Human embryonic stem cells (hESC) hold great promise as sources of tissue for regenerative medicine and therapeutics. In addition, their utility as tools to study the origins and biology of human disease must not be underestimated. hESC give rise to tissue-specific adult stem cells and, ultimately, to all mature tissues in the body. As such, disruptions to normal stem cell function can have catastrophic consequences and result in life-threatening or debilitating disease. Understanding how such diseases arise will afford novel insights into how we can better prevent and treat them. Laboratory based studies with hESC therefore stand to make extraordinary contributions to human health.
Human tumors, and in particular the cancers that affect children, often look like tissues that have not developed normally and whose growth has gone unchecked. In fact, recent studies have shown that, in many cases, tumors arise because genetic mutations in the DNA of normal stem cells lead to disordered development, resulting in formation of malignant rather than normal tissues. For example, leukemia can arise when a mutation occurs in a normal blood stem cell, thus inducing formation of cancerous rather than normal blood. Analogous situations exist in other human tissues and their respective tumors. However, because of the relative rarity of normal stem cells in other parts of the body and our inability to effectively isolate them, very little is yet known about how these stem cells go awry and create cancer. hESC, therefore, represent an invaluable resource for the generation of tissue-specific stem cells and for studies of the genesis of human, and in particular, pediatric cancer.
Several different human cancers are believed to arise either directly or indirectly from stem cells called neural crest stem cells (NCSC). NCSC exist in small numbers throughout the body and contribute to the formation of multiple different tissues including the peripheral nervous system and the pigment cells of our skin. It is our central hypothesis that NCSC-derived tumors arise because genetic mutations in NCSC lead to disordered tissue development and the initiation of cancer.
Ewing’s sarcoma family tumors (ESFT) are highly aggressive tumors that primarily affect children and young adults. ESFT have a specific mutation in their DNA and this mutation leads to the creation of a cancer-causing gene. We believe that expression of this abnormal gene in NCSC disrupts normal stem cell differentiation and development and, ultimately, leads to ESFT formation. In this proposal we will use hESC as tools to prove or disprove this theory.
Unfortunately, despite highly toxic and aggressive treatment, the survival rate for patients diagnosed with ESFT remains poor. By creating novel hESC-based models to study the origin and biology of ESFT we aim to gain novel insights into the origin and biology of these tumors that will aid in the development of more effective, less toxic therapies.
Human embryonic stem cells (hESC) represent a tremendous resource as tools to study numerous human diseases, including cancer. Cancer claims the lives of over 50,000 Californians, including over 300 children, annually. Laboratory based studies using hESC, such as those proposed in this application, stand to make extraordinary and unique contributions to our understanding of the origin and biology of human cancer. These contributions will ultimately aid in the development of novel therapeutic strategies designed to improve survival and quality of life of cancer patients.
In this proposal we will exploit the power of hESC to study the cellular origins of sarcomas. Sarcomas arise in the bones and soft tissues and primarily affect children and young adults. Despite intensive therapy, the survival rate of patients diagnosed with sarcoma remains poor. The proposed research will provide much needed insight into sarcoma biology and will enable development of novel sarcoma-targeted therapies. In addition, the hESC-derived models that we establish will be readily adaptable to and available for studies of other human cancers.
SYNOPSIS: Several highly aggressive human cancers, including melanoma, neuroblastoma and peripheral primitive neuroectodermal tumors—also known as Ewing’s sarcoma family tumors (ESFT)— are believed to be of neural crest origin. All ESFT express characteristic fusion oncogenes, most commonly EWS-FLI1. This proposal will test the hypothesis that expression of this oncogene in neural crest stem cells initiates Ewing’s Sarcoma by disrupting the normal pathway of neural crest differentiation. In Aim 1 the PI will use published protocols to induce human ES cells to undergo neural crest differentiation, and will develop tools to purify those cells. In Aim 2 hES and NC cells will be transduced with the EWS-FLI1 oncogene. The PI proposes to assess the effect of the oncogene on differentiation in vitro, and on tumorigenicity in vivo. Gene expression profiling will be used to determine the extent to which the resulting tumors resemble Ewing Sarcoma’s from pediatric patients.
INNOVATION AND SIGNIFICANCE: Studies on neural crest biology have been limited almost entirely to model organisms. These studies represent a pathway for generating tools to study human neural crest development and tumorigenesis. The goal of deriving neural crest stem cells (NCSCs) from hES cells, and then examining their response to an oncogene that causes Ewings sarcoma is novel and important. The PI makes a good case that NCSCs with physiological properties are unlikely to become available, except through derivation from hES cells. Therefore the general approach taken in this grant is right on the mark. Furthermore, one of the great values of hES cells lies in their ability to model disease. The goal of using hES-derived neural crest cells to study the origin and development of Ewing's sarcoma is significant and worthwhile and would also be of benefit for the development of new therapeutic agents.
STRENGTHS: The experiments described here offer a new opportunity for understanding the oncogenic mechanism of the EWS-FLI1 fusion oncogene, which remains poorly understood. The PI presents a strong argument that ESFT arise from the expression of this oncogene in progenitor cells, specifically those in the neural crest lineage. Whereas expression of EWS-FLI1 is lethal in most normal cells, progenitor cells that also express Bmi-1 are tolerant of EWS-FLI1 and indeed the two genes cooperate to cause oncogenic transformation.
The approach of deriving NCSC from hESC is a good approach and the assays proposed to isolate and identify NCSCs are the right ones, short of more extensive in vivo analyses
The PI and colleagues are highly qualified to perform the proposed experiments, the PI having assembled a team of scientists with complementary expertise in neural crest biology, in the biology and clinical oncology of neural crest-derived tumors, and in human stem cell biology. In Aim 2 the PI proposes to use gene expression profiling to determine the extent to which tumors generated after EWS-FLI1 expression in human stem cells resemble ESFT from pediatric patients. This will be greatly facilitated by ongoing NIH funded work in the PI’s laboratory to generate gene expression datasets from clinical ESFT samples.
WEAKNESSES: The PI is not yet established as a leader in this area, and has few publications since 1998. Nonetheless, the proposal suggests she is on the right track.
A minor consideration is that the PI of this grant is already performing virtually identical experiments in murine stem cells, and is fully funded for that work. The applicant states that the purpose of this new proposal to CIRM is to translate that work into humans, but this seems premature. It could be argued that it would make more sense to wait to see if the murine work is successful rather than trying to run the redundant programs concurrently.
Two issues that the PI should think about (if hasn't already) are the following.
1. It is possible that the ability to tolerate EWS-FL1 expression is caused by other mutations, such as deletion of Ink4a, Arf, or p53, rather than Bmi-1 expression or stem cell identity. Are any of these genes normally deleted in Ewing's sarcoma? These experiments might work a lot better if done in Ink4a/Arf or p53 deficient cells. The reviewer realizes that such hES cells are not yet available and offers this as a future option to think about, not as a criticism of this proposal.
2. It might work better to infect NCSCs with the EWS-FL1 lentivirus after derivation from hES cells, rather than infecting the hES cell first.
DISSCUSSION: The question was raised as to what are the in vivo assays for generating neural crest cells. Answer was transplantation into chick embryos; this was then followed by question as to whether this can be done with human cells. The reviewers did not know whether this had been done or would work with human cells but noted that thus far, those cells which differentiate and function in the in vitro assay have worked in the in vivo assay.
PROGRAMMATIC REVIEW: The Working Group voted to recommend this application for special consideration for funding if additional funds become available based on: 1) the applicant is a young investigator with a lot of potential and 2) the proposal is for a stem cell model of childhood cancer that was scientifically well regarded.