Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Development of Therapeutic Antibodies Targeting Human Acute Myeloid Leukemia Stem Cells

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

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.

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

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

Stem Cell-Mediated Oncocidal Gene Therapy of Glioblastoma (GBM)

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01426
ICOC Funds Committed: 
$19 162 435
Disease Focus: 
Brain Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
Brain tumors (BTs) are incurable, whether they start in the brain or spread there from other sites. Despite advances in surgical, radiation, pharmacologic, and gene therapies, survival with a BT remains dismal. Current therapies are limited by their inability to reach widely disseminated tumor cells that become dispersed within normal brain structures. Interestingly, the therapeutic property that is needed to overcome this major obstacle to effective treatment of BTs matches well with one of the better accepted attributes of neural stem cells (NSCs): an attraction for sites of pathology in the adult brain, including primary & metastatic cancer. If armed with a proper tumor-killing gene, NSCs (whether administered into the brain or into the bloodstream), that are drawn to cancers, will dramatically reduce tumor burden, and will track after even single migrating tumor cells. The NSCs perform this action without themselves becoming tumorigenic or augmenting the pre-existing tumor, and this can be assured by having NSCs express a suicide gene that can be activated and cause NSCs to die. The tumor homing phenomenon of NSCs was first revealed by researchers on this proposed team and, in fact, the central concepts presented here have since been extended to many other kinds of disease. In this proposal, we will use a number of authentic mouse models of primary BTs to pre-clinically test therapeutic NSCs. Human NSCs (hNSCs) will be derived from 3 distinct sources, with each having been proffered as therapeutic, but never having been compared head-to-head in treating tumors. Each of these hNSCs will be modified using two therapeutic genes: TRAIL, which is a protein that specifically kills tumor cells, but does not harm normal cells and tissues, and cytosine deaminase which converts a non-toxic chemical into a toxic chemotherapeutic. We expect our research to identify the best hNSC + therapeutic gene combination to advance for clinical trial in patients with BTs, following our obtaining regulatory approval for using hNSC therapy at the end of this project. Because immunocompatibility of the hNSCs with recipient patients is not a concern in BT therapy, a limited number of hNSC lines can be used for treating all prospective patients. Furthermore, BT treatment does not require long-term NSC survival and can be combined with commonly used BT therapies. Finally, NSCs can be imaged in patients and therefore monitored after administration. Developing this approach for treatment of BT patients offers an ideal setting and opportunity for achieving dramatic results from stem cell therapy, and the results of this project will likely be applicable to the treatment of other cancers.
Statement of Benefit to California: 
Brain tumors (BTs) are incurable, whether they start in the brain or spread there from other sites. Despite advances in surgical, radiation, drug, & gene therapies, survival with a BT is extremely short, because current therapies are limited by their inability to reach tumor cells that spread widely to normal brain structures. Interestingly, the therapeutic property that is needed to overcome this major treatment obstacle matches well with one of the better accepted attributes of neural stem cells (NSCs): an attraction for sites of disease in the adult brain, including primary & metastatic cancer. If engineered to be armed with a tumor-killing gene, NSCs (whether administered into the brain or into the bloodstream), that are attracted to cancers, could dramatically reduce patient tumor burden, and track after even single migrating tumor cells, in a manner that has never been achieved. The NSCs would perform this action without themselves causing tumors or increasing growth of the patient’s tumor, and this would be assured by engineering the NSCs to self-destruct. The tumor homing phenomenon of NSCs was first revealed by researchers on this proposed team and, in fact, the central concepts presented here have since been extended to many other kinds of disease. In this proposal, we will use a number of authentic mouse models of primary BTs to test therapeutic NSCs before testing them in humans. Human NSCs (hNSCs) will be derived from 3 distinct sources, with each having been proposed as therapeutic, but never having been compared head-to-head in treating cancer. Each of these stem cells will be modified using two different therapeutic genes: TRAIL, a protein that specifically kills tumor cells, but does not harm normal cells and tissues, and cytosine deaminase, which converts a non-toxic chemical into a chemotherapy drug that kills the tumor. We expect our research to identify the best hNSC + therapeutic gene combination to advance for evaluation in clinical trials in patients with intracranial BTs, after we have performed all necessary animal safety testing and submitted a complete plan for review by the US FDA and NIH. Members of this proposed team have experience in bringing cancer therapies to clinical trial, hold the IP surrounding the use of stem cells against cancer, have begun discussions with the FDA and NIH, and have enlisted a GMP facility. Because immune system compatibility between donor and recipient of the hNSCs with the recipient is not a concern in BT therapy, a small number of donors could be used to produce genetically modified hNSCs to treat all prospective patients. Developing this approach for treatment of BTs offers an ideal setting and opportunity for achieving dramatic results from stem cell therapy, and accomplishing substantial improvements in quantity and quality of life for BT patients would no doubt increase California's worldwide visibility in offering the best possible medical care for cancer patients.
Progress Report: 
  • During the first year of this project we have made substantial progress toward achieving the ultimate goal of developing a stem cell (SC) therapy for treating patients with recurrent glioblastoma (GBM). At the outset, we began investigating three SC candidates as the cellular vehicle to carry a therapeutic payload and disperse within the tumor of GBM patients: mesenchymal stem cells (MSCs); fetal neural stem cells (fNSCs); and adult neural stem cells (aNSCs). In addition, we were considering two therapeutic genes as the payload, cytosine deaminase (CD) and tumor necrosis factor related apoptosis-inducing ligand (TRAIL), and two routes of therapeutic SC administration for treating brain tumor patients, intravascular and direct intratumoral. Thus, at the start of the project, there were twelve possibilities (3 stem cell vehicles x 2 therapeutic genes x 2 routes of administration) to investigate and compare prior to determining the best combination to develop for a GBM clinical trial. From this starting point we have been able to rapidly eliminate the aNSCs from consideration due to their slow rate of proliferation that would limit their expansion to sufficient number for use in a clinical product for patients. Next we determined that SC access to intracranial tumor through intravascular injection was negligible, and that it is highly unlikely that SC administration by this route would result in a sufficient number of SCs reaching intracranial tumor for achieving therapeutic benefit in treating patients with recurrent GBM. Thus, our work to date has resulted in the narrowing options for SC + therapeutic gene + route of delivery to four: two cellular vehicle candidates (fNSCs and MSCs) and two therapeutic gene payloads (CD and TRAIL). During the first year of this project, each of the four combinations has been tested and have demonstrated anti-tumor activity. During early year 2 research we will determine the most effective combination based on preclinical testing results using multiple human GBM models. The decision regarding most effective therapeutic gene + stem cell vehicle will be achieved within six months, and from that point, in going forward, project emphasis will focus on the development of a specific product candidate, including manufacturing process and assay development, GLP/GMP product manufacturing, further preclinical animal studies to demonstrate efficacy and safety, and development of a clinical protocol. In association with the research accomplished to date we have developed and applied several approaches that will prove useful for SC research and clinical application in general. Foremost among these is the use of micron-sized particles of iron oxide (MPIOs) for labeling SCs prior to their injection into animal subjects, and then monitoring the movement of labeled SCs using magnetic resonance imaging (MRI). This is a powerful technique with implications for understanding the distribution and persistence of SCs in patients receiving SC therapies. For our project, this method is informing us about the distribution of labeled SCs within and around brain tumors, as well as with regard to how long the SCs remain in animal subjects. In addition to the MRI detection of iron particle labeled SCs, we have developed and refined a technique for determining the amount of human SC DNA in animal subject tissues, which has a sensitivity of detecting one human cell among more than a million host cells. Similar to the MRI detection of labeled SCs, the DNA detection method provides us a very sensitive approach for monitoring SC biodistribution and persistence in animal subjects, and it is broadly applicable to all SC research in which rodent models are used for pre-clinical investigation of SCs for treating disease. We are also developing novel approaches for the use of optical imaging to visualize stem cells labeled with fluorescent reporters, and for monitoring the anti-tumor activity of therapeutic stem cells administered to animal subjects. These novel approaches are contributing to the repertoire of techniques available to expedite the identification and application of therapeutic SCs in clinical settings. This project is a collaboration among outstanding scientists and clinicians at five of California’s leading medical research institutions: the Sanford-Burnham and Salk Institutes in La Jolla, and the San Francisco, Los Angeles, and San Diego campuses of the University of California (including Ludwig Institute at UCSD). By leveraging complementary expertise of these investigators, we have made rapid progress in the preclinical animal studies, design of the clinical trial protocol, and the product development studies that will lead to preparation of a gene-modified SC product for the clinical trial. These activities will culminate in an IND application to FDA that will allow us to test the safety and efficacy of our SC product in patients with this devastating illness.
  • This project was initiated in April of 2010, and was for comparing
  • • three types of stem cells
  • • two distinct therapeutic gene modifications of stem cells, and
  • • intravascular administration vs. direct tumor injection of stem cells
  • in order to identify the most efficacious stem cell + therapeutic gene + route of administration for treating patients with recurrent glioblastoma (GBM), a brain tumor that has a dismal prognosis, and that badly needs innovative approaches for improving treatment outcomes.
  • Major conclusions from this project, as concerns the objectives indicated above, are:
  • 1. Stem cells administered by the vascular route do not reach brain tumors established in rodent subjects, to an extent which demonstrable therapeutic stem cell anti-tumor activity should be anticipated. In most instances, intravascular administration results in no detectable stem cells in intracranial tumor in rodent models. Therefore, therapeutic stem cells need to be administered direct into brain tumors in order to achieve a sufficient number and concentration of stem cells for observing anti-tumor effect.
  • 2. Neural stem cells and mesenchymal stem cells delivered directly into intracranial tumor display similar extents of dispersion in tumor, indicating these stem cell types should perform comparably as concerns their ability to disseminate within, and deliver therapy to tumor.
  • 3. However, unmodified (non-immortalized) neural stem cells, derived from single adult or fetal sources, have insufficient proliferative capacity for production as therapeutic stem cells to be used in clinical trials that enroll multiple patients. Because of the ready availability of mesenchymal stem cells (MSCs), from many donors, combined with the proliferative capacity of MSCs, MSCs were determined as the preferred candidate for developing therapeutic stem cells to treat patients with recurrent GBM.
  • 4. Studies conducted with therapeutic stem cell + tumor cell mixtures indicated superior anti-tumor activity of cytosine deaminase modified stem cells + 5-fluorocytosine (FC), relative to secretable TRAIL modified stem cells, when anti-tumor activity is examined in liquid media (cell culture). The two types of therapeutic stem cells showed comparable anti-tumor activities when administered directly into brain tumor in animal (rodent) subjects.
  • 5. In relation to other types of therapies (e.g., chemotherapeutics, antibodies, liposomal drugs) being tested by members of this disease team, manufactured therapeutic stem cells displayed low (modest) anti-tumor activity in animal subjects with brain tumor.
  • Technical advances, discovery, and products developed in association this project, and that can be shared/transferred in support of other CIRM funded research, include:
  • • Development of approaches for delivering stem cells through distinct routes of administration in rodent subjects.
  • • Development of a method, based on the use of polymerase chain reaction, for detecting human cells in rodent tissues, with a sensitivity of detection of one human cell per 100,000 mouse cells.
  • • Development of a cell labeling approach that enables tracking of stem cell migration in rodent subjects.
  • • Development of a histochemical method for detection of labeled human cells in rodent tissues.
  • • Development and characterization of multiple, tumorigenic human glioblastoma xenograft models for use in therapeutic testing.

Stem Cell-mediated Therapy for High-grade Glioma: Toward Phase I-II Clinical Trials

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01421
ICOC Funds Committed: 
$18 015 429
Disease Focus: 
Brain Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Despite aggressive multimodal therapy and advances in imaging, surgical and radiation techniques, malignant brain tumors (high-grade gliomas) remain incurable, with survival often measured in months. Treatment failure is largely attributable to the diffuse and invasive nature of these brain tumor cells, ineffective delivery of chemotherapeutic agents to tumor sites, and toxic side-effects to the body, which limits the dose of drug that can be given. Therefore, new tumor-selective therapies are critically needed. Neural stem cells (NSCs) offer an unprecedented advantage over conventional treatment approaches because of their unique ability to target tumor cells throughout the brain. This ability allows NSCs to be used to deliver prodrug-activating enzymes to tumors, where these enzymes will generate high concentrations of powerful anti-cancer agents selectively at tumor sites. We will use an established human NSC line to develop a novel NSC-based product to deliver the enzyme carboxylesterase (CE), which will activate a systemically administered prodrug, CPT-11, to a powerful chemotherapeutic agent, SN-38, selectively at tumor sites, destroying invasive glioma cells while sparing normal tissues. Based on our preliminary data, we hypothesize that CE-expressing NSCs will home to tumor sites in the brain, and, in combination with CPT-11, will generate high concentrations of SN-38 specifically at tumor sites. Thus, in addition to potentially improving lifespan by concentrating the powerful chemotherapeutic agent selectively at tumor sites, this NSC-mediated treatment strategy should significantly decrease toxic side-effects to normal tissues, thus preserving or improving the patient’s quality of life. Our research, regulatory and clinical teams have the collective expertise and experience to conduct the preclinical studies necessary to optimize the efficacy of this innovative treatment approach. Specifically, we will determine the optimal dose and route of NSC administration; the optimal prodrug dosing regimen; and assess the safety of this treatment approach. We will perform these studies and analyses, generate clinical grade products, and file and obtain all appropriate regulatory documents and approvals. Completion of these activities will lead to the filing of a new Investigational New Drug (IND) proposal to the FDA, for a first-in-human Phase I clinical trial of this pioneering NSC-mediated treatment in patients with recurrent high-grade gliomas. Importantly, our NSC line can be further modified for tumor-localized delivery of a variety of therapeutic agents, and can be given serially or in combination to maximize therapeutic benefit. Thus, the potential medical impact of this innovative NSC-mediated therapeutic approach may be very far-reaching, as it can be developed for application to other types of malignant brain tumors, as well as for metastatic cancers.
Statement of Benefit to California: 
Despite aggressive multimodality therapy and advances in imaging, surgical and radiation techniques, high-grade gliomas remain incurable, with survival often measured in months. Approximately, 22,500 malignant brain tumors are diagnosed annually in the U.S., of which more than 2,600 cases are in California. New therapies are desperately needed to improve both the survival and quality of life of these brain tumor patients and to reduce the economic impact of billions of dollars in related healthcare costs. We propose to develop a novel neural stem cell (NSC)-based treatment method that will selectively target glioma cells with a potent chemotherapy agent, locally activated by the NSCs at tumor sites to destroy neighboring tumor cells. Our tumor-selective approach also has the advantage of minimizing toxicity to normal tissues, thereby decreasing systemic side effects and damage to normal brain. This new therapeutic strategy, therefore, not only has the potential to improve survival, but, by preserving cognitive function and quality of life, it could also enable adult Californians diagnosed with brain tumors to continue making societal contributions that would benefit all Californians. Important for clinical translation of this novel therapeutic approach, we have established the NSC line to be used in this study as a fully characterized cGMP Master Cell Bank. The NSC line is thus expandable, easily distributed to other medical centers, and cost-effective, which will allow this therapeutic approach to be quickly adopted. Importantly, this NSC line can be further modified for tumor-localized delivery of a variety of therapeutic agents, which may be given serially or in combination to maximize therapeutic benefit. There is tremendous potential for developing NSC-mediated treatment applications for other types of malignant brain tumors, as well as for metastatic solid tumors throughout the body. Therefore, the impact of these proposed studies to advance NSC-mediated treatment of glioma may be very far-reaching and may significantly contribute to reducing healthcare costs. Finally, the combined strengths and experience of our research team will enable us to advance this NSC-meditated therapeutic approach in a timely, streamlined, and cost-effective manner to submit a new IND application for initiating first-in-human clinical trials in California, providing benefit to state taxpayers by efficient use of tax dollars and initial access to this novel therapy. In addition, our CIRM Disease Team NSC-mediated cancer treatment studies would stimulate and advance collaborative partnerships and alliances with other cancer centers and affiliates, pharmaceutical companies, academic institutions, and philanthropic societies within California, which would further enhance local and state economies.
Progress Report: 
  • Primary brain tumors are among the most difficult cancers to treat. High-grade gliomas, the most common primary brain tumors in adults, remain incurable with current therapies. These devastating tumors present significant treatment challenges for several reasons: 1) surgical removal runs the risk of causing permanent neurologic damage and does not eliminate cancer cells that have migrated throughout the brain; 2) most anti-cancer drugs are prevented from entering the brain because of the presence of the blood-brain barrier, which often does not allow enough chemotherapy into the brain to kill the cancer cells; and 3) typically, the amount of chemotherapy that can be given to cancer patients is limited by intolerable or harmful side effects from these agents. If concentrated cancer therapies could be specifically localized to sites of tumor, damage to healthy tissues would be avoided.
  • The long-range goal of this research project is to develop a neural stem cell (NSC)-based treatment strategy that produces a potent, localized anti-tumor effect while minimizing toxic side effects. NSCs hold the promise of improved treatment for brain cancers because they have the natural ability to distribute themselves within a tumor, as well as seek out other sites of tumor in the brain. Because they can home to the tumor cells, NSCs may offer a new way to bring more chemotherapy selectively to brain tumor sites. After modifying the NSCs by transferring a therapeutic gene into them, NSCs can serve as vehicles to deliver anti-cancer treatment directly to the primary tumor, as well as potentially to malignant cells that have spread away from the original tumor site. With funding from CIRM, we are studying the ability of NSCs, that carry an activating protein called carboxylesterase (CE) to convert the chemotherapy agent CPT-11 (irinotecan) to its more potent form, SN-38, at sites of tumor in the brain.
  • During the first year of funding we have determined that 1) when administered directly into the brain or into a peripheral vein (intravenous injection) of mice with brain tumors, NSCs will travel to several different subtypes of gliomas; 2) we can engineer the NSCs to consistently produce high levels of more powerful forms of CE: rCE and hCE1m6; 3) glioma cells die when they are exposed to very low (nanomolar) concentrations of SN-38, and 4) although glioma cells survive when exposed to a relatively high concentration of CPT-11 alone, they do die when the same concentration of CPT-11 is administered in combination with either rCE or hCE1m6. These results suggest that the engineered NSCs are expressing relatively high levels of CE enzymes and that the CE enzymes are converting CPT-11 into SN-38. We have also been able to label our NSCs with iron particles, so that we can track their movement in real-time by magnetic resonance imaging (MRI), and follow their location and distribution in relation to the tumor.
  • All of our data thus far support the original hypothesis that effective, tumor-specific therapy for glioma patients can be developed using NSCs that express rCE or hCE1 and the prodrug CPT-11. During the second year of CIRM funding, we will further analyze our data to make a final determination regarding the best form of CE to develop towards clinical trials, and the best dose range and route of delivery of NSCs to achieve maximal tumor coverage. We will then begin our therapeutic studies and start discussions with the Food and Drug Administration, to define the safety studies necessary to obtain approval for testing this new treatment strategy in patients with brain tumors.
  • High-grade gliomas, the most common primary brain tumors in adults, have a poor prognosis and remain incurable with current therapies. These devastating tumors present significant treatment challenges: 1) surgery may cause permanent neurologic damage; 2) surgery misses cancer cells that have invaded beyond the edge of the tumor or to other sites in the brain; 3) many, if not most, chemotherapy drugs cannot enter the brain because of the blood-brain barrier; and 4) due to the highly toxic nature of chemotherapy agents the therapeutic window (the difference between the dose that kills the tumor and the dose that causes toxic side effects) is very small, resulting in undesirable side-effects. Therefore, if therapeutic agents could be localized and concentrated selectively to the tumor sites, treatment efficacy may be improved while toxic side effects are minimized.
  • The overarching goal of this project is to develop a human Neural Stem Cell (NSC)-based treatment strategy that produces potent localized anti-tumor effects while minimizing toxic side effects. NSCs hold the promise of improved treatment for brain cancers because they have an innate ability to distribute within and around a tumor mass and to seek out tumor cells that have invaded further into surrounding brain tissue. By homing to cancer cells, NSCs offer a way to selectively deliver concentrated chemotherapy to brain tumor sites. We are modifying NSCs to make the protein carboxylesterase (CE), which will convert a systemically administered prodrug, CPT-11 (irinotecan) to an active, potent anti-cancer drug, SN38 at the tumor sites.
  • Our second year of funding was highly productive and informative. We validated key elements of our system, successfully negotiating Go/No Go milestones, yielding substantial progress:
  • (1) We have selected the optimal genetically modified human CE to efficiently convert CPT-11 to SN-38. This CE is being developed for clinical grade use.
  • (2) We have determined the volume of tumor coverage by NSCs injected directly into the brain versus injecting them intravenously. We found that we achieve more tumor coverage with direct injection of the NSCs into the brain, and will focus on developing this approach for initial NSC.CE/CPT-11 clinical trials. However, following intravenous injections we found the NSCs localize prominently at the invasive tumor edges, which may prove therapeutically efficacious as well. Due to the significant clinical and commercial advantages that intravenous administration presents, this approach will also be developed toward patient trials. We have determined the starting NSC dose range for both approaches.
  • (3) We have shown that CPT-11 + CE is1,000 fold more toxic to glioma cells than CPT-11 alone. Importantly, microdialysis studies in our preclinical models have confirmed the conversion of CPT-11 to SN-38 by our CE-secreting NSCs in the brain.
  • (4) We have completed studies labeling our NSCs with iron (Feraheme) nanoparticles, which allows for non-invasive cell tracking by Magnetic Resonance Imaging (MRI). Safety studies for clinical use of this iron-labeling method were completed and submitted to the FDA, for consideration of use in brain tumor patients enrolled in our current NSC.CD/5-FC recurrent glioma clinical trial. This would be the first-in-human use of Feraheme-labeled stem cells for MRI tracking.
  • Our results to date robustly support the original hypothesis that an effective, glioma-specific therapy can be developed using NSCs that home to tumors and express CE to convert CPT-11 to the potent anti-cancer agent SN-38. Pre-clinical therapeutic efficacy studies to optimize CPT-11 regimens are now in progress.
  • High-grade gliomas, the most common primary brain tumors in adults, have a poor prognosis and remain incurable with current therapies. These devastating tumors present significant treatment challenges; 1) surgery may cause permanent neurologic damage; 2) surgery misses cancer cells that have invaded beyond the edge of the tumor or disseminated to other sites in the brain; 3) many, if not most, chemotherapy drugs cannot enter the brain because of the blood-brain barrier; and 4) due to the highly toxic nature of chemotherapy agents the therapeutic window (the difference between the dose that kills the tumor and the dose that causes toxic side effects) is very small. Therefore, if therapeutic agents could be concentrated and localized to the tumor sites, treatment efficacy may be improved while toxic side effects are minimized.
  • The overarching goal of this project is to develop a human Neural Stem Cell (NSC)-based treatment strategy that produces potent localized anti-tumor effects while minimizing toxic side effects. NSCs hold the promise of improved treatment for brain cancers because they have an innate ability to distribute within and around a tumor mass and to seek out other, secondary and smaller tumor nodules in the brain. By homing to cancer cells, NSCs offer a way to selectively deliver concentrated chemotherapy to brain tumor sites. After modifying NSCs by adding the gene to make the protein carboxylesterase (CE), NSCs deliver CE to convert the drug CPT-11 (irinotecan) to its more potent form, SN-38 at primary and secondary brain tumor sites.
  • The major milestone in our third year of funding was that we completed our pre-IND package and held our pre-IND meeting with the FDA. To this end, we validated the following:
  • (1) NSCs can potentiate the in vivo efficacy of irinotecan (CPT-11) using a low dose (7.5 mg/kg) daily x 5 schedule. Both real time Xenogen and integrated morphometric analysis of immunohistochemically stained sections of tumor were used to determine tumor volumes.
  • (2) In vivo pharmacokinetics demonstrated increased accumulation of SN-38 in tumor over that of tumor interstitium. The concentrations of tumor SN-38 were approximately 3-fold higher in tumor-bearing brain tissue than in corresponding normal tissue supporting the hypothesis that NSCs can direct toxic chemotherapy in a tumor localized manner.
  • (3) Following FDA approval of the incorporation of iron (Feraheme) into NSCs, three patients were treated with FeHe-labeled HB1.F3.CD, the first generation NSCs undergoing clinical trial. There were no adverse effects from the treatment demonstrating relative safety and lack of toxicity of this method.
  • Our results to date robustly support the original hypothesis that an effective, glioma-specific therapy can be developed using NSCs that home to tumors and express CE to convert CPT-11 to SN-38. During the fourth and coming year of CIRM funding, we will conduct experiments to determine the optimal schedule for NSC/CPT-11 therapy and demonstrate the safety and lack of toxicity of the treatment schema in rodents to fulfill requirements for IND submission and clinical trial in humans.
  • High-grade gliomas, the most common primary brain tumors in adults, have a poor prognosis and remain incurable with current therapies. These devastating tumors present significant treatment challenges; 1) surgery may cause permanent neurologic damage; 2) surgery misses cancer cells that have invaded beyond the tumor edge to other sites in the brain; 3) many, if not most, chemotherapy drugs cannot enter the brain because of the blood-brain barrier; and 4) chemotherapy drugs are toxic to normal tissues as well as tumor, causing undesirable side effects. Therefore, if therapeutic agents could be concentrated and localized to the tumor sites, treatment efficacy may improve while side effects are minimized.
  • Our goal is to bring to the clinic a human Neural Stem Cell (NSC)-based treatment strategy that produces potent localized anti-tumor effects while minimizing toxic side effects. NSCs have a natural ability to home to invasive brain tumor cells throughout the brain. NSCs, used as a delivery vehicle, offer a novel way to selectively target chemotherapy to brain tumor sites. NSCs are modified to express a certain enzyme (carboxylesterase; CE), that converts systemically administered prodrug (irinotecan) to a much more potent form (SN-38), that is up to 1000 times more effective at killing brain tumor cells.
  • Milestones reached in our fourth year include:
  • (1) receiving regulatory approval from the NIH/OBA following a public form in September, 2013.
  • (2) determining the dose and timing of NSC and irinotecan administration for optimal therapeutic efficacy in pre-clinical brain tumor models.
  • (3) demonstrating that the CE-expressing NSCs can increase concentrations of the toxic drug SN-38 by > 6-fold compared to giving irinotecan alone. Furthermore, SN-38 concentrations were dose proportional to administered irinotecan concentrations.
  • (4) Safety-toxicity studies required by the FDA for Investigational New Drug (IND) approval were completed. These studies demonstrated no significant toxicities and safety of our NSC treatment protocol in preclinical brain tumor models.
  • Our results to date support our hypothesis that a safe and effective NSC-mediated therapy can be developed for clinical use in patients with high-grade glioma, with potential application to other types of brain tumor and brain tumor metastases. We hope to initiate clinical trials with our CE-expressing NSCs and irinotecan by the end of 2014.

White matter neuroregeneration after chemotherapy: stem cell therapy for “chemobrain”

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06510
ICOC Funds Committed: 
$2 800 536
Disease Focus: 
Neurological Disorders
Brain Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Chemotherapy for cancer is often life saving, but it also causes a debilitating syndrome of impaired cognition characterized by deficits in attention, concentration, information processing speed, multitasking and memory. As a result, many cancer survivors find themselves unable to return to work or function in their lives as they had before their cancer therapy. These cognitive deficits, colloquially known as "chemobrain" or "chemofog," are long-lasting and sometimes irreversible. For example, breast cancer survivors treated with chemotherapy suffer from cognitive disability even 20 years later. These cognitive problems occur because chemotherapy damages the neural stem and precursor cells necessary for the health of the brain's infrastructure, called white matter. We have discovered a powerful way to recruit the stem/precursor cells required for white matter repair that depends on an interaction between the electrical cells of the brain, neurons, and these white matter stem/precursor cells. In this project, we will determine the key molecules responsible for the regenerative influence of neurons on these white matter stem cells and will develop that molecule (or molecules) into a drug to treat chemotherapy-induced cognitive dysfunction. If successful, this will result in the first effective treatment for a disease that affects at least a million cancer survivors in California.
Statement of Benefit to California: 
Approximately 100,000 Californians are diagnosed with cancer each year, and the majority of these people require chemotherapy. While cancer chemotherapy is often life saving, it also causes a debilitating neurocognitive syndrome characterized by impaired attention, concentration, information processing speed, multitasking and memory. As a result, many cancer survivors find themselves unable to return to work or function in their lives as they had before their cancer therapy. These cognitive deficits, colloquially known as "chemobrain" or "chemofog" are long-lasting; for example, cognitive deficits have been demonstrated in breast cancer survivors treated with chemotherapy even 20 years later. With increasing cancer survival rates, the number of people living with cognitive disability from chemotherapy is growing and includes well over a million Californians. Presently, there is no known therapy for chemotherapy-induced cognitive decline, and physicians can only offer symptomatic treatment with medications such as psychostimulants. The underlying cause of "chemobrain" is damage to neural stem and precursor cell populations. The proposed project may result in an effective regenerative strategy to restore damaged neural precursor cell populations and ameliorate or cure the cognitive syndrome caused by chemotherapy. The benefit to California in terms of improved quality of life for cancer survivors and restored occupational productivity would be immeasurable.
Progress Report: 
  • Cancer chemotherapy can be lifesaving but frequently results in long-term cognitive deficits. This project seeks to establish a regenerative strategy for chemotherapy-induced cognitive dysfunction by harnessing the potential of the interactions between active neurons and glial precursor cells that promote myelin plasticity in the healthy brain. In the first year of this award, we have made on-track progress towards establishing a working experimental model system of chemotherapy-induced neurotoxicity that faithfully models the human disease both in terms of the cellular damage as well as functional deficits in cognition. We have also been able to identify several therapeutic candidate molecules that we will be studying in the coming years of the project to ascertain which of these candidates are sufficient to promote OPC population repletion and neuro-regeneration after chemotherapy exposure.

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.

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.

Mechanisms of Hematopoietic stem cell Specification and Self-Renewal

Funding Type: 
New Faculty I
Grant Number: 
RN1-00557
ICOC Funds Committed: 
$2 286 900
Disease Focus: 
Blood Cancer
Cancer
Anemia
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
During an individual’s lifetime, blood-forming cells in the bone marrow called hematopoietic stem cells (HSCs) supply all the red and white blood cells needed to sustain life. These blood stem cells are unique because they can make an identical copy of themselves (self-renew). Disorders of the blood system can be terminal, but such diseases may be cured when patients are treated with a bone marrow transplant. Unfortunately, bone marrow is in short supply due to limited availability of donors, and it is not yet possible to expand HSCs outside of the human body; HSCs that are removed from their native environment, or niche, rapidly lose their ability to self-renew and thus cannot sustain hematopoiesis in a transplant recipient. Furthermore, attempts to make blood stem cells from embryonic stem cells (ESCs) have also proved unsuccessful to date because these “tailored HSCs” are defective in self-renewal as well. These problems suggest that our understanding of the biology of HSCs is not sufficient to foster their maintenance or generation. To address this issue, we propose to study hematopoietic stem cells in the context of mammalian development; the entire complement of a person’s HSCs is made in a very short time window during the first trimester of pregnancy. By increasing our understanding of how HSCs are made and acquire self-renewal in vivo, we hope to develop better methods of generating HSCs in vitro and learn to provide the missing cues to coax them into becoming fully functional, self-renewing hematopoietic stem cells. Specifically, we plan to investigate how the fate decision that delineates blood cells from their embryonic precursor, called specification, is maintained at the molecular level. Second, we are interested in what cell type human HSCs descend from so as to understand what precursor to look for when attempting to differentiate ESCs into blood stem cells. Finally, we plan to apply molecular analyses to the property of self-renewal by looking at cell populations that cover a spectrum with regards to self-renewal: HSCs, cultured HSCs (not self-renewing), HSC precursors (not self-renewing), and ESCs differentiated to non-self-renewing HSCs. These comparisons will help define the molecular regulation of self-renewal, and place ESC-derived progenitors on the spectrum of self-renewal. Through these studies, we hope to better understand blood stem cells as they are made and maintained during human development with the ultimate goal to provide wider access to stem cell-based therapies.
Statement of Benefit to California: 
Funding of research to understand hematopoietic stem cell (HSC) biology offers rewards beyond the pursuit of knowledge. HSCs are responsible for providing all of the blood cells in the body, including both red cells that carry oxygen and white cells that mediate immunity. Inherited disorders affecting HSCs and their progeny are responsible for diseases such as sickle cell anemia, Severe Combined Immunity Disorder (SCID), and leukemia; these devastating ailments change the lives of thousands of people in California every year, and currently most are incurable without a bone marrow or cord blood transplant. Due to the limited availability of donors, other alternatives, such as differentiating embryonic stem cells (ESCs) into HSCs, are being explored. One critical fault of ESC-derived progenitors is their inability to “self-renew”, i.e. produce more of themselves, thus eliminating their usefulness for transplantation. However, a deeper understanding of the developmental and molecular processes that create functional HSCs that can self-renew may ultimately make the goal of deriving HSCs from ESCs attainable. Research into the mechanisms of self-renewal may also improve treatments of cancers such as leukemia, as these diseases are a function of over-proliferation of cells caused by uncontrolled self-renewal; targeting genes or proteins involved in abnormal self-renewal programs may provide more specific cancer fighting drugs, and would likely foster collaborations with biotechnology companies. Furthermore, as all stem cells in the body have the ability to self-renew, a clear understanding of self-renewal mechanisms will benefit all stem cell research, and could have a positive effect in a wide range of biomedical specialties.
Progress Report: 
  • The goal of this grant is to investigate the cell intrinsic mechanisms that govern hematopoietic stem cell specification and self-renewal. During the second year of this award, we have further elucidated the regulatory mechanisms that dictate hematopoietic fate specification by validating the target genes that Scl/tal1 activates and represses in vivo (Aim 1). We have also shown that loss of Scl results not only results in loss of all blood cells, but also causes defective arterio-venous identity that precludes generation of hemogenic endothelium and hematopoietic stem cells. We have defined the phenotype of hemogenic endothelium and emerging HSCs in both mouse and human embryos (Aim 2), and identified novel markers that can be used to isolate developing HSCs at distinct stages, as well as to purify functional HSCs further (Aim 3). We have also established an inducible lentiviral based expression system that will now be used to test functionally candidate HSC regulators that were identified by comparing gene expression profiles between freshly isolated HSCs and dysfunctional HSCs that were expanded in culture or generated from human ES cells. We hope that these studies will provide better understanding of the key regulatory mechanisms that govern HSC properties, and ultimately lead to development of improved methods for generation of functional HSCs in culture.
  • Our work has focused on defining mechanisms that govern the specification and self-renewal of hematopoietic stem cells during mouse and human development. Using gene targeted mouse ES cells and mouse embryos, we defined the transcriptional programs that are regulated by Scl, the master regulator for blood formation. We discovered that Scl not only establishes the transcriptional programs that are critical for specifying hemogenic endothelium and hematopoietic stem cells, but it also represses heart development. Strikingly, in the absence of Scl, hemogenic endothelium in embryonic hematopoietic tissues becomes converted to cardiogenic fate, and gives rise to fully functional, beating cardiomyocytes.
  • In order to define the key programs that distinguish self-renewing HSCs from their downstream progenitors or the compromised HSPCs (hematopoietic stem/progenitor cells) that were generated in vitro, we performed microarray analysis for human phenotypic HSCs from various sources. We identified novel markers for human HSCs that can be used to purify transplantable HSCs to a higher purity. We have identified key molecular defects in HSCs that are expanded in culture, or generated from human ES cells. We have further validated that dysregulation of certain Hox genes is a major bottleneck for generating functional HSCs from human ES cells. Future studies are focused on establishing methods that would allow correction of the compromised HSC regulatory networks in cultured HSCs.
  • We have defined key regulatory mechanisms that are required for generation and maintenance of blood forming stem cells. We showed that transcription factor Scl is critical for specifying hemogenic endothelium from where blood stem cells emerge, and moreover, we discovered and unexpected repressive function for Scl to suppress cardiomyogenesis; in the absence of Scl, the blood vessels in start to generate beating cardiomyocytes. We have also identified factors that are critical for blood stem cells to maintain the unique properties: to self-renew (make more of themselves) and engraft (interact with the niche cells that support them). We will now continue to define how these key regulators act so that we can design better strategies to generate blood stem cells as well as heart muscle precursors for therapeutic applications.
  • The goal of this grant was to define mechanisms that govern blood stem cell specification and self-renewal. We have completed the studies on hematopoietic fate specification by defining how Scl/tal1 establishes hemogenic endothelium. We documented that, in addition to Scl’s critical function in activating blood cell regulators, Scl also has to repress heart factors to prevent the misspecification of blood precursors to heart muscle. We documented that Scl controls blood and heart regulators through enhancers that have been primed for activation prior to Scl action (Aim 1). We identified a new surface marker that is expressed in hemogenic endothelium and blood forming cells in the yolk sac (Lyve1), which provides new tools to investigate the origin of blood stem and progenitor cells during development (Aim 2). We identified GPI-80 as a novel marker for transplantable blood stem cells during human fetal development (Aim 2, 3). Taking advantage of this new marker for blood stem cells, we narrowed down the critical defects in the dysfunctional blood precursors that are generated from human ES cells, or expanded in culture from fetal liver blood stem cells (Aim 3). We showed that the inability to induce HOXA cluster genes and other novel blood stem cell regulators that cannot be sustained in culture hinder the generation of blood stem cells from pluripotent cells, and further validated these novel regulators using lentiviral knockdown and overexpression. These findings will now be used to develop novel strategies to generate blood stem cells in culture.

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.

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