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RN1-00561-1: Combinatorial Chemistry Approaches to Develop LIgands against Leukemia Stem Cells
Recommendation: Recommended for funding
Scientific Score: 94
First Year Funds Requested: $477,287
Total Funds Requested: $2,392,397
Public Abstract (provided by applicant)
Various cells and organs in the human body originate from a small group of primitive cells called stem cells. Human cancer cells were also recently found to arise from a group of special stem cells, called cancer stem cells (CSCs). At present, cancer that has spread throughout the body (metastasized) is difficult to treat, and survival rates are low. One major reason for therapeutic failure is that CSCs are relatively resistant to current cancer treatments. Although most mature cancer cells are killed by treatment, resistant CSCs will survive to regenerate additional cancer cells and cause a recurrence of cancer. As opposed to other human stem cells, CSCs have their own unique molecules on their cell surface. This project aims to develop agents that specifically target the unique cell surface molecules of CSCs. These agents will have the potential to eradicate cancer from the very root, i.e., from the stem cells (CSCs) that produce mature cancer cells. In this project, we will develop agents that specifically target leukemia stem cells to determine the feasibility of our approach. Leukemia is the fourth most common cause of cancer death in males and the fifth in females. If our approach is successful, we can use the same approach for other cancer types. To develop these specific agents, we will screen a library of billions of molecules to identify those that specifically target the unique cell surface molecules of leukemia stem cells (LSCs). After we identify these specific molecules, we will optimize their structure to increase their specific binding to LSCs. Specific binding to LSCs is crucial, as the optimized molecules will be able to uniquely kill LSCs and spare normal blood cells.
Many leukemia patients need stem cell transplantation during treatment. There are two approaches to harvesting stem cells for transplantation: those harvested from patients themselves and those harvested from healthy donors. Stem cells harvested from healthy donors need to genetically match patients’ cells. Otherwise, these transplanted cells from the donor recognize the recipient’s (host or patient) cells as non-self cells and attack these cells. This response leads to a serious disease called graft-versus-host disease (GVHD). It is often difficult to find matched donors. Stem cells harvested from patients are usually not used for the treatment of acute leukemia because they are contaminated with LSCs that will lead to recurrence of leukemia after transplantation. If this project is successful, the targeting agents developed in this project can be used to eliminate the contaminating LSCs and decrease the leukemia recurrence after transplantation.
Statement of Benefit to California (provided by applicant)
Acute leukemia is the sixth most common cause of cancer death in males and females in California. The outcome for acute leukemia is poor and over 70% of patients will die from this disease. This project aims to develop therapeutic agents that specifically target leukemia stem cells and therefore eradicate leukemia from its root. These agents can also be used for stem cell transplantation. Many leukemia patients need stem cell transplantation during treatment. There are two approaches to harvesting stem cells for transplantation: those harvested from patients themselves and those harvested from healthy donors. Stem cells harvested from healthy donors need to genetically match patients’ cells. Otherwise, these transplanted cells from the donor recognize the recipient’s (host or patient) cells as non-self cells and attack these cells. This response leads to a serious disease called graft-versus-host disease (GVHD). It is often difficult to find matched donors. This is especially true in California because of the genetically diversified population. Stem cells harvested from patients are usually not used because they are contaminated with leukemia stem cells that will lead to recurrence of leukemia after transplantation. If this project is successful, the targeting agents developed in this project can be used to eliminate the contaminated leukemia cells and decrease the likelihood of leukemia recurrence after transplantation.
The ligands developed in this project can be used for targeted therapy for leukemia. Since no such ligands have been identified so far that specifically target leukemia stem cells, these ligands can be patented and eventually commercialized. This may have huge financial benefits to California. If this project is successful, the same approach can be used to treat other cancers and for the development of more commercialized drugs.
If this grant is funded, it will secure my career as a physician-scientist in stem cell and cancer research. The physician-scientist is a diminishing breed in that it is difficult for physicians to do research while meeting the huge demands of the clinic. However, there is a huge gap between basic research and clinical applications. This gap is in part traced to the fact that it is difficult to find researchers who know and can integrate clinical needs with basic research. I consider myself a promising physician-scientist who has received extensive, rigorous and systematic training in medical science and basic research ([REDACTED]). If this grant is funded, I will not only carry out this important research, but this will also give me protected time for this research.
Review
SYNOPSIS: The goal of this application is to use combinatorial chemistry approaches to develop compounds that can be used to specifically delete cancer stem cells (CSCs) in the body or to purge these cells from potentially contaminated hematopoietic cell grafts. The Principal Investigator (PI) argues that non-cell-autonomous factors are involved in both the maintenance of stemness and differentiation (including cancer). While both stem cells and cancer stem cells require signaling from the extracellular environment to maintain “stemness” through self-renewal and to promote differentiation, CSCs have their own unique cell surface molecules. The hypothesis to be tested here is that distinct cell surface molecules on CSCs can be targeted for therapy.
The applicant will use technology developed by his/her previous mentor to generate novel molecules that can be selected for their binding to cancer stem cells. This will be done by isolating leukemia stem cells (LSCs) from the blood of patients, and counter-selecting against molecules that recognize normal hematopoietic stem cells (HSC) or other normal constituents of the bone marrow. The first aim of the proposal will be to identify molecules that bind LSCs. Sorted LSCs will be obtained through a collaboration, and non specific ligands targeting normal cells will be eliminated by subtraction against normal HSCs from healthy donors. Selection and counter-selection will be applied through multiple consecutive rounds. Any molecules that bind LSCs, but not normal HSC, will be checked for binding to other LSC subtypes and optimized for binding affinity. These optimized libraries will be screened again for binding to stem cells, and then positive hits will be analyzed further.
After generation of this library, the PI will test the efficacy of positive hits in two different strategies of tumor eradication. In the first, the PI will generate novel agents which can be used to evoke target-directed cellular cytotoxicity. Efficacy of these various agents will be measured in vitro and in vivo. In the second, the PI will use these agents in capture assays to test their ability to remove cancer stem cells from bone marrow grafts. These studies will be read out by in vitro cytogenetics and in vivo tumor formation assays. Finally, as these tumor-targeted agents recognize unique epitopes on cancer stem cells that may be interesting for cancer stem cell function, the PI will attempt to identify their molecular targets and begin the dissection of molecular pathways involved in generating LSCs using bioinformatics, inhibition studies, and labeling techniques.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Although bone marrow transplantation (BMT) is a successful therapy that has been used for many years, it can be accompanied by severe graft versus host disease which renders the patient in need of immunosuppressive therapy for life, even though the cancer may have been cured. Patient-specific stem cells will alleviate this problem and also allow patients with no bone marrow tissue match to undergo BMT. The advent of growth factor therapy to mobilize bone marrow cells into peripheral blood was a marked advance to this BMT as cancer therapy, but has not been available to patients with leukemia because of the obvious problem of having the leukemia stem cells contaminate the mobilized bone marrow cells. This is a highly promising approach because once the leukemia stem cells can be targeted, they can be eliminated. Targeted therapies may include the possibility of removing leukemia stem cells from the bone marrow cells of the afflicted patient, thus allowing the patient to go through bone marrow transplantation with autologous bone marrow cells. The successful accomplishment of the goals of this project will not only provide a platform for clinical and translational application of the findings, but also will provide molecular probes to investigate pathways involved in CSC differentiation, directly influencing basic research.
This project is both a highly significant and highly innovative. The PI uses a novel approach to generate molecules targeted at cancer stem cells and then applies them to test multiple potential therapeutic applications and to discover new aspects of the biology of hematopoietic malignancy. The strength of the proposal is based on the high throughput platform of screening for novel LSC binding molecules. While a large number of screens use synthetic chemical combinatorial libraries from synthetic as well as natural sources, this one uses an innovative molecular agent as opposed to a compound screen. This platform also provides the high-risk aspect of the work. However, the PI presents successful attempts in the context of other cell lines, and the fact that the PI was trained by the individual who pioneered these techniques provides confidence that this project should work. It is also important to note that by NIH criteria this project would not only be qualified as high-risk but also extremely overambitious, and perhaps not in line with the publication record of the PI. The significance of the successful accomplishment of this project is so high that it is certainly worth the investment.
Each of the four aims is carefully and completely described, and each experiment is feasible. The overall concern with these studies is, of course, that it is possible that no specific cancer-targeting agents will be uncovered, as this is a true discovery effort. Success in Aim 1 is obviously critical for success in the next 3 aims. This concern is allayed somewhat by the PI’s assertion of success in applying this strategy in a wide variety of other cancers, as well as normal tissue cells. If this broad-based approach does fail, the PI proposes a more focused strategy aimed specifically at a cell surface molecule that is expressed by a certain type of LSCs.
The PI’s approach of testing multiple potential therapeutic applications for cancer stem cell-directed peptides is commendable. The applicant also applies innovative approaches to the attempts to delete or purge cancer stem cells. Finally, the use of these molecules to directly identify novel cancer stem cell markers of potential functional importance is significant, though the precise strategy and specific experiments by which the PI will assess these functions is not clear. Another minor weakness of the proposal concerns the in vivo efficacy studies. These experiments may be complicated due to a need to optimize timing and dosage, which is not discussed.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant is an Assistant Professor in a department of internal medicine. S/he received a MD outside of the U.S., and a PhD in microbiology and immunology in 1999 at a U.S. institution. After internship and residency in internal medicine and a follow-up fellowship, the applicant trained with the individual who pioneered the proposed binding techniques, and mastered the technology to be applied in this grant application. The PI is a talented investigator with limited but important contributions to the field. S/he has seed funding from a number of private sources, and has published many clinical papers but few basic research articles.
The applicant outlines an excellent career development plan that emphasizes both basic research and clinical experience, and has the enthusiastic support of the departmental chairman. The PI’s career goals for the next years include both didactic training and personal mentoring to enrich his/her background in research and grantsmanship. The PI intends to become a highly productive clinician-scientist in the area of stem cell therapies, and the plan as outlined should accomplish this goal. The PI has recruited senior mentors to support both aspects of his/her career and has enrolled in a mentored clinical research training program, which will involve didactic sessions, research guidance, workshops, seminars, and retreats. The PI will also participate together with his/her students in a stem cell training program offered by the applicant institution to improve his/her mastery of stem cell science. The PI defines clear milestones for grants, papers, clinical service and teaching.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution is well-suited to support these studies, encompassing all of the necessary resources and centers to successfully execute the proposed specific aims. The institution has made a clear commitment to this young investigator. The PI cites 1500 sq ft of his/her own laboratory space plus 600 sq ft of office space, and 80% time protected for research. The PI has several excellent senior mentors who will collaborate on these studies and provide ongoing input and advice. The PI meets weekly with these mentors, one of whom is his/her former advisor. The applicant institution has approximately 100 faculty associated with its stem cell program, organized into disease-specific focus groups, and the institution has made major investments in training and core facilities of relevance to the current proposal. All facilities needed for this work are in place.
DISCUSSION: Reviewers concurred that this combinatorial chemistry proposal has a big picture view, with extreme logic used for compound identification. The significance of the proposal is extremely high. The PI is very appropriate for this RFA – s/he is a young physician scientist in a good environment, who wants to be involved in the stem cell field and stay in translational research. One takes a chance with any fishing expedition, but the libraries here haven’t been explored, the proof of ability is strong, and the PI proposes alternative approaches. Preliminary data provide proof of feasibility, there is a logical progression of the four aims, and the outcome analysis is well articulated; thus, reviewers are confident that the PI will get results and be successful. This grant could not be funded by NIH because it is too high-risk. The generation of these novel binding agents is very inventive, and once the agents are identified the PI will pursue multiple strategies for purging cancer stem cells. One panel member asked about the methods for identification of the leukemic stem cells. A reviewer responded that the identification strategy uses an appropriate combination of cell surface markers with functional assays.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None