In recent years, ground-breaking discoveries have been made in two important areas of stem cell biology. First, normal stem cells and cancer-initiating cells (or cancer stem cells) share many properties, among them a seemingly unlimited potential to self-renew. This has initiated a paradigm shift in our thinking about cancer; tumorigenesis may be driven by a small number of cancer stem cells, just like most tissues in our body are maintained by a small number of tissue-specific stem cells. Therefore, studying normal stem cell self-renewal may help us to understand cancer, and thus help us find a cure. Second, the breakthroughs in engineering embryonic stem (ES) cells without the need for embryo destruction, so-called iPS cells. With this discovery personalized cell-based therapies are much closer to reality. The progression from bench to bedside for this type of cell-based therapy will largely depend on achieving a full understanding of the true nature of ES/iPS cells and the consequences of inadvertently transferring them into patients. Equally important to the success of developing cell-based therapies, will be the elucidation of the molecular mechanisms that regulate adult-type stem cells. This will include generating stem cells de novo from ES/iPS cells, as well as manipulating stem cells into self-renewal while allowing them to simultaneously produce differentiated progeny.
Understanding stem cell regulation has always been hampered by the fact that these cells are rare. In most adult tissues, the frequency of stem cells is between 1 in every 104 to 105 cells. They are also difficult to identify and assay. The exception being the HSC population, which is relatively accessible and therefore well-characterized, both phenotypically and functionally. This has lead to the identification of many genes that control the different fates if HSCs. Despite the discovery of these “HSC regulatory genes” there is no comprehensive understanding of how they all fit together in one regulatory network that controls HSC fate.
Regulatory processes of HSCs. The fate of a stem cell, although executed by an intracellular gene transcription regulatory network, is determined by extracellular signals from the microenvironment (also called HSC niche). Proper HSC regulation depends on an intact intracellular gene regulatory network (1) and on proper extracellular signals (2 and 3). Together they balance self-renewal, differentiation, apoptosis, and senescence.
CREB binding protein (CBP)- and p300 deficient mice to study HSC regulation
CBP and p300 proteins 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. CBP and p300 are paralogues (i.e., derived from gene duplication), and 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 CBP and p300 in regulating HSCs. Neither gene is required for the initiation of HSCs, however, HSC self-renewal requires maximum expression of CBP, but not p300, though at least one allele of p300 is needed for optimal differentiation. Recent experiments show that a full dose of CBP is required in the HSC niche for optimal extracellular regulation of HSCs. Loss of one CBP allele in the HSC niche causes the HSC pool to decrease in size. Thus, CBP and p300 loss-of-function mouse models provide unique tools to delineate both intracellular and extracellular regulatory mechanisms of the HSC pool.
CBP+/- mice develop a myelodysplastic syndrome with age.
In addition to the HSC defects, CBP+/- mice shows abnormal myeloid differentiation and they have a high propensity of developing myeloid tumors. In short, CBP+/- mice suffer from a hematopoietic syndrome that is reminiscent of human Myelodysplastic syndrome (MDS). In humans, MDS represents a group of pre-leukemic diseases, associated with defective hematopoietic differentiation, cytopenias, and a high propensity of developing hematologic malignancies, specifically acute myeloid leukemia (AML). MDS is a disease of the elderly, but it is also associated with prior anti-cancer treatments. AML developing in the latter category of patients is associated with chromosomal translocations, some of which include CBP. Little is known about the etiology of MDS, other than that it is associated with genetic instability and defects in the stem cell compartment. Given the similarities between the phenotype of CBP+/- mice and human MDS, CBP+/- mice are ideally suited as a model to study MDS.
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 CBP [and not by the loss of p300]. Since loss of CBP is associated with loss of HSC self-renewal, these genes are thus potential regulators of HSC self-renewal. Similarly, by comparing CBP+/- stroma cells with wild type and p300+/- stroma, we have identified a set of genes expressed in stroma cells that are specifically affected by the loss of CBP 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. A variety of bioinformatics tools and functional testing of individual genes are being performed to validate these gene sets.
Genomic Integrity in normal and CBP+/- HSCs as a function of age.
In mice, perturbations in genes involved in DNA damage response signaling pathways and/or DNA repair are associated with an age-related 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. CBP interacts with many DNA repair and DNA damage response genes. In addition, CBP 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 CBP 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. In collaboration with Dr. Christi Walter (Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio), we are currently investigating if (a) genomic integrity is compromised in HSCs as part of a normal aging process and (b) if loss of CBP leads to decreased/improper DNA repair and therefore increased mutagenesis with age.