ATM , and ATR Characterization of Torin 2 , an ATP-Competitive Inhibitor of mTOR

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase–like kinase (PIKK) family kinases includingATM (EC50, 28 nmol/L), ATR (EC50, 35 nmol/L), and DNA-PK (EC50, 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase andmTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal– regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broadnumber of oncologic settingswheremTOR signaling has a pathogenic role. Cancer Res; 73(8); 2574–86. 2013 AACR. Introduction The mTOR is a highly conserved and widely expressed serine/threonine kinase that is a member of the phosphatidylinositol-3 kinase–like kinase (PIKK) family, which includes the serine/threonine kinases ATR, ATM, DNA-PK, and SMG-1 (1, 2). mTOR serves as a pivotal node in the PI3K/Akt/mTOR signaling pathway, which senses growth factor and nutrient signals and controls fundamental cellular processes such cell growth, autophagy, translation, and metabolism (3, 4). Hyperactivation of this pathway through loss of negative regulators, such as PTEN, or mutational activation of receptor tyrosine kinases of phosphoinositide 3-kinase (PI3K) is a frequent occurrence in cancer (5). mTOR exists in at least 2multiprotein complexes, which are named mTORC1 and mTORC2 (6, 7). mTORC1 contains mTOR, mLST8, and raptor as core components and regulates cell growth, protein synthesis, and autophagy through its downstream effectors, including S6K1, 4EBP1, and ATG13. mTORC2 consists of mTOR, mLST8, rictor, PRR5, and SIN1 as core components and regulates cell survival and actin organization through effectors such as Akt, SGK1, and PKCa. Through its inclusion in these 2 protein complexes mTOR functions both upstream of Akt through Authors' Affiliations: Departments of Cancer Biology and Medical Oncology; Departments of Medicine and Medical Oncology, Ludwig Center at Dana-Farber–Harvard Cancer Center; Lowe Center for Thoracic Oncology; Lurie Family Imaging Center, Dana-Farber Cancer Institute; Division of Genetics, Howard Hughes Medical Institute; Department of Medicine, Brigham and Women's Hospital; Department of Radiation Oncology, Massachusetts General Hospital; Departments of Biological Chemistry and Molecular Pharmacology and Genetics, Center for Cell Decision Processes, Department of Systems Biology, Harvard Medical School; Harvard Radiation Oncology Program, Boston; Whitehead Institute for Biomedical Research; Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology; Koch Center for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, Massachusetts; and ActivX Biosciences, Inc., La Jolla, California Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Q. Liu and C. Xu contributed equally to this work. Corresponding Author: Nathanael S. Gray, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115. Phone: 617-582-8590; Fax: 617582-8615; E-mail: Nathanael_Gray@dfci.harvard.edu doi: 10.1158/0008-5472.CAN-12-1702 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(8) April 15, 2013 2574 on April 30, 2013. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 22, 2013; DOI: 10.1158/0008-5472.CAN-12-1702

[1]  Andrew L. Kung,et al.  A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response , 2012, Nature.

[2]  J. Baselga,et al.  Dual Mtorc1/2 and Her2 Blockade Results in Antitumor Activity in Preclinical Models of Breast Cancer Resistant to Anti-her2 Therapy Statement of Translational Relevance , 2022 .

[3]  T. Gilmer,et al.  Combinations of BRAF, MEK, and PI3K/mTOR Inhibitors Overcome Acquired Resistance to the BRAF Inhibitor GSK2118436 Dabrafenib, Mediated by NRAS or MEK Mutations , 2012, Molecular Cancer Therapeutics.

[4]  P. Sorger,et al.  Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics* , 2012, The Journal of Biological Chemistry.

[5]  Sarat Chandarlapaty,et al.  mTOR kinase inhibition causes feedback-dependent biphasic regulation of AKT signaling. , 2011, Cancer discovery.

[6]  Aileen McHarg,et al.  PF-04691502, a Potent and Selective Oral Inhibitor of PI3K and mTOR Kinases with Antitumor Activity , 2011, Molecular Cancer Therapeutics.

[7]  N. Gray,et al.  In situ kinase profiling reveals functionally relevant properties of native kinases. , 2011, Chemistry & biology.

[8]  P. Houghton,et al.  Preclinical Characterization of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2: Distinct from Rapamycin , 2011, Molecular Cancer Therapeutics.

[9]  James R Bischoff,et al.  A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations , 2011, Nature Structural &Molecular Biology.

[10]  Mario Niepel,et al.  Adaptive informatics for multi-factorial and high content biological data , 2011, Nature Methods.

[11]  D. Sabatini,et al.  Mammalian Target of Rapamycin ( mTOR ) Inhibitor for Treatment of Cancer , 2011 .

[12]  L. Feldberg,et al.  Antitumor Efficacy of PKI-587, a Highly Potent Dual PI3K/mTOR Kinase Inhibitor , 2011, Clinical Cancer Research.

[13]  D. Sabatini,et al.  mTOR: from growth signal integration to cancer, diabetes and ageing , 2010, Nature Reviews Molecular Cell Biology.

[14]  L. Feldberg,et al.  PKI-179: an orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor. , 2010, Bioorganic & medicinal chemistry letters.

[15]  D. Sabatini,et al.  Discovery of 1-(4-(4-propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a highly potent, selective mammalian target of rapamycin (mTOR) inhibitor for the treatment of cancer. , 2010, Journal of medicinal chemistry.

[16]  Francesca Molinari,et al.  Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus. , 2010, The Journal of clinical investigation.

[17]  William Pao,et al.  A Pilot Study of Volume Measurement as a Method of Tumor Response Evaluation to Aid Biomarker Development , 2010, Clinical Cancer Research.

[18]  Bart S. Hendriks,et al.  DataPflex: a MATLAB-based tool for the manipulation and visualization of multidimensional datasets , 2010, Bioinform..

[19]  Kaushik Raha,et al.  Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin. , 2010, ACS medicinal chemistry letters.

[20]  R. Abraham,et al.  Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2. , 2010, Cancer research.

[21]  John R. Engen,et al.  Novel mutant-selective EGFR kinase inhibitors against EGFR T790M , 2009, Nature.

[22]  Kevin Curran,et al.  Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin. , 2009, Cancer research.

[23]  Pixu Liu,et al.  Targeting the phosphoinositide 3-kinase pathway in cancer , 2009, Nature Reviews Drug Discovery.

[24]  J. Blenis,et al.  Molecular mechanisms of mTOR-mediated translational control , 2009, Nature Reviews Molecular Cell Biology.

[25]  C. Chresta,et al.  Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR) , 2009, The Biochemical journal.

[26]  D. Sabatini,et al.  An ATP-competitive Mammalian Target of Rapamycin Inhibitor Reveals Rapamycin-resistant Functions of mTORC1* , 2009, Journal of Biological Chemistry.

[27]  D. Guertin,et al.  mTOR complex 2 is required for the development of prostate cancer induced by Pten loss in mice. , 2009, Cancer cell.

[28]  Robbie Loewith,et al.  Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2 , 2009, PLoS biology.

[29]  Ralph Weissleder,et al.  Effective Use of PI3K and MEK Inhibitors to Treat Mutant K-Ras G12D and PIK3CA H1047R Murine Lung Cancers , 2008, Nature Medicine.

[30]  J. Baselga,et al.  NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. , 2008, Cancer research.

[31]  W. Sellers,et al.  Drug discovery approaches targeting the PI3K/Akt pathway in cancer , 2008, Oncogene.

[32]  D. Neil Hayes,et al.  LKB1 modulates lung cancer differentiation and metastasis , 2007, Nature.

[33]  M. Meyerson,et al.  Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy. , 2007, Cancer cell.

[34]  David M Sabatini,et al.  Defining the role of mTOR in cancer. , 2007, Cancer cell.

[35]  L. Helman,et al.  Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism , 2007, Oncogene.

[36]  Vivienne Marsh,et al.  Biological Characterization of ARRY-142886 (AZD6244), a Potent, Highly Selective Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor , 2007, Clinical Cancer Research.

[37]  Helge Weissig,et al.  Functional interrogation of the kinome using nucleotide acyl phosphates. , 2007, Biochemistry.

[38]  Nesrin Asaad,et al.  Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts , 2005, Oncogene.

[39]  D. Guertin,et al.  Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex , 2005, Science.

[40]  Robert T Abraham,et al.  PI 3-kinase related kinases: 'big' players in stress-induced signaling pathways. , 2004, DNA repair.

[41]  D. Guertin,et al.  Rictor, a Novel Binding Partner of mTOR, Defines a Rapamycin-Insensitive and Raptor-Independent Pathway that Regulates the Cytoskeleton , 2004, Current Biology.

[42]  T. Hunter,et al.  The Protein Kinase Complement of the Human Genome , 2002, Science.

[43]  T. Jacks,et al.  Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. , 2001, Genes & development.

[44]  F. Casagrande,et al.  G1 phase arrest by the phosphatidylinositol 3‐kinase inhibitor LY 294002 is correlated to up‐regulation of p27Kip1 and inhibition of G1 CDKs in choroidal melanoma cells , 1998, FEBS letters.

[45]  Stuart L. Schreiber,et al.  Structure of the FKBP12-Rapamycin Complex Interacting with Binding Domain of Human FRAP , 1996, Science.

[46]  Paul Tempst,et al.  RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs , 1994, Cell.

[47]  Lisa L. Smith,et al.  AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. , 2010, Cancer research.

[48]  G. Mills,et al.  A vascular targeted pan phosphoinositide 3-kinase inhibitor prodrug, SF1126, with antitumor and antiangiogenic activity. , 2008, Cancer research.