Targeting Cancer Stem Cells for Immunotherapy

Funding Type: 
New Faculty II
Grant Number: 
RN2-00923
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Glioblastoma multiforme (GBM) is an aggressive, highly malignant type of brain tumor that resists all treatments developed and is highly lethal. Almost all patients with GBM die within one year after diagnosis. The proposed research seeks to develop a novel therapeutic approach to treating GBM and perhaps other malignant brain tumors by targeting particular types of stem cells that are believed to generate and/or repopulate tumors. Currently, there is no cure for GBM, and the prognosis is grim. Some patients respond differently to current common treatments for brain tumors, including chemotherapy and radiation therapy. However, the tumor virtually always grows back, even after complete surgical resection. Recent studies suggest that the reason may be that cancer stem cells (CSC) remain scattered in the brain, and are capable of completely repopulating the tumor even after treatments and surgical excision. The experiments proposed in this application may provide answers to these problems, and seek to address the basic mechanisms of cancer stem cells, how they respond to treatment, and how they might be targeted for destruction using the body’s natural defenses. In our proposed studies, we will first determine information about how CSC respond to chemotherapy in cell culture conditions. Next, we will determine how implanted tumors in mice respond to those same drugs, and evaluate proportions of CSC at baseline, and after treatment. We believe drug treatments will increase the number of CSC, because the drugs will preferentially kill non-CSC tumor cells, and therefore leave behind an enriched population of CSC that are highly resistant to treatment. Next we will directly manipulate the proportion of CSC in implanted tumors in mice. We will treat these mice with a vaccine therapy we have been developing that uses the tumor tissue to activate the immune system. Specifically, we take tumor tissue and treat dendritic cells (DCs) with that tumor tissue. DCs then activate cytotoxic T cells that kill tumor cells. However, thus far our vaccine has only shown limited success; we believe the reason is because CSC resist this therapy also. Therefore, we will attempt to more precisely target CSC by activating the immune system with dead CSC material. We will vary the proportion of CSC in the tumor material that we use to treat DCs with. Then we will test how effective this is in mice that have implanted tumors where we again control the proportion of CSC. If this proves only partially successful, we may attempt to combine vaccination treatment with chemotherapy, in the hopes that both treatments together can overwhelm the CSC and eradicate the tumors entirely. If successful, we may generate an entirely new way to treat GBM, and possibly one day other types of brain tumors also.
Statement of Benefit to California: 
The 2006 Central Brain Tumor Registry of the United States reported that the incidence rate in the state of California for primary malignant brain tumors and other CNS tumors is approximately 6 per 100,000 California residents every year. Malignant brain tumors like glioblastoma multiforme (GBM) have a devastating prognosis, and no cure or effective treatment has been developed to date. Recent studies suggest that GBM brain tumors may be derived from cancer stem cells (CSC), and that these CSC are resistant to common therapies including chemotherapy and radiation therapy. Cancer stem cells are thought to be responsible for tumor recurrence after therapy, and that could be a reason why treatments are ineffective. We propose that targeting these cancer stem cells using immunologic ways to destroy them may consititute a new way to treat GBM, and/or may improve the efficacy of current therapies. The proposed research will test that notion. Our research will provide novel information regarding the function of CSC and could generate an entirely new way to treat GBM. The research proposed in this application will benefit the people of California by potentially identifying a novel way of treating brain tumors, which will have a direct benefit for the patient and their families, and could also lower the financial burden associated with this devastating disease.
Progress Report: 
  • The world of small non-coding RNAs (ncRNA) is continuously expanding, reinforcing the biological importance of these species in both development and disease. Over the past year, our efforts funded by CIRM has been focused on studying the roles of these small ncRNAs in regulating stem cell self-renewal and differentiation. miRNAs are a class of novel, small ncRNAs that negatively regulate global gene expression at posttranscriptional level. Using expression studies, we have characterized the miRNA expression profiles in both ES cells and induced pluripotent stem cells (iPS cells). This effort led to the identification of multiple miRNAs whose levels of expression are either enriched or depleted during stem cell reprogramming. A key finding of the previous funding period is the identification of a novel miRNA, Esdmir-1, whose loss-of-function significantly promotes the reprogramming of iPS cells. This is an important finding, not only does it set up a paradigm for our future studies, it also provides an attractive methodology to improve iPS reprogramming in human. Our future effort in the next funding period (year 2) will be focused on completing the studies on Esdmir-1, evaluating the functions of additional candidate miRNAs in stem cell self-renewal and differentiation and identifying novel ncRNAs that regulate stem cell biology.
  • The world of small non-coding RNAs (ncRNA) is continuously expanding, reinforcing the biological importance of these species in both development and disease. Over the past year, our efforts funded by CIRM has been focused on studying the roles of these small ncRNAs in regulating stem cell self-renewal and differentiation. miRNAs are a class of novel, small ncRNAs that negatively regulate global gene expression at posttranscriptional level. Using expression studies, we have characterized the miRNA expression profiles in both ES cells and induced pluripotent stem cells (iPS cells). This effort led to the identification of multiple miRNAs whose levels of expression are either enriched or depleted during stem cell reprogramming. A key finding of the previous funding period is the identification of a family of novel miRNAs that promote differentiation in pluripotent stem cells. This is an important finding because we demonstrated that repression of these miRNAs significantly enhances reprogramming efficiency. miRNAs functions can be modulated by transient transfection of oligonucleotide based antagonist, therefore, our findings are likely to lead to an approach to greatly promote iPSC generation in clinical application. Our future effort in the next funding period (year 3) will be focused on functional characterizations of additional miRNAs in stem cell self-renewal and differentiation and identifying novel ncRNAs that regulate stem cell biology.
  • So far, our CIRM funded project have provided strong evidence suggesting miRNAs as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells, thus playing an important role in the generation of induced pluripotent stem cells (iPSCs). Several major progresses have been made for year 3. In short, we have carefully characterized the roles of miR-34 miRNAs in somatic reprogramming and indicated the importance of miR-34 inhibition in promoting stem cell self-renewal and somatic reprogramming. In addition, we completed two functional screens to identify miRNAs whose overexpression or inhibition regulates ES cell self-renewal and ES cell differentiation. Functional validation has been performed for majority of the hits from the screen. In particular, we have identified a miRNA, miR-meso, which specifically promotes mesoderm differentiation and represses ectoderm differentiation. Finally, using recombineering technology, we have constructed a BAC with Flag-tagged Lin28 at its endogenous locus. We have also constructed a piggy bac vector that allows us to generate stably integrated ES cell line that expresses Flag-tagged LIN28 for identification of novel LIN28 bound non-coding RNAs. These progresses significantly improved our understanding on the roles of non-coding RNAs in regulating stem cell self-renewal and pluripotency, and may lead to novel strategy for generating completely pluripotent human stem cells for clinical applications.
  • In 2011, a portion of our results funded by the CIRM project were published in Nature Cell Biology as a cover story, and we also filed a patent application reporting a novel strategy to increase somatic reprogramming efficiency.
  • Our CIRM funded project have provided strong evidence suggesting microRNAs (miRNA), and in a broader perspective, non-coding RNAs, as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells. Although non-coding RNAs, including miRNAs, do not have protein coding capacity, but they possess strong effects in regulating Using genomic approaches, embryonic stem cell culture and induced pluripotent stem cell culture and molecular biology, we were able to screen for, identify and characterize a number of non-coding RNAs, particularly, microRNAs, as important regulators for stem cell self-renewal and differentiation.Our results can have profound implications on the role of ncRNAs in the generation of induced pluripotent stem cells (iPSCs). Several major progresses have been made for the year 4 of our CIRM funded project. .
  • Last year, we have successfully carried out a functional screen to identify miRNAs with important roles in regulating ES cell self-renewal. Among the miRNA candidates emerged from this screen, we primarily focused on the functional and mechanistic characterization of several important miRNA regulators for ES cell pluripotency. We investigated roles of miR-34 miRNAs, whose deficiency significantly promotes somatic reprogramming. We studied the role of miR-34 in the epigenetic remodeling during somatic reprogramming. We also studied the function of mir-290-295 cluster, which constitute the majority of miRNA species in the ES cells. We generated ES cell deficient for the mir-290-295 miRNA cluster to characterize its effects on ES cell self-renewal, and we also explored the functional redundancy of the mir-290-295 family miRNAs by functionally characterized related miRNA family, including the mir17-92 miRNAs.
  • In the previous funding period, we also carried out a screen to identify miRNAs or non-coding RNA with important roles in regulating ES cell differentiation. Here, we functionally characterized the effects of miR-meso in mesoderm differentiation both in vitro and in vivo, using teratoma assays and KO ES cells. In addition, we identified a long ncRNA that exhibit an interesting pattern of expression during ES cell differentiation. We characterized its unique expression alteration during ES cell differentiation, and we are currently generating the knockout ES cells for this long ncRNA to further investigate its function. Finally, we characterized miRNA expression profiles during ES cell to Epiblast stem cell differentiation, and identified additional candidate miRNAs that regulate the exit of pluripotency of ES cells during their differentiation.
  • Finally, we successfully established the biological system to generate human iPSCs from human dermal fibroblasts, and will use this system to explore the role of specific ncRNAs in human somatic reprogramming. Our previous results using mouse ES cells and iPSCs have prepared us well in exploring the significance of our finding in human. This will be a main focus to achieve in our last funding period.
  • Our CIRM funded project have provided strong evidence suggesting miRNAs, and in a broader perspective, non-coding RNAs, as essential gene regulators in the self-renewal and pluripotency of embryonic stem cells. Consistently, these ncRNAs play an important role in the generation of induced pluripotent stem cells (iPSCs). Using expression studies, functional screening and candidate approaches, we have identified and characterized the roles of multiple miRNAs and long ncRNAs (lncRNAs) in regulating the self-renewal and differentiation of pluripotent stem cells. These non-coding RNAs are essential component of a regulatory network that regulate stem cell cell fate potential, self-renewal and differentiation potential.

© 2013 California Institute for Regenerative Medicine