Cdc7/ASK kinase as a novel target for anti-cancer drug development programmes.
Doctoral thesis, UCL (University College London).
Targeting Cdc7, a kinase essential for DNA replication initiation, results in potent cancer cell killing. Cancer cells in which CDC7 is silenced by RNAi enter an abortive S phase followed by apoptosis due to loss of a functioning DNA replication origin activation checkpoint. This checkpoint prevents normal cells from entering S phase (reversible G1 arrest) if the DNA replication initiation machinery is perturbed. The pre-clinical anti-cancer effects of CDC7 silencing have highlighted this kinase as an important target for new drug development. Expanding on published reports, I performed further target validation using molecular tools generated in the work of this thesis, including an affinity-purified antibody to the Cdc7 regulator ASK and functional recombinant Cdc7/ASK kinase complex. Making use of fibroblast and HL60 tissue culture model systems, I show that Cdc7 and ASK are amongst a group of essential replication initiation factors that are tightly downregulated to suppress proliferative capacity during exit from cycle into quiescent and differentiated states. This finding is further supported by low expression levels in normal liver and oral squamous epithelium and the lack of Mcm2 phosphorylation at serine 53, a well known Cdc7 target. In liver carcinoma and oral squamous cell carcinoma, on the contrary, the majority of cancer cells are expressing Cdc7 and ASK and show Mcm2 phosphorylation at Ser-53. Thus it can be postulated that Cdc7 inhibitors should selectively kill cancer cells, while normal proliferating cells are reversibly arresting in G1 and quiescent and differentiated cell populations are not affected due to downregulation of the target protein. To screen for compounds that selectively inhibit Cdc7, I developed a sensitive in vitro kinase assay and contributed to the successful transfer of this assay to a high-throughput screening platform and the generation of a structural model of the Cdc7 kinase domain allowing in silico predictions of the most potent inhibitors. On completion of the work for this thesis, the HTS assay and structural model fromed the core of an ongoing drug discovery programme run by Cancer Research Technology. Two series of novel, selective small molecule inhibitors which exhibit low nM activity against Cdc7 and cellular efficacy (apoptosis) have been developed and are currently being tested in mouse xenograft models. The work presented in this thesis provides a strong rationale for targeting the DNA replication initiation pathway, and in particular Cdc7. Future intend to treat clinical trials will establish the potential of pharmacological Cdc7 inhibitors for selective cancer cell killing in patients.
|Title:||Cdc7/ASK kinase as a novel target for anti-cancer drug development programmes|
|Open access status:||An open access version is available from UCL Discovery|
|Additional information:||Copyright restricted material comprising of published articles in appendices D, E and F has been removed from the e-thesis.|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Wolfson Institute and Cancer Institute Administration > Cancer Institute > Research Department of Pathology|
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