancer initiating stem cell is resistant to these therapies. Understanding the stem cell populations involved in repair of the lung and how these cells may give rise to lung cancer is important for potentially generating new therapeutic targets for lung cancer. We propose to study the stem cell populations of the lung that are crucial for normal airway repair and characterize the putative cancer initiating stem cell in the lung. We have also found stem cells in the blood that are critical for normal airway repair and we plan to test their role in the prevention of premalignant lung cancer lesions. We also plan to test whether levels of these stem cells in the blood may be used as a biomarker of lung cancer. Ultimately, the ability to perform a screening test to detect lung cancer at an early stage, and the development of new therapies for lung cancer will be of major benefit to the citizens of California.
Progress Report: 
  • We identified a putative tumor-initiating stem/progenitor cell that goes rise to smoking-associated non small cell lung cancer (NSCLC). We examined 399 NSCLC samples for this tumor-initiating stem/progenitor cell and found that the presence of this cell in the tumor gave rise to a significantly worse prognosis and was associated with metastatic disease. This stem/progenitor cell is known to be important for repair of the airway and is present in precancerous lesions. We believe that this cell undergoes aberrant repair after smoking injury, which leads to lung cancer. We are currently trying to identify the genetic and epigenetic mechanisms involved in this aberrant repair as a means to identify a novel therapy to prevent the development of lung cancer. The presence of these stem/progenitor cells may also be used as a biomarker of poor prognostic NSCLC even in early stage disease.
  • We have identified markers on these stem/progenitor tumor-initiating cells and identified sub-populations of these cells. We are now determining the stem cell capabilities of each of these sub-populations. We are using a model of the development of lung cancer to determine if giving a stem/progenitor cell sub-population for repair can prevent NSCLC from developing.
  • We examined the blood of patients diagnosed with a lung nodule for circulating epithelial stem/progenitor cells. We found that the presence of these cells in the blood of patients predicted the presence of a subtype of NSCLC as compared to a benign lung nodule. We are currently obtaining many more blood samples from patients to further determine whether circulating epithelial stem/progenitor cells could be used as a biomarker of early NSCLC.
  • We have found a stem cell that is important for lung repair after injury that is located in a protected niche in the airway. After repeated injury, for example in smokers, these stem cells persist in an abnormal location on the surface of the airway and replicate and form precancerous areas in the lung. The presence of these stem cells in lung cancer tumors was associated with a poor prognosis with an increased chance of relapse and metastasis.This was especially true in current and former smokers. We therefore believe we have found a putative stem cell that is a tumor initiating cell for lung cancer. We developed a method to isolate these lung stem cells and to profile these cells and developed in vitro and in vivo models to assess their stem cell properties. Finally, we examined human blood samples to assess levels of surrogate markers of these stem cells to assess whether we could use this as a biomarker to predict the presence or absence of lung cancer in patients with a lung nodule.
  • We found a stem cell that is important for lung repair after injury that we believe may form precancerous areas in the lung. We are characterizing these stem cells and identifying pathways involved in normal repair and aberrant repair that leads to lung cancer. We are also isolating this stem cell population and other cell populations from the airway and inducing genetic changes to determine the tumor initiating cell/s for lung cancer. We are also examining the effect the environment may have on the regulation of genes in these stem cells, in precancerous areas and in lung cancers. Finally, we are examining human blood samples to assess levels of surrogate markers of these stem cells to assess whether we could use this as a biomarker to predict the presence or absence of lung cancer in patients with a lung nodule.
  • During this period of funding we discovered a method to reproducibly recover stem cells from human airways and grow them in a dish into mature airway cells. We also discovered the role that certain metabolic cell processes play in regulating the repair after airway injury. We believe that an inability to shut off these processes leads to abnormal repair and lung cancer and are actively investigating this. We are also determining whether the stem cells we isolate from the airways are the stem cells for lung cancer and how they might give rise to lung cancer.
  • In the last year of funding we identified a novel mechanism that tightly controls airway stem cell proliferation for repair after injury. We found that perturbing this pathway results in precancerous lesions that can ultimately lead to lung cancer. Correcting the abnormalities in this pathway that are seen in smokers could allow the development of targeted chemoprevention strategies to prevent the development of precancerous lesions and therefore lung cancer in at risk populations. We also continued our work on trying to identify a cell of origin for squamous lung cancer and identifying the critical drive mutations that are required for squamous lung cancer to develop.

THERAPEUTIC OPPORTUNITIES TO TARGET TUMOR INITIATING CELLS IN SOLID TUMORS

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01477
ICOC Funds Committed: 
$19 979 660
Disease Focus: 
Solid Tumor
Cancer
Collaborative Funder: 
Canada
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Cancer is a major cause of human death worldwide. The vast majority of cancer patients suffer from solid tumors whose growth destroys vital organs. We propose to develop novel therapeutic drugs that target solid tumors affecting the brain, colon and ovaries. These cancers account for a significant proportion of currently intractable solid malignancies. Scientists have made great strides in understanding the molecular and cellular changes that cause cancer but the approval of new therapeutics that can specifically kill cancer cells has lagged behind. This disparity suggests that there must be critical bottlenecks impeding the process of turning a basic research discovery into a finished anti-cancer drug. Research over the past decade has given rise to the idea that one of these bottlenecks may be caused by the existence of cancer stem cells. According to the cancer stem cell hypothesis, there is a minor population of cancer stem cells that drives the growth of the entire tumor. However, cancer stem cells are very rare and hard to identify. Technical innovations have recently allowed the identification, isolation and growth of these cells in the laboratory, and it has become clear that they have properties that are distinct from both the bulk of tumor cells and the cancer cell lines usually used to test anticancer drug candidates. Furthermore, in the lab, cancer stem cells are resistant to the chemotherapy and radiation treatments used to kill most tumor cells. In a patient, cancer stem cells may not be killed by standard drugs and may eventually regrow the tumor, causing a cancer to relapse or spread. Thus, a drug that specifically targets cancer stem cells could dramatically improve the chances of treatment success. Our team is one of the few in the world that can identify cancer stem cells in brain, colon and ovarian tumors. Furthermore, we have developed assays that can accurately test the effectiveness of drug candidates in killing these cells. Our preliminary data suggest that our lead drug candidates can inhibit the growth of cancer stem cells in culture and block tumor initiation in animal models. Importantly, our drug candidates appear to work through mechanisms that are different from those employed by current chemotherapeutics, meaning that our drugs represent a fresh and potentially very effective approach to cancer treatment. Over the next several years, we propose to complete our development and preclinical studies of these drugs so that testing in cancer patients can begin.
Statement of Benefit to California: 
Our proposal may benefit the state of California in four important ways. First, solid tumors cause significant morbidity and mortality. We propose to develop 2-3 Investigational New Drugs (INDs) to treat colon, brain and ovarian tumors, which are often difficult to treat with conventional therapies and are associated with poor prognoses. Thus, the proposed INDs should lead to a decreased burden on the California health system. The second benefit arises from our novel approach to drug development, a route that other researchers may emulate. Most targeted cancer drugs fail in clinical trials, despite our growing knowledge of the molecular and cellular causes of cancer. These failures indicate that there are rate-limiting factors in the way basic research is currently translated to cancer drug discovery and development. One such factor may be related to a major new hypothesis in tumorigenesis, which states that a minor population of cancer initiating cells (CICs) drives bulk tumor growth. These CICs appear to survive existing therapies that kill most tumor cells, and so can go on to initiate relapses and metastases. A second rate-limiting factor may be the heterogeneity that exists both among and within different tumor types. Both of these “bottleneck” factors can be obviated by the molecular characterization and comparison of CICs and bulk tumor cells. Knowing the features that distinguish CICs from bulk tumor cells will facilitate a targeted drug development plan that optimizes chances for clinical success. We have devised such a strategy based on the integration of solutions to these limiting factors into a state-of-the-art drug discovery platform. This strategy may provide a foundation for the rapid extension of our approach to the treatment of other solid tumors. The third benefit is the linking of CIC identification to clinical outcome. The ability to isolate and propagate CICs from solid tumors is a recent innovation. We will perform a thorough genetic examination of the alterations in these cells that lead to oncogenesis. Because we intend to carry out this work in parallel with the characterization of tumor samples from patients with documented clinical outcomes, we will be able to correlate the nature of particular CICs with similarities/differences among human tumors in a way that identifies features statistically linked to poor outcomes. This information will allow the selection and validation of additional drugs so that a pipeline of ever more refined compounds is established even if initial attempts fail in the clinic. The fourth benefit falls directly in line with the focus of California’s robust biotechnology industry on drugs to address unmet medical needs. Our data and methods will be published and readily available, and so can be applied by existing and emerging biotech companies. Great advances in novel targeted therapeutics to treat solid tumors should be realized, expanding the drug development expertise of the state.
Progress Report: 
  • The objective of our collaborative project is the development of therapeutic candidates that will form the basis of IND submissions designed to test a novel class of drugs for the treatment of tumor initiating cells (TICs) in three solid human malignancies where TICs have been implicated in the pathogenesis of disease. The target profile is the TIC population in colon cancer, ovarian cancer and glioblastoma. The therapeutic compounds that have been developed in the course of the collaboration target a pair of serine-threonine kinases that act at the nexus of mitosis, hypoxia, and DNA repair. These enzymes are over-expressed in many forms of cancer and alterations in their expression patterns correlate with dysregulation of a number of genes that are significantly linked to poor patient outcome.
  • Compounds against the first target have been developed to the point at which a developmental candidate can be selected. The compounds show single digit nanomolar potency in vitro, adequate specificity, appropriate pharmacokinetics to support oral delivery, and the ability to trigger growth inhibition and cell death in a wide panel of tumor cell lines and TICs from the three targeted histologies. Recently completed dose and schedule studies have been used to design and implement tumor model studies. The compound that demonstrates the widest therapeutic index will be selected for IND enabling studies. These IND enabling studies will include synthetic scale-up, toxicity evaluations, combination studies, mechanism of action studies, and a biomarker identification program that will be used to identify a targeted population for optimal clinical trial design.
  • The medicinal chemistry program against the second target was started approximately 15 months after the initiation of the effort against the first target. Sufficient potency, specificity, and activity against tumor cell lines and TICs have been demonstrated with novel molecules. Current efforts are focused on improving the pharmacokinetic properties of the drug candidates.
  • A phospho-flow platform to measure mRNA levels, protein levels, and enzymatic activity using a mass spectrometric readout has also been tested. This system enables the simultaneous measurement of up to 35 different biomolecules. A data management system has been developed to facilitate the associated complex data analysis. Proof or principle experiments have demonstrated that this experimental paradigm can be used to reconstruct the developmental lineages of all progeny downstream of hematopoietic stem cells from human and mouse bone marrow. This approach has recently been applied to the analysis of ovarian cancer cells taken directly from patients. The results of these studies suggest that cancer cells are clearly heterogenous, but perhaps most importantly can be organized into developmental lineages that are formally similar to those seen in bone marrow development. Furthermore, this platform can assess the response of individual subcomponents of the oncological lineage to both approved and experimental drugs. We will be using this platform to gain insight into how tumors respond to individual drugs, including our drug candidates, and combination studies. It is reasonable to expect that it will be possible to not only assess the response of the cancer stem cells, but all subtypes of the tumor lineage.
  • Slamon Mak Cancer Stem Cell Abstract
  • Drug discovery programs against two different mitotic kinases are being pursued. Both programs follow the same general process flow in which lead optimization experiments culminate in the selection of a single small molecule candidate for advancement to preclinical development. The development candidate then proceeds through a standard series of evaluations to establish its suitability for an IND submission and use in subsequent clinical trials.
  • CFI-003 was selected as a clinical development candidate and is progressing through investigational new drug application (IND)-enabling studies. Chemistry activity in the past year has included the selection of the fumarate salt as the final salt form, and production of two kilogram-scale clinical batches, the first of which is scheduled to be released at the end of April. The compound is stable when stored under typical storage conditions, and has an impurity profile that is safe for clinical dosing. In cancer models, CFI-003 was shown to be particularly effective against tumors deficient for the tumor suppressor gene PTEN; this is important given that deficiencies in this gene are generally considered to be an indicator of poor prognosis in the clinic. Experiments are ongoing to determine biomarkers of response to CFI-003 for application in the clinic. Other work includes selection and management of contract research organizations (CROs) for critical IND-enabling studies. For example, Pharmatek has been engaged to assist in the development of a drug formulation that enhances the stability of CFI-003, and maximizes bioavailability of the compound when dosed orally. Other CRO work that is ongoing involves in vitro pharmacology experiments geared toward understanding how CFI-003 might interact with co-administered drugs, and performing key toxicology experiments for determination of a safe and effective clinical dose of the compound. An important milestone was reached in the previous reporting period in that the patent application covering CFI-003 was allowed by the US patent office. The CFI-003 IND development team will continue to move the project forward planning for a successful IND submission toward the end of Q1 2013.
  • The drug discovery efforts in the second program have been focused on improving the pharmacokinetic properties of the lead series molecules while maintaining excellent in vitro activity. Approximately 400 new chemical entities have been synthesized during the last reporting period. Progress to date has been measured by an increase in potency in the biochemical assay and improved anti-proliferative potency in cancer cell growth assays. Activity toward Aurora B has simultaneously been attenuated, and current compounds demonstrate improved selectivity against a diverse panel of kinases. Progress was aided by the acquisition of multiple co-complex x-ray structures which allowed for further refinement of binding models to the target’s active site. Compounds to be qualified for further study must continue to induce an aneuploidy phenotype at least an order of magnitude above the HCT116 (colon adenocarcinoma cell line) GI50, and importantly must also demonstrate adequate plasma levels upon oral dosing. A lead series compound has been shown to have oral efficacy in a cancer model. To follow up this result, additional compounds have been scaled up for testing. Experiments to determine the tolerability have been completed for the latest candidates and further efficacy studies have been initiated. Results from these efficacy studies will aid in the identification of a development candidate for subsequent IND enabling studies.
  • Drug discovery programs against two different mitotic kinases are being pursued. Both programs follow the same general process flow in which lead optimization experiments culminate in the selection of a single small molecule candidate for advancement to preclinical development. The development candidate then proceeds through a standard series of evaluations to establish its suitability for an IND submission and use in subsequent clinical trials.
  • CFI-400945 was selected as a clinical development candidate. The IND-enabling studies included the selection of the fumarate salt as the final salt form, and the production of two kilogram-scale clinical batches, which have been released during the past year. The compound is stable when stored under typical storage conditions, and has an impurity profile that is safe for clinical dosing. In cancer models in mice, CFI-400945 was shown to be particularly effective against specific subsets of tumor cell lines in both tumor cells grown in soft agar and in xenograft models. Experiments are ongoing to determine biomarkers of response to CFI-400945 for application in the clinic. Pharmatek was engaged to assist in the development of a drug formulation that enhanced the stability of CFI-400945, and maximized the bioavailability of the compound when dosed orally. Other CRO work that was completed included in vitro pharmacology experiments geared toward understanding how CFI-400945 might interact with co-administered drugs, and performing key toxicology experiments in animals for determination of a safe and effective clinical dose of the compound. This work culminated in an IND submission in the second quarter of 2013.
  • The drug discovery efforts in the second program has focused on improving the pharmacokinetic properties of the lead series molecules while maintaining excellent in vitro activity. Approximately 400 new chemical entities were synthesized and tested using a battery of biochemical and cell-based assays. Off target activity towards Aurora B has simultaneously been attenuated, and current compounds demonstrate improved selectivity against a diverse panel of kinases. Progress was aided by the acquisition of multiple co-complex x-ray structures which allowed for further refinement of binding models to the target’s active site. Compounds were qualified for in vivo study based on the induction of an aneuploid phenotype at an order of magnitude above the HCT116 (colon adenocarcinoma cell line) GI50, and importantly the demonstration high mouse plasma levels upon oral dosing. Mouse xenograft studies based on a number of tumor cell lines were used to select a short list of compounds. The aggregate data was then used to select a developmental candidate CFI-1870. IND enabling studies have been launched. In parallel, detailed dose and schedule studies are underway along with approaches to identify susceptible tumor subpopulations and associated biomarkers that will eventually support a targeted clinical trial.
  • The Slamon/Mak cancer stem cell drug discovery program funded by CIRM/CSCC has achieved two important milestones in the past year. Our first therapeutic candidate was approved by the FDA and first-in-human dosing of CFI-400945 has taken place as part of the Phase I clinical trial. In our second program we have selected a development candidate that is now in the midst of IND ennabling studies
  • The clinical trial is being carried out at Princess Margaret Cancer Centre (Principal Investigator, Dr Philippe Bedard) and UCLA (Principal Investigator, Dr Zev Wainberg). This clinical trial was initiated after a number of milestones were successfully met following the submission of the IND and CTA in 2013. These have included making improvements to the formulation of the CFI-400945 tablets resulting in the successful reduction of the appearance of a degradant that was slowly accumulating in the initial formulation. This enabled the manufacturing of the cGMP tablets for use in the clinic in September 2013. These formulation changes and the Certificates of Analysis of these tablets were submitted to the FDA and permission was granted to begin clinical evaluation. In December 2013, we were awarded the CIRM Disease Team III funding to continue the CFI-400945 program which enabled the planning and initiation of this Phase I clinical evaluation and additional non-clinical studies.
  • In our second program, Pyrazolo-pyrimidines have emerged as the most promising class of 3rd series TTK inhibitors. TTKis with potent in vitro activity, excellent oral exposure in rats and in vivo efficacy were identified. A short list of 5 pyrazolo-pyrimidines was identified as potential third series development candidates. After further characterization it was determined that 4 or 5 compounds met the preponderance of the selection criteria, 2 of which had outstanding PK properties. The TTK inhibitor CFI-402257 had the best balance of efficacy, PK and off-target activities and was selected as the development candidate. IND enabling studies with 402257 have been initiated, and will continue during the no cost extension period of the grant.

A Phase I dose escalation and expansion clinical trial in patients with advanced solid tumors

Funding Type: 
Disease Team Therapy Development III
Grant Number: 
DR3-07067
ICOC Funds Committed: 
$6 924 317
Disease Focus: 
Cancer
oldStatus: 
Closed
Public Abstract: 
Cancer is a major cause of morbidity and mortality worldwide. Many believe that progress in drug development has not been as rapid as one would have predicted based on the significant technological advancements that have led to improved molecular understanding of this disease. There are numerous explanations for the lag in clinical success with new therapeutics. However, work in the past decade has provided support for what has become known as the cancer stem cell hypothesis. This model suggests that there is a class of cells that are the main drivers of tumor growth that are resistant to standard treatments. In one model the cancer stem cells inhabit an anatomical “niche” that prevents drug efficacy. Another view is one in which tumors can achieve resistance by cell fate decisions in which some tumor cells are killed by therapeutics, while other cells avoid this fate by choosing to become cancer stem cells. These stem cells are thought to be capable of both cancer stem cell renewal and repopulation of the tumor. Our proposal aims to conduct a Phase I clinical trial of a first-in-class mitotic inhibitor. The target is a serine/threonine kinase that was originally selected because blocking this target affects both tumor cell lines and tumor initiating cells (TICs). Our data suggest that the target kinase functions at the intersection of mitotic regulation, DNA damage and repair, and cell fate decisions associated with stem cell renewal. Preclinical work has begun to segregate “sensitive” and “resistant” groups of tumor cell lines and TICs after treatment with the drug candidate as a single agent and in combination with standard-of-care therapeutics. Our data also support the model in which cancer stem cell resistance is likely to arise, at least in some cases, due to stem cell fate decisions that happen in response to therapeutic intervention. This grant is a natural progression of work partially funded by CIRM that enabled the isolation of Tumor Initiating Cells (TICs)from tumors in different tissue types. This facilitated the development and assessment of drug candidates that target both bulk tumor cells and TICs and has now led to the development of a potential anti-cancer drug which we are now preparing to test in humans. The goal of the Phase I trial is to determine the maximum tolerated dose, the recommended Phase II dose, and any dose-limiting toxicities. We will also characterize safety, pharmacokinetic, and pharmacodynamic profiles along with any antitumor activity. Once the maximum tolerated dose has been identified, a biomarker expansion cohort will be opened in order to determine whether appropriately selected biomarkers are associated with a predictable patient response. This will allow a rational approach to study single agent and combination studies that perturb this network and allow us the opportunity to facilitate a targeted clinical development plan.
Statement of Benefit to California: 
It has been estimated, by the California Department of Public Health, that in 2013 about 145,000 Californians will be diagnosed with cancer and more than 55,000 of these will ultimately succumb to their disease. Furthermore, more than 1.3 million Californians are living today with a history of cancer. Therefore, innovative research programs that are able to impact this devastating disease burden are likely to have a large potential benefit to the state of California and its residents. This grant application proposes a Phase I clinical trial for a first-in-class inhibitor of a target that has never been tested in patients. The aim of this trial is to determine the maximum tolerated dose in humans, the recommended dose for phase II trials, and evaluate any dose-limiting toxicities. The trial will also characterize safety, pharmacokinetics, and pharmacodynamic properties. It will also provide early insight into any antitumor activity. Our group has developed a comprehensive unbiased platform that facilitates the segregation of sensitive and resistant populations of cancer based on their molecular subtypes. This capability has the promise to improve the success rate and reduce the cost of oncology clinical trials by focusing on the subsets that are most likely to benefit while avoiding unnecessarily treating patients that would otherwise benefit from alternative treatments. Our preliminary pre-clinical work, funded by CIRM in the context of a Disease Team I award, suggests that this approach can be successfully applied to the networks associated with mitotic regulation, DNA repair, and stem-cell fate decisions. Our ongoing research has tested a number of chemical compounds that are able to block pathways that are critical to the growth and proliferation of many cancer models. These compounds have all been tested in multiple in vitro and in vivo systems and have been found to inhibit the ability of the cancer stem cell to repopulate. Now that our pre-clinical enabling studies have been completed, we have submitted an Investigational New Drug (IND) application to the FDA for a first-in-human phase I clinical trial. In the current proposal, we will be able to test our hypotheses in a clinical setting, which if successful will lead to confirmation of safety and the establishment of the appropriate dose with which to test in later stage trials. This trial will set the stage for a new class of agents that has not yet been tested in clinical settings. We believe that the proposal described herein has the promise to expand the reach of targeted therapies into mechanisms that in most cases have been resistant to innovation. Finally, it is reasonable to expect that our preclinical work and the proposed clinical trials will validate a number of potential biomarkers that will identify susceptible patient subpopulations.

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.

Pages

Subscribe to RSS - Cancer

© 2013 California Institute for Regenerative Medicine