Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$777 467
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The successful use of human embryonic stem cells (hESCs) as novel regenerative therapies for a spectrum of currently incurable diseases critically depends upon the safety of such cell transfers. hESCs contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of their genetic material. While many of these retroelements have been permanently silenced during evolution by crippling mutations, many remain active and capable of moving to new chromosomal locations potentially producing disease-causing mutations or cancer. More mature differentiated cells control retroelement movement (retrotransposition) by methylating the DNA comprising these elements. Strikingly, such DNA methylation is largely absent in hESCs because these cells must be able to develop into a wide spectrum of different tissues and organs. Thus, in order to protect the integrity of their genomes, hESCs must deploy an additional defense to limit retroelement retrotransposition. Recent studies of HIV and other exogenous retroviruses have identified the APOBEC3 family of genes (A3A-A3H) as powerful anti-retroviral factors. These APOBEC3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also used by retroelements for their successful retrotransposition. We hypothesize that one or more of the APOBECs function as guardians of genome integrity in hESCs. We propose to compare and contrast which APOBEC3s are expressed in one federally approved and nine nonapproved hESC lines and to assess the natural level of retroelement RNA expression occurring in each of these lines. Next we will test whether the knockdown of expression of these APOBEC3s in the hESCS lines by RNA interference leads to a higher frequency of retrolement retrotransposition. Finally, if higher levels of retrotransposition are detected, we will examine whether these cells display an impaired ability to differentiate into specific tissue types corresponding to the three germ cell layers (ectoderm, mesoderm, and endoderm) and whether increased retrotransposition is associated with a higher frequency of malignant transformation within the hESC cultures. These studies promise to provide important new insights into how genomic stability in is maintained in hESCs and could lead to the identification of specific GMP culture conditions that minimize the chances of such unwanted retrotransposition events in cells destined for infusion into patients. These studies are directly responsive to the CIRM request for application. If funded, these studies would allow the entry of my laboratory with extensive APOBEC experience, into the exciting field of stem cell biology.
Statement of Benefit to California: 
Harnessing the exciting potential of embryonic stem cells as therapies for a wide range of diseases like diabetes, Alzheimer’s disease, myocardial infarction among others first requires ensuring that the infusion of these cells into patients can be performed safely. Of note, human embryonic stem cells contain up to 3 million “jumping genes” or mobile genetic retroelements that can potentially move from location to another in the genome. Great harm could occur if the movement of these retroelements in human embryonic stem cells results in the mutation of key genes or the inactivation of tumor suppressor genes, the latter could facilitate the development of cancer in recipients of these cells. The safety of stem cell therapy thus depends on the rigorous maintenance of genomic integrity and stability within the embryonic stem cell during its manipulation. Strikingly, the major cellular defense against the movement of the retroelements to new genetic locations, DNA methylation, is greatly reduced in human embryonic stem cells. A general state of hypomethylation is likely required to permit these pluripotent cells to differentiate into multiple cell types. With DNA methylation no longer able to constrain the activity of these retroelements, we believe a second natural defense springs into action to protect these stem cells. We proposeto identify and characterize this defensive network. These studies could lead to new approaches for maintaining or even enhancing this defense when embryotic stem cells are manipulated in culture, thereby helping to ensure the safety of embryonic stem cells destined for therapeutic transfer. Thus, the results of these studies will have both scientific and practical value. As such, we believe these studies will benefit the citizens of California certainly at a societal level and potentially at a personal level.
Progress Report: 
  • Human embryonic stem cells contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of human genome. While many of these retroelements have been silenced during evolution by crippling mutations, many remain active and capable of jumping to new chromosomal locations potentially producing disease-causing mutations or cancer. In tissues, mobility of these elements is suppressed by DNA methylation, which inactivates expression of the retroelement RNAs. In sharp contrast, embryonic stem cells exhibit very dynamic changes in DNA methylation, where the methylation patterns are gained and lost at high rates. During periods of low DNA methylation, retroelement RNA expression likely increases. Accordingly, hESCs must deploy other defensive strategies in order to maintain genomic integrity. Recent studies have identified the APOBEC3 family of genes (A3A-A3H) as powerful antiviral factors. These A3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also employed by retroelements for their successful retrotransposition. We hypothesized that one or more of the APOBECs function as guardians of genome integrity in hESCs. In the last two years we have found that six out of the seven human A3 genes located in a tandem array on chromosome 22 are expressed in hESCs. A3A, which in prior studies was suggested to exert the greatest anti-retroelement effects, surprisingly is not expressed in hESCs. Further, we find that the A3 proteins decrease when pluripotent cells differentiate into somatic cells suggesting an important function of these A3 proteins in pluripotent hESCs. We established a LINE1 retrotransposition assay in hESCs that allows us to visualize genetic jumping of this class of “marked” retroelements via flow cytometry. Using this assay we have found that LINE1 elements effectively jump in hESCs. To test our central hypothesis, namely that A3 proteins guard the genome in hESCs, we have established experimental conditions for RNAi knock-down of all expressed A3 genes. By combining the knock-down and the retrotransposition assay we demonstrated that the knock-down of one member of the A3 protein family leads to a 3.5-fold increase in LINE1 retrotranspositon. This finding highlights a protective role for the A3 family of cytidine deaminases that helps safeguard the genome integrity of hESCs.

Genetic Enhancement of the Immune Response to Melanoma via hESC-derived T cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00203
ICOC Funds Committed: 
$642 501
Disease Focus: 
Melanoma
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The overall goal of the proposed studies is to utilize human gene therapy approach using human embryonic stem cells to direct our body’s defenses to specifically attack melanoma tumor cells. Current technologies try to accomplish this by genetically manipulating certain circulating T lymphocytes, such that they will target tumor cells. T lymphocytes are the major cell type of our body’s immune system. However it is likely that this type of approach will not result in the presence of stable, lifelong genetically modified T cells. In contrast, a potentially more long-lasting approach would be to genetically modify human embryonic stem cells with the same therapeutic gene. Stem cells have the ability to form any type of blood cell, including T cells. Importantly, stem cells can persist for the life of the individual, and thus have the potential to produce genetically modified T cells for many years. In addition, these new tumor specific cells should expand in the body in response to the presence of the tumor, thus a large supply of tumor-fighting cells should be available as long as needed. This project proposes to develop novel means to introduce the anti-cancer gene into human embryonic stem cells. These stem cells will then be differentiated to generate tumor specific T cells utilizing animal model systems. We will then use several laboratory and mouse models to determine if the T cells derived from these genetically modified stem cells have anti-tumor activity. If successful, we will have provided proof-of principle that long-lived stem cells have the potential be utilized as a means of producing anti-cancer T cells. In the long run, these results could provide important information for design of future clinical trials designed to produce life-long improved anti-cancer immune responses.
Statement of Benefit to California: 
We propose to use human embryonic stem cells to develop a novel, yet potentially very effective method to treat invasive melanoma. Melanoma is a serious type of skin cancer which, if not removed early, spreads internally and is usually fatal. Overall melanoma is the 6th most common cancer in males and 7th in females and the incidence of this form of cancer is currently increasing at an epidemic rate. Although melanomas may occur in areas of skin that are not normally exposed to sunlight, sun exposure is believed to be a factor in about 70% of new cases. California’s mild winters and high number of sunny days provide opportunities for a number of occupational and recreational outdoor activities, and people in California are exposed to more than average levels of solar radiation. Consequently, there is a higher risk of developing this disease. As a matter of fact, California is one of the five US states with the highest predicted incidence of new cases of melanoma. According to the California Cancer Registry, each year 4,700 new cases of invasive melanoma and over 800 deaths related to this disease are reported in California, with the incidence rate increasing by 15% over the last decade. While the white population is at the greatest risk of developing this disease, it was recently reported that the rates of invasive melanoma have risen substantially in Hispanic people living in California as well. If our proposal is successful, our work could pave the way to the development of a new and effective form of melanoma therapy, one which would clearly benefit all the people of California affected by this disease.
Progress Report: 
  • In this grant we proposed to genetically engineer human embryonic stem cells (hESC) and hematopoietic stem cells (HSC) and to use them to produce T cells with enhanced ability to kill melanoma cells. Our proposal consists of several steps. In the first year of the grant, we completed the first step and introduced the genes for a melanoma specific T cell receptor (TCR) into hESC and HSC. In this, second year of funding we were able to generate genetically modified T cells from hESC and HSC and to characterize the HSC-derived cells in more details. We found that HSC-derived T cells carrying the new TCR are indistinguishable from normal T cells, based on the cell surface expression of other T cell specific proteins. Also, we found that they can kill human melanoma cells in a Petri dish. We are currently evaluating their ability to destroy tumors in experimental animals transplanted with human melanoma cells. This is a more relevant approach as it mimics the potential treatment of melanoma patients. We are also trying to obtain larger numbers of the genetically modified hESC-derived T cells and analyze them in the same types of assays. Our data are encouraging and suggestive of possible clinical application of these cells in future.

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

Funding Type: 
Early Translational II
Grant Number: 
TR2-01789
ICOC Funds Committed: 
$3 341 758
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Cancer is the leading cause of death for individuals under 85. Relapse and metastatic disease are the leading causes of cancer related mortality. Anti-apoptotic BCL2 family member overexpression has been shown to promote disease progression in both chronic myeloid leukemia (CML) and prostate cancer. Andr., the emergence of cancer stem cells (CSC) promotes apoptosis resistance in the bone marrow metastatic microenvironment. While targeted therapy with BCR-ABL inhibitors has improved survival of patients with chronic phase CML, the prevalence has doubled since 2001 with over 22,000 people living with CML in the US in 2009. Unfortunately, a growing proportion of patients become intolerant or simply cannot afford full dose BCR-ABL inhibitor therapy and thus, progress to advanced phase disease with a 5 year survival rate of less than 30%. Although prostate cancer prevalence was high at 2.26 million in 2007, distant disease was relatively rare at 5%. However, like blast crisis CML, metastatic prostate cancer survival was only 30% over 5 years. 
Overexpression of B-cell lymphoma/leukemia-2 (BCL2) family genes has been observed in human blast crisis CML and advanced prostate cancer and may fuel CSC survival. Recent RNA sequencing data demonstrate that human CSC express a panoply of anti-apoptotic Bcl-2 isoforms in response to extrinsic signals in vivo, indicating that a pan BCL2 inhibitor will be required to abrogate CSC survival. Through binding and anti-tumor studies, a potent inhibitor of BCL2 pro-survival family proteins, BI-97C1, has been identified which inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl1-1 with nanomolar IC50 values. Notably, BI-97C1 potently inhibits growth of human prostate cancer in a xenograft model as well as blast crisis CML CSC engrafted in RAG2-/-c-/- mice while exerting minimal cytotoxicity toward bax-/-bak-/- cells. Because BI-97C1 inhibits all six anti-apoptotic Bcl-2 family members including Bcl-2, Mcl-1 (myeloid cell leukemia 1), Bcl-XL (BCL2L1), Bfl-1 (BCL-2A1), Bcl-W (BCL2L2) and Bcl-B (BCL2L10) proteins, with improved chemical, plasma and microsomal stability relative to apogossypol, we anticipate that it will have clinical utility for targeting apoptosis resistant human CSC in two malignancies with proven reliance on BCL2 signaling – blast crisis CML and advanced prostate cancer. 
Thus, anti-apoptotic BCL2 family member inhibition with BI-97C1 could represent a vital component of a potentially curative strategy for advanced malignancies that may obviate the need for costly continuous tyrosine kinase inhibitor therapy by increasing sensitivity to therapy. Elimination of CSC contributing to therapeutic resistance, the primary cause of cancer death, is of high clinical importance and thus, development of a small molecule pan-BCL2 inhibitor would fulfill a vital unmet medical need, fuel California biotechnology stem cell R&D efforts and decrease health care costs for patients with cancer.

Statement of Benefit to California: 
Cancer is the leading cause of death for individuals under 85 and usually results from metastatic disease in the setting of therapeutic recalcitrance. Anti-apoptotic BCL2 family member overexpression has been shown to promote disease progression in both chronic myeloid leukemia and prostate cancer. Moreover, the emergence of quiescent cancer stem cells promotes apoptosis resistance in the bone marrow niche for. While targeted BCR-ABL inhibition has resulted in improved survival of patients with chronic phase CML, the prevalence has doubled since 2001 with over 22,000 people living with CML in the US in 2009 (http://www.leukemia-lymphoma.org). Unfortunately, a growing proportion of patients become intolerant or simply cannot afford full dose BCR-ABL inhibitor therapy as a result of spiraling annual costs and thus, progress to advanced phase disease with a 5 year survival rate of less than 30%. Although prostate cancer prevalence was high at 2.26 million in 2007, distant disease was relatively rare at 5%. Like CML, metastatic prostate cancer survival was only 30% over 5 years (http://seer.cancer.gov/statfacts/html/prost.html#prevalence <http:> ). 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&amp;D efforts and decrease health care costs for patients with cancer.</http:>
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.

CD61-driven stemness program in epithelial cancer

Funding Type: 
Basic Biology V
Grant Number: 
RB5-06978
ICOC Funds Committed: 
$1 161 000
Disease Focus: 
Solid Tumor
Cancer
Stem Cell Use: 
Cancer Stem Cell
oldStatus: 
Closed
Public Abstract: 
Tumors contain a heterogeneous mix of cancer cells with distinct features, including subsets of particularly aggressive stem-like cells. Since a single cancer stem cell can self-renew, divide, and differentiate to reconstitute the heterogeneity of an entire tumor, the ability of one cell to evade therapy or surgical resection could lead to tumor re-growth and disease relapse. Few, if any, individual markers have been capable of identifying cancer stem cells among distinct tumor types. It is therefore remarkable that we have detected enrichment of CD61 on stem-like cells within tumor biopsies from many different drug-resistant samples of lung, breast, pancreatic, and brain tumors from mice or humans. CD61 promotes a stem-like reprogramming event, since ectopic expression CD61 induces stemness, including self-renewal, tumor-forming ability, and resistance to therapy. CD61 drives these behaviors by activating a signaling pathway which can be inhibited to reverse stemness and sensitize tumors to therapy. Our project is focused on learning how CD61 drives this cancer stem cell program, and how the increase in CD61 could be prevented or reversed. If successful, our work will provide valuable new insight into a cancer stem cell program that is unexpectedly shared among a variety of solid tumor types.
Statement of Benefit to California: 
The American Cancer Society estimates 171,330 new cancer cases will be diagnosed in California this year, a 10th of the national total. As part of an NCI-designated comprehensive cancer, we are uniquely positioned to translate our basic science research into clinical impact for the cancer patients within our community. From a clinical perspective, the understanding gained from our proposed studies will broadly benefit patients in California who will be diagnosed with an epithelial cancer this year, including 25,360 new breast cancer patients and 18,720 new lung cancer patients. Gaining fundamental insight into how these cancers are reprogrammed to become more stem cell-like as they acquire resistance to therapy will facilitate development of new strategies to prevent or reverse this behavior to benefit these large numbers of patients who live in California. In addition, our work will also yield new diagnostic tools that could identify which patients might respond to certain therapies. At the basic science level, our project will also serve to elucidate the mechanisms by which cancer stem cells contribute to cancer progression and response to therapy. During the course of our project, we will be able to train more people in California to work on this cutting-edge research, and to establish a foundation for the logical design of anti-cancer therapies targeting this unique cancer stem cell population.

Recombinant Bispecific Antibody Targeting Cancer Stem Cells for the Therapy of Glioblastoma

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05373
ICOC Funds Committed: 
$109 750
Disease Focus: 
Brain Cancer
Cancer
oldStatus: 
Closed
Public Abstract: 
Glioblastoma multiforme is the most prevalent and aggressive type of brain tumor, and devastating to any patient unfortunate enough to receive its diagnosis. As the most populous state in the nation, more Californians are diagnosed with glioblastoma multiforme than any other state. Over the past 20 years, surgery, radiation therapy and chemotherapy have been utilized with frustrating results. Today, even with the most advanced treatments available, survival rates average only 14-15 months. Our proposed research focuses on a new theory that brain tumor cells are initiated and maintained by a small fraction of cells with stem cell-like properties. This “cancer stem cell” hypothesis states that if this small subset of cancer stem cells could be eliminated then the tumor would cease to grow. Cancer stem cells in glioblastoma have been identified using CD133, a well known marker for isolating normal neural stem cells. The fact that CD133 is present on normal stem cells means that only targeting this molecule would be potentially dangerous. To enhance targeting, we reasoned that a cancer-specific alteration found in glioblastoma could be used as a potential marker for cancer stem cells. EGFRvIII is a specific variant of the normal EGF receptor and is widely found in glioblastoma but is rarely present in normal tissues. We have now shown that tumor cells that express both CD133 and EGFRvIII have the most cancer stem cell properties—more so than cells that have CD133 or EGFRvIII alone. We then developed a “bispecific” antibody that simultaneously recognizes both of these markers and we have shown that this bispecific selectively kills the cancer cells in glioblastoma tumors that express both CD133 and EGFRvIII. However, the bispecific did not kill normal stem cells. These results are very promising and suggest that bispecific can be tested as a therapeutic for glioblastoma. To move this into patients, we will produce large quantities of the bispecific and perform rigorous tests to ensure that it is uniform and has the required properties. We will also determine that it is safe through a combination of cell based and animal studies. Extensive planning will be made for the correct format for the clinical trial to test this molecule. Once the properties of the bispecific are certified and plans for the clinical trial are finalized, we will submit the drug to the FDA for an Investigational New Drug application. Once approved by the FDA, we can then move forward with testing this compound in glioblastoma patients. We are particularly excited about the bispecific as it could serve as the paradigm for a new class of drugs that specifically target cancer stem cells.
Statement of Benefit to California: 
Glioblastoma is a devastating diagnosis. The most common and malignant form of brain cancer, the most aggressive treatments currently available yield an average survival of only 14-15 months. As the most populous state in the nation, more Californians are diagnosed with glioblastoma each year than any other state, with a consequent significant economic toll to the state as well as its emotional toll. As the leader in cutting edge biomedical research, California through CIRM has recognized the unmet need to provide a roadmap for the translation of stem cell research to clinical applications. Through CIRM there is an unparalleled opportunity to foster clearly-defined discovery that will not only benefit Californians with glioblastomas, but potentially those with many other cancers, and ultimately all Californians, through healthier citizens, increased employment opportunities, and reduced economic burdens. We have previously shown that two markers of cancer stem cells, CD133 and EGFRvIII, are tightly associated in glioblastoma tumors. We created a recombinant bispecific antibody (BsAb) selectively targeting CD133 and EGFRvIII. This antibody selectively kills glioblastoma tumor cells but not healthy cells. When glioblastoma cells pre-treated with BsAb were injected into mice, tumor formation was significantly reduced, strongly suggesting that targeting of the EGFRvIII/CD133 cancer stem cell population can inhibit glioblastoma formation. The key objective of our project is to identify efficient and high yield methods for BsAb production, identify an effective dose and route of delivery for the treatment of brain tumors, and evaluate any potential effects on cells/tissues that express CD133. Our goal is to ready the BsAb for investigational new drug-related development. Californians will benefit from this research project in several significant ways. 1) Most importantly, this research has the promise to dramatically extend the long-term survival rates for Californians with glioblastomas, with potential applications to multiple other human cancers. 2) The research will take place in California with direct benefit to the California economy through the hiring of employees and purchase of supplies and reagents. 3) With successful completion of the proposed project, a clinical trial will be the direct next step, requiring additional employees along with associated expenditures. 4) If the therapeutic BsAb generated is commercialized, profits derived from the production of the BsAbs by CIRM policy will result in improved treatments to insured patients and lower cost treatments to the uninsured, thus ultimately benefiting all Californians. 5) Finally, funding this research will help raise awareness of California’s prominence as a national and international leader in stem cell research with the potential to benefit glioblastoma patients world-wide.
Progress Report: 
  • During the funding period, we were able to identify a project manager, Mauri Okamoto-Kearney, MBA who was then able to engage various consultants for all areas needed to write the Disease Team Proposal. We held various meetings with several CROs and CMOs to identify the best facility and processes for carrying out the manufacture and testing of the product. Following our fact gathering process, we used further personnel to write and assemble the final proposal.

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.

Mechanisms of Hematopoietic stem cell Specification and Self-Renewal

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

Stem Cells in Lung Cancer

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

Stem Cells for Immune System Regeneration to Fight Cancer

Funding Type: 
New Faculty II
Grant Number: 
RN2-00902
ICOC Funds Committed: 
$3 072 000
Disease Focus: 
Melanoma
Cancer
oldStatus: 
Active
Public Abstract: 
This proposal will define the biology of stem cell engineering to produce a cancer-fighting immune system. The immune system protects our body against most outside threats. However, it frequently fails to protect us from cancer. The T cell receptor (or TCR), a complex protein on the surface of an immune cell (or lymphocyte), allows to specifically recognize cancer cells. The TCR functions like a steering wheel for lymphocytes, allowing them to travel around the body and specifically find and attack cancercells. The goal of this research is to put TCR genes into stem cells to generate a renewable source of cancer-fighting lymphocytes. The studies in mice provide compelling evidence that inserting TCR genes into stem cells has several advantages for the progeny lymphocytes, allowing them to better fight cancer. The next step is to bring this approach to patients with cancer. The main reason is that the TCR genes inserted into stem cells allow the generation of a larger army of TCR re-directed cancer-fighting killer lymphocytes. I have dedicated most of my prior work to make the transition from studies in mice to the bedside. I have gaind the expertise to conduct clinical trials using cells as targeted drugs from patients. This experience has allowed me to design and start working on the clinical trials that will test the concept of inserting TCR genes into progenitors of lymphocytes and give them to patients. With my collaborators at other institutions, we have raised the adequate resources from private foundations and the NIH to initiate clinical trials inserting TCR genes into lymphocytes. I request additional funds from CIRM to allow me to extract the most information from the clinical trials and then help take them one step further by ultimately testing the use of hematopoietic stem cells (HSC) and induced pluripotent cells (iPS) to engineer a cancer-fighting immune system. There are several challenges tha need to be addressed, including what is the best approach to generate both immediate and long-term cancer fighting cells, what are the optimal stem cells to target, and how they should be manipulated and given to patients in the clinic. The study of samples obtained from patients participating in pilot clinical trials will provide information how to design new clinical trials using the method of inserting the cancer-specific TCR genes into stem cells. The experience of regenerating a cancer-fighting immune system in humans could then be applied to multiple cancer types and to infectious diseases that currently lack good treatment options.
Statement of Benefit to California: 
Preclinical studies have validated the concept that the immune system can be harnessed to fight cancer. However, clinical testing has failed short of expectations. I propose to genetically program the immune system starting from stem cells with the hope of advancing cancer immunotherapy. Malignant melanoma will be the cancer for the initial testing of this approach. Melanoma has a track record of being “immune-sensitive” and there are well-defined antigens against which the immune system can be targeted. Melanoma is the cancer with the fastest rising incidence in the U.S. This disease impacts heavily in our society, since it strikes adults at the prime years of life (30-60 years old). In fact, melanoma is the second cancer cause of lost of productive years given its incidence early in life and its high mortality once it becomes metastatic. The problem is particularly worrisome in areas of the world like California, with large populations of persons originally from other latitudes with much lower sun exposure and with skin types unable to handle the increased exposure to ultraviolet (UV) light in California. Although most frequent in young urban Caucasians, melanoma also strikes other ethnicities. The incidence of acral melanoma (non-UV light induced melanoma that develops in the palms and soles) has also steadily increased in Hispanics and Blacks over the past decades. Early melanoma can be cured with surgery. Therefore, programs aimed at early detection have the highest impact in this disease. Once it becomes metastatic, melanoma has no curative standard therapy. Despite this grim outlook, it has been long known that occasional patients participating in experimental immunotherapy protocols have long remissions and are seemingly cured. This proposal aims at incorporating the most current knowledge arising from preclinical research and prior clinical experimentation of immunotherapy strategies to engineer the immune system genetically to better fight metastatic melanoma. Bringing new science from the laboratory to the bedside requires well-designed, well-organized and informative clinical trials. It is not enough to show some responses, we need to understand how they develop and why some patients respond and other do not. Therefore, the analysis of stem cell-based immune system engineering within clinical trials proposed herein requires thorough analysis of patient-derived samples to inform the follow-up clinical testing. Information resulting from the genetic engineering of the immune system in patients with melanoma will help develop studies to direct the immune system to fight other cancers and infectious diseases like HIV. Once optimized, I envision the ability to clone T cell receptor (TCR) genes specific for tumor or infectious disease antigens expressed by different cancers or infectious agents, and use these TCRs to genetically program the patient’s immune system to attack them.
Progress Report: 
  • The awarded grant supports a patient-oriented research project to genetically engineer the human immune system to become cancer-targeted and provide benefit to patients with metastatic melanoma, a deadly form of skin cancer currently devoid of successful treatment options.
  • During the first funding period we initiated a clinical trial where patients with metastatic melanoma receive immune cells that have been re-directed by gene engineering techniques to become melanoma-specific. The immune cells are obtained from the patient’s own blood and they are manipulated in an in-house clinical grade facility for one week to insert into the cells two genes (T cell receptor or TCR genes) that turn them specific melanoma killer cells, called the. The genetic reprogramming of the immune system cells to express TCR genes is done using a crippled virus called a gene transfer vector. These cells undergo extensive testing to meet the standards of the Food and Drug Administration (FDA) before they can be given back to patients.
  • We give back the TCR re-directed immune cells to patients after receiving a chemotherapy preparative regimen to partially deplete their own immune system so the new cells have the ability to expand. In addition, the patients receive a treatment called high dose interleukin-2 (IL-2) to further allow these cells to expand. Furthermore, these patients receive three doses of dendritic cell vaccines, also generated from the patient’s own blood cells, which further helps the TCR re-directed immune cells to attack the melanoma lesions.
  • Seven patients have been enrolled onto this study at this time. Two patients are too early to evaluate and in the other patients we have early encouraging evidence of antitumor activity. We are conducting studies to determine how these cells behave in the patients by analyzing if they acquire ability to persist long term, what we call T memory stem cells. These are ongoing studies that will continue to the next funding period.
  • Finally, we have initiated the work to set up a follow up clinical trial where we will genetically modify patient’s blood stem cells, which we hypothesize will allow the continuous generation of TCR re-directed immune cells starting from the stem cells. This would provide means for immune system regeneration that would have applications to other cancers and non-cancer diseases like infectious diseases and autoimmune diseases. To this end, a new gene transfer vector has initiated clinical grade production to allow us to use it in the proposed next generation clinical trial.
  • The awarded grant supports a patient-oriented research project to genetically engineer the human immune system to become cancer-targeted and provide benefit to patients with metastatic melanoma, a deadly form of skin cancer currently devoid of successful treatment options.
  • During the second funding period we continued to conduct a clinical trial where patients with metastatic melanoma receive immune cells that have been re-directed by gene engineering techniques to become melanoma-specific. The immune cells are obtained from the patient’s own blood and they are manipulated in an in-house clinical grade facility for one week to insert into the cells two genes (T cell receptor or TCR genes) that turn them specific melanoma killer cells, called the. The genetic reprogramming of the immune system cells to express TCR genes is done using a crippled virus called a gene transfer vector. These cells undergo extensive testing to meet the standards of the Food and Drug Administration (FDA) before they can be given back to patients.
  • Ten patients have been enrolled onto this study at this time. In nine of them there has been evidence of tumor shrinkage, demonstrating the strong therapeutic activity of TCR redirected lymphocytes. However, these have been transient beneficial effects. Our ongoing studies point to a loss of function of the TCR transgenic cells over time. Therefore, it is of key importance to develop means to optimize the presence of long lasting memory cells. As proposed in the initial grant we are conducting studies to characterize the presence of T memory stem cells, which are cells able to self-replicate and maintain a cancer-fighting immune system for long periods of time. These are ongoing studies that will continue to the next funding period.
  • In addition, we have put a lot of work to set up a follow up clinical trial where we will genetically modify patient’s blood stem cells, which we hypothesize will allow the continuous generation of TCR re-directed immune cells starting from the stem cells. This would provide means for immune system regeneration that would have applications to other cancers and non-cancer diseases like infectious diseases and autoimmune diseases. To this end, we have generated new gene transfer vectors that are being studied for optimal function in relevant animal models to then allow an informed decision on the vector to take for clinical grade production and use it in the proposed next generation clinical trial.
  • The awarded grant supports patient-oriented research with the ultimate goal of reconstituting a cancer-fighting immune system. The research is conducted in samples obtained from patients with metastatic melanoma, a deadly form of skin cancer, and using preclinical models.
  • During the third funding period we have introduced modifications to enhance the ability of immune cell long term persistence within a clinical trial where patients with metastatic melanoma receive immune cells that have been re-directed by gene engineering techniques to become cancer-fighter cells. The immune cells are obtained from the patient’s own blood and they are manipulated in an in-house clinical grade facility for one week to insert into the cells two genes (T cell receptor or TCR genes) that turn them specific melanoma killer cells. The genetic reprogramming of the immune system cells to express TCR genes is done using a crippled virus called a gene transfer vector. These cells undergo extensive testing to meet the standards of the Food and Drug Administration (FDA) before they can be given back to patients.
  • When using a higher number of the TCR genetically engineered lymphocytes that are not frozen before their infusion to patients we are now detecting a higher ability of these cancer-fighting immune system cells to persist for long periods of time. This may be because the protocol modifications were guided to foster a higher ability to generate immune system cells that have long term memory and ability to self-renew (termed T memory stem cells). The detection of these cells is one of the research projects in this grant, since there is no defined set of markers for them. We have been testing several strategies to detect these cells and these are ongoing studies that will continue to the next funding period.
  • In addition, we have continued to move forward to set up a follow up clinical trial where we will genetically modify patient’s blood stem cells, which we hypothesize will allow the continuous generation of TCR re-directed immune cells starting from the stem cells. This would provide means for immune system regeneration that would have applications to other cancers and non-cancer diseases like infectious diseases and autoimmune diseases. To this end, we have tested the performance of two candidate gene transfer vectors for optimal function in humanized animal models. The results of these studies have demonstrated that one of the vectors is better suited for continued testing and it is the one that we plan to take into clinical grade production with the pre-IND activities being completed during the next funding period.
  • This grant proposed the conduct of pre-clinical work to support the use of stem cells to regenerate a cancer-fighting immune system in mice and humans, and bedside-to-bench work to analyze populations of cells with potential ability to function as long term repopulating T lymphocytes obtained from patients treated within a phase 1 clinical trial. During this past year we have made progress to continue our study the biology of T cells with characteristics of long term memory immune cells, termed T memory stem cells (TMSC). We have recently studied the ability to use specific small molecule targeted inhibitors to increase the fraction of mature T cells with TMSC characgteristics, which will improve our ability to characterize them. We have also advanced our studies to test the transplantation of hematopoietic stem cells (HSC) genetically engineered to express T cell receptors (TCR) and provide a continuous progeny of TCR transgenic mature T cells in humanized mouse models. This work provides the rationale to allow us advancing our plans to conduct a clinical trial based on the transplantation of HSC genetically engineered to express TCR to regenerate a cancer-fighting immune system. We have successfully competed for a CIRM disease team award and have gone through a pre-IND meeting with the FDA to adequately plan for such a clinical trial to be started in approximately two years.
  • We have continued to make progress to reach the proposed goals of this grant:
  • We have further characterized immune cells that naturally express three transcription factors that transform normal cells into pluripotent stem cells. We are interested in determining if these immune cells with pluripotency transcription factors are long term memory cells able to maintain immune responses.
  • In parallel, we have continued to advance our studies to bring a new approach to the clinic based on the genetic modification of blood stem cells to regenerate a cancer-fighting immune system. In the past year we have discussed our plans with the Food and Drug Administration and we have proceeded to follow their recommendations on what needs to be provided to open such a clinical trial. This clinical trial will be further developed within a newly approved CIRM disease team grant.

Generation of clinical grade human iPS cells

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00681
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Melanoma
Cancer
Muscular Dystrophy
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The therapeutic use of stem cells depends on the availability of pluripotent cells that are not limited by technical, ethical or immunological considerations. The goal of this proposal is to develop and bank safe and well-characterized patient-specific pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have the potential to develop into any cell type or tissue type in the body. Importantly, the generation of these cells does not require human embryos or human eggs. Since these cells can be derived directly from patients, they will be genetically identical to the patient, and cannot be rejected by the immune system. This concept opens the door to the generation of patient-specific stem cell lines with unlimited differentiation potential. While the current iPS cell technology enables us now to generate patient-specific stem cells, this technology has not yet been applied to derive disease-specific human stem cell lines for laboratory study. Importantly, these new cells are also not yet suitable for use in transplantation medicine. For example, the current method to make these cells uses retroviruses and genes that could generate tumors or other undesirable mutations in cells derived from iPS cells. Thus, in this proposal, we aim to improve the iPS cell reprogramming method, to make these cells safer for future use in transplant medicine. We will also generate a large number of iPS lines of different genetic or disease backgrounds, to allow us to characterize these cells for function and as targets to study new therapeutic approaches for various diseases. Lastly, we will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases.
Statement of Benefit to California: 
Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have, similar to embryonic stem cells, the potential to develop into any cell type or tissue type in the body. This new technology holds great promise for patient-specific stem-cell based therapies, the production of in vitro models for human disease, and is thought to provide the opportunity to perform experiments in human cells that were not previously possible, such as screening for compounds that inhibit or reverse disease progression. The advantage of using iPS cells for transplantation medicine would be that the patient’s own cells would be reprogrammed to an embryonic stem cell state and therefore, when transplanted back into the patient, the cells would not be attacked and destroyed by the body's immune system. Importantly, these new cells are not yet suitable for use in transplantation medicine or studies of human diseases, as their derivation results in permanent genetic changes, and their differentiation potential has not been fully studied. The goal of this proposal is to develop and bank genetically unmodified and well-characterized iPS cell lines of different genetic or disease backgrounds that can be used to characterize these cells for function and as targets to study new therapeutic approaches for various human diseases. We will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases. Taken together, this would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from such research. Californians will also benefit greatly as these studies should speed the transition of iPS cells to clinical use, allowing faster development of stem cell-based therapies.
Progress Report: 
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases, and that are not limited by technical, ethical or immunological considerations. To that end, we proposed to establish protocols for generation of human induced pluripotent stem cells (hiPSC) that would not involve viral vector integration, and that would be compatible with Good Manufacturing Processes (GMP) standards. To establish baseline characteristics of hiPSCs, we performed a complete molecular characterization of all existing hiPSCs in comparison to human embryonic stem cells (hESCs). We found that all hiPSC lines created to date, regardless of the method by which they were reprogrammed, shared a common gene expression signature, distinct from that of hESCs. The functional role of this gene expression signature is still unclear, but any lines that are generated under the guise of this grant will be subjected to a similar analysis to set the framework by which these new lines are functionally characterized. Our efforts to develop new strategies for the production of safe iPS cells have yielded many new cell lines generated by various techniques, all of which are safer than the standard retroviral protocol. We are currently expanding many of the hiPSCs lines generated and will soon demonstrate whether their gene expression profile, differentiation capability, and genomic stability make them suitable for banking in our iPSC core facility. Once fully characterized, these cells will be available from our bank for other investigators.
  • For hiPSC technology to be useful clinically, the procedures to derive these cells must be robust enough that iPSC can be obtained from the majority of donors. To determine the versatility of generation of iPS cells, we have now derived hiPSCs from commercially obtained fibroblasts derived from people of different ages (newborn through 66 years old) as well as from different races (Caucasian and mixed race). We are currently evaluating medium preparations that will be suitable for GMP-level use. Future work will ascertain the best current system for obtaining hiPSC, and establish GMP-compliant methodologies.
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. To speed this process, we are taking approaches that are not limited by technical, ethical or immunological considerations. We are establishing protocols for generation of human induced pluripotent stem cells (hiPSCs) that would not involve viral vector integration, and that are compatible with Good Manufacturing Practices (GMP) standards. Our efforts to develop new strategies for the production of safe hiPSC have yielded many new cell lines generated by various techniques. We are characterizing these lines molecularly, and have found hiPSCs can be made that are nearly indistinguishable from human embryonic stem cells (hESC). We have also recently found in all the hiPSCs generated from female fibroblasts, none reactivated the X chromosome. This finding has opened a new frontier in the study and potential treatment of X-linked diseases. We are currently optimizing protocols to generate hiPSC lines that are derived, reprogrammed and differentiated in the absence of animal cell products, and preparing detailed standard operating procedures that will ready this technology for clinical utility.
  • This project was designed to generate protocols whereby human induced pluripotent stem cells could be generated in a manner consistent with use in clinical trials. This required optimization of protocols and generation of standard operating procedures such that animal products were not involved in generation and growth of the cells. We have successfully identified such a protocol as a resource to facilitate widespread adoption of these practices.

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