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AT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

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AT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity Published OnlineFirst July 10, 2012; DOI: 10.1158/1078-0432.CCR-11-3313 Clinical Cancer Cancer Therapy: Preclinical Research AT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity Timothy A. Yap1, Mike I. Walton1, Kyla M. Grimshaw2, Robert H. te Poele1, Paul D. Eve1, Melanie R. Valenti1, Alexis K. de Haven Brandon1, Vanessa Martins1, Anna Zetterlund1, Simon P. Heaton1, Kathrin Heinzmann1, Paul S. Jones3, Ruth E. Feltell2, Matthias Reule2, Steven J. Woodhead2, Thomas G. Davies2, John F. Lyons2, Florence I. Raynaud1, Suzanne A. Eccles1, Paul Workman1, Neil T. Thompson2, and Michelle D. Garrett1 Abstract 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); 1–12. Ó2012 AACR. Introduction threonine kinases, including several members of the AGC The class I phosphoinositide 3-kinases (PI3K) are key kinase family, such as AKT, also known as protein kinase B mediators of intracellular signaling between the membrane- (PKB), phosphoinositide-dependent kinase 1 (PDK1), bound receptor tyrosine kinases (RTKs) and downstream p70S6 kinase (p70S6K), p90 ribosomal S6 kinase (RSK), effector molecules, which control many vital cellular func- serum- and glucocorticoid-induced kinase (SGK), and Rho tions, including survival, growth, proliferation, and motility kinase (ROCK; refs. 3, 4). The PI3K-AKT axis of this signaling (1, 2). Downstream of these PI3Ks lies a network of serine/ network is hyperactivated in multiple cancers through dif- ferent mechanisms, including the deregulation of upstream RTKs, for example, insulin-like growth factor-1 receptor 1 Authors' Affiliations: Cancer Research UK Cancer Therapeutics Unit, (IGF-1R), and genetic alterations of PIK3CA, PTEN, or AKT Division of Cancer Therapeutics, The Institute of Cancer Research, Sutton; 2Astex Pharmaceuticals, Cambridge; and 3Cancer Research UK Drug genes, AKT1, 2, and 3 (1, 2). Thus, pharmacologic inhibition Development Office, Research Operations and Funding, London, United of this pathway is an area of great therapeutic interest (5). Kingdom Several drugs targeting the PI3K-AKT pathway are cur- Note: Supplementary data for this article are available at Clinical Cancer rently in clinical development, including inhibitors of PI3K, Research Online (http://clincancerres.aacrjournals.org/). AKT, and mTORC1/2 (5, 6). However, inhibiting PI3K-AKT Corresponding Author: Michelle D. Garrett, Cancer Research UK Cancer signaling at a single node has shown relatively limited Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SM2 clinical efficacy to date. There are several possible explana- 5NG, United Kingdom. Phone: 44-20-8722-4352; Fax: 44-20-8722-4126; tions for this. First, AKT inhibition has been shown to E-mail: [email protected] relieve feedback suppression of RTK expression and activity, doi: 10.1158/1078-0432.CCR-11-3313 which may attenuate antitumor activity (7). Second, PI3K Ó2012 American Association for Cancer Research. deregulation may promote cancer through both AKT- www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst July 10, 2012; DOI: 10.1158/1078-0432.CCR-11-3313 Yap et al. ies that characterize the underlying molecular mechanisms Translational Relevance of action of AT13148, including compensatory feedback Deregulated phosphoinositide 3-kinase (PI3K)-AKT loops that reveal differences compared with a more selective pathway signaling through AGC kinases is implicated AKT inhibitor. On the basis of these studies, AT13148 is in many cancers. The simultaneous inhibition of mul- now undergoing preclinical development before entry into tiple AGC kinases may increase antitumor activity and phase I clinical trials. minimize clinical resistance compared with a single kinase target. The clinical candidate AT13148 is a novel, Materials and Methods oral, multi-AGC kinase inhibitor, which has potent Cell culture and reagents pharmacodynamic and antitumor activity in human All human cancer cell lines were purchased from the tumor xenografts with clinically relevant genetic defects American Type Culture Collection and grown in their in vitro and in vivo, and shows a distinct mechanism of recommended culture medium, containing 10% FBS at action from selective AKT inhibitors. AT13148 showed 37C in an atmosphere of 5% CO and passaged for less linear pharmacokinetics, achieved therapeutically active 2 than 6 months. AT13148 (16), CCT128930 (17), and drug concentrations, and induced biomarker changes LY294002 (Calbiochem, Merck Biosciences) were made up consistent with AGC inhibition in human tumor xeno- as 10 mmol/L stocks in dimethyl sulfoxide (DMSO). grafts. Moreover, we show for the first time that induc- tion of AKT phosphorylation at serine 473 by AT13148, In vitro kinase assays as reported for other ATP-competitive inhibitors of AKT, AT13148 was assayed against 40 kinases (National Centre is not a therapeutically relevant reactivation step for this for Kinase Profiling, Dundee, UK) and the percentage inhi- compound. These detailed preclinical and mechanistic bition at 10 mmol/L of AT13148 was determined. Individual data will facilitate the forthcoming first-in-human phase IC values were measured for selected kinases using ATP I trial of AT13148. 50 concentrations equivalent to the Km for each enzyme (Invitrogen). Protein immunoblotting and immunoassay dependent and AKT-independent mechanisms, the latter Cells were harvested, lysates prepared, protein estima- involving the AGC kinases PDK1 and SGK (8). Third, tions conducted, and Western blots undertaken as described inhibition of a single node such as PI3K or AKT may allow (18), using the following antibodies: pSer473 AKT, AKT, clinical resistance as reported for the selective BRAF inhib- pSer9 GSK3b, GSK3b, pSer235/236 S6 ribosomal protein itor vemurafenib (PLX4032, Roche; ref. 9). Furthermore, (S6RP), S6RP, pSer330 NDRG, NDRG, pSer157 VASP, concurrent blockade of multiple components of the PI3K VASP, pSer19 MLC2, MLC2, pThr24 FOX01/pThr32 network may have greater therapeutic value than inhibition FOX03a, FOX01, PRAS40, cleaved PARP, IRS2, pThr1135 of any single target (10). Thus, the simultaneous inhibition Rictor, Rictor, cyclin E2, c-MYC (Cell Signaling Technolo- of several essential nodes of the PI3K signaling network may gy), PIK3IP1 (Abcam), pThr246 PRAS40 (Upstate), cleaved provide greater overall suppression of key pathways, with caspase-3 (Epitomics), cyclin D1, p27, and glyceraldehyde- the potential for improved therapeutic efficacy across a 3-phosphate dehydrogenase (GAPDH; Neomarkers). broader range of cancer types with less opportunity for Western blot analysis of MES-SA cells was conducted as resistance to develop. above, except that samples were lysed in 40 mmol/L Tris/ A possible strategy for such a targeted combinatorial HCl pH 7.5, 274 mmol/L NaCl, 2% Triton-X-100, and 20% blockade is the development of a drug that inhibits AKT glycerol. Samples were resolved by SDS-PAGE, blotted onto together with other key AGC kinases that form part of the nitrocellulose filters, blocked with Odyssey blocking buffer PI3K signaling network. In this paper, we present the first (LI-COR Biosciences) and incubated overnight with the detailed pharmacologic characterization of the novel, oral respective antibodies (pSer9 GSK3b, GSK3b, pSer240/244 clinical drug candidate AT13148, a potent multi-AGC S6RP, S6RP, pThr1462 tuberin, tuberin, and cleaved PARP kinase inhibitor. AT13148 was identified using high- (Cell Signaling Technology). Primary antibodies were throughput X-ray crystallography and fragment-based lead labeled with infrared IRDye-labeled
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