This application for a K08 award describes a multidisciplinary 5-year training program to facilitate Dr. Michael Rothenberg's career development as an independent academic physician-scientist. Dr. Rothenberg recently completed a clinical fellowship in Gastroenterology and now seeks to build on his research experience to attain his long-term career goal of becoming an independent academic investigator, directing a research program in intestinal stem cell biology. This K08 award will provide Dr. Rothenberg with the support and protected time necessary to accomplish the following training objectives: (1) to become an expert in intestinal stem cell and niche biology, (2) to master advanced techniques of cell culture, flow cytometry, and gene expression analysis, (3) to acquire expertise in the manipulation of mouse models for the study of intestinal stem cells, and (4) to submit an R01 application during year 4. To achieve these goals, Dr. Rothenberg has assembled a strong mentoring team of experts in stem cells, intestinal biology, intracellular signaling, and bioinformatics-all of whom have successfully mentored young scientists. His primary mentor, Dr. Michael Clarke, is a leader in the field of normal and cancer stem cells. Dr. Rothenberg's co-mentor, Dr. Harry Greenberg, is an expert in intestinal physiology and virology. The advisory committee consists of Dr. Irv Weissman (a pioneer in stem cell and niche biology), Dr. Rob Tibshirani (a leader in gene expression analysis), and Dr. Ken Weinberg (an expert in intracellular signaling and cKit). Dr. Rothenberg's career development plan includes regular meetings with the mentoring team, didactics, and attendance and presentation at local and international meetings. Building on his recent discovery of a cKit+ colonic goblet cell that supports intestinal stem cells, Dr. Rothenberg hypothesizes that bidirectional signaling between these cells and adjacent intestinal stem cells coordinates colonic epithelial homeostasis.
Aim I : To characterize the effects of acute depletion of cKit+ cells from the colonic epithelium.
This aim (years 1-3) will start with in-vitro studies, later moving to in-vvo work. It will make use of monoclonal cKit blocking antibodies, cytotoxins targeted to cKit+ cells, and small molecule cKit inhibitors.
Aim 2 : To identify novel regulators of colon stem cell physiology by profiling cKit+ cells and testing selected secreted factors in both in-vitro and in-vivo assays.
This aim (years 2-4) will test in-vitro effects of two growth factors that are highly enriched in cKit+ goblet cells, and will use purified protein, blocking antibodies, and lentiviral knockdown in cKit+ cells. Additional factors will be identified with gene expression analysis, validated, and tested both in-vitro and in-vivo.
Aim 3 : To characterize a cKit+ crypt base population in human colon epithelium.
This aim (years 3-5) will use single cell gene expression profiling on fresh human colon and will also use in-vitro organoid culture. Preliminary data support the feasibility of all 3 aims. Ultimately, an improved understanding of the colon stem cell niche may enable development of novel targeted treatments for digestive diseases.
Because they drive the continuous regeneration of the colonic epithelium, colon stem cells are likely to play important roles in colonic diseases. These include inflammatory bowel disease (IBD) and colorectal cancer, diseases that are generally treated with non-targeted therapies that have significant toxicity: immunosuppressive agents and chemotherapy, respectively. The research proposed here will deepen our understanding of the molecular signals regulating colonic epithelial regeneration, and may lead to the development of novel, targeted therapies.