Purpose: In the current study, we examined the in vivo effects of AZD1152, a novel and specific inhibitor of Aurora kinase activity (with selectivity for Aurora B). Experimental Design: The pharmacodynamic effects and efficacy of AZD1152 were determined in a panel of human tumor xenograft models. AZD1152 was dosed via several parenteral (s.c. osmotic mini-pump, i.p., and i.v.) routes. Results: AZD1152 potently inhibited the growth of human colon, lung, and hematologic tumor xenografts (mean tumor growth inhibition range, 55% to z100%; P < 0.05) in immunodeficient mice. Detailed pharmacodynamic analysis in colorectal SW620 tumor-bearing athymic rats treated i.v. with AZD1152 revealed a temporal sequence of phenotypic events in tumors: transient suppression of histone H3 phosphorylation followed by accumulation of 4N DNA in cells (2.4-fold higher compared with controls) and then an increased proportion of polyploid cells (>4N DNA, 2.3-fold higher compared with controls). Histologic analysis showed aberrant cell division that was concurrent with an increase in apoptosis in AZD1152-treated tumors. Bone marrow analyses revealed transient myelosuppression with the drug that was fully reversible following cessation of AZD1152 treatment. Conclusions: These data suggest that selective targeting of Aurora B kinase may be a promising therapeutic approach for the treatment of a range of malignancies. In addition to the suppression of histone H3 phosphorylation, determination of tumor cell polyploidy and apoptosis may be useful biomarkers for this class of therapeutic agent. AZD1152 is currently in phase I trials.

The Aurora kinases have been the subject of considerable interest as targets for the development of new anticancer agents. While evidence suggests inhibition of Aurora B kinase gives rise to the more pronounced antiproliferative phenotype, the most clinically advanced agents reported to date typically inhibit both Aurora A and B. We have discovered a series of pyrazoloquinazolines, some of which show greater than 1000-fold selectivity for Aurora B over Aurora A kinase activity, in recombinant enzyme assays. These compounds have been designed for parenteral administration and achieve high levels of solubility by virtue of their ability to be delivered as readily activated phosphate derivatives. The prodrugs are comprehensively converted to the des-phosphate form in vivo, and the active species have advantageous pharmacokinetic properties and safety pharmacology profiles. The compounds display striking in vivo activity, and compound 5 (AZD1152) has been selected for clinical evaluation and is currently in phase 1 clinical trials.

Summary Oncogenes and oncosuppressors can derugulate cell replication in tumours, and recently have been shown to influence the probability of apoptosis. The effects of human c-myc and mutated (T24) Ha-ras oncogenes on susceptibility to apoptosis were investigated by introducting them into immortalised rat fibroblasts. The resulting family of transfectants showed closely similar measures of proliferation, but widely divergent rates of apoptosis, differing by up to fifteen-fold, that correlated inversely with population expansion rates in vitro. T24-ras transfectants with moderate or high p2lras expression showed reduced apoptosis, and this was reversed by pharmacological inhibition of membrane localisation of p2l's by mevinolin. In contrast, c-myc stimulated apoptosis, and this was further enhanced by serum deprivation. Inducibility of effector proteins represents one possible mechanism of genetic control of the susceptibility to apoptosis, and its investigation showed that c-myc was associated with expression by viable cells of latent calcium/magnesium sensitive endonuclease activity characteristic of apoptosis. In contrast, endonuclease activity was not detected in viable cells of a T24-ras transfectant expressing high levels of p2lras. Thus, there appeared to be differential regulation of susceptibility to apoptosis, positively by c-myc and negatively by activated ras, and this was associated with availability of endonuclease activity. Genetic modulation of apoptosis in human neoplasms is likely to influence net growth rate, retention of cells acquiring new mutations and response to certain chemotherapeutic agents.

The synthesis of a novel series of quinazolines substituted at C4 by five-membered ring aminoheterocycles is reported. Their in vitro structure-activity relationships versus Aurora A and B serine-threonine kinases is discussed. Our results demonstrate that quinazolines with a substituted aminothiazole at C4 possess potent Aurora A and B inhibitory activity and excellent selectivity against a panel of various serine-threonine and tyrosine kinases, as exemplified by compound 46. We found also that the position and nature of the substituent on the thiazole play key roles in cellular potency. Compounds with an acetanilide substituent at C5' have the greatest cellular activity. The importance of the C5' position for substitution has been rationalized by ab initio molecular orbital calculations. Results show that the planar conformation with the sulfur of the thiazole next to the quinazoline N-3 is strongly favored over the other possible planar conformation. Compound 46 is a potent suppressor of the expression of phospho-histone H3 in tumor cells in vitro as well as in vivo, where 46, administered as its phosphate prodrug 54, suppresses the expression of phospho-histone H3 in subcutaneously implanted tumors in nude mice.

AZD6738 is a potent and selective orally bioavailable kinase inhibitor of ataxia telangiectasia and rad3 related (ATR). Here we report the pre-clinical in vitro and in vivo and biological profile of AZD6738. ATR is a serine/threonine protein kinase involved in DNA damage response signalling caused by DNA replication associated stress. Activation of ATR at stalled replication forks leads to suppression of replication fork origin firing, promotes repair and S/G2-cell cycle checkpoints to prevent premature mitosis and maintain genomic integrity. Failure to resolve damage leads to genomic instability and if sufficiently high, cell death. Stalled replication forks may collapse leading to formation of DNA double stranded breaks and activation of the ataxia telangiectasia mutated (ATM) kinase. ATM works in conjunction with ATR to efficiently resolve replication associated DNA damage creating a co-dependency with loss of one leading to a greater reliance on the other to maintain genomic stability. ATM is frequently inactivated across B-cell malignancies, head and neck, breast and lung cancers through chromosomal deletion, promoter hypermethylation or mutation. ATM-deficient tumours are hypothesised to be more reliant on ATR for survival and specific inhibition of ATR may lead enhanced anti-tumour activity while minimizing normal tissue toxicity. AZD6738 demonstrates this preclinical profile. AZD6738 inhibits the phosphorylation of direct downstream substrate CHK1 while increasing the phosphorylation of ATM-dependent substrate CHK2 and DNA damage marker γH2AX. This is associated with impaired S-phase cell cycle progression with prolonged inhibition causing cell death, indicative of replication fork stalling, collapse and irreversible damage. AZD6738 is active as a single agent across cancer cell line panels but shows enhanced sensitivity in cell lines with ATM-pathway defects. AZD6738 when used in combination with DNA damaging inducing agents’ gemcitabine, cisplatin or ionising radiation (IR) shows enhanced synergistic cell killing activity. In vivo, AZD6738 monotherapy treatment leads to significant anti-tumour activity in ATM-deficient but not ATM-proficient xenograft models at equivalent, tolerated doses. When AZD6738 is used in combination with carboplatin or IR anti-tumour growth inhibitory activity or regression is observed. AZD6738 in vivo activity is also associated with a persistent increased γH2AX staining in tumour tissue but only a transient increase in normal bone marrow or gut tissue suggesting a favourable therapeutic index can be achieved. Early pre-clinical safety studies support these findings with target related bone marrow suppression, neutropenia and GI tract impact only at high-doses. These data merit further investigation of AZD6738 as a monotherapy or in combination with chemo or radiotherapy. Citation Format: Sylvie M. Guichard, Elaine Brown, Rajesh Odedra, Adina Hughes, Dan Heathcote, Jen Barnes, Alan Lau, Steve Powell, Clifford D. Jones, Willem Nissink, Kevin M. Foote, Philip J. Jewsbury, Martin Pass. The pre-clinical in vitro and in vivo activity of AZD6738: A potent and selective inhibitor of ATR kinase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2013-3343

The DNA damage response (DDR) comprises a range of mechanisms that ensure the integrity of the genome. As part of the DDR and in response to specific DNA damage, the master kinases ATM, ATR and DNA-PK are activated in a coordinated fashion inducing cell cycle arrest and repair of the damage before resuming DNA replication. Alternatively they may induce apoptosis if the DNA damage is incompatible with cell viability. Due to their genetic instability, tumour cells may be more reliant on the DNA damage response and so inhibiting specific DDR components may lead to antitumour activity while minimizing toxicity in normal tissue. ATR (Ataxia telangectasia and Rad 3 mutated) is recruited at specific sites of DNA damage, namely ssDNA (single stranded DNA) coated with RPA (Replication Protein A). ATR activates downstream substrates that regulate replication fork progression, cell cycle checkpoint, and DNA repair proteins. Here we report the discovery of AZ20, a novel potent and selective inhibitor of ATR kinase identified by screening a subset of the AZ compound collection against the target enzyme. In vitro, AZ20 decreases pChk1 Ser345, pChk1 Ser317 and pChk1 Ser296 levels in a concentration-dependent manner. Prolonged exposure with AZ20 increases γH2AX pan-nuclear staining, indicative of replication stress. This is associated with S-phase arrest and increase in phospho-histone H3. AZ20 induces growth inhibition and cell death in vitro and its profile of activity is distinct from other cytotoxic agents such as platinum derivatives, permetrexed or docetaxel. The cytotoxic effect of AZ20 can be increased in combination with the selective ATM inhibitor KU-60019. AZ20 induces significant tumour growth inhibition in vivo at well tolerated doses. This is associated with a persistent elevation of γH2AX pan-nuclear staining in xenograft tissue, but a transient increase in mouse bone marrow at therapeutic doses, suggesting a favourable therapeutic index. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1823. doi:1538-7445.AM2012-1823

The PARP inhibitor olaparib acts through both inhibition of DNA single-strand-break repair and trapping of PARP-DNA complexes creating DNA lesions which cause replication fork stalling and collapsed fork DNA breaks. Cells which have lost BRCA-dependent homologous recombination repair are highly sensitive to olaparib treatment and this has led to its approval in patients with tumors carrying BRCA mutations. In addition, ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia mutated and Rad3 related) dependent DNA repair processes are hypothesized to be important survival pathways to PARP inhibitor treatment. In pre-clinical studies cancer cells which have defects in either ATM or ATR have been shown to be sensitive to PARP inhibitors. Here we present data that the orally bioavailable ATR inhibitor AZD6738 (in Phase-I clinial trials) combines synergistically with olaparib leading to cell death and anti-tumour activity in pre-clinical models. The combinations are effective across a panel of gastric and lung cancer cell lines in vitro. In addition results from isogenic ATM−/-/− knockout versus ATM+/+/+FaDu head and neck cancer cell line pairs show enhanced combination activity in ATM knockout cells versus ATM wild-type cells. Studies in vivo show that through intermittent dosing the combination is tolerated while demonstrating significant anti-tumour efficacy and regressions across multiple human patient derived primary explant models. Together, these data support the notion of development of AZD6738 and olaparib combinations for the treatment of ATM-deficient cancers. Citation Format: Alan Lau, Elaine Brown, Andrew Thomason, Rajesh Odedra, Victoria Sheridan, Elaine Cadogan, Shirlian Xu, Andy Cui, Paul R. Gavine, Mark O'Connor. Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C60.

Purpose-PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA mutant (BRCA MUT ) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression.Experimental Design-Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776) or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cellcycle, genome instability and apoptosis were evaluated in BRCA1/2 MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2 MUT patient-derived-xenograft (PDX) model.Results-PARPi monotherapy resulted in a decrease in BRCA MUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum-tolerated dose in a BRCA MUT PDX. PARPi treatment increased pATR and pCHK1 indicating activation of the ATR-CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared to single agent treatments in BRCA MUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCA MUT PDX with the PARPi-ATRi combination inducing tumor regression and in most cases, complete remission.Conclusions-PARPi causes increased reliance on ATR/CHK1 for genome stability and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCA MUT models.