The Akt/PKB pathway: molecular target for cancer drug ... - Nature

Oncogene (2005) 24, 7482–7492

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The Akt/PKB pathway: molecular target for cancer drug discovery Jin Q Cheng*,1, Craig W Lindsley2, George Z Cheng3, Hua Yang1 and Santo V Nicosia1 1

Departments of Pathology and Interdisciplinary Oncology, H Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, 12902 Magnolia Drive, SRB3, Tampa, FL 33612, USA; 2Department of Medicinal Chemistry, Merck & Co., West Point, PA 19486, USA; 3Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA

The serine/threonine kinase Akt/PKB pathway presents an exciting new target for molecular therapeutics, as it functions as a cardinal nodal point for transducing extracellular (growth factor and insulin) and intracellular (receptor tyrosine kinases, Ras and Src) oncogenic signals. In addition, alterations of the Akt pathway have been detected in a number of human malignancies. Ectopic expression of Akt, especially constitutively activated Akt, is sufficient to induce oncogenic transformation of cells and tumor formation in transgenic mice as well as chemoresistance. Akt has a wide range of downstream targets that regulate tumor-associated cell processes such as cell growth, cell cycle progression, survival, migration, epithelial–mesenchymal transition and angiogenesis. Blockage of Akt signaling results in apoptosis and growth inhibition of tumor cells with elevated Akt. The observed dependence of certain tumors on Akt signaling for survival and growth has wide implications for cancer therapy, offering the potential for preferential tumor cell killing. In the last several years, through combinatorial chemistry, high-throughput and virtual screening, and traditional medicinal chemistry, a number of inhibitors of the Akt pathway have been identified. This review focuses on ongoing translational efforts to therapeutically target the Akt pathway. Oncogene (2005) 24, 7482–7492. doi:10.1038/sj.onc.1209088 Keywords: Akt; cancer; therapeutics; inhibitor

Akt proteins: overview and rationale as antitumor targets Akt was originally discovered as an oncogene transduced by the acute transforming retrovirus (Akt-8), which was isolated from an AKR thymoma (Staal et al., 1977; Staal, 1987), and subsequently found to encode a serine/threonine protein kinase (Bellacosa et al., 1991). Akt is also known as protein kinase B (Coffer and Woodgett, 1991) and RAC-PK (Jones et al., 1991). Viral akt highly activated and oncogenic due to the fact that v-akt is associated with the cell membrane through a myristylated Gag protein sequence fused to the Nterminus of Akt (Bellacosa et al., 1991). The important *Correspondence: JQ Cheng; E-mail: chengjq@moffitt.usf.edu

role of Akt in transformation and cancer was shortly thereafter strengthened by the cloning of the AKT2 gene (Cheng et al., 1992) and the discovery that AKT2 is frequently amplified and overexpressed in human cancers (Cheng et al., 1992, 1996; Bellacosa et al., 1995). To date, three Akt family members have been identified in mammals, designated Akt1/PKBa, Akt2/ PKBb and Akt3/PKBg (Testa and Bellacosa, 2001). The members of Akt family share similar domain structure and are activated by various stimuli in a phosphatidylinositol 3-kinase (PI3K)-dependent manner (Burgering and Coffer, 1995; Franke et al., 1995; Liu et al., 1998; Shaw et al., 1998). Activation of Akt depends on the integrity of the pleckstrin homology (PH) domain, which mediates its membrane translocation, and on the phosphorylation of Thr308 in the activation loop and Ser473 (Chan et al., 1999; Datta et al., 1999; Testa and Bellacosa, 2001; Brazil et al., 2002). Phosphoinositides, PtdIns-3,4-P2 and PtdIns-3,4,5-P3, produced by PI3 K bind directly to the PH domain of Akt, driving a conformational change in the molecule, which enables the activation loop of Akt to be phosphorylated by PDK1 at Thr308 (Alessi et al., 1997). Full activation of AKT1 is also associated with phosphorylation of Ser473 (Alessi et al., 1996) within a C-terminal hydrophobic motif characteristic of kinases in the AGC kinase family. Although the role of PDK1 in Thr308 phosphorylation is well established, the mechanism of Ser473 phosphorylation is controversial. A number of candidate enzymes responsible for this modification have been put forward, including integrin-linked kinase (Persad et al., 2001), PDK1 when in a complex with the kinase PRK2 (Balendran et al., 1999), Akt itself, through autophosphorylation (Toker and Newton, 2000), PKCa (Partovian and Simons, 2004), PKCbII (Kawakami et al., 2004), DNA-dependent kinase (Feng et al., 2004), and the rictor-mTOR complex (Sarbassov et al., 2005). The activity of Akt is negatively regulated by tumor suppressor PTEN, which is frequently mutated in human malignancy (Li et al., 1997; Steck et al., 1997; Parsons, 2004). PTEN encodes a dual-specificity protein and lipid phosphatase that reduces intracellular levels of PtdIns-3,4,5-P3 by converting them to PtdIns-4,5-P2, thereby inhibiting the PI3K/Akt pathway (Stambolic et al., 1998). Akt phosphorylates and/or interacts with a number of molecules to exert its normal cellular functions, which include roles in cell proliferation, survival and differentiation (Chan et al., 1999; Datta

Akt as target for anticancer drug discovery JQ Cheng et al

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et al., 1999; Testa and Bellacosa, 2001; Brazil et al., 2002). Gene knockout studies have defined the biological importance of Akt members in normal cells. In particular, Akt2-null mice develop typical type II diabetes (Cho et al., 2001a, b), while Akt1- and Akt3deficient mice do not display a diabetic phenotype but exhibit a decrease in the sizes of all organs and a selective impairment of brain development, respectively (Chen et al., 2001; Cho et al., 2001a, b; Easton et al., 2005; Tschopp et al., 2005). Moreover, although Akt1and Akt3-deficient brains are reduced in size to approximately the same degree, the absence of Akt1 reduces neuronal cell number, whereas the lack of Akt3 results in smaller and fewer cells in which mTOR signaling is attenuated (Easton et al., 2005). Several lines of evidence suggest that Akt is a critical target for anticancer drug discovery. First, Akt sits at the crossroads of multiple oncogenic and tumor suppressor signaling networks (see related Reviews in this issue). Almost all known oncogenic growth factors, angiogenic factors and cytokines activate Akt by binding to cognate receptors on cell surface. Further, Akt is also activated by steroid hormones, such as estrogen and androgen through a mechanism independent of their nuclear receptors (Sun et al., 2001a; Sun et al., 2003). In addition, Akt is shown to be activated by constitutively active Ras and Src (Datta et al., 1996; Liu et al., 1998). Second, frequent deregulations of many components of the Akt signaling pathway have been observed in human cancer (see review by Altomare and Testa in this issue). Of the Akt family, only the AKT2 gene is frequently amplified in human cancer. Further, overexpression of AKT2 RNA and/or protein is also more commonly observed in human cancer than are AKT1 and AKT3 (Testa and Bellacosa, 2001; Kim et al., 2005). However, recurrent activation of the three Akt family members has been detected in a variety of types of human malignancy (Yuan et al., 2000; Sun et al., 2001a, b, 2003; Altomare et al., 2003, 2004, 2005; Balsara et al., 2004). Activation of Akt is primarily the result of aberrant upstream molecules of Akt, which include overproduction of growth factors, upregulation and/or mutation of receptor tyrosine kinases, Ras and Src as well as PIK3CA and PTEN. While a point mutation of AKT2 has been reported in familial diabetes (George et al., 2004), a dominant mutation of Akt has not been identified in human tumor. Third, ectopic expression of constitutively active Akt and even wildtype Akt2 results in oncogenic transformation in vitro and in vivo (Cheng et al., 1997; Hutchinson et al., 2001; Malstrom et al., 2001; Mende et al., 2001; Sun et al., 2001a, b; Majumder et al., 2003). Furthermore, a number of studies have shown that overexpression and/or activation of Akt render tumor cells resistant to chemotherapeutic drugs and signal molecule inhibitors such as Gleevec, Iressa, Herceptin and retinoid acid (Cheng et al., 2002; Arlt et al., 2003; Knuefermann et al., 2003; Yuan et al., 2003; Nagata et al., 2004). In addition, Akt targets many signal molecules to regulate tumor development-associated cell processes such as apoptosis, cell proliferation, differentiation, migration

and angiogenesis. Finally, knockdown of Akt by antisense or siRNA significantly reduces tumor growth and invasiveness and induces apoptosis and cell growth arrest only in tumor cells overexperssing Akt (Cheng et al., 1996; Chen et al., 2001; Asnaghi et al., 2004; Remy et al., 2004; Tabellini et al., 2005). These observations make Akt an attractive target for anticancer drug discovery, and it has been postulated that inhibition of Akt alone or in combination with standard cancer chemotherapeutics will reduce the apoptotic threshold and preferentially kill cancer cells. The development of specific and potent inhibitors will allow this hypothesis to be tested in animals. The majority of small molecule inhibitors in this nascent field are classic ATP-competitive inhibitors, which provide little specificity. Phosphatidylinositol (PI) analogs have been reported to inhibit Akt, but these inhibitors may also have specificity problems with respect to other PH domain containing proteins and may have poor bioavailability. Recently, small chemical compounds triciribine/Akt/protein kinase B inhibitor-2 (API-2) and allosteric inhibitors have been reported which are PH domain dependent, and the latter also exhibit Akt isozyme selectivity. In addition, inhibitors toward upstream regulators and downstream targets of Akt have also been tested for their capability of reversing the phenotype of cancer cells expressing altered Akt. This review focuses on the ongoing efforts to therapeutically target individual components of Akt pathway including Akt itself as well as its upstream regulators and downstream effectors (Figure 1). Some of these efforts involve AKT-specific inhibition based on structurebased analyses (see also the Review by Kumar and Madison in this issue).

Therapeutic targeting of upstream regulators of Akt PDK1 inhibitors PDK1 is a serine/threonine protein kinase that can phosphorylate and activate a number of kinases in the AGC kinase superfamily (Mora et al., 2004). The first identified and best characterized PDK1 substrates are the three members of the Akt family (Mitsiades et al., 2004). PDK1 phosphorylates the activation loop of Akt (also called the T-loop) on residue Thr308, which primarily regulates Akt activation (Alessi et al., 1997). Therefore, a PDK1 inhibitor should significantly block activation of Akt. Three potent PDK1 inhibitors, BX-795, BX-912 and BX-320 (Figure 2a), recently identified by screening of compound libraries, have IC50 between 11 and 30 nM (Feldman et al., 2005). The inhibitors blocked PDK1/ Akt signaling in tumor cells resulting in the inhibition of anchorage-independent growth and the induction of apoptosis in a variety of tumor cell lines. A number of cancer cell lines with elevated Akt activity were >30fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/ Oncogene


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PTEN PI-4,

5-P3

PI 3-kinase

p85

p110

Src TRK inhibitor

PIP3 competitor

PI-3,4,

5-P2

PH Kinase domain

Akt inhibitor

-T308-p

PDK1

RAS

Akt

RD -S473-p

FTI/GGTI

PI3K inhibitor

PDK1 inhibitor

mTOR inhibitor

mTOR

p70S6K

4E-BP1

5’top mRNA translation Cap dependent translation Figure 1

Therapeutic targeting of the Akt pathway including Akt itself as well as its upstream regulators and downstream effectors

Figure 2

Compound structures for inhibitors of PDK1 (a) and PI3K (b)

Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents. The staurosporine derivative UCN-01 (7-hydroxystaurosporine), a drug now in clinical trials, has been shown to potently inhibit PDK1 (IC50 ¼ 33 nM) in vitro. UCN-01-induced PDK1 inhibition was also observed in Oncogene

human tumor xenografts (Sato et al., 2002). Overexpression of constitutively active Akt diminished the cytotoxic effects of UCN-01, suggesting that UCN-01 may in part exert its cytotoxicity by inhibiting PDK1/ Akt survival pathway. Crystal structure analyses showed that staurosporine and UCN-01 form a complex with the kinase domain of PDK1 (Komander et al., 2003). Although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group (Figure 2a), which is not present in staurosporine, generates direct and water-


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mediated hydrogen bonds with active-site residues. This moiety is hydrogen-bonded directly to Thr222 and indirectly via an ordered water molecule to Gln220 of PDK1 (Zhao et al., 2002; Johnson and Pinto, 2002; Komander et al., 2003). A different water-mediated hydrogen-bonding network is also observed in other UCN-01 complexes and might serve as a starting point for further structure-based optimization. In addition, recent studies show that UCN-01 inhibits other kinases such as PKC and Chk1 and transcriptionally upregulates the cyclin-dependent kinase inhibitor p21waf1/cip1 (Senderowicz, 2003a, b). PI3K inhibitors Dissection of PI3K/Akt signaling pathway has been aided greatly by two pharmacological PI3K inhibitors, wortmannin and LY294002 (Figure 2b). Wortmannin is a fungal metabolite and a potent inhibitor of type I PI3K, with an IC50 range for inhibition of PI3K from 2 to 4 nM (Arcaro and Wymann, 1993; Vlahos et al., 1994). Wortmannin inhibits PI3K activity by binding covalently to a conserved lysine residues in the ATPbinding site of the enzyme (Wymann et al., 1996). Wortmannin has antitumor activity in vitro and in vivo, suggesting that it might offer a valuable approach to treat cancer. However, a major disadvantage of the use of wortmannin is its stability in an aqueous environment. Wortmannin is soluble in organic solvents, which may limit its use in clinical trials. Currently, watersoluble wortmannin conjugates are being developed to circumvent this issue. LY294002 is a flavonoid derivative and a reversible, ATP-competitive inhibitor with IC50 for recombinant PI3K in the low micromolar range. A number of in vitro studies have shown that LY294002 alone has antiproliferative and proapoptotic activities (Wetzker and Rommel, 2004). Relatively, few in vivo studies have been conducted to demonstrate the efficacy of LY294002 on the inhibition of tumor growth, but these studies showed that administration of LY294002 in human cancer xenografts inhibited tumor growth and induced apoptosis (Semba et al., 2002; Fan et al., 2003). Although inhibition of the PI3K/Akt pathway by wortmannin or LY294002 alone may inhibit cell proliferation, promote apoptosis and/or inhibit tumor growth, the combination of wortmannin or LY294002 with traditional cytotoxic drugs or radiation enhances the effectiveness of these treatments (Wetzker and Rommel, 2004). It is noteworthy that neither wortmannin nor LY294002 displays selectivity for different members of the class I PI3K (Finan and Thomas, 2004). Wortmannin also inhibits class III PI3K to reduce autophagy, a class II programmed cell death (Shintani and Klionsky, 2004). LY294002 also inhibits casein kinase 2 with similar potency to PI3K. At higher concentrations, wortmannin inhibits PI3K-related enzymes, such as mTOR, ATM and PI4-kinase b (Finan and Thomas, 2004). Moreover, methylxanthines, such as caffeine and theophylline, inhibit p110d, even though their activity is rather weak (Arcaro and Wymann, 1993; Abraham, 2004).

Recently, several new compounds have been described to have some selectivity for individual members of PI3K (Figure 2b). PIramed have described several imidazopyridine derivatives that exhibit excellent PI3K inhibitory activities, especially against p110a. A series of morpholino-substituted compounds related LY294002 have shown isoform selectivity. Quinolone and pyridopyrimidine (Kinacia) are approximately 100-fold more potent against p110a/b as compared to p110g (Finan and Thomas, 2004). ICOS Corporation has recently claimed a new PI3K inhibitor, IC87114, which selectively inhibits p110d with IC50 ¼ 0.5 mM and >50-fold selectivity over the other class PI3K isoforms (Sadhu et al., 2003). In addition, Novartis has described 5phenylthiazole derivatives as PI3K inhibitors (Finan and Thomas, 2004). However, antitumor activity of these compounds needs further investigation both in vitro and in vivo. It is noteworthy that use of PI3K inhibitors may be associated with undesirable side effects because of the many important cellular targets of this lipid kinase. Inhibitors of the prenylation Protein prenylation, including farnesylation and geranylgeranylation, is a lipid post-translational modification required for the cancer-causing activity of proteins such as the GTPase Ras. Farnesyltransferase and geranylgeranyltransferase I inhibitors (FTIs/GGTIs) represent a new class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without significant toxicity to normal cells, especially FTIs are currently in clinical trials. However, the mechanism of FTI and GGTI antitumor activity remains elusive. It has been shown that FTIs inhibit PI3K/Akt-mediated growth factor- and adhesion-dependent survival pathways and induce apoptosis in human cancer cells that overexpress Akt (Jiang et al., 2000; Prendergast, 2000; Sebti and Der, 2003). Furthermore, overexpression of Akt, but not oncogenic HRas, sensitizes NIH3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not Akt-transformed NIH3T3 cells. A constitutively active form of Akt rescues human cancer cells from FTI-277induced apoptosis. Integrin-dependent activation of Akt is also blocked by FTI-277. In addition, GGTI-298 and GGTI-2166 have also been shown to inhibit PI3K/Akt pathway, resulting in apoptosis in human cancer cells (Dan et al., 2004). Thus, a mechanism for FTIs and GGTIs inhibition of human tumor growth is by inducing apoptosis through inhibition of the PI3K/Akt pathway. However, neither FTIs nor GGTIs directly inhibits PI3K/ Akt, suggesting that the unidentified prenylated proteins that activate PI3K/Akt are the targets of FTIs and/or GGTIs. Recent studies suggest that RhoB could be a candidate to mediate this action (Liu and Prendergast, 2000; Adini et al., 2003; Jiang et al., 2004). RTK inhibitors Among RTKs, EGFR and Her2/Neu/ErbB2 are frequently altered in human cancer and primarily activate Oncogene


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the PI3K/Akt pathway. Two major approaches have been used to target the ErbB family, that is, smallmolecule tyrosine kinase inhibitors and humanized antibodies against the receptor extracellular domains (Yu and Hung, 2000; Chen et al., 2003). In general, antibodies bind to the extracellular domain of the receptors, inhibiting their activation by ligand, and promoting receptor internalization and downregulation, whereas small molecules competitively inhibit ATP binding to the receptor, thereby hindering autophosphorylation and kinase activation. At present, the most advanced of the newer therapies in clinical development are anti-EGFR monoclonal antibody IMC-C225 (cetuximab, Erbitux; Imclone), anti-ErbB2 and the reversible small-molecule inhibitors of EGFR, ZD-1839 (gefitinib, Iressa; AstraZeneca) and OSI-774 (erlotinib, Tarceva; OSI Pharmaceuticals). Both ZD-1839 and OSI-774 have been through phase I and phase II trials. Promising single-agent clinical antitumor activity has been reported in advanced NSCLC, head and neck cancer and prostate carcinoma. Furthermore, humanized monoclonal antibodies, IMC-C225 against EGFR and trastuzumab (Herceptin) targeted ErbB2, are also in phase II and phase III trials. These agents potently inhibit EGFR and ErbB2 resulting in the reduction of Akt kinase activity (Yakes et al., 2002; Mitsiades et al., 2004). A recent study has shown that patients who become resistance to Herceptin have elevated levels of Akt due to loss of PTEN, suggesting that elevated Akt activation is responsible for Herceptin resistance (Nagata et al., 2004).

Akt inhibitors Akt antibody Antibodies and antibody-based reagents have been used for the treatment of cancer. As described above, the humanized IgG1 trastuzamab (Herceptin) is an effective treatment for breast cancers that overexpress ErbB2 (Yu and Hung, 2000). Genetic engineering of antibodies can be used to modify and enhance antibody efficacy. For example, mouse monoclonal antibodies can be chimerized by such approaches to prevent the production of human antimurine antibodies when administered to immune-competent patients (Clark, 2000). An alternative strategy is to replace the antibody gene present in mouse B cells with human antibody genes. These modified B cells can then be used to produce hybridoma cell lines that express fully humanized monoclonal antibodies that avoid cross-species immune response (Fishwild et al., 1996). Over a decade ago, McCafferty et al. (1990) demonstrated that recombinant antibody fragments could be displayed on the tip of M13 bacteriophage. Some of the advantages of phagedisplayed recombinant antibodies over the conventional polyclonal or monoclonal antibodies are quick generation time, cheap production cost, and, importantly, accessibility to the antibody DNA for further genetic manipulations. Recently, Shin et al. (2005) developed Oncogene

novel recombinant anti-Akt single-chain antibodies by panning a mouse phage-displayed scFv recombinant antibody library using GST-Akt1 fusion protein. To generate a membrane-permeable version of the antiAkt1-scFv, the scFv gene was subcloned into a GST expression vector carrying a membrane-translocating sequence (MTS) from Kaposi fibroblast growth factor. A purified GST-anti-Akt1-MTS fusion protein accumulates intracellularly and inhibits activation of all three Akt family members. Interestingly, in vitro kinase assay shows that GST-anti-Akt1-MTS also inhibits constitutively active forms of Myr-Akt1, -Akt2 and -Akt3 as well as phosphomimetic mutant of Akt-DD, where Thr308 and Ser473 are replaced with aspartic acid. Furthermore, GST-anti-Akt1-MTS induces apoptosis in cancer cell lines that express constitutively active Akt. In addition, anti-Akt scFv exhibits antitumor activity in PyVmT-expressing transgenic tumors implanted in mouse dorsal window chambers (Shin et al., 2005). These data indicate that GST-anti-Akt1-MTS is a cellpermeable inhibitor of Akt and that this approach can be used to generate compounds that target tumor cells dependent on aberrant Akt for their growth. PI analog inhibitors As PtdIns(3,4,5)P3 directly binds to the PH domain of Akt and PDK1 and is required for activation of Akt, the development of a PtdIns(3,4,5)P3 analog would be a reasonable approach to develop an Akt inhibitor. This mode of inhibition would prevent Akt translocation to the plasma membrane and activation. The feasibility of this approach was suggested by the demonstration that D-3-deoxy-myo-inositols inhibited the growth of transformed cells (Powis et al., 1991). It was subsequently found that the inositol derivative DPI had an IC50 of 35 mM against H-29 colon cancer cell growth (Kozikowski et al., 1995). A recent study examined 24 modified phosphatidylinositol ether lipid analogues (PIAs) and found that five of them, PIA5, 6, 23, 24, and 25 (Figure 3), with modifications at two sites on the inositol ring, inhibited Akt with IC50o5 mM (Castillo et al., 2004a, b). PIAs decreased phosphorylation of many downstream targets of Akt without affecting upstream kinases, such as PI3K or PDK1. Importantly, PIAs selectively induced apoptosis in cancer cell lines with high levels of endogenous Akt activity. These findings identify PIAs as effective Akt inhibitors, and provide proof of principle for targeting the PH domain of Akt. However, whether PIAs are effective in vivo and whether PIAs affect other PH-domain containing proteins are currently unknown. Perifosine is a novel orally bioavailable alkylphospholipid and structurally resembles naturally occurring phospholipids (Figure 3). Perifosine is known to be a CDK inhibitor and has displayed significant antiproliferative activity in vitro and in vivo in several human tumor model systems (Senderowicz, 2003a, b; Vink et al., 2005). It has been shown that perifosine can cause cell cycle arrest with induction of p21WAF1/CIP1 in a p53-independent fashion. By searching for the under-


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Figure 3 Compound structures for representive Akt inhibitors

lying mechanism, Kondapaka et al. (2003) has demonstrated that perifosine inhibits Akt phosphoylation by decreasing the plasma membrane localization of Akt, and this is substantially relieved by Myr-Akt along with relief of downstream drug effect on induction of p21WAF1/CIP1. While perifosine does not directly affect PI3K, PDK1 or Akt activity, its antitumor activity is, at least in part, mediated by inhibition of the Akt pathway (Kondapaka et al., 2003). In a recent phase I clinical trial, 1 patient had a partial response to perifosine and 2 patients had stable disease among 42 patients with incurable solid malignancies, thus justifying additional investigation of this agent in a phase II trial (Van Ummersen et al., 2004).

ATP-competitive inhibitors Through the elucidation of the sequence and structural composition of kinase active sites, coupled with the solution of numerous ATP-competitive ligand complex structures, significant advances have been made in developing inhibitors that are highly selective. This has been the case not only for kinases that are divergent in primary structure, but also for isoforms with highly conserved structure and ATP-binding sites. However, due to the fact that there is a high degree of homology in the ATP-binding pocket between Akt, PKA and PKC (Yang et al., 2002), many typical PKA and PKC inhibitors have been identified as inhibitors of Akt (Reuveni et al., 2002). So far, no ATP-competitive inhibitors specific against Akt have been identified. While a recent report described the design and synthesis of three Akt-selective inhibitors, their ability to inhibit Akt has not been tested in either cell culture or animal models (Breitenlechner et al., 2005).

Pseudosubstrate inhibitors In contrast to ATP-competitive inhibitors, pseudosubstrate peptide inhibitors bind to the peptide/protein substrate sites of the catalytic domain and have been proven to be selective and potent for many kinases (Luo et al., 2004). This selectivity derives from the much larger peptide-kinase contact region that has evolved to discriminate between various protein substrates. A 14-mer peptide (AKTide-2T) was identified that binds to the substrate binding site of Akt1 and inhibits Akt1 activity (Luo et al., 2004). A hybrid peptide was recently constructed between this sequence and a sequence (amino acids 16–24) of the forkhead transcription factor FOXO3. The resulting 20-mer peptide construct inhibits Akt1 with a Ki of 1.1 mM. Replacement of a putative phosphorylation site serine (Ser) in the sequence with an alanine (Ala) moiety resulted in a further 10-fold improvement of potency. Efforts to truncate the peptide sequence showed that the 20-mer could be truncated to a 17-mer with only slight loss of potency but further chain shortening resulted in larger losses of potency. These peptides are highly selective versus p70S6K, p90S6K PKA, Cdc2, Src, and PKC. Interestingly, the original 20-mer was equally active against SGK but the subsequent Ala for Ser replacement resulted in a highly selective inhibitor. Fusion peptides were also constructed to allow cell uptake and demonstrated dose-dependent inhibition of GSK3b phosphorylation. While these peptides are potent and selective inhibitors of Akt, their size makes them poor leads for small molecule inhibitor development. Significant truncation of the peptides and incorporation of peptidomimetic functionality would likely be needed to lead to cell-permeable molecules with good pharmacokinetics. Oncogene


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API-2/triciribine By screening of the National Cancer Institute Diversity Set 2000-compound library, which are derived from approximate 140 000 compounds, a small molecule Akt pathway inhibitor, API-2, has recently been identified (Yang et al., 2004). API-2 suppressed the kinase activity and phosphorylation level of all three Akt family members. The inhibition of Akt kinase resulted in suppression of cell growth and induction of apoptosis in human cancer cells that harbor constitutively activated Akt due to overexpression of Akt or other genetic alterations such as PTEN mutation. API-2 is highly selective for Akt and does not inhibit PI3K, PDK1, PKC, SGK, PKA, Stat3, Erk-1/2 or JNK. Furthermore, API-2 potently inhibited tumor growth in nude mice of human cancer cells in which Akt is aberrantly expressed/ activated but not of those cancer cells in which it is not. Recent data suggest that API-2 inhibition of Akt depends on the interaction with the PH domain of Akt. API-2 is a synthetic small molecule compound identified previously and named triciribine (TCN, NSC-154020; Figure 3) or tricyclic nucleoside (Chung et al., 1980; Wotring et al., 1986). Its 5’phosphate ester, triciribine monophosphate (TCN-P, NSC-280594) is the chemical entity advanced into clinical trials because it is more soluble than the parent drug (Wotring et al., 1986). TCN and TCN-P have antiviral and antineoplastic activity at low micromolar or submicromolar concentrations (Migawa et al., 2005). By the early 1980s, TCNP had been identified as an inhibitor of DNA and protein synthesis, and shown preclinical activity against leukemias and carcinomas. A number of phase I and II clinical trials of TCN/API-2 have been conducted in patients with advanced tumors, including carcinomas of the breast, colon, bladder, ovary, pancreas and lung (Cobb et al., 1983; Mittelman et al., 1983; Feun et al., 1984, 1993; Powis et al., 1986; Schilcher et al., 1986; Hoffman et al., 1996). Owing to the fact that TCN-p was used as a cytotoxic drug, the majority of clinical trials were carried out with high doses of the drug in order to achieve maximal clinical efficacy. While it exhibited antitumor activity in some patients, TCN/ API-2 had significant side effects at high doses, including hepatotoxicity, hypertriglyceridemia, thrombocytopenia, and hyperglycemia, which hampers its application in the clinic. As TCN/API-2 specifically targets Akt, it is reasonable to speculate that a given tumor with high levels of Akt activity will be more sensitive/responsive to TCN/ API-2 treatment and that lower doses of TCN/API-2 should achieve clinical efficacy without significant side effects in patients whose tumors exhibit elevated Akt levels. In fact, a recent study has shown that TCN/API-2 effectively induces apoptosis and cell growth arrest in tumor cells with activation of Akt at a concentration of 10 mM. In xenograft experiments, no detectable side effects were observed in 50 mice treated with TCN/API2 at a concentration of l mg/kg/day, which significantly inhibited tumor growth in cancer cells overexpressing Akt (Yang et al., 2004). These findings indicate that Oncogene

TCN/API-2 at low dose could achieve antitumor growth without significant side effects in tumors with hyperactive Akt. Therefore, future clinical trials for additional assessment of TCN/API-2 must incorporate careful patient selection based on the Akt status of the tumor. Allosteric Akt kinase inhibitors Recent studies have identified novel allosteric Akt kinase inhibitors by screening a collection of approximately 270 000 compounds and application of an iterative analog library synthesis approach (Barnett et al., 2005a, b; DeFeo-Jones et al., 2005; Lindsley et al., 2005; Zhao et al., 2005). These inhibitors display an unprecedented level of specificity. Not only are they specific with respect to other kinases, but they are also isozyme specific, even though no compound specifically targets Akt3. The detailed development of these inhibitors has been described by recent reviews (Barnett et al., 2005a, b; Lindsley et al., 2005; Zhao et al., 2005). Inhibitors specifically targeting Akt1, Akt2 or both Akt1 and Akt2 have been named Akti-1, Akti-2 and Akti-1,2, respectively (Figure 3). These inhibitors all contain a well-known GPCR privileged structure, a piperidinyl benzimidazolone, that is crucial for improving potency (Barnett et al., 2005a, b; DeFeo-Jones et al., 2005; Lindsley et al., 2005). In addition to inhibiting kinase activity, these inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1. Akti-1, Akti-2 and Akti-1,2 are reversible inhibitors and have no inhibitory effects on Akt mutants lacking the PH domain, suggesting that they, similar to TCN/API-2, bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region abrogated the inhibition of Akt by these inhibitors. Several interesting observations have been made with these inhibitors. First, in multiple cell lines, maximal induction of caspase-3 activity is achieved when both Akt1 and Akt2 are inhibited, that is, Akti-1 or Akt1-2 alone induces moderate levels of caspase-3 activity (8 units and 10 units after 6 h treatment, respectively). In contrast, simultaneous treatment with both inhibitors resulted in strong synergy of caspase-3 activation (45 units) (DeFeo-Jones et al., 2005). Second, as these inhibitors depend on the integrity of the PH domain, constitutively active myr-Akt1 and myr-Akt2 were capable of protecting against caspase activation induced by these compounds. However, the programmed cell death induced by Akti-1 or/and Akti-2 as well as Akti-1/ 2 could not be reversed by overexpression of functionally active Akt3, suggesting that Akt3 is not able to compensate for loss of Akt1/2. Third, these inhibitors selectively sensitize tumor cells, but not normal cells, to apoptotic stimuli, suggesting a potential therapeutic window for cancer therapy. Finally, these Akt inhibitors are broadly active chemosensitizers. When used as single agents, Akt1/2 dual inhibitors (Akti-1/Akti-2 or Akti1,2) show limited proapoptotic activity in cell culture.


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Maximal caspase induction is seen only when combining Akt1/2 dual inhibitors with chemotherapeutics such as the topoisomerase inhibitor camptothecin, or biologics, such as the death receptor ligand, TRAIL. Thus, Akt inhibitors combined with conventional chemotherapy or radiation therapy may provide a more effective strategy to improve patient outcome. Although Akti-1, Akti-2 and Akti-1,2 have a great potential for pharmacologically targeting Akt for anticancer drug discovery, further investigations are required to test their antitumor efficacy in animal models. In fact, a mouse pharmacodynamic study has been undertaken to determine if Akti-1,2 could also inhibit Akt phosphorylation in vivo (DeFeo-Jones et al., 2005; Lindsley et al., 2005). Despite poor physical properties, Akti-1,2 achieved 1.5–2.0 mM plasma levels when dosed i.p. (50 mpk, three doses, every 90 min). Following dosing, mice were tail vein-injected with IGF to stimulate Akt phosphorylation. This experiment demonstrated that Akti-1,2 could inhibit the phosphorylation of both basal and IGF-stimulated Akt1 and Akt2 in mouse lung, with no effect on Akt3. Additional in vivo studies with Akti1,2 could not be conducted due to the poor solubililty and pharmacokinetics of the compound (DeFeo-Jones et al., 2005; Lindsley et al., 2005). Nevertheless, these inhibitors represent the first generation of isozymespecific small molecule compounds antagonizing Akt and further modification is warrented. Other inhibitors Previous studies have shown that TCL1 interacts with Akt and functions as an Akt kinase co-activator (Laine et al., 2000; Pekarsky et al., 2000). Based on the binding domain of TCL1 with Akt (Auguin et al., 2004), Hiromura et al. (2004) have recently synthesized a peptide (NH2-AVTDHPDRLWAWEKF-COOH-TCL110-24, encompassing the bA strand of human TCL1) that is named ‘Akt-in’ (Akt inhibitor). ‘Akt-in’ interacts with the PH domain of Akt resulting in conformational changes on the variable loop 1 of Akt, the site mediating phosphoinositide binding. Consistently, interaction of ‘Akt-in’ with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, ‘Akt-in’ inhibited not only cellular proliferation and antiapoptosis in vitro but also in vivo tumor growth without any observable adverse effect (Hiromura et al., 2004). In addtion, a recent report showed that KP3721(OLT Inc., Canada) inhibited Akt kinase activity and phosphorylation by an unkown mechanism. In cell culture, KP372-1 induces apoptosis and cell growth arrest in thyroid cancer cells, in which Akt is activated (Mandal et al., 2005).

Therapeutic intervention at level of signaling molecules downstream of Akt Several dozens of downstream targets of Akt have been identified and are thought to mediate Akt cellular

function. Therefore, it is difficult to conceptualize how inhibitor(s) of only one or two of Akt downstream targets abrogates aberrant Akt in human cancer. However, a number of studies have shown that mTOR inhibitors, rapamycin and its derivatives CCI-779 and RAD001, effectively induce cell cycle arrest and/or apoptosis in the tumors exhibiting activation of Akt (Altomare et al., 2004; Chan, 2004; deGraffenried et al., 2004). In particular, prostate intraepithelial neoplasia (PIN) phenotype developed in Akt1 transgenic mice was completely reversed by administration of the rapamycin derivative RAD001 for 2 weeks (Majumder et al., 2004), suggesting that mTOR mediates crucial aspects of Aktinduced oncogenesis. RAD001 not only inhibited proliferation but also induced programmed cell death in the transgenic mice. Given the number of Aktregulated molecules that prevent apoptosis independent of mTOR, the prominent role of mTOR in mediating antiapoptotic Akt signals came as something of a surprise. Recent studies have shown that mTOR is not only a major downstream target of Akt but also a critical activator of Akt by forming complex with rictor (Sarbassov et al., 2005). The rictor-mTOR complex directly phosphorylated Akt on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1 (Figure 4). In addition, it has been demonstrated that prolonged, but not acute, treatment of certain human cells with rapamycin inhibits Akt phosphorylation (Edinger et al., 2003). The rictor-mTOR feedback activation of Akt may explain why during long-term treatment rapamycin could eventually sequester many of the newly synthesized mTOR molecules within cells. Thus, as the rictormTOR complex turns over, rapamycin interferes with its reassembly or over time become part of the new complexes, which would explain why rapamycin is particularly effective at inducing apoptosis and suppressing the proliferation of tumor cells with hyperactive Akt. Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus, which binds FKBP-12. Thereby, the rapamycin–FKBP12 complex can inhibit mTOR. Rapamycin is used alone or in combination with cyclosporine as an immunosuppressive drug to

PDK1

T308

PH

Kinase domain

S473

RD

rictor Other Targets

mTOR

GβL Figure 4 Schematic diagram of feedback regulation of Akt by rictor-mTOR Oncogene


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prevent renal graft rejection. Rapamycin analogues currently selected for clinical development are CCI-779 (intravenous formulation currently in phase III from Wyeth Ayest), RAD001 (oral formulation currently in phase I-II from Novartis Pharma) and AP23573 (intravenous formulation currently in phase I from Ariad Pharma). In clinical settings, using intermittent administration of CCI-779, RAD001 or AP23576, no evidence of immunosuppressive effects has been observed (Meric-Bernstam and Mills, 2004; Rao et al., 2004). Currently, targeted disruption of mTOR signaling by these mTOR inhibitors is being tested for efficacy against a broad range of refractory tumors. In conclusion, the Akt pathway represents an attractive target for anticancer drug discovery. While our understanding of Akt biology, mechanisms of Akt cellular function and the design of small molecule Akt inhibitors has grown tremendously, key issues regarding Akt specificity and Akt isozyme selectivity remain. Even though the recent discovery of allosteric Akt kinase inhibitors has begun to address these issues, their antitumor activity in vivo, pharmacodynamics and

toxicity are currently unknown. It also remains to be determined if a compound that only inhibits an individual Akt family member will have an improved therapeutic window when compared to an inhibitor of all three Akt kinases, which may depend on the genetic alterations in individual tumors. For example, a tumor with activation of Akt resulting from the PTEN mutation should differ from a tumor with overexpression/amplification of a member of the Akt family. In addition, inhibition of the Akt pathway at multiple sites or in combination with inhibitors of different signaling pathways may allow the development of an acceptable therapeutic index for cancer management. Further, inhibition of the Akt pathway combined with conventional chemotherapy and/or radiation therapy may provide a more effective strategy to improve patient outcome. Acknowledgements We thank Dr Joseph R Testa for his comments and critical reading. This work was supported by grants from the National Cancer Institute and Department of Defense (JQC).

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