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Title: The investigation of AT7519 (CDK inhibitor) and AT13387 (HSP90 inhibitor) as novel therapies for the treatment of pancreatic ductal adenocarcinoma
Author: Thomas, A. J.
ISNI:       0000 0004 6060 018X
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is usually a systemic disease and so requires chemotherapy even when surgical resection is possible. Current agents such as gemcitabine are only effective in a minority of patients and chemoresistance is an important issue faced in pancreatic cancer. There is an urgent need for more effective therapies; Novel agents may offer an avenue for this. The CDK family of protein kinases are pivotal in cell cycle regulation that is often deranged in cancer. HSP90 is a molecular chaperone affecting multiple key cellular signaling pathways of importance in pancreatic cancer. OBJECTIVES: To determine the efficacy of AT7519, a novel CDK inhibitor, and AT13387, a novel HSP90 inhibitor in pancreatic cancer models in vivo and in vitro both as single agents and in combination with gemcitabine. METHODS: Cell proliferation was measured using the EZ4U assay. Cell cycle analysis was performed with flow cytometry. Apoptosis analysis was using the Caspase-Glo 3/7 luminescent assay. Western blotting assessed expression of client proteins and phosphorylation. A murine xenograft model was employed assessing tumour volumes with external calipers. Experiments were performed using AT7519 and AT13387 as single agents and in combination with gemcitabine. RESULTS: AT7519 inhibited proliferation in cell lines including a gemcitabine resistant line (SUIT-2 GR) with IC50 values ranging 5-2000nM. Cell cycle analysis showed actions in line with CDK inhibition. At 24 hours induction of caspase 3/7 was significantly increased in AT7519 treated cells compared with those in control media (p=0.01). Phosphorylation of client proteins were inhibited. Isobolar analysis of AT7519 combined with gemcitabine suggested an additive effect. AT7519 was tolerated as a single agent and in combination with gemcitabine in a xenograft model and resulted in slowed tumour growth compared to control (p = 0.0446). AT13387 inhibited proliferation of all cell-lines with IC50 values of 29nM-325nM including a cell line with acquired gemcitabine resistance. AT13387 treated cells accumulated in G0/G1 and G2/M phases of the cell cycle (p < 0.05). Late apoptosis was seen after 40 hours post treatment with At13387. AT13387 treatment resulted in down-regulation of HSP90 client proteins and up-regulation of the HSP70 co-chaperone. AT13387 was tolerated and moderately efficicacous as a singe agent in a murine xenograft model but was poorly tolerated in combination with gemcitabine and conferred no benefit over single agents. CONCLUSIONS: AT7519 is a promising agent for combination therapy in pancreatic cancer, as acquired resistance to gemcitabine does not give AT7519 resistance. AT13387 showed promising effects in vitro but was poorly tolerated in an in vivo combination with standard therapy therefore may offer a therapeutic avenue for non-responders to conventional therapies.
Supervisor: Ghaneh, P. ; Greenhalf, W. Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral