Murine embryonic stem (ES) cells are derived from the inner cell mass of the developing embryonic blastocyst. These cells can self renew which allows them to be propagated indefinitely in the laboratory and they can differentiate into cell types derived from all three germ layers. Manipulation of the mouse genome using gene targeting techniques in conjunction with ES cell technology has provided valuable insights into embryonic development and cell lineage specification. KIT is a trans-membrane receptor tyrosine kinase (RTK) that dimerises upon binding to its ligand, stem cell factor (SCF) resulting in the auto-phosphorylation of intracellular kinase domains. This activity is crucial for the transmission of signals from the cell surface to the nucleus. KIT is expressed on stem and progenitor cells of many lineages and defects in the SCF/KIT signaling pathway causes detrimental effects at both the cellular and physiological level. This project aimed to investigate the role of the SCF/KIT signalling pathway during murine ES cell differentiation and survival. To assess the role of SCF/KIT signalling in ES cell proliferation and survival, we knocked out the c-kit gene in mouse ES cells to produce heterozygous (KitW-lacZ/+) and KIT Null (KitW-lacZ/W-lacZ) cell lines. The self renewal and differentiation profile of these cell lines revealed an auxiliary role for SCF/KIT during ES cell self renewal and an absolute role in survival upon in vitro differentiation. This phenotype of apoptosis upon differentiation was recapitulated in wild type E14 ES cells treated with a KIT neutralising antibody (ACK2). Wild type cells that were treated with the JNK inhibitor, SP600125 had a comparable phenotype to KIT null cells indicating that this could be one of the mediators of KIT signalling that has a protective role in the survival of differentiating ES cells. We hypothesised that blocking classical apoptotic pathways might prevent the death on differentiation observed in KIT null cells. However, neither blocking the pro-apoptotic P38 pathway with the chemical inhibitor PD169316 nor over-expressing the pro-survival protein BCL2 in KIT Null cells could prevent their apoptosis upon differentiation phenotype. This strongly suggests that these pathways are not involved in KIT mediated survival of differentiating ES cells. Although compensatory mechanisms are thought to exist for defective KIT signaling in vivo, an absolute role is assigned to KIT during ES cell differentiation. Further analysis of micro array data comparing gene expression from wild type E14 and KIT Null cell lines may reveal the specific mechanisms of KIT mediated survival during differentiation onset.

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Chronic Myelogenous Leukemia (CML) is a blood cancer that arises when hematopoietic cells acquire an abnormal protein known as BCR-ABL. Current therapies for CML include drugs that inhibit BCR-ABL. However, these drugs only suppress the disease and do not cure it. One reason is that BCR-ABL drugs fail to kill the primitive population of CML cells, referred to as leukemia stem cells (LSCs), which are responsible for initiating and propagating CML. Since LSCs are not killed, the cancer is not cured and many affected patients eventually relapse. Recent studies suggest that LSCs are protected from current therapies by the bone marrow micro-environment where they reside. There, cytokine signaling molecules are present, which mediate processes that protect LSCs from BCR-ABL drugs. The stem cell factor (SCF) is one of these signaling molecules. It activates the receptor c-Kit located on the surface of LSCs, and this activation in turn allows proliferating LSCs to resist BCR-ABL drugs, even without prior exposure to these drugs, i.e., innate resistance is observed. In this thesis, the mechanism of this innate resistance is investigated, so that a suitable treatment strategy can be developed. To this end, a co-agent approach based on synthetic antibodies (sABs) is proposed to inhibit the receptor c-Kit, with the goal of disrupting its activation by the ligand SCF. This disruption should in turn block the SCF-mediated innate resistance, thus potentially restoring BCR-ABL drug apoptotic activity. The method for this disruption involves targeting the c-Kit structural susceptibility. Specifically, the sABs are designed via antibody phage display technology to target the D1–D2–D3 domains representing the SCF binding sites, hence preventing downstream pathway activation. The hypothesis is that, by blocking the SCF-mediated innate resistance, a suitable combination of such an sAB co-agent and a BCR-ABL drug should be conducive to suppressing LSCs, thereby providing a potential means to improve CML treatment. In addition, to assess the performance of the proposed treatment strategy, a set of in vitro tests is conducted, focusing on performance behaviors such as cell binding, cell death, and the progenitor inhibition. The experimental results support the hypothesis that the proposed combinatorial strategy is indeed a promising approach to mitigate the innate resistance, thus restoring BCR-ABL drug apoptotic activity.