Vivienne Rebel, MD, PhD
Associate Professor of Cellular & Structural Biology
Hematopoietic Stem Cell Regulatory Mechanisms in Relation to Myelodysplastic Syndrome
The goal of our laboratory is to understand the nature of the molecular events that lead to the development of Myelodysplastic Syndrome (MDS). The incidence of MDS in children is low (~ 4 / million per year), however, after chemotherapeutic treatment as many as 2-6% will develop MDS, with a median survival of only 13 months! Thus, MDS is a serious and common side-effect of anti-cancer treatment, one for which there are currently very few options of effective treatments.
MDS is a hematopoietic stem cell (HSC) disease associated with defective hematopoietic differentiation, cytopenias, and a high propensity of progressing into hematologic malignancies, specifically acute myeloid leukemia (AML). MDS is furthermore associated with genomic instability and although little is known about the mechanisms responsible for the genomic instability, it has been linked to deficient DNA repair pathways.
MDS is not only associated with prior anticancer therapy, but also with old age. Childhood MDS and MDS in adults have many features in common, although there are also some distinct differences. One of the chromosomal perturbations that have been found in both children’s and adult-type MDS involves the CREBBP gene. Our studies of the hematopoietic system of mice with only one intact copy of the CREB binding protein locus (Crebbp+/-) show that these mice display, with age, all the characteristic features of MDS, including abnormal myeloid differentiation, HSC defects, and a high probability of developing leukemia.
CREBBP and its paralogue EP300 function as molecular integrators of various transcriptional signals. When recruited to promoters by transcription factors, they function as co-activators of transcription through multiple mechanisms, including chromatin remodeling, acetylation of associated proteins, and recruitment of the basal transcription machinery. CREBBP and EP300 a are highly homologous on a structural level, with up to 93% identity within certain protein-binding domains. Despite this homology, our loss-of-function mouse models demonstrated different roles for CREBBP and EP300 in regulating HSCs. Neither gene is required for the initial formation of HSCs during embryology, however, HSC self-renewal requires maximum expression of CREBBP, but not EP300, though at least one allele of EP300 is needed for optimal differentiation. Recent experiments show that a full dose of CREBBP is required in the HSC niche for optimal extracellular regulation of HSCs, as well as for control of myeloid differentiation. Loss of one Crebbp allele in the HSC niche causes the HSC pool to decrease in size and gives rise to excessive myelopoiesis. Thus, CREBBP and EP300 loss-of-function mouse models provide unique tools to delineate both intracellular and extracellular regulatory mechanisms of normal hematopoiesis, as well as those underlying MDS development.
Identifying novel molecular mechanisms of (a) intracellular HSC self-renewal and (b) microenvironment-mediated (extracellular) HSC regulation.
Using comparative microarray analysis we identified a set of HSC genes that are specifically affected by the loss of Crebbp [and not by the loss of Ep300]. Since Crebbp+/- HSCs show a problem with self-renewal, these genes are thus potential regulators of HSC self-renewal and contributing to MDS development. Similarly, by comparing Crebbp+/- stroma cells with wild type and Ep300+/- stroma, we have identified a set of genes expressed in stroma cells that are specifically affected by a half dose of CREBBP in stroma cells; these genes encode cell surface molecules or secreted proteins that may be important in the HSC niche to maintain an optimal number of HSCs, as well as normal myeloid differentiation. A variety of bioinformatics tools and functional testing of individual genes are being performed to validate these gene sets.
Genomic Integrity in normal and CREBBP+/- HSCs.
Children with hereditary diseases, such as Bloom syndrome and Fanconi’s anemia that are associated with defective DNA repair have an increased risk of developing MDS. In mice, perturbations in genes involved in DNA damage response signaling pathways and/or DNA repair are associated with hematopoietic failure and loss of HSCs. This suggests that the inability to properly respond to DNA damage interferes with normal HSC regulation, including self-renewal. CREBBP interacts with many DNA repair and DNA damage response genes. In addition, CREBBP is a histone acetylating agent. Loss of histone acetylation may alter the DNA structure and thereby alter sensitivity to DNA damage and access of the repair machinery. Therefore a likely connection between CREBBP heterozygosity, loss of HSCs and the development of MDS/AML is decreased DNA repair. Indeed, several genes known to play a role in DNA damage response and - repair were among the set of potential HSC self-renewal genes we identified as being perturbed in Crebbp+/- HSCs. We are currently investigating (a) if loss of CREBBP leads to decreased/improper DNA repair and therefore increased mutagenesis, ultimately leading to MDS and (b) since CREBBP levels in hematopoietic cells decrease with age, if in genomic integrity is compromised in HSCs as part of a normal aging process.
With the number of long-term cancer survivors increasing, an ever more import aspect of cancer treatment is restoring tissue damage resulting from the anti-cancer treatment. E.g., replacing muscle, bone, cardiac valves, eyes, etc; i.e., tissues that were damaged by radiation, chemotherapy and/or radical surgery. Regenerative medicine is the field of medicine involved with the development and implementation of such replacement therapies. Since stem cells are the basis of regenerating and maintaining the differentiated cells that together make up tissues and organs, it is of great importance to understand the basic biology of stem cells in different tissues. Moreover, recent breakthroughs in engineering embryonic stem (ES) cell lines without the need for embryo destruction, so-called induced pluripotent stem (iPS) cells, hold a promise of a near-unlimited source of stem cells for the development of such cell-based replacement therapies.
Some types of stem cell-based replacement therapies have been in practice for decades (e.g., skin replacement and bone marrow transplantations), while other tissue replacement therapies are still in its infancy. Depending on the targeted patient group (in addition to cancer survivors, trauma victims and the elderly (in which tissue functions can be severely degenerated) are also important candidates for cell-based tissue replacement therapies), as well as the tissue involved and the source of the stem cell population used, each novel therapeutic protocol faces its own particular sets of hurdles. To facilitate the development of cell-based replacement therapies, we have initiated a Stem Cell Think Tank; meetings are held every first business Monday of the month at the Greehey Children’s Cancer Research Institute. The goal of the meetings is to provide a forum to discuss the science and clinical aspects of stem cell biology/regenerative medicine in the hope to learn from each other and start working together on intriguing problems associated with stem cells and regenerative medicine.