Blood Cancer

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

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.

The Innovation-Alpha Clinic for Cellular Therapies (I-ACT) – A Program for the Development and Delivery of Innovative Cell-based Treatments and Cures for Life-threatening Diseases.

Funding Type: 
Alpha Stem Cell Clinics
Grant Number: 
AC1-07659
ICOC Funds Committed: 
$8 000 000
Disease Focus: 
Blood Disorders
Blood Cancer
Cancer
HIV/AIDS
Solid Tumor
Stem Cell Use: 
Adult Stem Cell
Public Abstract: 
As the largest provider of bone marrow cell transplants in California, and the second largest in the nation, our institution has great expertise and an excellent record of safety in the delivery of stem cell treatments. We now propose to create the Alpha Clinic for Cell Therapy and Innovation (ACT-I) in which new, state-of-the-art, stem cell treatments for cancer and devastating blood-related diseases will be conducted and evaluated. As these experimental therapies prove to be effective, and become routine practice, our ACT-I Program will serve as the clinical center for delivery of these treatments. ACT-I will be an integral part of our Hematologic Malignancy and Stem Cell Transplantation Institute, placing it in the center of our institutional strengths, expertise, infrastructure and investment over the next decade. To move quickly once the CIRM award is made, ACT-I can be launched within our institution’s Day Hospital, a brand new, outpatient blood stem cell transplantation center opened in late 2013 with California Department of Health approval for 24 hour a day operation. This will ensure that ACT-I will have all the clinical and regulatory expertise, trained personnel, state-of-the-art facilities and other infrastructure in place to conduct first-in-human clinical trials and to deliver future, stem cell-based therapies for cancer and blood-related diseases, including AIDS. When our new Ambulatory Treatment Center is complete in 2018, it will double our capacity for patient visits and allow for expansion of the ACT-I pipeline of new stem cell products in a state-of-the-art facility. Beyond our campus, we operate satellite clinics covering an area that includes urban, suburban and rural sites. More than 17.7 million people live in this area, and represent some of the greatest racial and ethnic diversity seen in any part of the country. Our ACT-I is prepared to serve a significant, diverse and underserved portion of the population of California. CLINICAL TRIALS. Our proposal has two lead clinical trials that will be the first to be tested in ACT-I. One will deliver transplants of blood stem cells that have been modified to treat patients suffering from AIDS and lymphoma. The second will use neural stem cells to deliver drugs directly to cancer cells hiding in the brain. These studies represent some of the new and exciting biomedical technologies being developed at our institution. In addition to the two lead trials, we have several additional clinical studies poised to use and be tested in this special facility for clinical trials. In summary, ACT-I is well prepared to accommodate the long list of clinical trials and begin to fulfill the promise of providing new stem cell therapies for the citizens of California.
Statement of Benefit to California: 
California’s citizens voted for the California Stem Cell Research and Cures Act to support the development of stem cell-based therapies that treat incurable diseases and relieve human suffering. To achieve this goal, we propose to establish an Alpha Clinic for Cellular Therapies and Innovation (ACT-I) as an integral part of our Hematological Malignancies and Stem Cell Transplantation Institute, and serve as the clinical center for the testing and delivery of new, cutting-edge, cellular treatments for cancer and other blood-related diseases. Our institution is uniquely well-suited to serve as a national leader in the study and delivery of stem cell therapeutics because we are the largest provider of stem cell transplants in California, and the second largest in the country. According to national benchmarking data, our Hematopoietic Cell Transplantation program is the only program in the nation to have achieved survival outcomes above expectation for each of the past nine years. This program currently offers financially sustainable, research-driven clinical care for patients with cancer, HIV and other life-threatening diseases. CIRM funding will allow the ACT-I clinic to ramp up quickly, drawing upon institutionally established protocols, personnel and infrastructure to conduct first-in-human clinical trials for assessment of efficacy. As CIRM funding winds down, ACT-I will have institutional support to offer proven cellular therapeutics to patients. The lead studies at the forefront of the ACT-I pipeline of clinical trials focus on treatments for HIV-1 infection and brain tumors, two devastating and incurable conditions. These first trials are closely followed by a robust queue of other stem cell therapeutics for leukemia, lymphoma, prostate cancer, brain cancers and thalassemia. Our long list of proposed treatments addresses diseases that have a major impact on the lives of Californians. Thalassemia is found in up to 1 in 2,200 children born in California; prostate cancer affects 211,300 men, and HIV-1 infection occurs in 111,000 of our citizens. From 2008 to 2010, 6,705 Californians were diagnosed with brain cancers, 4,580 of whom died. In considering hematological malignancies during this same period, 2,800 patients were diagnosed with Hodgkin lymphoma (416 died), 20,351 with non-Hodgkin lymphoma (6,241 died), 13,358 with leukemia (6,961 died), 3,900 with acute myelogenous leukemia (2,972 died), 2,129 with acute lymphoblastic leukemia (648 died) and 4,198 with chronic lymphocytic leukemia (1,271 died). Standard of care fails in many cases; mortality rates for patients with hematological malignancies range from 25% to 76%. Successful stem cell therapeutics hold the promise to reduce disease-related mortality while improving disease-related survival and quality of life for the citizens of California, and for those affected by these diseases worldwide.

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

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

Clinical Investigation of a Humanized Anti-CD47 Antibody in Targeting Cancer Stem Cells in Hematologic Malignancies and Solid Tumors

Funding Type: 
Disease Team Therapy Development III
Grant Number: 
DR3-06965
ICOC Funds Committed: 
$12 726 396
Disease Focus: 
Cancer
Solid Tumor
Blood Cancer
Collaborative Funder: 
UK
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Most normal tissues are maintained by a small number of stem cells that can both self-renew to maintain stem cell numbers, and also give rise to progenitors that make mature cells. We have shown that normal stem cells can accumulate mutations that cause progenitors to self-renew out of control, forming cancer stem cells (CSC). CSC make tumors composed of cancer cells, which are more sensitive to cancer drugs and radiation than the CSC. As a result, some CSC survive therapy, and grow and spread. We sought to find therapies that include all CSC as targets. We found that all cancers and their CSC protect themselves by expressing a ‘don’t eat me’ signal, called CD47, that prevents the innate immune system macrophages from eating and killing them. We have developed a novel therapy (anti-CD47 blocking antibody) that enables macrophages to eliminate both the CSC and the tumors they produce. This anti-CD47 antibody eliminates human cancer stem cells when patient cancers are grown in mice. At the time of funding of this proposal, we will have fulfilled FDA requirements to take this antibody into clinical trials, showing in animal models that the antibody is safe and well-tolerated, and that we can manufacture it to FDA specifications for administration to humans. Here, we propose the initial clinical investigation of the anti-CD47 antibody with parallel first-in-human Phase 1 clinical trials in patients with either Acute Myelogenous Leukemia (AML) or separately a diversity of solid tumors, who are no longer candidates for conventional therapies or for whom there are no further standard therapies. The primary objectives of our Phase I clinical trials are to assess the safety and tolerability of anti-CD47 antibody. The trials are designed to determine the maximum tolerated dose and optimal dosing regimen of anti-CD47 antibody given to up to 42 patients with AML and up to 70 patients with solid tumors. While patients will be clinically evaluated for halting of disease progression, such clinical responses are rare in Phase I trials due to the advanced illness and small numbers of patients, and because it is not known how to optimally administer the antibody. Subsequent progression to Phase II clinical trials will involve administration of an optimal dosing regimen to larger numbers of patients. These Phase II trials will be critical for evaluating the ability of anti-CD47 antibody to either delay disease progression or cause clinical responses, including complete remission. In addition to its use as a stand-alone therapy, anti-CD47 antibody has shown promise in preclinical cancer models in combination with approved anti-cancer therapeutics to dramatically eradicate disease. Thus, our future clinical plans include testing anti-CD47 antibody in Phase IB studies with currently approved cancer therapeutics that produce partial responses. Ultimately, we hope anti-CD47 antibody therapy will provide durable clinical responses in the absence of significant toxicity.
Statement of Benefit to California: 
Cancer is a leading cause of death in the US accounting for approximately 30% of all mortalities. For the most part, the relative distribution of cancer types in California resembles that of the entire country. Current treatments for cancer include surgery, chemotherapy, radiation therapy, biological therapy, hormone therapy, or a combination of these interventions ("multimodal therapy"). These treatments target rapidly dividing cells, carcinogenic mutations, and/or tumor-specific proteins. A recent NIH report indicated that among adults, the combined 5-year relative survival rate for all cancers is approximately 68%. While this represents an improvement over the last decade or two, cancer causes significant morbidity and mortality to the general population as a whole. New insights into the biology of cancer have provided a potential explanation for the challenge of treating cancer. An increasing number of scientific studies suggest that cancer is initiated and maintained by a small number of cancer stem cells that are relatively resistant to current treatment approaches. Cancer stem cells have the unique properties of continuous propagation, and the ability to give rise to all cell types found in that particular cancer. Such cells are proposed to persist in tumors as a distinct population, and because of their increased ability to survive existing anti-cancer therapies, they regenerate the tumor and cause relapse and metastasis. Cancer stem cells and their progeny produce a cell surface ‘invisibility cloak’ called CD47, a ‘don’t eat me signal’ for cells of the native immune system to counterbalance ‘eat me’ signals which appear during cancer development. Our anti-CD47 antibody counters the ‘cloak’, enabling the patient’s natural immune system to eliminate the cancer stem cells and cancer cells. Our preclinical data provide compelling support that anti-CD47 antibody might be a treatment strategy for many different cancer types, including breast, bladder, colon, ovarian, glioblastoma, leiomyosarcoma, squamous cell carcinoma, multiple myeloma, lymphoma, and acute myelogenous leukemia. Development of specific therapies that target all cancer stem cells is necessary to achieve improved outcomes, especially for sufferers of metastatic disease. We hope our clinical trials proposed in this grant will indicate that anti-CD47 antibody is a safe and highly effective anti-ancer therapy that offers patients in California and throughout the world the possibility of increased survival and even complete cure.

Therapeutic Eradication of Cancer Stem Cells

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

RUNNING TITLE: Stem Cell Gene Therapy for HIV in AIDS Lymphoma Patients

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05327
ICOC Funds Committed: 
$74 195
Disease Focus: 
Blood Cancer
Cancer
HIV/AIDS
oldStatus: 
Closed
Public Abstract: 
The Human Immunodeficiency Virus (HIV) is still a major health problem. In both developed and underdeveloped nations, millions of people are infected with this virus. HIV infects cells of the immune system, becomes part of the cell’s genetic information, stays there for the rest of the life of these cells, and uses these cells as a factory to make more HIV. In this process, the immune cells get destroyed. Soon a condition called AIDS, the Acquired Immunodeficiency Syndrome sets in where the immune system cannot fight common infections. If left untreated, death from severe infections occurs within 8 to 10 years. Although advances in treatment using small molecule drugs have extended the life span of HIV infected individuals, neither a cure for HIV infection nor a well working vaccine could be developed. Drug treatment is currently the only option to keep HIV infected individuals alive. Patients have to take a combination of drugs daily and reliably for the rest of their lives. If not taken regularly, HIV becomes resistant to the drugs and continues to destroy immune cells. What makes this situation even more complicated is the fact that many patients cannot take these drugs due to severe side effects. Stem cell gene therapy for HIV may offer an alternative treatment. Blood forming stem cells, also called bone marrow stem cells make all blood cells of the body, including immune system cells such as T cells and macrophages that HIV destroys. If “anti-HIV genes” were inserted into the genetic information of bone marrow stem cells, these genes would be passed on to all new immune cells and make them resistant to HIV. Anti-HIV gene containing immune cells can now multiply in the presence of HIV and fight the virus. In previous and current stem cell gene therapy clinical trials for HIV, only one anti-HIV gene has been used. Our approach, however, will use a combination of three anti-HIV genes which are much more potent. They will not only prevent HIV from entering an immune cell but will also prevent HIV from mutating, since it would have to escape the anti-HIV effect of three genes, similar to triple combination anti-HIV drug therapy. To demonstrate safety and effectiveness of our treatment, we will perform a clinical trial in HIV lymphoma patients. In such patients, the destruction of the immune system by HIV led to the development of a cancer of the lymph nodes called B cell lymphoma. High dose chemotherapy together with the transplantation of the patient’s own bone marrow stem cells cures B cell lymphoma. We will insert anti-HIV genes in the patient’s bone marrow stem cells and then transplant these gene containing cells into the HIV infected lymphoma patient. The gene containing bone marrow stem cells will produce a new immune system and newly arising immune cells will be resistant to HIV. In this case, we have not only cured the patient's cancer but have also given the patient an HIV resistant immune system which will be able to fight HIV.
Statement of Benefit to California: 
As of September 30, 2010, over 198,883 cumulative HIV/AIDS cases were reported in California. Another 40,000 un-named cases of HIV were also reported before 2006 although some of them may be duplicates of the named HIV cases. Patients living with HIV/AIDS totaled 108,986 at the end of September 2010. These numbers continue to grow since new cases of HIV and AIDS are being reported on a daily basis and patients now live much longer. In fact, after New York, California has the second highest number of HIV cases in the nation. Although the current and improved anti-retroviral small molecule drugs have prolonged the life of these patients, they still have to deal with the emotional, financial, and medical consequences of this disease. The fear of side effects and the potential generation of drug resistant strains of HIV is a constant struggle that these patients have to live with for the rest of their lives. Furthermore, not every patient with HIV responds to treatment and not every complication of HIV dissipates upon starting a drug regimen. In fact, the risk of some AIDS-related cancers still remains high despite the ongoing drug therapy. Additionally, in the current economic crisis, the financial burden of the long term treatment of these patients on California taxpayers is even more obvious. In 2006, the lifetime cost of taking care of an HIV patient was calculated to be about $618,900. Most of this was related to the medication cost. With the introduction of new HIV medications that have a substantially higher price and with the increase in the survival of HIV/AIDS patients, the cost of taking care of these patients can be estimated to be very high. The proposed budget cuts and projected shortfall in the California AIDS assistant programs such as ADAP will make the situation worse and could result in catastrophic consequences for patients who desperately need this of kind of support. Consequently, improved therapeutic approaches and the focus on developing a cure for HIV infected patients are issues of great importance to the people of California. Our proposed anti-HIV stem cell gene therapy strategy comprises the modification of autologous hematopoietic blood forming stem cells with a triple combination of potent anti-HIV genes delivered by a single lentiviral vector construct. This approach would engineer a patient’s immune cells in a way to make them completely resistant to HIV infection. By transplanting these anti-HIV gene expressing stem cells back into an HIV infected patient, the ability of HIV to further replicate and ravage the patient’s immune system would be diminished. The prospect of such a stem cell based therapy which may require only a single treatment to cure an HIV infected patient and which would last for the life of the individual would be especially compelling to the HIV community and the people of California.
Progress Report: 
  • HIV is still a major health problem. In both developed and underdeveloped nations, millions of people are infected with this virus. If left untreated, death from severe infections occurs within 8 to 10 years. Although advances in treatment using small molecule drugs have extended the life span of HIV infected individuals, neither a cure for HIV infection nor a well working vaccine could be developed. Drug treatment is currently the only option to keep HIV infected individuals alive. Patients have to take a combination of drugs daily and reliably for the rest of their lives. If not taken regularly, HIV becomes active again and may even become resistant to the drugs and continues to destroy immune cells. What makes this situation even more complicated is the fact that many patients cannot take these drugs due to severe side effects. Stem cell gene therapy for HIV may offer an alternative treatment. If “anti-HIV genes” were inserted into the genetic information of bone marrow stem cells, these genes would be passed on to all new immune cells and make them resistant to HIV. Anti-HIV gene containing immune cells can now multiply in the presence of HIV and fight the virus. In our approach, we are planning to use a combination of three anti-HIV genes which are much more potent. They will not only prevent HIV from entering an immune cell but will also prevent HIV from mutating, since it would have to escape the anti-HIV effect of three genes, similar to triple combination anti-HIV drug therapy. To demonstrate safety and effectiveness of our treatment, we have proposed a clinical trial in HIV lymphoma patients with stem cell gene therapy incorporated into their routine treatment with high dose chemotherapy together with the transplantation. The fund provided by CIRM (California Institute for Regenerative Medicine) gave us the opportunity to put together a panel of experts within the University of California at Davis and another panel of international experts in the area of gene therapy (an external advisory board). Intense discussion in multiple meeting with members of these two panels as well as many other meetings with individual researches within our institution resulted in the design of a clinical trial for treating patients with HIV disease using our gene therapy approach. It further helped us to identify the necessary means needed to support such a regulatory intensive gene therapy trial. To be able to recruit enough patients for such a trial, we used the funds from this planning grant for several presentations to our colleagues in other institutions for a multi-institutional clinical trial approach. The funds provided to us through this grant helped to calculate the budget required to 1) finish our application with Federal Drug Administration (FDA) to obtain the appropriate license for starting such a trial and 2) to manufacture the target drug and 3) to run the actual clinical trial. Finally, with the help of this grant, we have put together a CIRM disease grant proposal and have applied for necessary funds based on the above calculation.
  • The original progress report was submitted to the CIRM on March 1st 2012. The no cost extension was requested to perform the necessary work related to further development of our clinical trial before submission to RAC. During this period, in multiple meetings we rewrote our clinical trial based on the comments of our external advisory board and other consultants. We submitted our clinical trial protocol and Appendix M to RAC committee and after receiving their preliminary comments, we formulated our response. As the last step, we presented our clinical trial to the members of RAC committee and received a unanimous approval to move forward with the IND application to FDA.

Forming the Hematopoietic Niche from Human Pluripotent Stem Cells

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05217
ICOC Funds Committed: 
$1 375 983
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop. To approach this question we will use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation. Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells. The findings from these studies will have broad applicability to the production of other types of tissues from pluripotent stem cells, all of which have stem cells that require interaction with a specialized niche. In addition to the biological questions explored in this proposal, our focus on the blood system has direct clinical relevance to the field of bone marrow and cord blood transplantation. The development of a human hematopoietic niche from pluripotent stem cells could potentially be used to expand hematopoietic stem cells from adult tissues like cord blood. Most importantly, the ability to control differentiation from pluripotent stem cells into the blood lineage could provide an unlimited source of matched cells for transplantation for patients with leukemia and other diseases of the bone marrow and the immune system who currently lack suitable donors.
Statement of Benefit to California: 
The unique combination of pluripotentiality and unlimited capacity for proliferation has raised the hope that pluripotent stem cells will one day provide an inexhaustible source of tissue for transplantation and regeneration. Diseases that might be treated from such tissues affect millions of Californians and their families. However, much is still to be learned about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The clinical potential of pluripotent stem cells for regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and better controlled than is currently the case. The research proposed in this application has broad potential benefits for Californians both through the biological questions it will answer and the relevance of these studies for clinical translation. Our goal is to understand the way the microenvironment influences tissue production from pluripotent stem cells, a critical issue for the field of stem cell biology. Specifically we will explore the question- Do the cell types produced during differentiation of pluripotent stem cells produce an adequate microenvironment for the differentiation of tissue or are some cells inhibitory to tissue production? Our approach to these questions will be to use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation and offers a range of unique technical tools with which to study these questions rigorously. However, the fundamental concepts formed from these studies will have great relevance for the clinical production of other types of tissues from pluripotent stem cells, such as islets, neural cells and cardiac muscle. In addition to the broad biological questions explored in this proposal, our focus on the blood system has direct clinical relevance to the field of bone marrow and cord blood transplantation. One goal in the proposal is to generate a cellular platform from pluripotent stem cells that will create an environment in which adult blood stem cells can grow and be expanded. Cell numbers collected from cord blood at birth are often insufficient for transplantation in adult patients and older children. The development of a human cell culture system that could expand the number of cord blood stem cells would provide new opportunities for transplantation for patients with leukemia and other diseases of the bone marrow and the immune system who currently lack suitable donors. All scientific findings and technical tools developed in this proposal will be made available to researchers throughout California, under the guidelines from the California Institute of Regenerative Medicine.
Progress Report: 
  • The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop.
  • To approach this question we use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation.
  • Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells.
  • During the first year of support, we have made significant progress in the first two specific aims. We have developed a method that allows us to track the common origin of the blood forming cells and their microenvironment. We also have identified subsets of cells generated from pluripotent cells that have distinct functions in blood formation. Our plan during the next year is to fully characterize these subsets to understand how they function, and to improve our methods to expand them in culture.
  • The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop.
  • To approach this question we use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells (HSC) survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation.
  • Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells.
  • During the second year of support, we have made significant progress in all three specific aims. We continue to refine our method that allows us to track the common origin of the blood forming cells and their microenvironment during development. We have identified subsets of cells generated from pluripotent cells that can support cord blood HSC and now we are determining the mechanisms by which these cells act and how they can be best used to support HSC that develop from PSC.
  • The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop.
  • To approach this question we use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells (HSC) survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation.
  • Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells.
  • During the third year of support, we have made significant progress in all three specific aims. We have now completed our studies that track the common origin of the blood forming cells and their microenvironment. We have performed functional studies to identify which of the cell types that we generate from pluripotent cells support HSC when grown in culture, and which do not. Finally we have performed gene expression analyses on these different cell types to understand the molecular pathways that they use to support HSC in culture.

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

Funding Type: 
Early Translational II
Grant Number: 
TR2-01816-A
ICOC Funds Committed: 
$3 607 305
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Adult Stem Cell
Cancer Stem Cell
Public Abstract: 
Leukemia is the most frequent form of cancer in children and teenagers, but is also common in adults. Chemotherapy has vastly improved the outcome of leukemia over the past four decades. However, many patients still die because of recurrence of the disease and development of drug-resistance in leukemia cells. In preliminary studies for this proposal we discovered that in most if not all leukemia subtypes, the malignant cells can switch between an “proliferation phase” and a “quiescence phase”. The “proliferation phase” is often driven by oncogenic tyrosine kinases (e. g. FLT3, JAK2, PDGFR, BCR-ABL1, SRC kinases) and is characterized by vigorous proliferation of leukemia cells. In this phase, leukemia cells not only rapidly divide, they are also highly susceptible to undergo programmed cell death and to age prematurely. In contrast, leukemia cells in “quiescence phase” divide only rarely. At the same time, however, leukemia cells in "quiescence phase" are highly drug-resistant. These cells are also called 'leukemia stem cells' because they exhibit a high degree of self-renewal capacity and hence, the ability to initiate leukemia. We discovered that the BCL6 factor is required to maintain leukemia stem cells in this well-protected safe haven. Our findings demonstrate that the "quiescence phase" is strictly dependent on BCL6, which allows them to evade cell death during chemotherapy treatment. Once chemotherapy treatment has ceased, persisting leukemia stem cells give rise to leukemia clones that reenter "proliferation phase" and hence initiate recurrence of the disease. Pharmacological inhibition of BCL6 using inhibitory peptides or blocking molecules leads to selective loss of leukemia stem cells, which can no longer persist in a "quiescence phase". In this proposal, we test a novel therapeutic concept eradicate leukemia stem cells: We propose that dual targeting of oncogenic tyrosine kinases (“proliferation”) and BCL6 (“quiescence”) represents a powerful strategy to eradicate drug-resistant leukemia stem cells and prevent the acquisition of drug-resistance and recurrence of the disease. Targeting of BCL6-dependent leukemia stem cells may reduce the risk of leukemia relapse and may limit the duration of tyrosine kinase inhibitor treatment in some leukemias, which is currently life-long.
Statement of Benefit to California: 
Leukemia represents the most frequent malignancy in children and teenagers and is common in adults as well. Over the past four decades, the development of therapeutic options has greatly improved the prognosis of patients with leukemia reaching 5 year disease-free survival rates of ~70% for children and ~45% for adults. Despite its relatively favorable overall prognosis, leukemia remains one of the leading causes of person-years of life lost in the US (362,000 years in 2006; National Center of Health Statistics), which is attributed to the high incidence of leukemia in children. In 2008, the California Cancer Registry expected 3,655 patients with newly diagnosed leukemia and at total of 2,185 death resulting from fatal leukemia. In addition, ~23,300 Californians lived with leukemia in 2008, which highlights that leukemia remains a frequent and life-threatening disease in the State of California despite substantial clinical progress. Here we propose the development of a fundamentally novel treatment approach for leukemia that is directed at leukemia stem cells. While current treatment approaches effectively diminish the bulk of proliferating leukemia cells, they fail to eradicate the rare leukemia stem cells, which give rise to drug-resistance and recurrence of the disease. We propose a dual targeting approach which combines targeted therapy of the leukemia-causing oncogene and the newly discovered leukemia stem cell survival factor BCL6. The power of this new therapy approach will be tested in clinical trials to be started in the State of California.
Progress Report: 
  • Leukemia is the most frequent form of cancer in children and teenagers, but is also common in adults. Chemotherapy has vastly improved the outcome of leukemia over the past four decades. However, many patients still die because of recurrence of the disease and development of drug-resistance in leukemia cells. In preliminary studies for this proposal we discovered that in most if not all leukemia subtypes, the malignant cells can switch between an "expansion phase" and a "dormancy phase". The "expansion phase" is often driven by oncogenic tyrosine kinases (e. g. FLT3, JAK2, PDGFR, BCR-ABL1, SRC kinases) and is characterized by vigorous proliferation of leukemia cells. In this phase, leukemia cells not only rapidly divide, they are also highly susceptible to undergo programmed cell death and to age prematurely. In contrast, leukemia cells in "quiescence phase" divide only rarely. At the same time, however, leukemia cells in "domancy phase" are highly drug-resistant. These cells are also called 'leukemia stem cells' because they exhibit a high degree of self-renewal capacity and hence, the ability to initiate leukemia.
  • Progress during Year 1: During the first year of this project, we discovered that the BCL6 factor is required to maintain leukemia stem cells in this well-protected safe haven. Our findings during year 1 demonstrate that the "dormancy phase" is strictly dependent on BCL6, which allows them to evade cell death during chemotherapy treatment. Once chemotherapy treatment has ceased, persisting leukemia stem cells give rise to leukemia clones that reenter "proliferation phase" and hence initiate recurrence of the disease. Pharmacological inhibition of BCL6 using inhibitory peptides or blocking molecules leads to selective loss of leukemia stem cells, which can no longer persist in a "dormancy phase" .
  • In year 1, we have performed screening procedures to identify novel therapeutic BCL6 inhibitors to eradicate leukemia stem cells: We have found that dual targeting of oncogenic tyrosine kinases ("expansion phase" ) and BCL6 ("dormancy phase") represents a powerful strategy to eradicate drug-resistant leukemia stem cells and prevent the acquisition of drug-resistance and recurrence of the disease.
  • Goal for years 2-3: Targeting of BCL6-dependent leukemia stem cells may reduce the risk of leukemia relapse and may limit the duration of tyrosine kinase inhibitor treatment in some leukemias, which is currently life-long.

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

Funding Type: 
Early Translational II
Grant Number: 
TR2-01816-B
ICOC Funds Committed: 
$3 607 305
Disease Focus: 
Blood Cancer
Cancer
Collaborative Funder: 
Germany
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Adult Stem Cell
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Leukemia is the most frequent form of cancer in children and teenagers, but is also common in adults. Chemotherapy has vastly improved the outcome of leukemia over the past four decades. However, many patients still die because of recurrence of the disease and development of drug-resistance in leukemia cells. In preliminary studies for this proposal we discovered that in most if not all leukemia subtypes, the malignant cells can switch between an “proliferation phase” and a “quiescence phase”. The “proliferation phase” is often driven by oncogenic tyrosine kinases (e. g. FLT3, JAK2, PDGFR, BCR-ABL1, SRC kinases) and is characterized by vigorous proliferation of leukemia cells. In this phase, leukemia cells not only rapidly divide, they are also highly susceptible to undergo programmed cell death and to age prematurely. In contrast, leukemia cells in “quiescence phase” divide only rarely. At the same time, however, leukemia cells in "quiescence phase" are highly drug-resistant. These cells are also called 'leukemia stem cells' because they exhibit a high degree of self-renewal capacity and hence, the ability to initiate leukemia. We discovered that the BCL6 factor is required to maintain leukemia stem cells in this well-protected safe haven. Our findings demonstrate that the "quiescence phase" is strictly dependent on BCL6, which allows them to evade cell death during chemotherapy treatment. Once chemotherapy treatment has ceased, persisting leukemia stem cells give rise to leukemia clones that reenter "proliferation phase" and hence initiate recurrence of the disease. Pharmacological inhibition of BCL6 using inhibitory peptides or blocking molecules leads to selective loss of leukemia stem cells, which can no longer persist in a "quiescence phase". In this proposal, we test a novel therapeutic concept eradicate leukemia stem cells: We propose that dual targeting of oncogenic tyrosine kinases (“proliferation”) and BCL6 (“quiescence”) represents a powerful strategy to eradicate drug-resistant leukemia stem cells and prevent the acquisition of drug-resistance and recurrence of the disease. Targeting of BCL6-dependent leukemia stem cells may reduce the risk of leukemia relapse and may limit the duration of tyrosine kinase inhibitor treatment in some leukemias, which is currently life-long.
Statement of Benefit to California: 
Leukemia represents the most frequent malignancy in children and teenagers and is common in adults as well. Over the past four decades, the development of therapeutic options has greatly improved the prognosis of patients with leukemia reaching 5 year disease-free survival rates of ~70% for children and ~45% for adults. Despite its relatively favorable overall prognosis, leukemia remains one of the leading causes of person-years of life lost in the US (362,000 years in 2006; National Center of Health Statistics), which is attributed to the high incidence of leukemia in children. In 2008, the California Cancer Registry expected 3,655 patients with newly diagnosed leukemia and at total of 2,185 death resulting from fatal leukemia. In addition, ~23,300 Californians lived with leukemia in 2008, which highlights that leukemia remains a frequent and life-threatening disease in the State of California despite substantial clinical progress. Here we propose the development of a fundamentally novel treatment approach for leukemia that is directed at leukemia stem cells. While current treatment approaches effectively diminish the bulk of proliferating leukemia cells, they fail to eradicate the rare leukemia stem cells, which give rise to drug-resistance and recurrence of the disease. We propose a dual targeting approach which combines targeted therapy of the leukemia-causing oncogene and the newly discovered leukemia stem cell survival factor BCL6. The power of this new therapy approach will be tested in clinical trials to be started in the State of California.
Progress Report: 
  • During the past reporting period (months 18-24 of this grant), we have made progress towards all three milestones. Major progress in Milestone 1 was made by identifying 391 compounds in 10 lead classes that will be developed further in a secondary fragment-based screen. While the goal of identifying lead class compounds with BCL6 inhibitory activity has already been met, we propose to run a secondary, fragment-based screen to refine the existing lead compounds and prioritize a small number for cell-based validation in Milestone 2. The success in Milestone 1 was based on computational modeling, HTS of 200,000 compounds and Fragment-based drug discovery (FBDD).
  • For Milestone 2, we have successfully established POC analysis tools for validation of the ability of compounds to bind the BCL6 lateral groove and already produced 300 mg of BCL6-BTB domain protein needed for biochemical binding assays. Progress in Milestone 2 is based on surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) assays. In the coming months, we will use crystallographic fragment screening using a subset of our fragment library in addition to SPR and NMR, since crystallographic fragment screens have been shown to yield complimentary hits. For Milestone 3, we have now set up a reliable method to measure disease-modifying activity of BCL6-inhibitory compounds based on a newly generated knockin BCL6 reporter mouse model, in which transcriptional activation of the endogenous BCL6 promoter drives expression of mCherry. This addresses a main caveat of these measurements was that they were strongly influenced by the copy number of lentivector integrations. The BCL6fl/+-mCherry knockin BCL6 reporter system will provide a stable platform to study BCL6-expressing leukemia cells and effects of BCL6 small molecule inhibitors on survival and proliferation on BCL6-dependent leukemia cell populations. This will be a key requirement to measure disease-modifying activity of inhibitory compounds in large-scale assays in Milestone 3. Other requirements (e.g. leukemia xenografts) are already in place. 
  • During the past two years of this grant, we have generated compounds that have the ability to block the function of BCL6. In previous work, we had identified BCL6 as a key requirement for persistence of leukemia stem cells, which are the root cause of leukemia relapse and drug-resistance in patients. Over the past six months, we have focused on validating the new compounds based on functional tests that allow us to measure the depth and durability of BCL6 blockade in cell-based assay. To this end, we designed a large-scale petri-dish system in which we measured the efficacy of 11 lead compounds and their derivatives to abrogate the ability of leukemia cells to form colonies, a capability that reflects the activity of leukemia stem cells. This assay allowed us to prioritize 4 compounds for further testing. In parallel, we developed a biological assay to verify that the compounds are actually hitting their target, i.e. BCL6, by measuring the activity of genes that are typically regualted by BCL6. These genes include tumor suppressors like p53 and Arf and we measured the ability of our compounds to re-instate p53 and Arf expression. We found that p53 and Arf were reinstated only by 2 of our 4 lead candidates, so current trouble-shooting efforts will attempt to clarify why this is the case and whether we can modify these two compounds to improve their on-target efficacy. The other two compounds will move forward in the next derivative screen, in which we perform a fragment-based, screen, i.e. test multiple derivative based on addition and removal of small structural changes (fragments). Other caveats to address in the next year will be stability (half-life) of the lead compounds, bioavailability (how much and how long the compound will be available in the blood stream) and toxicity (how much of the compound will be tolerated by mice, is there indication of damage to tissues upon long-term treatment?).The goal of these studies will be to make a strong case for IND-enabling studies, i.e. to enter a formal, government-regulated process to convert the strongest of our compound into an FDA-approved drug for potential clinical testing in patients with drug-refractory AML and ALL.

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

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

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