Blood Cancer

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

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.

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. 

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.

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.

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