Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology
Department of Dermatology
University of Colorado Denver
Mail Stop 8320
12800 E. 19th Avenue
Aurora, CO 80045
Graduate School and Center Affiliations:
Associate Professor, Department of Dermatology, University of Colorado Denver
Member, Charles C. Gates Regenerative Medicine and Stem Cell Program
Integrated Department of Immunology, The University of Colorado Health Sciences Center
Program in Molecular Biology, The University of Colorado Health Sciences Center
Basic Sciences Program, The University of Colorado Health Sciences Center
MSTP Program, The University of Colorado Health Sciences Center
Full member, UCHSC Cancer Center
Member, UCHSC Center for AIDS Research (CFAR)
B.A., University of Pennsylvania, Philadelphia, PA. (Biology), cum laude, 1994
Ph.D., Harvard University, Cambridge, MA. Committee on Immunology (1999).
Postdoctoral fellow, laboratory of Dr. J. Michael Bishop, UCSF, 2000–2003
Functions of proto-oncogenes in health and diseases of hematopoietic lineages.
- Our laboratory is interested in defining the molecular mechanisms involved in the initiation, establishment and maintenance of lymphoid tumors. We have based our work on the hypothesis that lymphoid neoplasms arise as a result of the dysregulation of signals that normally control lymphoid function and homeostasis. Our work suggests that lymphoid neoplasms may arise upon the disruption of lymphocyte proliferation and survival pathways. The work in our lab focuses on three broad areas that relate the functions of proto-oncogenes in the regulation of lymphoid and hematopoietic stem cell physiology under normal and aberrant conditions. Our work aims to address three major questions. First, what is the nature and identity of antigen-receptor derived signals that can cooperate with dysregulated oncogenes in the context of lymphoid neoplasia? We believe that those molecules will provide an important and novel source of molecular targets with therapeutic potential for the treatment of lymphoid malignancies. Second, we are interested in determining the mechanisms by which the proto-oncogene MYC is able to regulate lymphoid tolerance. The further understanding of the role of MYC in lymphoid homeostasis and function may allow us to improve vaccination strategies on a molecular level as well as determine new pathways that may prove useful in the context of autoimmune lymphoproliferative diseases. Third, we are interested in the contribution of the proto-oncogene MYC to the regulation of self-renewal, proliferation and differentiation of long-term hematopoietic stem cells (HSCs). These findings will provide novel insights on the regulation of HSC homeostasis, longevity and susceptibility to transformation that may ultimately allow us to propose new approaches for the treatment of congenic and aquired immune deficiencies and leukemias. We use a combined approach to address these questions, that involve genetic and biochemical approaches that ultimately come to bear on animal models of disease.
- What is the contribution of the proto-oncogene MYC to the regulation of self-renewal, proliferation and differentiation of long-term hematopoietic stem cells (HSCs)?
- Aging is associated with the continuous erosion of some of the unique characteristics of the hematopoietic stem cell (HSC) compartment. One of the effects of the senescence of HSCs is the inability of the individual to mount productive primary humoral immune responses. A number of studies have suggested that a major cause of immunological aging is a failure of aged HSCs to give rise to a fully functional B-cell compartment. Several lines of evidence have shown this to be a cell-intrinsic defect of the aged HSCs. We wanted to generate immortalized HSC cell lines in order to study the defect responsible for the defective B-lymphopoiesis observed in aged mice. When we induced the function of MYC-ER with tamoxifen, we observed that the bone marrow transplant recipient mice that were given HSCs derived from a young donor developed leukemia within three weeks, whereas those recipients that were given HSCs derived from an aged donor only developed leukemia after three months. The same HSC compartment that was shown to have a differentiation defect was also more resistant to transformation by defined oncogenic lesions.
- The bulk of the experiments exploring the nature of the leukemic stem cell (LSC) as well as the genetic lesions required for their transformation have been carried out in bone marrow cells derived from young mice. In addition, most of those experiments were carried out with C57/BL6 mice, where the effects of aging have not been readily apparent in prior studies. Those two instances would preclude the observation of the more subtle effects of aging on the ability of HSCs to self-renew, differentiate, proliferate, survive, and undergo transformation upon exposure to defined oncogenic lesions. This application seeks to expand on our observations in order to address three major questions regarding the generation of leukemic stem cells from aged HSCs.
- While leukemias occur primarily in older individuals, the bulk of the research aimed at determining the molecular basis of the generation of LSCs has been carried out in HSCs derived from young donors. Our findings should a) improve our understanding of the effects of aging on the ability of oncogenes to render HSCs tumorigenic, b) provide a better insight into the possibility that the defects in differentiation and oncogene-driven transformation may be linked and ultimately controlled by a common mechanism and c) help identify novel therapeutic targets for the treatment of leukemias and autoimmune diseases that result from aging.
- What is the nature and identity of antigen-receptor derived signals that can cooperate with dysregulated oncogenes in the context of lymphoid neoplasia?
- Our long-term goal is to determine how a continuous signal from the B-cell antigen receptor can cooperate with a dysregulated oncogene in the genesis of lymphoid tumors. We have used mouse models to demonstrate that a clonal B-cell antigen receptor (BCR) can contribute to the genesis of lymphomas. Our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen, and reveal a previously unappreciated physiological effect of MYC – the breach of immune tolerance. These data suggest that MYC overexpression is required to establish and maintain the breach of immunological tolerance. These findings demonstrate a critical role for MYC in the response of B-cells to antigen and expand the potential contributions of MYC to the genesis of lymphomas.
- The models we have developed will prove valuable for the further study of the mechanisms of lymphomagenesis, and for preclinical testing of new therapeutics. We propose to test the hypothesis that specific BCR-induced signaling effector molecules mediate the cooperation with elevated levels of MYC in lymphomagenesis. This is an appealing hypothesis, since these signaling mediators may also play an important role in the establishment and maintenance of B-cell tolerance, and may prove to be attractive therapeutic targets for lymphoproliferative diseases and lymphoid neoplasia.
- By identifying molecules involved in mediating the lymphomagenic signals that arise from the cooperation between the BCR-derived signal and MYC, we hope to aid in the discovery of new therapies to treat lymphoid malignancies and lymphoproliferative diseases. In addition, the details surrounding the mechanism of cooperation between BCR-derived signals and MYC may provide further insights into the role of MYC in the regulation of B-cell responses to antigen and homeostasis of the B-cell compartment.
- What are the mechanisms by which the proto-oncogene MYC is able to regulate lymphoid tolerance?
- Our goal is to define the underlying mechanisms by which a surfeit of MYC expression in the context of auto-reactive B-cells is able to break immune tolerance. We have observed that mice that would otherwise be tolerant to a transgenic autoantigen mounted an immune response to the antigen if MYC was vigorously expressed in the B-cell lineage. The responsive B-cells converted to an activated phenotype and produced copious amounts of autoantibody that engendered immune complex disease of the kidney. MYC was required to both establish and maintain the breach of tolerance. These mice also developed lymphomas, as a result of the cooperation between signals derived from their BCR and MYC. These effects may be due to the ability of MYC to serve as a surrogate for T-cell help. We found that the gene could mimic the effects of cytokines on both B-cell proliferation and survival, and indeed, was required for those effects. These findings demonstrate a critical role for MYC in the response of B-cells to antigen and expand the potential contributions of MYC to the genesis of lymphomas. These data suggest that MYC overexpression is required to establish and maintain the breach of immunological tolerance. These findings also demonstrate a critical role for MYC in the response of B-cells to antigen and expand the potential contributions of MYC to the genesis of lymphomas. Our observations may aid in the understanding of the etiology of AIDS-associated Non-Hodgkin’s Lyphomas (NHLs). In that instance, B-cell tumors arise in individuals that have lost their CD4+ T-cells due to the effects of HIV. Their tumors are composed of mature, activated B-cells that resemble antigen-experienced lymphocytes. The ability of overexpressed MYC to render B-cells independent of T-cell help may provide a possible explanation for the development of those tumors.
- The models we have developed should prove valuable for the further study of the mechanisms of lymphomagenesis, and for preclinical testing of new therapeutics. We propose to test the hypothesis that MYC is both a necessary and sufficient effector for the T helper-cell derived signals that regulate B-cell function in normal immune reponses and in the genesis of lymphomas upon overexpression. This is an appealing hypothesis, since the same signaling mediators that are likely to be involved in the MYC-dependent regulation of B-cell tolerance and homeostasis are at play in the oncogenic functions of MYC, and may prove to be attractive therapeutic targets for lymphoproliferative diseases and lymphoid neoplasia.
Refaeli, Y., Field, K.A., Turner, B.C., Trumpp, A., and Bishop, J.M. (2005). Overexpression of MYC can break B-cell tolerance. Proc Natl Acad Sci USA, 102: 4097-102.
Trumpp, A., Refaeli, Y., Martin, G., and Bishop, J.M. (2001) The proto-oncogene c-MYC regulates body mass and the rate of cellular proliferation. Nature, 414: 768-773.
Turner, B.C., Eves, T., and Refaeli, Y. (2008). Small molecule inhibitors of Bcl-2 family proteins are able to induce tumor regression in a mouse models of pre-B cell acute lymphocytic leukemia. DNA and Cell Biology, 27, 133-42.
Refaeli, Y., Young, R.M, Turner, B.C., Duda, J., Field, K.A., and Bishop, J.M. (2008). Contributions of Antigen and Antigen Receptor to the genesis of B-cell tumors in mice. PLoS Biology 6, e152. Accompanying Primer on this article authored by Tak Mak, PLoS Biology 6, e120. PMCID: PMC2435152.
Field, K.A, Charoenthongtrakul, S., Bishop, J.M., and Refaeli, Y. (2008). Farnesyl transferase inhibitors induce extended remissions in transgenic mice with acute B cell lymphomas. Molecular Cancer 7, 39-49. PMCID: PMC2409375.
Young, R.M., Hardy, I.R., Clarke, R.L., Lundy, N., Turner, B.C., Potter, T.A., and Refaeli, Y. Syk is a novel target for the treatment of Non-Hodgkin’s lymphoma. (2009). Blood, 113, 2508-16. PMID: 18981293 (Accompanying editorial authored by P. Leif Bergsagel).
Zeman, R.L., Briones, N., Young, S.K., Malcolm, K.C., Refaeli, Y., Downey, G.P., and Worthen, S.G. (2008). A novel method for long term bone marrow culture and genetic modification of murine neutrophils via retroviral transduction. J Immunol Methods, 340, 102-15.
Refaeli, Y., Bhoumik, A., Roop, D.R., and Ronai, Z. (2009). Melanoma Initiating Cells – A Compass Needed. EMBO Reports, In Press. PMID: 19680286
Young, R.M., Polsky, A., and Refaeli, Y. (2010). TC-PTP is required for the maintenance of MYC-driven B-cell lymphomas. Blood, In Press. PMID: 19755676
Turner, B.C., Bird, G.A., and Refaeli, Y. (2009). The biology of MYC in health and disease: a high altitude view. Chapter in “Medical Biostatistics: Analysis of Complex Diseases”. Eds. F. Emmert-Streib and M. Dehmer. Wiley, In Press.