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Stem Cell Survival in the Face of Genomic Instability

Geiselhart, Anja

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Hematopoietic stem cells (HSCs) sustain the life-long production of blood and maintain the integrity of the hematopoietic system. Therefore, they possess self-renewal capacity and are able to differentiate into all the mature blood cell lineages. These are finely tuned processes, which constantly involve dynamic HSC fate decisions and an imbalance may result in misregulation associated with cellular transformation or other types of diseases. In the hematopoietic disorder Fanconi Anemia (FA), such an imbalance in the HSC pool results in bone marrow failure, a collapse of the entire hematopoietic system. In addition, FA patients are susceptible to developing hematologic malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). This is the result of a loss of function in the FA DNA repair machinery, which renders HSCs genetically unstable as they are unable to repair certain forms of DNA damage. Murine knockout models for individual FA pathway members fully recapitulate the FA HSC DNA repair defect seen in patients and demonstrate a severe engraftment defect in competitive transplantation experiments. We could recently show, that in response to proliferative stress, such as during transplantation and reconstitution, FA HSCs accumulate DNA damage and are quickly lost due to apoptotic cell death, which is a key driver of bone marrow failure. FA HSCs are also subject to cellular transformation, which may result in the outgrowth of a leukemic founder clone. The identification of novel therapeutic targets would likely provide insight into the underlying mechanism of the FA HSC defect and the associated pathology of FA. In this respect, forward genetic screens using insertional mutagenesis have proven to be a powerful screening method for the identification of genes with the potential to influence stem cell kinetics when upregulated or disrupted. We have employed retroviral insertional mutagenesis in the context of FA HSC biology using a murine transplantation model in order to try and identify novel factors with the potential to rescue the inherent FA HSC transplantation defect. In this respect, we show that retroviral vector integrations trigger the expansion of nonmalignant dominant FA clones in transplanted mice and allow the retrospective identification of nearby genes whose deregulation had caused clonal expansion in the face of genomic instability. We identified four candidate drivers of clonal dominance in the FA HSCs: Osgin1, Evi1, Taf1b and Grhl1, which we further characterized in the context of HSC biology under homeostatic and stress conditions as well as during development. In this respect, we identified the oxidative stress-induced growth inhibitor (Osgin1) as a promising candidate target gene, which is expressed in FA and WT HSCs under homeostatic conditions as well as in hemogenic endothelium/HSPCs during developmental specification from normal embryonic stem cells. Furthermore, we provide insight into the hematopoietic role of Osgin1, which displays differential expression levels during the individual steps of HSC commitment and their response to physiologic stress. We conclude that Osgin1 is an essential factor for hematopoiesis and we will further characterize Osgin1 using the tools and assays that we have developed to assess whether it impacts on FA HSC fate decisions in vitro and during hematopoietic reconstitution in vivo. Furthermore, we provide some insight into how the FA HSCs may become a dominant clone and suggest likely candidate mechanisms with the potential to compensate the inherent FA HSC defect. According to our hypothesis, the identified candidate target genes may either prevent the acquisition of DNA damage or repair it or, alternatively, block the apoptotic cell death, which is a likely cell fate outcome for FA HSCs. Our findings hold great potential for HSC research with implications for normal as well as FA HSC biology.

Document type: Dissertation
Supervisor: Milsom, Dr. Michael Dennis
Date of thesis defense: 20 May 2015
Date Deposited: 29 May 2015 11:07
Date: 2015
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
DDC-classification: 500 Natural sciences and mathematics
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