Funding opportunities

Combinatorial Chemistry Approaches to Develop LIgands against Leukemia Stem Cells

Funding Type: 
New Faculty I
Grant Number: 
RN1-00561
Principle Investigator: 
Funds requested: 
$2 392 397
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
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 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 PI proposes a combination of combinatorial peptide libraries as well as phage library display. The applicant will use combinatorial chemistry-based technology developed by his previous mentor to generate novel compounds 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 with acute myeloid leukemia (AML), and counter-selecting against compounds that recognize normal HSC or other normal constituents of the bone marrow. The first aim of the proposal will be to use a phage display library to identify peptides that bind AML stem cells (CD34+CD38-CD123+). The sorted AML cells will be obtained through a collaboration with Jan Nolta, and non specific ligands targeting normal cells will be eliminated by subtraction of the library with normal HSCs from healthy donors. Selection and counter-selection will be applied through four consecutive rounds. Any clones that bind AML stem cells by in vitro bead-based adhesion assay, but not normal HSC, will be sequenced and checked for binding to other AML subtypes, and finally optimized for binding affinity. Optimization will first determine which amino acids are critical for binding, and then will use One-Bead-One-Compound (OBOC) synthesis technology to replace non-critical residues with natural and unnatural amino acids. These optimized libraries will be screened again for binding to stem cells, and then positive hits will be sequenced. After generation of this library of peptides, the PI will test their efficacy in two different strategies of tumor eradication. In the first, he will conjugate these peptides to an Fc backbone to generate "chemibodies", which can be used to evoke target-directed cellular cytotoxicity. Efficacy of various chemibodies will be measured in vitro by co-culture with human NK cells, and in vivo by injection into AML-engrafted NOD/SCID/g-/- mice. In the second, he will use these 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 peptides 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, antibody inhibition, and photoaffinity labeling. 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 by targeted therapy, including the possibility of removing leukemia stem cells from the bone marrow cells of the afflicted patient 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 using peptides as ligands. While a large number of screens use synthetic chemical combinatorial libraries from synthetic as well as natural sources, this one uses an innovative peptide as opposed to compound screen. This same attribute also provides the high-risk aspect of the work. However since the PI presents successful attempts made by this library in the context of other cell lines, and the fact that he was trained by Dr. Lam--who has pioneered these techniques, provides confidence that this project should work. It is also important to note that by NIH RO1 grant 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 peptides 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 that he has been successful 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 targeted strategy aimed specifically at CD123, which is expressed by AML stem cells. The PI's approach of testing multiple potential therapeutic applications for cancer stem cell-directed peptides is commendable. He also applies innovative approaches to his 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 injections into AML-engrafted NOD/SCID/g-/- mice. 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: Dr. Pan is an Assistant professor in the Department of Internal Medicine, UC Davis School of Medicine. He received his MD from Fudan University in Shanghai and his PhD in microbiology and immunology from Loyola University Chicago in 1999. After internship and residency in Internal Medicine and a follow-up fellowship, he trained with Dr. Kit Lam and mastered the OBOC technology, which he will apply in this grant application. Dr. Pan is a talented investigator with limited but important contributions to his field. 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 he has the enthusiastic support of his chairman, Dr. Fred Meyers. His career goals for the next years include both didactic training and personal mentoring to enrich his background in research and grantsmanship. He intends to become a highly productive clinician-scientist in the area of stem cell therapies, and his plan as outlined should accomplish this goal. He has recruited senior mentors to support both aspects of his career and has enrolled in a mentored clinical research training program (NIH K30), which will involve didactic sessions, research guidance, workshops, seminars, and retreats. He will also participate together with his students in a Stem Cell Training Program offered by UC Davis to improve his mastery of stem cell science. He defines clear milestones for grants, papers, clinical service and teaching. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: UC Davis is the best institution for 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. He cites 1500 sq ft of his own laboratory space plus 600 sf of office space in the Oak Park Research Research Building, and 80% of his time is protected for research. He has several excellent senior mentors who will collaborate on his studies and provide ongoing input and advice. He meets weekly with these mentors, one of whom is his former advisor. UC Davis has approximately 100 faculty associated with their 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 - he is a young physician scientist in a good environment who wants to be involved in the stem cell field and wants to 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. Binding the ligands to Fc is very inventive, and once the peptides are identified he will pursue multiple strategies for purging cancer stem cells. One panel member asked about the identification of the leukemic stem cells. A reviewer responded that it is by using a good combination of cell surface markers (CE34+, CD38-, CD123+) with functional assays.
Conflicts: 

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