Steven Pruitt, PhD
The overarching theme of work in this laboratory is to understand the mechanisms by which somatic stem cells maintain tissue homeostasis and the consequences of dysfunction in these mechanisms for age related disease. In particular, we are addressing fundamental issues concerning the relationship between cell proliferation, the role of errors in DNA replication on genetic damage accumulation, and the mechanisms by which cells and tissues manage this damage. Since somatic stem cells are critical for ongoing tissue maintenance and regeneration after injury in most tissues of vertebrates, this area of research intersects with a large number of basic biological processes and diseases of aging including cancer.
A central concept underlying much current thinking on the relationship between caner and other age related dysfunction is that there is a trade-off between the benefits of cell proliferation in tissue maintenance and the negative consequences of errors in DNA replication on genetic damage accumulation and sequelae of DNA damage responses leading to cell death or senescence. Two mouse model systems that directly address this relationship have been developed in this laboratory. In one model, the rate of replication related genetic damage accumulation is accelerated due to a reduction in the expression of a key component of the DNA replication licensing complex, mini-chromosome maintenance protein 2 (Mcm2). These mice are remarkably cancer prone and succumb with early onset and complete penetrance (Pruitt et al., 2007; Kunnev et al., 2010). The insight provided by this model is that simply reducing the efficiency with which DNA replication origins are licensed leads to high rates of cancer. This leads to a number of important questions concerning the mechanism by which insufficient replication licensing results in genetic damage, whether specific locations within the genome are more susceptible and whether conditions arise within normal tissues or hyperplastic lesions that result in replication licensing deficits. In a second model, cell proliferation in vivo has been brought under conditional control through tetracycline dependent expression of the cyclin dependent kinase inhibitor Cdkn1b (p27kip1). These mice exhibit an inducible progeroid phenotype that can be directly linked to insufficient cell proliferation (Pruitt et al., 2013).
Major questions that are under investigation are:
1. To what degree does sequence specificity in the sites at which pre-replicative complexes assemble influence the locations at which replication related genetic damage is likely to arise? This question is being addressed through genome-wide assessment of replication origins by next-generation sequencing of nascent strand DNAs. We have developed a new approach to defining replication origins genome wide and, using this methodology, we have shown that Mcm2 deficiency has a disproportionate effect on specific subsets of origins. Further, these sites are coincident with sites of recurrent DNA damage in the tumors arising in these mice. Ongoing studies are aimed at identifying the genetic and epigenetic elements that determine sites of origin licensing and defining the subset of these elements that are differentially sensitive to deficiency for Mcm2 or other components of the DNA replication licensing complex. This model also allows us to address the relationship between the locations and densities of replication origins and the timing of replication during S-phase. Further, congenic mice carrying the Mcm2 deficiency allele are being used to address the roles of cis acting polymorphisms in replication origin sequence elements on sites of replication initiation. Results from these studies may allow prediction of the likelihood of genetic damage occurring at critical locations in the genome that may predispose to cancer or other disease states.
The consequences of deficient replication licensing that we have documented in the mouse are likely to extend to human disease. Using data from The Cancer Genome
Atlas project, we have found that about half of all cancers, and a remarkable 98% of ovarian cancers, exhibit heterozygous deletion or mutation of one or more replication licensing genes and reduced expression similar to the situation in the Mcm2 deficient mouse model. We expect that, as in the mouse model, reduced expression contributes to accumulation of replication related genetic damage and, in a collaborative study with Carl Morrison, we have found that mutation in one component of the replication licensing complex, Mcm4, is correlated with human bladder tumors exhibiting higher levels of chromothripsis (Morrison et al., 2014). Further, a number of studies have now linked replication timing to sites of genome damage in human tumors where there is a strong correlation between late replication and focal CNV or mutation rate. However, late replicating regions are largely devoid of genes involved in oncogenesis and much of the damage observed in these regions is likely not to contribute to cancer. A potentially important implication of our ongoing work is that, once a disruption in the replication licensing system occurs, a subset of otherwise early replicating regions are predicted to become late replicating with a corresponding increase in the rate of loss or mutation of the tumor suppressors and oncogenes carried within these regions.
Are there specific genetic factors that modify the impact of Mcm2 deficiency? In a collaborative study with John Schimenti, we have found that reduction in Mcm3 expression compensates for reduced Mcm2 levels and delays tumor onset (Chuang et al., 2012). This effect may be mediated in part through the presence of a nuclear export signal on the Mcm3 protein. Additional factors that alter the efficiency of replication licensing, and impact genome stability and disease, may exist. To identify such factors, analysis of additional specific candidates, genetic mapping, and high throughput shRNA genetic screens are being utilized to define genes that modulate the effect of Mcm2 deficiency.
What is the role of cell proliferation in tissue and genome aging? Development of the Tet-inducible Cdkn1b transgenic mouse model provides the ability to directly test the role that cell proliferation plays in genetic damage accumulation and age related tissue dysfunction. These studies (Pruitt et al., 2013) have shown that when cell proliferation is reduced in adult mice there is a marked reduction in cancer incidence and expression of markers of genetic damage such as γH2AX is decreased. However, these mice also exhibit a profound progeriod syndrome. Remarkably, a very different result occurs when cell proliferation is suppressed in neonatal mice. These mice exhibit delayed growth but the progeroid symptoms are ameliorated, supporting that an important determinant of tissue maintenance during aging is the relationship between the rate of cell proliferation and the mass of tissue that must be maintained. Ongoing studies utilize this model to test approaches to delaying or reverse age related tissue changes though modulation of cell proliferation or stem cell transplantation.