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Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial. Clin Cancer Res; 18(14); 3912–23. ©2012 AACR.

The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3β and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a single-agent anticancer strategy. Mol Cancer Ther; 9(5); 1100–10. ©2010 AACR.

The AGC kinase AKT is a key component of the phosphatidylinositol 3-kinase (PI3K) pathway, which is frequently deregulated in cancer, making AKT a target of major therapeutic interest. However, PI3K signaling through both AKT-dependent and AKT-independent mechanisms involving other AGC kinases, such as p70S6K, PKA, SGK and ROCK, is important in a range of cancers. Hence, the pharmacological inhibition of these multiple AGC kinases may increase response rates and minimize clinical resistance compared with targeting AKT alone. The clinical drug candidate AT13148 is a multi-AGC kinase, ATP-competitive inhibitor, identified utilizing high-throughput X-ray crystallography and fragment-based lead discovery techniques. Screening of this oral small molecule against a panel of kinases at 10μM revealed >80% inhibition of the structurally related AGC kinases AKT, PKA, ROCK2, p70S6K, MSK, RSK1/2, and SGK. We demonstrate that AT13148 has antiproliferative activity in a range of in vitro models harboring relevant genetic abnormalities, including PTEN, KRAS, PIK3CA and HER2 aberrations. AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induction of apoptosis in both a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects both in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP competitive inhibitors of AKT, is not a therapeutically relevant reactivation step for this compound. We used gene expression microarray studies to characterize the underlying molecular mechanisms of action of AT13148 and the selective AKT inhibitor CCT128930, and observed the induction of upstream regulators including insulin receptor substrate-2 and PIK3IP1 due to compensatory feedback loops, consistent with blockade of AKT signaling. These studies also showed that AT13148 and CCT128930 have distinct molecular effects in cancer cells: AT13148 had a predominant effect on apoptosis genes and caused a greater apoptotic phenotype, while CCT128930 modulated genes in the network regulating cell cycle. This finding emphasizes the functional differences of AT13148 as a multi-AGC kinase inhibitor in contrast to a more AKT-selective inhibitor. In view of the potential mechanistic advantages detailed above, and the potent antitumor activity observed at well tolerated doses against established human tumor xenografts with clinically relevant genetic drivers, the clinical utility of such an AGC kinase inhibitor strategy will now be assessed in a first-in-human Phase I trial of AT13148. 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 928. doi:1538-7445.AM2012-928

The tumour microenvironment promotes selective outgrowth of cells carrying mutations in oncogenes or tumour suppressors, enabling the activated pathways to drive tumour growth. Two such pathways are the PI3-K/AKT and RAS/RAF/MEK signaling pathways, which demonstrate a high frequency of mutations in human cancer. These pathways represent important therapeutic targets, and a number of targeted agents are now in clinical trials. Despite being pertinent to the clinical situation, the role that the tumour microenvironment plays in modifying responsiveness to therapeutic agents targeted against these pathways is poorly understood. A major challenge faced during the development of PI3-K/AKT and RAS/RAF/MEK targeted agents is to demonstrate inhibitor selectivity and efficacy in a patient-relevant cell based system. Isogenic ‘X-MAN’ paired cell lines help address this by providing a system in which the endogenous wild-type or mutant alleles of the parental cell line are selectively activated or silenced via targeted homologous integration, providing a pair of human cell-lines in which the only difference between the cells is the mutation of interest. This approach therefore overcomes the issues that may occur when over-expression methods are employed. We therefore set out to investigate how changes in the tumour microenvironment altered cell responses to PI3-K and MEK inhibitors. We employed isogenic HCT116 human colon cancer cells where the parental cells harbor pre-existing H1047R PIK3CA and G13D KRAS alleles and compared these to isogenic clones that retain either the PIK3CA or KRAS wild-type alleles. Inhibitors were profiled under a range of defined conditions chosen to model the tumour microenvironment, including low glucose, low serum and hypoxia, and the activity of the compounds investigated. Under standard culture conditions, we found that PI3-K and MEK inhibitors showed minimal selectivity for cells with activating mutations compared to wild-type cells. When grown under defined culture conditions which more closely mimic the tumour microenvironment, the effects of the driving mutations became dominant and a differential between wild-type and mutant cell responses to inhibitors was observed. The selective PI3-K inhibitors GDC-0941 and PI-103 showed a >10-fold increase in potency when tested in anti-proliferative assays in mutant vs, wild-type cell lines. However, the dual PI3-K/mTOR inhibitor BEZ-235 did not show selectivity in this system. We show that by using isogenic cell lines under conditions which model the tumour microenvironment, differences in activity profiles between specific inhibitors can be quickly and definitively identified, highlighting the advantages of using this system in profiling molecularly targeted agents. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1651.

Deregulated AKT signalling is implicated in cancer. The preclinical characterization of AKT inhibitors and development of PD biomarkers are vital prior to clinical trials. Invasive techniques for clinical PD analyses pose ethical and logistical issues, hence hair follicles may represent a non-invasive option. The pyrazole AT7867 and pyrrolopyrimidine CCT128930 are novel ATP-competitive AKT inhibitors from different chemical series developed at The Institute of Cancer Research in collaboration with Astex Therapeutics (Cambridge, UK). AT7867 and CCT128930 have IC50 values against AKT2 of 17nM and 6nM respectively. Growth inhibitory IC50 (GI50) values were 2.4μM and 6.3μM in PTEN-null U87MG glioblastoma cells as measured by SRB assay. Cellular studies of both compounds in U87MG cells were carried out by Meso Scale Discovery (MSD) ELISA, western blot (WB) and immunofluorescence. These showed an initial induction of phosphorylated (p) Ser473 AKT as expected with ATP-competitive AKT inhibitors, and inhibition of downstream AKT targets, including pSer9 GSK-3β, pThr246 PRAS40, pSer127 YAP and pSer235/236 S6RP, indicating AKT pathway blockade. Interestingly, CCT128930 inhibited phosphorylation of AKT targets GSK-3β, S6RP and PRAS40 at lower concentrations (0.5-1µM), compared to AT7867 (5-10µM) in U87MG cells. Phosphorylation was also inhibited at earlier time points with equipotent doses (3XGI50) of CCT128930 versus AT7867. CCT128930 also caused a predominant G0/G1 phase arrest, while AT7867 resulted in a predominant G2 arrest in U87MG cells at equipotent doses (3XGI50), using BrdU and PI staining and flow cytometry. We report for the first time in vivo efficacy with intraperitoneally (ip) or orally administered AT7867 in PTEN-null MES-SA uterine sarcoma and U87MG mouse xenograft models; and ip administered CCT128930 in HER2-overexpressing BT474 breast cancer and U87MG xenografts, which correlated with in vivo pharmacokinetics (PK) and PD biomarker modulation of pSer9 GSK-3β and pSer235/236 S6RP using WB and MSD. Hair follicles were developed as a robust surrogate PD biomarker, with pThr246 PRAS40 as a PD readout. This assay was validated with healthy volunteer hair follicles, which were treated ex vivo with CCT128930. A significant decrease in pThr246 PRAS40 (p In conclusion, by employing an integrated PK and PD biomarker-driven drug discovery strategy, we have developed 2 novel and potent AKT inhibitors with antitumor activity, and have validated a robust surrogate biomarker assay for AKT inhibition for use in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4481.