Human development from the egg to the fully-grown adult is an epigenetic process. That is, with few exceptions, development does not involve changes to the structure of the gene (or DNA sequence). Importantly, epigenetic processes are reversible as shown by the ability to de-differentiate a mature adult cell back to an early embryonic cell. De-differentiation has now been accomplished by several mechanisms including somatic cell nuclear transfer (SCNT), fusion of adult cells to embryonic cells, and the transduction of virally expressed factors. All of these methods of de-differentiatiton turn back the clock, epigenetically speaking, from an adult cell to that of an embryonic cell.
Epigenetics also plays an important role in disease. For example, there is good evidence that deviant epigenetic modifications promote and maintain cancers. Therefore, it may be possible suppress cancer by reversing these modifications. Support for this comes from the success of ongoing clinical trials using drugs that can suppress proteins that direct these modifications. Furthermore we have used SCNT to globally reset the epigenetics of tumor cells, analogous to resetting the state of the mature adult to the embryonic stem cell. Amazingly, following SCNT, the nucleus from the former cancer cell was able to produce functional adult tissues. This finding strongly suggests that resetting the epigenetic state of tumor cells can partially, if not fully suppress further tumor growth.
There were two limitations to the SCNT studies. First, the work was limited to the mouse model system. Second, it remained unclear which of the many epigenetic modifications that were reset by SCNT were originally responsible for supporting growth of the cancer. In this grant, we propose to address these two limitations by extending our work to humans and by following the epigenetic events at a more detailed level. We will first optimize SCNT in rhesus macaques, a nonhuman primate model. Next we propose to translate our experience with the rhesus macaque SCNT to human SCNT. We also propose alternative techniques to SCNT in case we find SCNT infeasible. We will use these techniques to reprogram the epigenetics of human cancers. We will then evaluate how this resetting of epigenetics influences cancer growth. Finally, we will aim to identify which are the sentinel epigenetic events supporting tumor growth.
The tools we develop should be broadly applicable to the study of epigenetics in human disease. Furthermore, identification of the sentinel epigenetic modifications in cancer should identify excellent targets for cancer therapeutics.
Statement of Benefit to California:
This grant proposes to develop methods for reprogramming cancer cells into embryonic stem cells. The techniques developed through these experiments should be broadly applicable to the production of disease specific embryonic stem cell lines. The production of such lines will provide an unlimited source of cells to study the biology, genetics, and epigenetics of disease. Furthermore the cells could be used in drug screens for identification of novel therapeutics.
This grant also proposes to identify and follow epigenetic aberrations that lead to tumor formation and maintenance. Knowledge of such aberrations would provide targets that are reversible by nature and hence potentially very promising drug targets for therapeutic development.
Such advances could have a significant impact on the public health and the economy of the State of California. For example, the development of drugs that specifically target aberrant epigenetic events could alter tumor growth without the harmful side effects associated with most current cancer therapeutics. Such a drug would be a dramatic improvement in the care of the cancer, the second leading cause of death in the United States.
SYNOPSIS: The Principal Investigator (PI), an assistant professor of Pathology at the University of California San Francisco, wishes to understand how epigenetic modifications regulate stem cell differentiation and tumor formation. His prior work with Dr. Jaenisch focused on the reversibility of the cancer phenotype through the use of nuclear transplantation. Those studies had been performed in the mouse. He now proposes to do Somatic Cell Nuclear Transfer (SCNT) in a non-human primate in order to prepare to do SCNT reprogramming of human cancers. In addition he will utilize the 4 factor reprogramming cocktail of Dr. Yamanaka to attempt to reprogram human cancer cell lines. If a substantial portion of the epigenetic contribution to cancer is demonstrated, the option will then be open to use modulation of epigenetics to treat cancers. He describes a modification of the Yamanaka somatic reprogramming approach in which he does not select for reprogrammed cells but merely inspects. For epigenetic preclinical studies, he proposes to use a mouse APML model with which to perform SCNT.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The question about the reversibility of the cancer phenotype by epigenetic modification is important. It is to be anticipated however that in many (or most) epithelial cancers there are multiple somatic mutations--hence, the ultimate impact of the research on therapeutics is unknown, though worth pursuing. The research plan is solid only if there were preliminary data to back it up. The first goal of Aim 1 is to improve SCNT in non-human primates is challenging and high risk, but might greatly assist attempts to perform SCNT with human material. The approaches described are feasible and within the range of expertise of the PI, who has lined up suitable collaborators for his research. Aim 1 seems to have started, but no data is shown - only a few morula-stage SCNT embryos have been produced. The PI does not adequately justify these experiments in terms of what specific biological questions require the further development of ES cell lines from Rhesus macaques, and the aim as written is entirely technical with no follow-up. The second part of this aim is to develop new ES cell lines by transfer of human somatic nuclei into non-human primate oocytes. The justification for this technically and biologically risky approach seems suspect. An unpredictable biology may occur with an uncertain contribution of the non-human cytoplasm and especially the mitochondria to cellular phenotypes, which will preclude their serious use as disease models.
The second aim is to ask whether human cancer cell nuclei can be reprogrammed by SCNT into human oocytes. As proposed, this aim is totally dependent on aim 1 and since aim one’s feasibility at this point is low, the level of enthusiasm is not high. The basic question being asked here has already been answered in mice, and the answer is sometimes yes and sometimes no. The experiments proposed here don’t go beyond that very descriptive approach and therefore will not result in a significant advance, even if successful. Moreover, the methods that will be used to assess whether the transferred nuclei remain tumorigenic are unclear and apparently superficial. The PI merely says he will look for dysplasia or an altered nuclear/cytoplasmic ratio in in vitro cultures. Its hard to see what can be learned from this. The alternative approach, making cell fusions between human ES cells and human cancer cells is very poorly developed. How will tumorigenicity be evaluated in these tetraploid or binucleate cells? The PI seems to overlook the possibility of complementation of tumor suppressor mutations, which has nothing to do with epigenetic reprogramming.
The third aim is the best developed of the three. Here the PI will use the four transcription factor approach to ask whether APML cells can be reprogrammed to pluripotency. He will test their pluripotency in vivo in chimeric mice. Moreover, the oncogene responsible for APML is known to interact with many epigenetic modifying enzymes and specific epigenetic changes have been identified in APML’s. Thus, this system affords the opportunity to ask whether those epigenetic changes are reversed during reprogramming. While aim 3 is possible to complete, there are certain pitfalls, like using myc to reprogram cancer cells, that can make the interpretation of the data very difficult. Also, the contribution of these epigenetic changes to the tumorigenic phenotype is not known, and there are no experiments here to pursue that question. In fact, the PI mentions that the APML reprogramming model may not be the most suitable form of cancer in which to test his hypotheses.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI is a superbly trained physician-scientist pathologist who is addressing multiple aspects of stem cell biology in his research. The PI earned his MD and PhD degrees from Wisconsin-Madison and did his postdoc, until 2005 in the Jaenisch lab at the Whitehead. He was moderately productive as a postdoc, publishing an important co-first author paper in G&D on the reprogramming of a mouse melanoma cell line by nuclear transplantation. He became an Assistant Professor in the Department of Urology at UCSF in 2005 and a member of their stem cell biology program. Beyond this proposal he is also engaged in study of miRNAs, particularly with respect to neural stem cell differentiation. He has an excellent publication record and has already obtained two RO1 grants to study neural development and the role of miRNAs in ES cells, and is a Pew Scholar. The career development plan is excellent.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: UCSF and the department of Pathology is highly committed to this new investigator. He has all the requisite facilities available to him at UCSF and appropriate local experts in hES cells to assist in his work. The PI occupies 1000 square feet of independent lab space within the UCSF human embryonic stem cell center, and is adjacent to a CIRM-funded ES cell training center. He has received a generous start-up package from UCSF and as well as generous ongoing support in terms of salary, research funds and seed grants. His colleagues and mentors are outstanding, and UCSF has an outstanding record of training top academic investigators.
DISCUSSION: Reviewers concurred that this is a situation in which the proposal is not as strong as the investigator. The research plan is solid, only if there are preliminary data to back it up. The major concern is that it is not clear that nuclear transfer in primates will work; this area of study is not easy, and it is not easy to obtain blastocysts with normal cells. Reviewers wanted to see some evidence from this lab that they can produce blastocysts. Reprogramming has been done before, by the same investigator in mice, and the issue is what biological questions will be answered by work in monkeys? Another caution is that cancer stem cells have many mutations, and regulation may not be a purely epigenetic phenomenon. It was noted that the PI is Assistant Professor at UCSF with epigenetics expertise, but one reviewer asked, how much of the proposal is really focussed on epigenetics? While there are some new technical innovations and the idea is good (especially Aim 3), the enthusiasm was dampened by the biological risk associated with insufficient preliminary data.