PL02-01

The clinical response to DNA-damaging anticancer therapies may be compromised by cellular DNA repair processes, and agents that impede DNA repair can thus lead to the potential therapeutic endpoints of radio- and chemo-sensitisation. The phosphatidylinositol (PI) 3-kinase related kinases (PIKKs) are a family of six protein serine/threonine kinases, structurally different from classical protein kinases, and whose kinase domains more closely resemble those of the phosphatidylinositol 3-kinase family of phospholipid kinases [1]. Two prominent members of the PIKK family, DNA-dependent protein kinase (DNA-PK) and ATM (ataxia telangiectasia mutated), play key roles in the cellular response to DNA damage through the detection and repair of DNA double-strand breaks (DSBs) [2-4]. DNA-PK comprises a large (465 kDa.) catalytic subunit DNA-PKcs, and a heterodimeric DNA targeting subunit termed Ku, with subunits of ~ 70 kDa (Ku70) and ~ 83 kDa (Ku80). DNA-PK plays a pivotal role in the non-homologous end-joining component of the DSB repair pathway, and in the site-specific end joining process of V(D)J recombination. The 345 kDa phosphoprotein ATM coordinates the cellular response to ionizing radiation-induced DSBs, by phosphorylating multiple downstream response factors involved in cell cycle regulation and DNA repair, including p53, CHK2, NBS1, and BRCA1.
 Importantly, human cell lines defective in either DNA-PK or ATM function are hypersensitive to agents that elicit DNA DSBs, and the non-selective PIKK inhibitors wortmannin and LY294002 sensitize tumor cells to ionizing radiation and DSB-inducing cancer chemotherapeutic agents, as well as inhibiting DNA-DSB repair. Taken together, these data provide compelling evidence that selective small-molecule inhibitors of DNA-PK and ATM may have a therapeutic role as radio- and chemo-sensitizers in the treatment of cancer. Kinase-specific small molecule inhibitors would also aid in elucidating the roles played by PIKKs in the cellular response to DNA damage. Our ongoing studies, conducted in collaboration with KuDOS Pharmaceuticals, are directed towards the development of potent and selective DNA-PK and ATM inhibitors for clinical evaluation as agents to enhance the cytotoxicity of DNA-damaging therapies in the treatment of cancer. In the absence of crystal structures of DNA-PK or ATM, the chromenone LY294002 was employed as a structural lead for the development of potent and selective inhibitors of these kinases. These studies resulted in the identification of potent and selective small molecule inhibitors of DNA-PK and ATM, exemplified by NU7441 (DNA-PK, IC50 = 12 nM) and KU-0055993 (ATM, IC50 = 10 nM), respectively [5,6]. These inhibitors effectively sensitized several human tumour cell lines to the cytotoxic effects of both ionizing radiation, and DNA-damaging drugs at submicromolar concentrations in vitro, and preliminary in vivo studies proved promising [7,8].
 A major determinant of poor prognosis and therapeutic resistance in B-cell chronic lymphocytic leukemia (CLL) is dysfunction in the p53-dependent DNA damage response pathway, with defects in p53 (including 17p deletions [del(17p)] and p53 mutation) conferring an extremely poor prognosis [9]. Recently, we have found that the mutational status of ATM and associated 11q deletions [del(11q)] predict reduced overall and treatment-free survival. Since ATM is a key upstream regulator of p53, defects in either ATM or p53 lead to an impaired DNA damage response that correlates with therapeutic resistance and reduced survival. Interestingly, NU7441 was demonstrated to sensitize CLL cells to chlorambucil and fludarabine, including those cells having p53 or ATM gene deletions or dysfunction, and expressing high levels of DNA-PK. NU7441 was also shown to increase fludarabine-induced DSBs. Levels of DNA-PK activity were consistently higher in del(17p) and del(11q) cases, with high DNA-PK levels and chlorambucil resistance predicting for reduced treatment-free interval. These observations suggest that a DNA-PK inhibitor may demonstrate therapeutic efficacy in poor prognosis CLL patients.
 The further development of NU7441 and KU-0055993 has been hampered by poor aqueous solubility. Current studies centre on optimisation of the biological and pharmaceutical properties of these chemotypes as a prelude to the selection of clinical candidates.
 References
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AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics-- Oct 22-26, 2007; San Francisco, CA