Potent and Selective Inhibitors of Akt Kinases Slow the Progress of Tumors in Vivo

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Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo

Yan Luo,1 Alexander R. Shoemaker,1 Xuesong Liu,1 inhibition may be useful in cancer therapy, but significant Keith W. Woods,1 Sheela A. Thomas,1 metabolic toxicities are likely dose limiting. [Mol Cancer Ron de Jong,4 Edward K. Han,1 Tongmei Li,1 Ther 2005;4(6):977–86] Vincent S. Stoll,2 Jessica A. Powlas,1 1 1 1 Anatol Oleksijew, Michael J. Mitten, Yan Shi, Introduction Ran Guan,1 Thomas P. McGonigal,1 Vered Klinghofer,1 Eric F. Johnson,1 Akt activity is elevated in a large proportion of human malignancies, where it plays a central role in inducing a Joel D. Leverson,1 Jennifer J. Bouska,1 malignant phenotype by both promoting cell growth and Mulugeta Mamo,2 Richard A. Smith,2 2 3 decreasing apoptosis (1, 2). Akt1 is a serine/threonine Emily E. Gramling-Evans, Bradley A. Zinker, protein kinase that was first discovered as the human 3 3 Amanda K. Mika, Phong T. Nguyen, homologue of the transforming gene in the AKT-8 onco- 2 1 1 Tilman Oltersdorf, Saul H. Rosenberg, Qun Li, genic virus, which was isolated from a spontaneous and Vincent L. Giranda1 thymoma in the AKR mouse (3, 4). Since the discovery of human Akt1 (also called protein kinase B), two additional 1 2 3 Cancer Research, Structural Biology, and Metabolic Disease mammalian Akt isoforms, Akt2 and Akt3, have been Research, Abbott Laboratories, Abbott Park, Illinois and 4Idun Pharmaceuticals, San Diego, California identified (5–8). Akt is downstream of phosphatidylinositol 3-kinase (PI3K) and is a critical node in this signal transduction Abstract pathway. The activation of Akt by PI3K is antagonized by The Akt kinases are central nodes in signal transduction the tumor suppressor PTEN (9). Thus, the increased Akt pathways that are important for cellular transformation activity that is observed in most human malignancies and tumor progression. We report the development of a could be the result of (a) an increased Akt expression, (b) series of potent and selective indazole-pyridine based Akt increased PI3K activity, or (c) decreased PTEN activity inhibitors. These compounds, exemplified by A-443654 (10). Correlative evidence for all three of these mechanisms (Ki = 160 pmol/L versus Akt1), inhibit Akt-dependent has been found in human tumors. Akts are overexpressed signal transduction in cells and in vivo in a dose- in a variety of human tumors (7, 11–13), and at the responsive manner. In vivo, the Akt inhibitors slow the genomic level, AKT1 and AKT2 have been shown to be progression of tumors when used as monotherapy or in amplified in a number of cancer types (7). PI3K activity is combination with paclitaxel or rapamycin. Tumor growth increased by numerous growth factors, many of which are inhibition was observed during the dosing interval, and the themselves targets for cancer therapy (e.g., KDR and tumors regrew when compound administration was HER2; refs. 14–16). PTEN mutations that result in ceased. The therapeutic window for these compounds is increased Akt activity have likewise been described in a narrow. Efficacy is achieved at doses f2-fold lower than wide variety of malignancies (17–19). Alterations that the maximally tolerated doses. Consistent with data from increase Akt activity (e.g., PTEN loss, PI3K up-regulation, knockout animals, the Akt inhibitors induce an increase in and increased Akt expression) rival alterations in the p53/ insulin secretion. They also induce a reactive increase in p16 pathway as the most common changes found in Akt phosphorylation. Other toxicities observed, including malignant tumor cells (10, 20). malaise and weight loss, are consistent with abnormalities In addition to the correlative data, there is also consid- in glucose metabolism. These data show that direct Akt erable experimental evidence for the importance of PI3K, Akt, and PTEN in neoplasia. The constitutive expression of active Akts promotes tumorigenesis when these Akt- expressing clones are inoculated into nude mice (21–23). Mice heterozygous for Pten deletions, where Akt activity is Received 1/6/05; revised 3/16/05; accepted 4/11/05. dramatically increased, develop neoplasms in colon, testis, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked thyroid, prostate, endometrium, and liver. These animals advertisement in accordance with 18 U.S.C. Section 1734 solely to also have an increased incidence of lymphoma and indicate this fact. leukemia (24, 25). In humans, there are three closely related Note: R. de Jong is currently at Syrrx, Inc., 10410 Science Center Drive, and inherited cancer syndromes caused by mutations in the San Diego, CA 92121. PTEN locus: Cowden disease (26), Lhermitte-Duclos Requests for reprints: Vincent L. Giranda, Cancer Research, Abbott Laboratories, 100 Abbott Park Road, IL 60064-6117. Phone: 847-937- disease (26), and Bannayan-Zonana syndrome (27). Patients 0268; Fax: 847-938-2365. E-mail: [email protected] with these disorders have multiple benign tumors and Copyright C 2005 American Association for Cancer Research. greatly increased incidence of carcinomas.

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In Drosophila, a germ line deletion of dAkt reverses the cKit (544-976; ProQinase, Freiburg, Germany) were com- large cell phenotype in dPten/ flies (28). More recently, mercially obtained. His-tagged Akt1 (S378A, S381A, T450D, deletion of Akt1 has been shown to reverse the aggressive S473D; 139-480), Chk1 (1-269), KDR (789-1354), Flt-1 (786- growth phenotype of Pten/ mouse embryonic stem cells 1338), and phosphatidylinositide-dependent kinase 1 (1- (29). These data suggest the tumorigenic phenotype 396), were expressed using the FastBac bacculovirus caused by loss of PTEN can be largely reversed by expression system (Life Technologies, Gaithersburg, MD) inhibition of Akt. and purified using either nickel (his-tag) or glutathione The prevalence of Akt activation in human tumors, S-transferase affinity chromatography. Peptides substrates coupled with the genetic experiments showing that PTEN had the general structure biotin-Ahx-peptide with the deletions or active Akt can induce tumor formation, following sequences: Akt, EELSPFRGRSRSAPPNLWA- suggests that inhibition of Akt may be useful in the AQR;PKA,LRRASLG;PKCg and PKCy, ERMRPRK- treatment of neoplastic diseases. Indeed, there have been RQGSVRRRV; CK2, RRADDSDDDDD; cyclin-dependent attempts to modulate the activity of this pathway. kinase 2, LPPCSPPKQGKKENGPPHSHTLKGRRAAFD- Inhibition of PI3K directly has been reported to decrease NQL; GSK3h, YRRAAVPPSPSLSRHSSPHQS(p)EDEEE; the progression of tumors in rodents (30, 31). Likewise, MAPK-AP2, KKLNRTLSVA; RSK2, KKKNRTLSVA; extra- using rapamycin or one of its analogues to inhibit the PI3K cellular signal–regulated kinase 2, KRELVEPLTPSGE- pathway downstream at mammalian target of rapamycin APNQALLR; Chk1, AKVSRSGLYRSPSMPENLNRPR; (mTOR) has been shown to reduce tumor growth in mouse phosphatidylinositide-dependent kinase 1, KTFCGTPEY- models (several compounds are currently in human clinical LAPEVRREPRILSEEEQEMFRDFDYIADWC; KDR, Flt-1, trials; refs. 32, 33). There have also been reports of and cKit, AEEEYFFLFA-amide. For Src assays, the bio- compounds that inhibit the activation of Akt [e.g., heat tinylated substrate PTK-2 (Promega, Madison, WI) was shock protein 90 inhibitors (34), pleckstrin homology used. Inhibition of kinase activity was assessed using a domain inhibitors (35, 36), and others (37)]. Here we report radioactive FlashPlate-based assay platform as previously on the discovery of potent compounds that directly inhibit described (40). the kinase activity of Akt. We also report the effects of these Alamar Blue CellViabilityAssay compounds on transformed cells in vitro as well as on The cells on 96-well plates were gently washed with tumor progression in vivo. 200 AL of PBS. Alamar Blue reagent (Biosource Interna- tional, Carmarillo, CA) was diluted 1:10 in normal growth media. The diluted Alamar Blue reagent (100 AL) Materials and Methods was added to each well on the 96-well plates and Crystallization and X-ray Analysis incubated until the reaction was complete as per Protein kinase A (PKA) was purified (38), concentrated to manufacturer’s instructions. Analysis was done using an 20 mg/mL, and complexed with peptide inhibitor of PKA fmax Fluorescence Microplate Reader (Molecular Devices, for 1 hour and complexed with A-443654 (39). Crystals Sunnyvalle, CA), set at the excitation wavelength of were transferred to cryosolutions that contained well 544 nm and emission wavelength of 595 nm. Data were solution plus increasing amounts of glycerol, soaking for analyzed using SOFTmax PRO software provided by the 1 minute in 5%, 15%, and 25% glycerol. Crystals were then manufacturer. frozen in a stream of 100jK nitrogen. Diffraction data were Protein Extraction and Western Blot Analysis recorded using a MAR-165 CCD detector system on a Cells were treated with 0 to 30 Amol/L of Akt inhibitors Rigaku RU-2000 rotating anode X-ray generator (Danvers, for 2 hours. All drug samples were adjusted to contain MA) operating at 100 mA and 50 kV. Diffraction data were equal volumes of the vehicle (DMSO). Cells were har- reduced using HKL2000 (HKL Research, Inc., Charlottes- vested, sonicated for 5 minutes in ice-cold insect cell lysis ville, VA) and the protein model (accession number 1YDT) buffer [BD PharMingen, San Diego, CA; 10 mmol/L Tris- with the inhibitor (H89) and the phosphorylation sites HCl (pH 7.5), 130 mmol/l NaCl, 1% Triton X-100, 10 omitted for initial phasing. Generation of initial electron mmol/L NaF, 10 mmol/L NaPi, 10 mmol/L NaPPi and density maps and structure refinement was achieved using 1 Ag/mL microcyctin LR plus the protease inhibitors CNX program package. Electron density maps were aprotinin (10 Ag/mL), leupeptin (10 Ag/mL), and phenyl- generated using the program QUANTA 97/2001 (Accelrys, methylsulfonyl fluoride (1 mmol/L)], and centrifuged at San Diego, CA), and compound A-44654 was fit to the 15,300 rpm for 10 minutes. The concentrations of the total electron density. The structure was determined to a lysate protein were determined using a standard Bradford ˚ resolution of 2.7 A and refined to Rwork = 24.41% and assay (Bio-Rad, San Diego, CA). RFree = 30.18 %. MiaPaCa-2 pancreatic tumor–bearing mice were treated In vitro Kinase Assays with control vehicle or A-443654 at 7.5 or 75 mpk for Recombinant CK2, PKCg, and PKCy (Calbiochem, San 2 hours. Tumors were isolated and immediately frozen in Diego, CA); PKA (Panvera, Madison, WI); cyclin-dependent liquid nitrogen. Frozen tumor tissues were sliced in thin kinase 2/CyclinA, GSK3h, MAPK-AP2, Src, and RSK2 (Upstate sections and resuspended in 500 AL of lysis buffer. Biotechnology, Charlottesville, VA); extracellular signal– This suspension was homogenized with a Polytron regulated kinase 2 (New England Biolabs, Beverly, MA), PT 1200C (Kinematica AG, Luzern, Switzerland) for

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2 minutes. The homogenized tissues were centrifuged at Tu m o r E f f i c a c y S t u d i e s 15,300 rpm for 10 minutes, supernatants were collected, Animal studies were conducted following the guide- and the protein concentrations were determined with the lines of the internal Institutional Animal Care and Use Bradford method. Committee. Immunocompromised male scid mice (C.B-17- For Western blot analysis, 40 Ag of protein from the total Prkdcscid) were obtained from Charles River Laboratories cell lysate were electrophoresed by SDS-PAGE. The (Wilmington, MA) at 6 to 8 weeks of age. MiaPaCa-2 and proteins were then electrotransfered onto immobilon-P PC-3 cells were obtained from the American Type Culture membranes (Millipore, Bedford, MA), using transfer buffer Collection (Manassas, VA). The 3T3-Akt1 cell line was (25 mmol/L Tris, 190 mmol/L glycine, and 10% methanol). developed and characterized in our laboratory (23). The 1 Membranes were treated with blocking buffer (50 mmol/L 106 3T3-Akt1 or 2 106 MiaPaCa-2 and PC-3 cells in Tris, 200 mmol/L NaCl, 0.2% Tween 20, 5% nonfat dry 50% Matrigel (BD Biosciences, Bedford, MA) were inocu- milk) for 1 hour at room temperature and incubated with lated s.c. into the flank. For early treatment studies, mice 1:1,000 dilution of antibodies (phospho-GSK3 a/h Ser21/9, were randomly assigned to treatment groups and therapy phospho-TSC2 Thr1462, phospho-FOXO1A/3A Thr24/32, was initiated the day after inoculation. Ten animals were phospho-ribosomal protein S6 Ser235/236 phospho-Akt1 assigned to each group, including controls. For established Ser473, and phospho-mTOR Ser2448; all from Cell Signaling tumor studies, tumors were allowed to reach a designated Technology, Beverly, MA) for 16 hours at 4jC. After size and mice were assigned to treatment groups of equal washing with blocking buffer without 5% nonfat dry milk tumor size (n = 10 mice per group). Tumor size was (washing buffer) for 30 minutes, the membranes were evaluated by twice weekly measurements with digital incubated with horseradish peroxidase–linked anti-rabbit calipers. Tumor volume was estimated using the formula: donkey serum (1:1,000; Amersham, Arlington Heights, IL) V = L W2 / 2. A-443654 was given s.c. in a vehicle of for 1 hour. Membranes were then washed with washing 0.2% HPMC. A-674563 was given orally in a vehicle of 5% buffer and immune detection was done using the dextrose. Gemcitabine and paclitaxel were obtained from enhanced chemiluminescence Western blotting detection Eli Lilly and Company (Indianapolis, IN) and Bristol- system (Amersham). Quantitation of Western blots was Myers Squibb Co. (Princeton, NJ), respectively, and given done using a phospho-imager (Amersham Biosciences, according to the manufacturers’ guidelines. Piscataway, NJ). For the determination of plasma drug concentrations, FL5.12Akt1-,Akt2-, orAkt3-Overexpressing Cell Lines proteins were precipitated with two volumes of acidified Stable transfectants of FL5.12 cells expressing full-length methanol. Tissue samples were taken from the same human Akt1, Akt2, or Akt3 were generated by electro- animals and homogenized with 2 volumes of saline, then poration with a pCIneo plasmid (Promega) containing Akt precipitated with 2 volumes of acetonitrile. Precipitated cDNAs with an NH2-terminal HA-tag (41). Single clones proteins were removed by centrifugation and super- were isolated by limiting dilution and selection for G418 natants were stored at 20jC until analysis by UV-liquid resistance (Invitrogen, Carlsbad, CA). Expression was chromatography or liquid chromatography-mass spec- confirmed by immunoblotting with Akt isoform-specific trometry.Plasmaandtissueextractswereinjected antibodies. FL5.12/Akt cell lines were maintained in RPMI directly on a C18 reversed phase column (YMC-AQ 5 1640 supplemented with 10% fetal bovine serum, 10% A; Waters Co., Milford, MA) and eluted using combina- WEHI-3B conditioned medium as a source of IL-3, tions of acetonitrile, methanol and 8 mmol/L triethyl- nonessential amino acids, 1 mmol/L sodium pyruvate, amine acetate buffer (pH 4) with UV detection 2 mmol/L glutamine, 50 Amol/L h-mercaptoethanol, 50 (Shimadzu 10A/VP, Shimadzu Scientific, Columbia, Ag/mL G418, and 2.5 mmol/L HEPES (pH 7.5) at 37jCina MD) or with acetonitrile and 0.1% acetic acid in water humidified atmosphere containing 5% CO2. for mass spectrometer analysis (LCQ Duo, Thermoquest, FOXO3A Translocation Assay San Jose, CA). Pharmacokinetic variables were calculated The pFOXO3A-hrGFP construct was engineered by using WinNonlin software.(Pharsight Co., Mountain inserting a FOXO3A fragment encoding NH2-terminal View, CA). amino acids 1 to 400 into plasmid phrGFP (Stratagene, La Immunohistochemistry Jolla, CA) using standard PCR and DNA recombinant 3T3-Akt1 and MiaPaCa-2 tumors were fixed for 48 techniques. This sequence includes the nuclear import and hours in Streck Tissue Fixative (Streck Laboratories, export signals and Akt phoshorylation sites (42). HeLa Omaha, NE), processed, and embedded in paraffin; 5-Am cells were plated into 6-well plates at a density of 0.3 tissue sections were blocked with streptavidin/biotin 6 j 10 cells per well and incubated at 37 Cat5%CO2 over complex followed by 0.3% bovine serum albumin before night. Cells were transiently transfected with pFOXO3A- incubation with primary antibody (caspase-3: rabbit anti- hrGFP plasmid using Effectene (Qiagen, Valencia, CA). At active Caspase-3 at 1:400, BD PharMingen; phospho-Akt: 40 hours post-transfection, cells were treated with com- rabbit anti-phospho-Akt at 1:200, Cell Signaling Technol- pounds added to culture medium lacking phenol red. ogy) for 1 hour at room temperature. Secondary antibody Equal amounts of DMSO were added to cells to a final was used at 1:250 to 300 followed by streptavidin-biotin concentration of 0.5%. After 6 hours, images of fluorescent horseradish peroxidase and 3,3V-diaminobenzidine color live cells were captured by microscopy. development.

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Results observed with all of the Akt inhibitors tested and seems a Discovery of Indazole-Pyridine Series of Akt Inhibitors sensitive marker of Akt inhibition. However, in spite of the The initial lead in this series was obtained from a high- increased Akt activation, the ability of Akt to phosphory- throughput screen (Fig. 1A, compound 1). This compound late its downstream targets was markedly decreased in weakly inhibited Akt (Ki =5Amol/L). The potency was the presence of the inhibitors (Fig. 3A). The addition improved by addition of an indole ring to the aliphatic side chain (Fig. 1A, compound 2), and still further improved by constraining rotatable bonds in the linker between the central and distal pyridine rings (Fig. 1A, compound 3). Addition of a second hydrogen bonding interaction to the hinge-binding region resulted in A-443654, that exhibited a Ki of 160 pmol/L, a 30,000-fold improvement in potency versus the initial lead molecule. Finally, a compound with oral activity was achieved by replacing the indole with a phenyl moiety (Fig. 1A, A-674563). The series of indazole-pyridine compounds bind to the ATP site of Akt. These compounds are potent, ATP competitive, and reversible inhibitors of the Akt catalyzed phosphorylation activity. The three-dimensional structures of these compounds complexed with a closely related protein kinase, PKA (Fig. 1B-C), were determined by X-ray crystallography. Three hydrogen bond sets are critical to maintain potency. The first is a canonical hydrogen bond to the hinge region backbone found with most ATP-competitive kinase inhibitors. The second is formed between the inhibitor’s central pyridine ring and a conserved lysine (Lys72 in PKA and Lys181 in Akt1). The third set of important hydrogen bonding interactions form between the primary amino group of the inhibitor’s aliphatic side chain and the PKA residues Asn171 and Asp184 (Asn279 and Asp292 in Akt1). This position is normally occupied by a divalent cation of Mg2+ ATP. In vitro Activity A-443654 is a pan Akt inhibitor and has equal potency against Akt1, Akt2, or Akt3 within cells (Fig. 2). A-443654 is, however, very selective for Akt versus other kinases (Table 1). It is the least selective towards several closely related kinases in the AGC family, including PKA and protein kinase C (PKC). Even so, A-443654 is 40-fold selective for Akt over PKA. A-674563 is somewhat less selective, particularly against the cyclin-dependent kinases in the CMGC family. However, in spite of the selectivity differences between these two classes of Akt inhibitors, their behavior is similar in cells and in vivo (see below). The phosphorylation of Akt downstream targets such as GSK3a/h, FOXO3, TSC2, and mTOR were measured as an indication of Akt activity within cells. The Akt inhibitors reduced the phosphorylation of all of these proteins in a dose-dependent fashion (Fig. 3A). As expected, we also observed that FOXO3 translocates into the nucleus upon the reversal of its phosphorylation Figure 1. A, a schematic representation of the discovery of the indazole- induced by Akt inhibition (Fig. 3B). Phosphorylation of pyridine series of Akt inhibitors. B, the X-ray structure of A-443654 bound to the ATP binding site of PKA. The structures of both Akt and signaling molecules further downstream in the Akt PKA contain very similar ATP binding sites, with only four amino acid pathway, such as the S6 protein, was also reduced by the differences within the inhibitor binding sites and minimal differences Akt inhibitors. between the protein backbones. Dotted lines, hydrogen bonds between Together with the decrease in phosphorylation of Akt A-443654 and PKA. C, the line drawing also illustrates the A-443654 308 mode of binding, with the indazole binding to the hinge region, the targets, we observed a concomitant increase in the Thr pyridine N binding to a conserved lysine, and the indole ring binding and Ser473 phosphorylation of Akt. This increase has been under the glycine-rich loop.

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Figure 2. A-443654 inhibits Akt1, Akt2, or Akt3 equally within cells. Murine FL5.12 cells were stably transfected with constitutively active myristoylated human Akt1 (.), Akt2 (E), or Akt3 (n). There is no appreciable phosphorylation of GSK3 in the parental cell lines, whereas there are high levels of phospho-GSK3 (P-GSK3) observed in all three Akt overexpressing lines. A-443654 reduces the P-GSK3 in a dose-responsive manner in all three cell lines. The broad-spectrum kinase inhibitor staurosporine was added at 1 Amol/L as a positive control. The P-GSK3 was quanti- tated and normalized to the total GSK3. Plot of the resulting inhibition for each overexpressing cell line (right).

of 20 Amol/L of the PI3K inhibitor Ly294002 together with can be given is limited to 14 days due to severe injection A-443654 eliminates the Akt inhibitor induced increase in site irritation. Nevertheless, in spite of the differences Akt phosphorylation (data not shown), suggesting in- between these compounds, both compounds behave creased phosphatidylinositol 3,4,5-triphosphate is required similarly in vivo. for the A-443654 induced increase in Akt phosphorylation. The effect of the inhibitors was examined in several Akt- To investigate the effects of Akt inhibition on cell dependent models. We tested a genetically engineered proliferation, we did an 3-(4,5-dimethylthiazol-2-yl)-2,5- model, where 3T3 murine fibroblasts stably express a diphenyltetrazolium bromide assay. The Akt inhibitors constitutively active form of Akt1. In contrast to the slowed proliferation of tumor cells with an EC50 of 0.1 parental 3T3 cell line, the 3T3-Akt1 cells have a dramat- Amol/L for A-443654 and 0.4 Amol/L for A-674563 (Fig. 3C). ically increased ability to form colonies in soft agar as well In vivo Antitumor Activity as tumors in immunocompromised mice, suggesting the We tested both A-443654 and A-674563 for their ability to active Akt1 is responsible for the transformed phenotype inhibit tumor growth in vivo (Fig. 4). A-443654 is more (23). In this model, A-443654 inhibited the growth of the potent and more selective than A-674563 but is not tumors (Fig. 4A); whereas the 2-methyl analogue of A- available orally. Furthermore, the length of time A-443654 443654, which is 5,000-fold less active versus Akt1, showed no effect in this model. We also tested the compounds in a MiaPaCa-2 human pancreatic cell xenograft model, where we compared the efficacy with Table 1. Selectivity of Akt inhibitors for selected kinases gemcitabine. MiaPaCa-2 is a human pancreatic carcinoma line with constitutively active Akt (43). Here again, the Family Kinase A-443654 fold A-674563 fold Akt inhibitor significantly slowed the growth of the A A (Ki, mol/L) (Ki, mol/L) tumors (Fig. 4B). A-443654 also showed antitumor activity in two rat orthotopic syngeneic models of tumor growth, a AGC Akt1 1 (0.00016) 1 (0.011) MatLyLu prostate carcinoma and a 9L glioblastoma (data PKA 40 (0.0063) 1.4 (0.016) not shown). PKCg 150 (0.024) 110 (1.2) PKCy 200 (0.033) 32 (0.36) In all models tested, the tumors rapidly regrew after PDK1 >120,000 (>20) >1,800 (>20) compound administration was stopped. The cytostatic TK KDR 19,000 (3.1) >330 (>3.7) behavior of the inhibitors suggests that continuous dosing cKIT 7,300 (1.2) >530 (>5.8) may lead to increased efficacy. The number of consecutive SRC 16,000 (2.6) 1,200 (13) days the orally available compounds could be dosed, Flt1 22,000 (3.6) >200 (>2.2) unlike the parenteral compounds, was not limited by CMGC CDK2 150 (0.024) 4.2 (0.046) injection site toxicity. Nevertheless, the oral compounds ERK2 2,100 (0.34) 24 (0.26) could only be given for 15 to 25 days, after which time h GSK3 260 (0.041) 10 (0.11) the animals became moribund. In all cases, the com- CK2 15,000 (2.4) 490 (5.4) pounds were given at their respective maximally tolerated CAMK Chk1 15,000 (2.3) 235 (2.6) RSK2 68 (0.011) 53 (0.58) doses. MAPK-AP2 21,000 (3.3) 100 (1.1) Activation of the Akt pathway is thought to inhibit apoptosis induced by a variety of cytotoxic agents and has

NOTE: The fold selectivity versus Akt1 is shown for each kinase and the K i been reported to be an effective survival mechanism in is in parentheses. many tumor cells (44–47). Therefore, we attempted to Abbreviations: PDK1, phosphatidylinositide-dependent kinase 1; MAPK, mitogen-activated protein kinase; GSK3, glycogen synthase kinase 3; ERK2, combine the Akt inhibitors with the antimitotic agent extracellular signal – regulated kinase 2. paclitaxel in a PC-3 xenograft model (48, 49). PC-3 is a

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PTEN-deficient human prostate carcinoma cell line, where We tested the pharmacokinetics and pharmacodynamics resistance to chemotherapeutic agents is at least partially to insure that we were inhibiting Akt in vivo (Fig. 5). dependent on Akt activity (50). When given in combina- When the inhibitors were given at their maximally tion, A-674563 increased the efficacy of paclitaxel in a PC-3 tolerated doses on a twice daily (bid) schedule, their xenograft model (Fig. 4C). concentration in plasma remained above the cellular EC50 for f5 hours (Fig. 5A). Concentrations in tumors were significantly higher than those in plasma, and drug concentrations in tumor remained above the cellular EC50 for the entire 12-hour dosing interval (Fig. 5B). In the tumors, as we have seen in the cellular assays, the inhibition of GSK3 and S6 phosphorylation are inhibited in a dose responsive manner by the Akt inhibitors (Fig. 5C). As also seen in the cellular studies, the increase in Akt phosphorylation seems the most sensitive marker for Akt inhibition. This suggests that the cells, in vivo, are also sensitive to Akt inhibition, and attempt to compensate for the loss of the activity. We also examined caspase-3 activation (a measure of apoptosis) and Akt phosphorylation by immunohistochemistry. As expected, at 8 hours after a single dose of A-443654, we observed an increase in apoptotic cells (Fig. 5D), suggesting that at least part of the antitumor activity of the Akt inhibitors is due to apoptosis induction. Normal liver and colon tissues showed no increase in apoptosis following treatment (data not shown). Corrobo- rating the Western blot analysis, we also observed that the phosphorylation of Akt increased when inhibitors are given (Fig. 5E). As seen in Fig. 5C, doses near the MTD do not maximally inhibit Akt within tumors. Furthermore, drug washout studies in cells suggested that compounds were more effective at inducing apoptosis when maintained above their cellular EC50 for at least 12 hours (data not shown). As stated above, the plasma concentrations of A-443654 fall below this EC50 after about 5 hours. In an attempt to increase the efficacy of A-443654 by intensi- fying the dosing regime, the compound was given thrice in a single day, 3 hours apart, for a total of 50 mg/kg/d. The animals could not tolerate 50 mg/kg/d every day; thus, compound was given every fourth day. This regime maintains the plasma concentrations of A- 443654 above its cellular EC50 for >12 hours. This thrice daily (tid)/q4d schedule proved to be more efficacious than the bid/daily schedule (Fig. 4D). Note that for the tid schedule, dosing was started on established tumors

Figure 3. Akt inhibitors affect the phosphorylation and localization of cellular Akt substrates. A, MiaPaCa-2 cells were treated for 2 h with various concentrations of Akt inhibitors A-443654 and A- 674563. Western blot analyses were done to assess the phosphorylation of Akt, GSK3 a/h, TSC2, mTOR, and ribosomal protein S6. B, HeLa cells transiently transfected with pFOXO3A-hrGFP were treated with A-443654 for 6 h. Images of live cells taken by fluorescence microscopy. Transcription factor FOXO3A is retained in the cytoplasm when phosphorylated by Akt. DMSO vehicle and 20 mmol/L LY 294002 (an inhibitor of PI3K) were used as negative and positive controls, respectively. C, MiaPaCa-2 cells were treated with A-443654 or A-674563 for 48 h. Alamar blue cell viability assays were done to measure the cell growth inhibition. Points, average of three determinations; bars, SD.

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Figure 4. Akt inhibitors slow tumor growth in vivo. A, A-443654 inhibits tumor growth in the 3T3-Akt1 flank tumor model. The scid mice bearing established 3T3-Akt1 tumors were treated s.c. bid for 14 d with vehicle (4), A-443654 at 7.5mg/kg/d (n), or the inactive analogue, 2- methyl A-443654 at 7.5 mg/kg/d (5). Therapy was initiated 21 d after tumor inoculation when the mean tumor volume of each group was f245 mm3. A-443654 was statistically different from vehicle from day 26 onward (P < 0.02). B, A-443654 inhibits tumor growth in the MiaPaCa-2 xenograft tumor model. scid mice were inoculated with cells on day 0 and therapy was begun on day 1. A-443654 at 7.5 mg/kg/d (E) or vehicle (4) was given s.c., bid for 14 d. Gemcitabine at 120 mg/kg/d (n) or vehicle (5) was given i.p., qd on days 3, 6, 9, and 12. A-443654 and gemcitabine both significantly inhibited tumor growth (P < 0.03). C, A-674563/paclitaxel combination therapy in the PC-3 prostate cancer xenograft model. scid mice bearing established PC-3 tumors were treated with A-674563 (4) p.o., bid at 40 mg/kg/d for 21 d; paclitaxel (E) i.p., qd at 15 mg/kg/d on days 20, 24, and 28; the combination (n); or the combination vehicle (5). Therapy was initiated 20 d after tumor inoculation when the mean tumor volume for each group was f270 mm3. Although A-674563 showed no significant monotherapy activity, the efficacy of the combination therapy was significantly improved compared to paclitaxel monotherapy (P < 0.002). D, A-443654 and rapamycin efficacy in the MiaPaCa-2 pancreatic cancer xenograft model. scid mice bearing established tumors were treated with A-443654 (n) s.c., tid at 50 mg/kg/d on days 16, 20, and 24; rapamycin (5) i.p., qd at 20 mg/ kg/d for 15 d; the combination of rapamycin plus A-443654 (y); s.c. vehicle (E); or combination vehicle (4). Therapy was initiated 16 d after tumor inoculation when the mean tumor volume for each group was f255 mm3. Each monotherapy showed significant activity throughout the study (P < 0.01) and the combination was statistically better than either monotherapy from day 23 onward (P < 0.01). whereas for the bid schedule dosing was started Metabolic Consequences of Akt Inhibition immediately after tumor cell inoculation. Similar to what Akt is downstream of the insulin receptor and was observed with the bid/daily schedule, when treated transduces the insulin response in vivo. We therefore on the tid/q4d schedule, the tumors regrew soon after measured blood sugar and insulin responses related to dosing was stopped. the inhibition of Akt. We examined random blood Akt inhibition was compared with inhibition further sugars during multiple tumor studies and have never downstream in the PI3K pathway at mTOR, using observed any differences between the treated and rapamycin (Fig. 4D). When dosed, both the Akt control groups. We also measured both blood sugar inhibitors and rapamycin are efficacious in the Mia- and plasma insulin levels in glucose challenge tests. PaCa-2 model. However, rapamycin is significantly less Blood sugars were not changed upon administration of toxic and has a much larger therapeutic window than Akt inhibitors, even when supertherapeutic doses were the Akt inhibitors. Furthermore, rapamycin therapy can given (data not shown). We did, however, observe be maintained longer, leading to more durable significant increases in plasma insulin following admin- responses. The function of mTOR in tumor progression istration of a single dose of Akt inhibitors at therapeu- does not seem completely epistatic to that of Akt, as the tic doses (Fig. 5F). This data is consistent with a efficacy observed for the combination of the Akt and homeostatic response, where the animals increase mTOR inhibitors was better than that observed for either insulin secretion to maintain blood glucose concentra- agent alone. tions. This is also what has been observed in Akt2

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Figure 5. The effects of Akt inhibitors after in vivo administration. Plasma (A)andtumor(B) concentrations of A-443654 are plotted versus the time after a final dose of A-443654 was given. Animals were treated at (.)3.8,(n)7.5,or(E) 15 mg/kg bid for 3 d before measurement. C, 2 h after A-443654 administration, the phosphorylation of Akt (solid columns), S6 Protein (open columns), and GSK3 (cross-hatched columns) were measured via Western blotting (bottom). Akt phosphorylation increases in response to the Akt inhibitor. In spite of this increase in P-Akt, phosphorylation of targets downstream from Akt is inhibited in a dose-responsive manner. D, A-443654 induces apoptosis in 3T3- Akt1 flank tumors. The scid mice bearing established 3T3-Akt1 tumors were given a single s.c. dose of A-443654 at 50 mg/kg or vehicle. Tumors were removed 8 h after treatment and apoptosis was examined by immunohistochemical staining with an antibody specific for the activated form of caspase-3. E, A-443654 treatment leads to increased levels of phosphorylated Akt1 in MiaPaCa-2 tumors. The scid mice bearing established MiaPaCa-2 flank tumors were given a single s.c. 30 mg/kg dose of A-443654. One hour after treatment, tumors were removed and the level of phospho-Akt was examined by immunohistochemical staining. F, A-674563 and A-443654 increase plasma insulin in an oral glucose tolerance test. Fasted animals administered vehicle (n), 20 mg/kg A-674563 (.), 100 mg/kg A-674563 (o), or 20 mg/kg A- 443654 (E) 30 min before the 1 g/kg glucose challenge (time 0). Insulin levels were elevated in all A-674563- and A-443654-treated animals. Similar increases in insulin responses were seen in the sham-challenged (no glucose challenge) animals after treating with Akt inhibitors (data not shown).

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knockout animals (51, 52). When young, these animals several cell lines to inhibit apoptosis in cancer cells (50). have normal blood sugars but increased plasma insulin. Thus, using these Akt inhibitors with other agents may Only when older, after islet cell failure, do the animals produce the desired improvement in therapeutic window lose their ability to maintain normal blood glucose by improving efficacy without increasing toxicity. concentrations. Finally, the Akt inhibitors, along with rapamycin, illus- While determining the maximum tolerated dose, we trate that different nodes in the PI3K signal transduction found that the Akt inhibitors induce a dose- and time- pathway can be inhibited to slow tumor progression. The dependent weight loss in treated animals. This weight loss efficacy of the Akt and mTOR inhibitors suggests that, as is typically >10% of total body mass when compounds are monotherapy, other inhibitors of the PI3K pathway will also given at thrice their maximum tolerated dose (data not be efficacious. However, the differences in toxicity suggest shown). Whereas weight loss is a general toxicity, it is that inhibiting at different nodes in the PI3K pathway might consistent with the expectation that the compounds may yield very different therapeutic windows. There are a interfere with the utilization of glucose by interfering with number of potential targets in this pathway, including the insulin pathway. PI3K, phosphatidylinositide-dependent kinase 1, ILK, Akt1, Akt2, Akt3, mTOR, p70S6K, and 4E-BP1, all of which are under investigation for utility in human cancer therapy. The Discussion results of these investigations will ultimately determine We have shown that inhibitors of Akt, when dosed to where to target the PI3K pathway to provide the best levels that inhibit Akt in vivo, can slow the tumor growth efficacy and therapeutic window for the treatment of cancer. in laboratory models of cancer. Several compounds have been tested and have shown efficacy in a number of tumor models. In all of these models, the dosing period is Acknowledgments limited due to toxicity of the therapy. In addition, in all We thank Lisa Li and Jennifer Hensel for their technical assistance with models, the tumors regrew rapidly when dosing is many of the cellular assays and Stephen Fesik for his assistance in editing stopped. The Akt inhibitors show efficacy in the same this article. models where activity is observed with rapamycin. However, the Akt inhibitors have a narrower therapeutic References window than does rapamycin. The dose-limiting toxicities for the oral compounds, 1. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489 – 501. malaise and weight loss, are consistent with a mechanism- 2. Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in based toxicity, where the inhibitors interfere with the human cancer: rationale and promise. Cancer Cell 2003;4:257 – 62. metabolism of glucose (the limiting toxicity for parenteral 3. Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA. compounds is an injection site irritation). However, weight Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A 1991;88: loss is a general toxicity seen with many anticancer agents 4171 – 5. that do not inhibit Akt. Also consistent with mechanism- 4. Bellacosa A, Testa JR, Staal SP, Tsichlis PN. A retroviral oncogene, based toxicity is the observation that compounds with akt, encoding a serine-threonine kinase containing an SH2-like region. substantially different selectivity profiles, but similar Akt Science 1991;254:274 – 7. inhibitory activity, elicit the same types of toxicity. The 5. Nakatani K, Sakaue H, Thompson DA, Weigel RJ, Roth RA. Identification of a human Akt3 (protein kinase B g) which contains the increase in plasma insulin attendant with Akt inhibition also regulatory serine phosphorylation site. Biochem Biophys Res Commun suggests metabolic toxicities may be dose limiting. Further 1999;257:906 – 10. studies with more widely divergent Akt inhibitors will be 6. Masure S, Haefner B, Wesselink JJ, et al. Molecular cloning, required to ascertain if Akt inhibition as cancer therapy will expression and characterization of the human serine/threonine kinase Akt-3. Eur J Biochem 1999;265:353 – 60. be limited by mechanism-based metabolic toxicities. 7. Staal SP. Molecular cloning of the akt oncogene and its human Whereas the efficacy observed in the tumor models is homologues AKT1 and AKT2: amplification of AKT1 in a primary human promising, it is clear that inhibition of Akt as a therapy for gastric adenocarcinoma. Proc Natl Acad Sci U S A 1987;84:5034 – 7. cancer would benefit from a widening of the therapeutic 8. Cheng JQ, Godwin AK, Bellacosa A, et al. AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is window. A number of active investigations are under way amplified in human ovarian carcinomas. Proc Natl Acad Sci U S A 1992; to further improve the therapeutic window. The inhibitors 89:9267 – 71. described here are pan-Akt inhibitors, and in cells have 9. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, equal potency inhibiting Akt1, Akt2, or Akt3 within cells. dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998;273:13375 – 8. Akt2 loss seems to predominate in toxicity related to 10. Di Cristofano A, Pandolfi PP. The multiple roles of PTEN in tumor insulin signaling (51, 52), whereas Akt1 has been reported suppression. Cell 2000;100:387 – 90. to be more important in cancer cell survival. Thus, 11. Nakatani K, Thompson DA, Barthel A, et al. Up-regulation of Akt3 in generation of inhibitors more selective for Akt1 over Akt2 estrogen receptor-deficient breast cancers and androgen-independent might improve therapeutic window. Furthermore, it is clear prostate cancer lines. J Biol Chem 1999;274:21528 – 32. 12. Sun M, Wang G, Paciga JE, et al. AKT1/PKB a kinase is frequently from the paclitaxel study that Akt inhibitors may collabo- elevated in human cancers and its constitutive activation is required for rate with chemotherapeutics that induce apoptosis. Akt has oncogenic transformation in NIH3T3 cells. Am J Pathol 2001;159:431 – 7. recently been shown to collaborate with Bcl2/BclXL in 13. Yuan ZQ, Sun M, Feldman RI, et al. Frequent activation of AKT2 and

Mol Cancer Ther 2005;4(6). June 2005

induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt 34. Basso AD, Solit DB, Munster PN, Rosen N. Ansamycin antibiotics pathway in human ovarian cancer. Oncogene 2000;19:2324 – 30. inhibit Akt activation and cyclin D expression in breast cancer cells that 14. Gerber HP, McMurtrey A, Kowalski J, et al. Vascular endothelial overexpress HER2. Oncogene 2002;21:1159 – 66. growth factor regulates endothelial cell survival through the phosphatidy- 35. Meuillet EJ, Mahadevan D, Vankayalapati H, et al. Specific inhibition linositol 3-kinase/AktV signal transduction pathway. Requirement for Flk-1/ of the Akt1 pleckstrin homology domain by D-3-deoxy-phosphatidyl-myo- KDR activation. J Biol Chem 1998;273:30336 – 43. inositol analogues. Mol Cancer Ther 2003;2:389 – 99. 15. Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL. 36. Martelli AM, Tazzari PL, Tabellini G, et al. A new selective AKT Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt is pharmacological inhibitor reduces resistance to chemotherapeutic drugs, required for antibody-mediated effects on p27, cyclin D1, and antitumor TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemia action. Cancer Res 2002;62:4132 – 41. cells. Leukemia 2003;17:1794 – 805. 16. Datta K, Bellacosa A, Chan TO, Tsichlis PN. Akt is a direct target of 37. Yang L, Dan HC, Sun M, et al. Akt/protein kinase B signaling the phosphatidylinositol 3-kinase. Activation by growth factors, v-src and inhibitor-2, a selective small molecule inhibitor of Akt signaling with v-Ha-ras, in Sf9 and mammalian cells. J Biol Chem 1996;271:30835 – 9. antitumor activity in cancer cells overexpressing Akt. Cancer 2004;64: 17. Steck PA, Pershouse MA, Jasser SA, et al. Identification of a candidate 4394 – 9. tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is 38. Shen J, Smith RA, Stoll VS, et al. Characterization of protein kinase A mutated in multiple advanced cancers. Nat Genet 1997;15:356 – 62. phosphorylation: multi-technique approach to phosphate mapping. Anal 18. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine Biochem 2004;324:204 – 18. phosphatase gene mutated in human brain, breast, and prostate cancer. 39. Engh RA, Girod A, Kinzel V, Huber R, Bossemeyer D. Crystal Science 1997;275:1943 – 7. structures of catalytic subunit of cAMP-dependent protein kinase in 19. Cairns P, Okami K, Halachmi S, et al. Frequent inactivation of PTEN/ complex with isoquinolinesulfonyl protein kinase inhibitors H7, H8, and MMAC1 in primary prostate cancer. Cancer Res 1997;57:4997 – 5000. H89: structural implications for selectivity. J Biol Chem 1996;271: 26157 – 64. 20. Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/ 40. Luo Y, Smith RA, Guan R, et al. Pseudosubstrate peptides inhibit Akt AKT pathway. Proc Natl Acad Sci U S A 1999;96:4240 – 5. and induce cell growth inhibition. Biochemistry 2004;43:1254 – 63. 21. Cheng JQ, Altomare DA, Klein MA, et al. Transforming activity and 41. Plas DR, Talapatra S, Edinger AL, Rathmell JC, Thompson CB. mitosis-related expression of the AKT2 oncogene: evidence suggesting a Akt and Bcl-xL promote growth factor-independent survival through link between cell cycle regulation and oncogenesis. Oncogene 1997;14: distinct effects on mitochondrial physiology. J Biol Chem 2001;276: 2793 – 801. 12041 – 8. 22. Mende I, Malstrom S, Tsichlis PN, Vogt PK, Aoki M. Oncogenic 42. Biggs WH, Meisenhelder J, Hunter T, Cavenee WK, Arden KC. Protein transformation induced by membrane-targeted Akt2 and Akt3. Oncogene kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the 2001;20:4419 – 23. winged helix transcription factor FKHR1. Proc Natl Acad Sci U S A 1999; 96:7421 – 6. 23. Liu X, Powlas J, Shi Y, et al. Rapamycin inhibits Akt-mediated oncogenic transformation and tumor growth. Anticancer Res 2004;24: 43. Fahy BN, Schlieman M, Virudachalam S, Bold RJ. AKT inhibition is 3899 – 905. associated with chemosensitisation in the pancreatic cancer cell line MIA- PaCa-2. Br J Cancer 2003;89:391 – 7. 24. Di Cristofano A, Pesce B, Cordon Cardo C, Pandolfi PP. Pten is essential for embryonic development and tumour suppression. Nat Genet 44. Nakashio A, Fujita N, Rokudai S, Sato S, Tsuruo T. Prevention of 1998;19:348 – 55. phosphatidylinositol 3-kinase-AktV survival signaling pathway during top- otecan-induced apoptosis. Cancer Res 2000;60:5303 – 9. 25. Suzuki A, de la Pompa JL, Stambolic V, et al. High cancer susceptibility and embryonic lethality associated with mutation of the 45. Ng SS, Tsao MS, Nicklee T, Hedley DW. Wortmannin inhibits pkb/akt PTEN tumor suppressor gene in mice. Curr Biol 1998;8:1169 – 78. phosphorylation and promotes gemcitabine antitumor activity in orthotopic human pancreatic cancer xenografts in immunodeficient mice. Clin 26. Liaw D, Marsh DJ, Li J, et al. Germline mutations of the PTEN gene in Cancer Res 2001;7:3269 – 75. Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet 1997;16:64 – 7. 46. Gru¨nwald V, DeGraffenried L, Russel D, Friedrichs WE, Ray RB, Hidalgo M. Inhibitors of mTOR reverse doxorubicin resistance conferred by 27. Marsh DJ, Dahia PL, Zheng Z, et al. Germline mutations in PTEN are PTEN status in prostate cancer cells. Cancer Res 2002;62:6141 – 5. present in Bannayan-Zonana syndrome [letter]. Nat Genet 1997;16:333 – 4. 47. Asselin E, Mills GB, Tsang BK. XIAP regulates Akt activity 28. Gao X, Neufeld TP, Pan D. Drosophila PTEN regulates cell growth and and caspase-3-dependent cleavage during cisplatin-induced apoptosis proliferation through PI3K-dependent and -independent pathways. Dev in human ovarian epithelial cancer cells. Cancer Res 2001;61: Biol 2000;221:404 – 18. 1862 – 8. 29. Stiles B, Gilman V, Khanzenzon N, et al. Essential role of AKT-1/ protein kinase B a in PTEN-controlled tumorigenesis. Mol Cell Biol 2002; 48. Mitsuuchi Y, Johnson SW, Selvakumaran M, Williams SJ, Hamilton 22:3842 – 51. TC, Testa JR. The phosphatidylinositol 3-kinase/AKT signal transduction pathway plays a critical role in the expression of p21(WAF1/CIP1/SDI1) 30. Bondar VM, Sweeney-Gotsch B, Andreeff M, Mills GB, McConkey DJ. induced by cisplatin and paclitaxel. Cancer Res 2000;60:5390 – 4. Inhibition of the phosphatidylinositol 3V-kinase-AKT pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivo. Mol Cancer 49. Shingu T, Yamada K, Hara N, et al. Synergistic augmentation Ther 2002;1:989 – 97. of antimicrotubule agent-induced cytotoxicity by a phosphoinositide 3-kinase inhibitor in human malignant glioma cells. Cancer Res 2003; 31. Semba S, Itoh N, Ito M, Harada M, Yamakawa M. The in vitro and 63:4044 – 7. in vivo effects of 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a specific inhibitor of phosphatidylinositol 3V-kinase, in human colon cancer 50. Yang CC, Lin Ho P, Chen CS, et al. Bcl-xL mediates a survival cells. Clin Cancer Res 2002;8:1957 – 63. mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells. J Biol Chem 2003;278:25872 – 8. 32. Huang S, Houghton PJ. Inhibitors of mammalian target of rapamycin as novel antitumor agents: from bench to clinic. Curr Opin Investig Drugs 51. Cho H, Mu J, Kim JK, et al. Insulin resistance and a diabetes mellitus- 2002;3:295 – 304. like syndrome in mice lacking the protein kinase Akt2 (PKB h). Science 2001;292:1728 – 31. 33. Sekulic A, Hudson CC, Homme JL, et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian 52. Garofalo RS, Orena SJ, Rafidi K, et al. Severe diabetes, age- target of rapamycin in mitogen-stimulated and transformed cells. Cancer dependent loss of adipose tissue, and mild growth deficiency in mice Res 2000;60:3504 – 13. lacking Akt2/PKB h. J Clin Invest 2003;112:197 – 208.

MolCancerTher2005;4(6).June2005

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