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Drug Resistance Updates 11 (2008) 32–50

Targeting the PI3K/Akt/mTOR pathway: Effective combinationsand clinical considerations

Jaclyn LoPiccolo, Gideon M. Blumenthal, Wendy B. Bernstein, Phillip A. Dennis ∗Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20889, United States

Received 2 November 2007; received in revised form 19 November 2007; accepted 19 November 2007

bstract

The PI3K/Akt/mTOR pathway is a prototypic survival pathway that is constitutively activated in many types of cancer. Mechanisms forathway activation include loss of tumor suppressor PTEN function, amplification or mutation of PI3K, amplification or mutation of Akt,ctivation of growth factor receptors, and exposure to carcinogens. Once activated, signaling through Akt can be propagated to a diverse arrayf substrates, including mTOR, a key regulator of protein translation. This pathway is an attractive therapeutic target in cancer because iterves as a convergence point for many growth stimuli, and through its downstream substrates, controls cellular processes that contribute tohe initiation and maintenance of cancer. Moreover, activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy,nd is a poor prognostic factor for many types of cancers. This review will provide an update on the clinical progress of various agents thatarget the pathway, such as the Akt inhibitors perifosine and PX-866 and mTOR inhibitors (rapamycin, CCI-779, RAD-001) and discusstrategies to combine these pathway inhibitors with conventional chemotherapy, radiotherapy, as well as newer targeted agents. We will alsoiscuss how the complex regulation of the PI3K/Akt/mTOR pathway poses practical issues concerning the design of clinical trials, potential
oxicities and criteria for patient selection.ublished by Elsevier Ltd.
eywords: PI3 kinase; Akt; Combination; Chemotherapy; Cancer; Wortmannin; PX-866; Perfosine; mTOR inhibitors; Rapamycin; RAD-001; PI-103;riciribine (API-2)

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. Introduction

.1. Activation of the PI3K/Akt/mTOR pathway

Signaling through the PI3K/Akt/mTOR pathway can benitiated by several mechanisms, all of which increase acti-ation of the pathway in cancer cells. Once activated, theI3K/Akt/mTOR pathway can be propagated to variousubstrates, including mTOR, a master regulator of proteinranslation. Initial activation of the pathway occurs at the
ell membrane, where the signal for pathway activations propagated through class IA PI3K (Fig. 1). Activationf PI3K can occur through tyrosine kinase growth factor
∗ Corresponding author at: NCI/Navy Medical Oncology, Building 8,oom 5101, 8901 Wisconsin Avenue, Bethesda, MD 20889, United States.el.: +1 301 496 0929; fax: +1 301 496 0047.

E-mail address: [email protected] (P.A. Dennis).

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eceptors such as epidermal growth factor receptor (EGFR)nd insulin-like growth factor-1 receptor (IGF-1R), celldhesion molecules such as integrins, G-protein-coupledeceptors (GPCRs), and oncogenes such as Ras. PI3Katalyzes phosphorylation of the D3 position on phos-hoinositides to generate the biologically active moietieshosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3) andhosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Uponeneration, PI(3,4,5)P3 binds to the pleckstrin homologyPH) domains of PDK-1 (3′-phosphoinositide-dependentinase 1) and the serine/threonine kinase Akt, causing bothroteins to be translocated to the cell membrane where theyre subsequently activated. The tumor suppressor PTENphosphatase and tensin homolog deleted on chromosome0) antagonizes PI3K by dephosphorylating PI(3,4,5)P and
3PI(3,4)P2), thereby preventing activation of Akt and PDK-1.
Akt exists as three structurally similar isoforms, Akt1,kt2 and Akt3, which are expressed in most tissues (Zinda

mailto:[email protected]

dx.doi.org/10.1016/j.drup.2007.11.003

J. LoPiccolo et al. / Drug Resistance Updates 11 (2008) 32–50 33

Fig. 1. Pharmacological inhibition of the PI3K/Akt/mTOR pathway. Receptor tyrosine kinases (RTKs) such as IGF-IR and EGFR, integrins, and G-protein-coupled receptors (GPCRs) can all stimulate PI3K. PI3K phosphorylates PI(4)P and PI(4,5)P2 at the 3′-position to generate PI(3,4)P2 and PI(3,4,5)P3,respectively. PTEN opposes the function of PI3K by removing 3′-phosphate groups. LY294002 is a reversible small molecule inhibitor of PI3K, whilewortmannin and its analogue, PX-866, are irreversible inhibitors. Generation of 3′-phosphoinositides activates both Akt and PDK-1, which phosphorylates Aktat T308. Akt propagates its signal to affect transcription, apoptosis, and cell cycle progression. Perifosine and phosphatidylinositol ether lipid analogues (PIAs)are lipid-based Akt inhibitors that interact with the PH domain of Akt and prevent its translocation to the membrane, while API-2, also known as triciribine,is a small molecule that inhibits the kinase activity of Akt. Akt can activate mTOR directly by phosphorylation at S2448 or indirectly, by phosphorylation andinactivation of TSC2. When TSC2 is inactivated, the GTPase Rheb is maintained in its GTP-bound state, allowing for increased activation of mTOR. mTOR(TORC1) activates S6 kinase-1, which activates ribosomal protein S6 and leads to increased protein translation. TORC1 also phosphorylates 4EBP-1, causingi the traa protein

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t to dissociate from eIF4E, and freeing eIF4E to participate in formation ofnalogues, RAD-001 and CCI-779, all of which bind to the FK506-binding

t al., 2001). Activation of Akt1 occurs through two crucialhosphorylation events, the first of which occurs at T308 inhe catalytic domain by PDK-1 (Andjelkovic et al., 1997;

alker et al., 1998). Full activation requires a subsequenthosphorylation at S473 in the hydrophobic motif, which cane mediated by several kinases such as PDK-1 (Balendrant al., 1999), integrin-linked kinase (ILK) (Delcommenne etl., 1998; Lynch et al., 1999), Akt itself (Toker and Newton,000), DNA-dependent protein kinase (Feng et al., 2004; Hillt al., 2002), or mTOR (when bound to Rictor in so-calledORC2 complexes (Santos et al., 2001)). Phosphorylation ofomologous residues in Akt2 and Akt3 occurs by the sameechanism. Phosphorylation of Akt at S473 is also controlled

y a recently described phosphatase, PHLPP (PH domaineucine-rich repeat protein phosphatase), that has two iso-orms that preferentially decrease activation of specific Aktsoforms (Brognard et al., 2007). In addition, amplificationf Akt1 has been described in human gastric adenocarcinomaStaal, 1987), and amplification of Akt2 has been describedn ovarian, breast, and pancreatic carcinoma (Bellacosa et al.,995; Cheng et al., 1996). Although mutation of Akt itself
s rare, Carpten et al. recently described somatic mutationsccurring in the PH domain of Akt1 in a small percentage ofuman breast, ovarian, and colorectal cancers (Carpten et al.,007).
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nslation initiation complex. Inhibitors of mTOR include rapamycin and its, FKBP-12, which then binds and inhibits mTOR.

.2. Downstream substrates of activated Akt

Akt recognizes and phosphorylates the consensusequence RXRXX(S/T) when surrounded by hydrophobicesidues. Because this sequence is present in many proteins,umerous Akt substrates have been identified and validatedObenauer et al., 2003). These substrates control key cellularrocesses such as apoptosis, cell cycle progression, tran-cription, and translation. For instance, Akt phosphorylateshe FoxO subfamily of forkhead family transcription factors,hich inhibits transcription of several pro-apoptotic genes,

.g., Fas-L, IGFBP1 and Bim (Datta et al., 1997; Nicholsonnd Anderson, 2002). Additionally, Akt can directly regulatepoptosis by phosphorylating and inactivating pro-apoptoticroteins such as BAD, which controls release of cytochromefrom mitochondria, and ASK1 (apoptosis signal-regulatinginase-1), a mitogen-activated protein kinase kinase involvedn stress- and cytokine-induced cell death (Datta et al., 1997;el Peso et al., 1997; Zha et al., 1996). In contrast, Akt canhosphorylate IKK, which indirectly increases the activity ofuclear factor kappa B (NF-kB) and stimulates the transcrip-
ion of pro-survival genes (Ozes et al., 1999; Romashkova and
akarov, 1999; Verdu et al., 1999). Cell cycle progressionan also be effected by Akt through its inhibitory phosphory-ation of the cyclin-dependent kinase inhibitors, p21WAF1/CIP1

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nd p27KIP1 (Liang et al., 2002; Shin et al., 2002; Zhou etl., 2001), and inhibition of GSK3� by Akt stimulates cellycle progression by stabilizing cyclin D1 expression (Diehlt al., 1998). Recently, a novel pro-survival Akt substrate,RAS40 (proline-rich Akt substrate of 40 kDa), has beenescribed (Vander Haar et al., 2007), whereby phosphory-ation of PRAS40 by Akt attenuates its ability to inhibit

TORC1 kinase activity. It has been suggested that PRAS40ay be a specific substrate of Akt3 (Madhunapantula et al.,

007). Thus, Akt inhibition might have pleiotropic effects onancer cells that could contribute to an antitumor response.

The best-studied downstream substrate of Akt is theerine/threonine kinase mTOR (mammalian target ofapamycin). Akt can directly phosphorylate and activateTOR, as well as cause indirect activation of mTOR by

hosphorylating and inactivating TSC2 (tuberous sclerosisomplex 2, also called tuberin), which normally inhibitsTOR through the GTP-binding protein Rheb (Ras homolog

nriched in brain). When TSC2 is inactivated by phosphoryla-ion, the GTPase Rheb is maintained in its GTP-bound state,llowing for increased activation of mTOR. mTOR existsn two complexes: the TORC1 complex, in which mTOR isound to Raptor, and the TORC2 complex, in which mTORs bound to Rictor. In the TORC1 complex, mTOR signalso its downstream effectors S6 kinase/ribosomal protein S6nd 4EBP-1/eIF4E to control protein translation. AlthoughTOR is generally considered a downstream substrate ofkt, mTOR can also phosphorylate Akt when bound to Rictor

n TORC2 complexes, perhaps providing a level of positiveeedback on the pathway (Sarbassov et al., 2005). Finally,he downstream mTOR effector S6 kinase-1 (S6K1) can alsoegulate the pathway by catalyzing an inhibitory phospho-ylation on insulin receptor substrate (IRS) proteins. Thisrevents IRS proteins from activating PI3K, thereby inhibit-ng activation of Akt (Harrington et al., 2004; Shah et al.,004).

.3. Rationale for targeting the PI3K/Akt/mTORathway

In addition to preclinical studies, many clinical obser-ations support targeting the PI3K/Akt/mTOR pathway inuman cancer. First, immunohistochemical studies usingntibodies that recognize Akt when phosphorylated at S473ave shown that activated Akt is detectable in cancers such asultiple myeloma (MM), lung cancer, head and neck cancer,

reast cancer, brain cancer, gastric cancer, acute myeloge-ous leukemia, endometrial cancer, melanoma, renal cellarcinoma, colon cancer, ovarian cancer, and prostate can-er (Alkan and Izban, 2002; Choe et al., 2003; Dai et al.,005; Ermoian et al., 2002; Gupta et al., 2002; Horiguchi etl., 2003; Hsu et al., 2001; Kanamori et al., 2001; Kreisberg
t al., 2004; Kurose et al., 2001; Malik et al., 2002; Min et al.,004; Nakayama et al., 2001; Nam et al., 2003; Perez-Tenoriond Stal, 2002; Roy et al., 2002; Schlieman et al., 2003; Sun etl., 2001; Terakawa et al., 2003; Yuan et al., 2000). Immuno-
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e Updates 11 (2008) 32–50

istochemical analysis has also been used to demonstraterognostic significance of Akt activation. Phosphorylation ofkt at S473 has been associated with poor prognosis in can-

ers of the skin (Dai et al., 2005), pancreas (Schlieman et al.,003; Yamamoto et al., 2004), liver (Nakanishi et al., 2005),rostate (Kreisberg et al., 2004), breast (Perez-Tenorio andtal, 2002), endometrium (Terakawa et al., 2003), stomachNam et al., 2003), brain (Ermoian et al., 2002), and bloodMin et al., 2004). Tsurutani et al. recently extended thesetudies by using antibodies against two sites of Akt phos-horylation, S473 and T308, to show that Akt activation iselective for NSCLC tumors versus normal tissue and is a bet-er predictor of poor prognosis in NSCLC tumors than S473lone (Tsurutani et al., 2006). In addition, amplification ofkt isoforms has been observed in some cancers, albeit at a

ower frequency (Bellacosa et al., 1995; Cheng et al., 1996;uggeri et al., 1998; Staal, 1987).

Another frequent genetic event that occurs in humanancer is loss of tumor suppressor PTEN function. PTENormally suppresses activation of the PI3K/Akt/mTOR path-ay by functioning as a lipid phosphatase. Loss of PTEN

unction in cancer can occur through mutation, deletion, orpigenetic silencing. Multiple studies have demonstrated aigh frequency of PTEN mutations or deletions in a varietyf human cancers, including brain, bladder, breast, prostate,nd endometrial cancers (Ali et al., 1999; Aveyard et al.,999; Dahia, 2000; Dreher et al., 2004; Li et al., 1997;asheed et al., 1997), making PTEN the second most fre-uently mutated tumor suppressor gene (Stokoe, 2001). Inumor types where PTEN mutations are rare, such as lungancer, epigenetic silencing may occur (Forgacs et al., 1998;ohno et al., 1998; Yokomizo et al., 1998). Several studiesave also demonstrated the prognostic significance of PTENoss in multiple human cancers, where mutation, deletion, orpigenetic silencing of PTEN correlates with poor prognosisnd reduced survival (Bertram et al., 2006; Perez-Tenorio etl., 2007; Saal et al., 2007; Smith et al., 2001; Yoshimotot al., 2007). Collectively, these studies have established thathe loss of PTEN is a common mechanism for activation ofhe PI3K/Akt/mTOR pathway and poor prognostic factor inuman cancer.

Finally, activation of PI3K has been described in humanancers. It can result from amplification, overexpression orrom mutations in the p110 catalytic or p85 regulatory sub-nits. Amplification of the 3q26 chromosomal region, whichontains the gene PIK3CA that encodes the p110� catalyticubunit of PI3K, occurs in 40% of ovarian (Shayesteh et al.,999) and 50% of cervical carcinomas (Ma et al., 2000).omatic mutations of this gene have also been detected ineveral cancer types and result in increased kinase activity ofhe mutant PI3K relative to wild-type PI3K. Mutations in theegulatory p85 subunit have also been detected (Jimenez et
l., 1998; Philp et al., 2001). Because any of these alterationsn individual components would result in activation of theathway, these studies suggest that pathway activation is onef the most frequent molecular alterations in cancer.

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.4. PI3K/Akt/mTOR pathway and chemotherapeuticesistance

The rationale for targeting the PI3K/Akt/mTOR pathwayn combination therapy comes from data describing con-titutive or residual pathway activation in cells that haveeveloped resistance to conventional chemotherapy and radi-tion (West et al., 2002), as well as to other targetedherapies such as EGFR antagonism. In these cases, com-ining chemotherapy or radiation with a pathway inhibitoran overcome acquired resistance to EGFR tyrosine kinasenhibitors (TKIs). Some standard chemotherapeutic agentsppear to directly inhibit Akt in vitro, and the cytotoxicityay be a direct consequence of inhibition of Akt signaling

Asselin et al., 2001; Hayakawa et al., 2002). Because Akt isntegrally involved in cellular survival, many groups havenvestigated the effects of combining chemotherapy withathway inhibitors. Preclinical studies that have investigatedhis concept will be discussed below.

. Preclinical combination data

.1. Combining pathway inhibitors with conventionalhemotherapy and radiation

.1.1. PI3 kinase inhibitors: LY294002 and wortmanninTargeting PI3 kinase, the most proximal pathway compo-

ent, has advantages over targeting more distal componentsuch as Akt and mTOR. Inhibitors of PI3K diminish signal-ng to Rac as well as Akt, providing a broader inhibitionf downstream signaling than distal inhibition. The phar-acologic agents LY294002 and wortmannin both target

he p110 catalytic subunit of PI3K. Although these com-ercially available inhibitors effectively inhibit PI3K, poor

olubility and high toxicity have limited their clinical applica-ion. However, these compounds provide powerful preclinicalools to study the cellular consequences of pathway inhibi-ion. Both of these inhibitors of PI3K sensitize cancer cellso various types of conventional chemotherapy. LY294002ncreases cytotoxicity induced by antimicrotubule agentsuch as taxanes and vinca alkaloids in glioma, ovarian can-er, esophageal cancer, and lung cancer cells in vitro andn vivo (Hu et al., 2002; Mabuchi et al., 2002; Nguyent al., 2004; Shingu et al., 2003). Wortmannin has alsoeen shown to enhance apoptosis of several cell lines whensed in combination with paclitaxel (Hu et al., 2002), cis-latin (Asselin et al., 2001), gemcitabine (Ng et al., 2000),r 5-fluorouracil (Wang et al., 2002), where potentiationf apoptosis caused by wortmannin was associated withnhibition of Akt activation. In another study, wortmanninnhanced cytotoxicity of etoposide in eight tumorigenic cell
ines, predominantly through inhibition of PI3K-dependenthosphorylation of protein kinase C zeta (PKC�) (Reis etl., 2005; Skladanowski et al., 2007). Wortmannin can alsoncrease the efficacy of chemotherapeutic agents in vivo. For
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e Updates 11 (2008) 32–50 35

xample, gemcitabine-induced apoptosis of orthotopic pan-reatic cancer in xenografts was potentiated by treatmentith wortmannin and was associated with decreased Akthosphorylation (Ng et al., 2001). In addition, the treatmentf human ovarian cancer xenografts with wortmannin plusaclitaxel increased apoptosis and decreased tumor burdenompared to either agent alone (Hu et al., 2002). Wort-annin combined with cisplatin increased the efficacy of

isplatin in an ovarian cancer model where cancer cellsere injected into the peritoneum of nude mice (Ohta et al.,006). In this study, wortmannin increased cisplatin-inducedpoptosis and inhibition of intra-abdominal disseminationf cancer cells. Additionally, several studies have identi-ed PI3K inhibitors as radiosensitizers and augmentationf radiation-induced cytotoxicity has been observed withanomolar doses of wortmannin (Edwards et al., 2002; Guptat al., 2003; Sarkaria et al., 1998). Although wortmanninnd LY294002 are not clinically useful, newer inhibitorsf PI3K such as PX-866 are being developed, but nonef these have been combined with traditional chemothera-ies.

.1.2. Akt inhibitors

.1.2.1. Perifosine. Due to feedback activation of Akt thatesults from mTOR inhibition, inhibiting Akt directly mayave advantages over targeting more distal components ofhe pathway. To date, the most developed inhibitor of Akt iserifosine, a lipid-based inhibitor. In vitro, perifosine inhibitsranslocation of Akt to the cell membrane, and inhibits therowth of melanoma, lung, prostate, colon, and breast cancerells in association with inhibition of Akt activity (Crul etl., 2002; Kondapaka et al., 2003). Additional in vitro dataemonstrates synergistic effects of perifosine and traditionalhemotherapeutic agents such as etoposide in leukemia cellsNyakern et al., 2006), doxorubicin in MM cells (Hideshimat al., 2006), and temozolomide in glioma cells (Momota etl., 2005). In the latter study, the combination of perifosinend temozolomide was more effective than temozolomidelone in inhibiting growth of glioma xenografts. Perifosineas also been found to sensitize cancer cells to apoptosisnd cell cycle arrest induced by radiation in vitro and inivo (Caron et al., 2005; Ruiter et al., 1999; Vink et al.,006a).

.1.2.2. PIAs. A relatively new group of lipid-based Aktnhibitors are the phosphatidylinositol ether lipid analoguesPIAs). PIAs were designed to interact with the PH domainf Akt and are structurally similar to the products of PI3inase. Although less clinically developed, PIAs are well-haracterized in vitro. In addition to Akt inhibition, Gillst al. recently identified several molecular targets that con-ribute to the cytotoxicity of PIAs, including activation of
he stress kinase p38�, and PIA-induced cytotoxicity corre-ates with inhibition of Akt phosphorylation in the NCI60ell line panel (Gills et al., 2006, 2007). PIAs mitigate drugesistance to several traditional chemotherapies and ionizing

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adiation in vitro (Martelli et al., 2003; Tabellini et al., 2004;an Meter et al., 2006). PIAs also enhance the apoptosis

nduced by other agents such as tumor necrosis factor-relatedigand (TRAIL), all-trans-retinoic acid (ATRA) and motex-fin gadolinium (Martelli et al., 2003; Puduvalli et al., 2005;amos et al., 2006).

.1.2.3. Triciribine (API-2). API-2, also known asriciribine-phosphate, was identified as an Akt inhibitor aftercreening the National Cancer Institute (NCI)’s structuraliversity set. Triciribine inhibits Akt2 phosporylation atoth sites (T309 and S474) and inhibits EGF-inducedhosphorylation of all three isoforms of Akt in vitro. In vivo,reatment with low doses of triciribine stimulated apoptosisn xenografts with constitutively activated Akt or PTEN

utations, but not in tumors with low Akt activity (Yang etl., 2004). Triciribine has not been preclinically combinedith standard chemotherapies or radiation.

.1.3. mTOR inhibitors: rapamycin and its analoguesThe PI3K/Akt/mTOR pathway inhibitors that are most

linically developed, target a more distal pathway compo-ent, mTOR. Rapamycin, the prototypic mTOR inhibitor,as discovered in 1975 as a potent anti-fungicide, and is pro-uced naturally by Streptomyces hygroscopicus (Sehgal et al.,975; Vezina et al., 1975). The ability of rapamycin to inhibithe proliferation of cancer cell lines was shown over 20 yearsgo (Douros and Suffness, 1981; Eng et al., 1984; Houchenst al., 1983). More recently, rapamycin analogues such asCI-779 and RAD-001 have been explicitly designed forevelopment as anticancer drugs. These inhibitors of mTORind to the FK506-binding protein, FKBP-12, which theninds and inhibits mTOR. Inhibition of mTOR decreaseshosphorylation of two downstream targets, 4E-BP1 and6K, resulting in inhibition of protein synthesis.

Rapamycin and its analogues have been studied in combi-ation with standard chemotherapies. For example, treatmentf orthotopic neuroblastoma-bearing mice with rapamycinnd vinblastine resulted in inhibition of tumor growth andngiogenesis, with an increase in survival compared to eitherrug alone (Marimpietri et al., 2005, 2007). Similar resultsere observed in hepatocellular carcinoma (Ribatti et al.,007). In vitro, synergy has been observed with rapamycinnd paclitaxel, carboplatin, or vinorelbine. In lymphomaodels, RAD-001 demonstrates in vitro synergy with ritux-

mab, doxorubicin, and vincristine (Haritunians et al., 2007;anner et al., 2006), predominantly through induction of cell

ycle arrest. Combinations of RAD-001 and anti-estrogengents tamoxifen and letrozole also demonstrated enhancedevels of apoptosis than with either drug alone (Boulay et al.,005; Treeck et al., 2006). Interestingly, RAD-001 sensitizesumor cells to cisplatin-induced apoptosis in a p53-dependent

anner via inhibition of mTOR function, resulting in reduced21 translation (Beuvink et al., 2005). CCI-779, anotherapamycin analogue, has been successfully combined withisplatin, gemcitabine, and camptothecin in vitro and in vivo

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e Updates 11 (2008) 32–50

Geoerger et al., 2001; Ito et al., 2006; Thallinger et al.,007a,b; Wu et al., 2005).

Rapamycin and RAD-001 are also potent radiosensitiz-rs through mTOR-dependent enhancement of radiation-nduced autophagy (Albert et al., 2006; Kim et al., 2006;aglin et al., 2005; Moretti et al., 2007). In a recent study,AD-001 sensitized PTEN wild-type and PTEN-null cancerells to ionizing radiation, but induced more cytotoxic-ty in PTEN-null cells (Cao et al., 2006). RAD-001 alsonhances radiation-induced damage of tumor vasculature inivo through induction of apoptosis of vasculature endothe-ial cells (Shinohara et al., 2005). Taken together, these datandicate that combining mTOR inhibition with chemother-py or radiation could be a potentially effective approach inancer treatment.

.2. Combining pathway inhibitors with other targetedherapies

Because signaling of multiple receptor tyrosine kinasesRTKs) is propagated through Akt, simultaneous inhibitionf RTKs such as IGF-IR or erbB family members with path-ay components such as Akt or mTOR may circumvent

eedback activation seen with either approach alone. Suchn approach can be considered proximal and distal signalingnhibition (Fig. 2, left panel). Based on the observed feed-ack activation of Akt by mTOR inhibitors, it is possible thathey might be most effective when combined with proximalathway inhibitors. For example, synergistic effects betweenapamycin and LY294002, an upstream inhibitor of PI3K, areommonly observed in vitro (Breslin et al., 2005; Sun et al.,005; Takeuchi et al., 2005). Most recently, Fan et al. showedhat a dual inhibitor of PI3K� and mTOR, PI-103, was ableo inhibit Akt activity as well as proliferation in glioma cells,egardless of PTEN or EGFR status (Fan et al., 2006). PI-103as effective in inhibiting the growth of glioma xenografts

n the absence of toxicity, most likely through a cytostaticechanism.Another possible approach is to combine inhibition of the

I3K/Akt/mTOR pathway with inhibition of a parallel pro-urvival signaling pathway such as the MEK/ERK pathwayTortora et al., 2007) (Fig. 2, middle panel). This approachbrogates compensatory activation of other pro-survival path-ays when the PI3K/Akt/mTOR pathway is inhibited. For

xample, combining an inhibitor of PI3K with an inhibitorf MEK causes a synergistic increase in apoptosis in bothTEN mutant and wild-type cells (She et al., 2005). Bothpproaches will be discussed below.

.2.1. Pathway inhibitors and inhibitors of receptoryrosine kinases (RTKs).2.1.1. Combinations with EGFR antagonists. Perhaps the
ost extensive data on proximal and distal signaling
nhibition exists for combining PI3K/Akt/mTOR pathwaynhibitors with EGFR antagonists. The epidermal growth fac-or receptor (EGFR/erbB1) is overexpressed or amplified in

J. LoPiccolo et al. / Drug Resistance Updates 11 (2008) 32–50 37

Fig. 2. Combinatorial approaches with inhibitors of the PI3K/Akt/mTOR pathway. Several approaches can be employed when combining pathway inhibitors withother targeted therapies. Inhibition of proximal pathway components, such as receptor tyrosine kinases (RTKs) and oncogenes, combined with distal inhibitiono vation ti redundc of histo(

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f Akt or mTOR may be an effective approach to circumvent feedback actinhibition of parallel signaling pathways prevents compensatory activation ofombined with several other types of targeted therapies, including inhibitionCOX-2) (right panel).

variety of tumor types and is a major target in cancer ther-py. Patients who respond to EGFR TKIs eventually developesistance and progressive disease. Elucidated mechanismsf resistance in NSCLC include a somatic T790M muta-ion in the kinase domain of EGFR (Engelman et al., 2006),pithelial to mesenchymal transition (EMT) (Irie et al., 2005;homson et al., 2005; Yauch et al., 2005), amplification of theet oncogene (Engelman et al., 2005, 2007) and downregula-

ion of BIM activity (Cragg et al., 2007; Gong et al., 2007). Allf these mechanisms of resistance are associated with main-enance and continued activation of the PI3K/Akt/mTORathway.

Cancer cell lines with mutant PTEN, which have high lev-ls of Akt are resistant to EGFR antagonists such as gefitinib.TEN reconstitution can restore sensitivity to EGFR inhibi-

ion. She et al. showed that this approach inhibited growthf breast cancer xenografts, which was not seen with eitherGFR inhibition or PTEN induction alone (She et al., 2005).imilar results have been observed in NSCLC, prostate, and

eukemia cell lines, thereby linking PTEN status and Aktctivity with sensitivity to EGFR inhibition (Festuccia etl., 2005; Janmaat et al., 2003, 2006; Kokubo et al., 2005;i et al., 2006). In PTEN-null gefitinib-resistant cells, re-

ntroduction of PTEN function or treatment with LY294002estores gefitinib sensitivity (She et al., 2003).

Many different PI3K inhibitors can restore sensitivity toGFR inhibitors (Bianco et al., 2003; She et al., 2003).ordella et al. found that NSCLC cells transfected withefitinib-sensitizing EGFR mutations had increased levels
f activated Akt, and these cells were more sensitive thanheir wild-type counterparts not only to gefitinib, but also toY294002 (Sordella et al., 2004). In another study with PX-66, a PI3K inhibitor selective for p110�, PX-866 was able
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hat could occur with distal inhibition alone (left panel). Alternatively, dualant pro-survival pathways (middle panel). Finally, pathway inhibition can bene deacetylase complexes (HDACs), the proteasome and cyclooxygenase-2

o abolish gefitinib resistance in NSCLC xenografts. Toxic-ties associated with PX-866 administration were decreasedlucose tolerance and hyperglycemia, both of which wereeversed upon discontinuation of drug treatment (Ihle et al.,005).

Synergistic effects of rapamycin and EGFR TKIs haveeen observed in several in vitro systems, including glioblas-oma multiforme (Hjelmeland et al., 2007; Rao et al., 2005;

ang et al., 2006), prostate cancer (Buck et al., 2006;asiello et al., 2007), pancreatic cancer (Buck et al., 2006),

quamous cell carcinoma (Jimeno et al., 2007), renal cell car-inoma (Costa et al., 2007; Gemmill et al., 2005), leukemiaMohi et al., 2004), cervical carcinoma (Birle and Hedley,006), and non-small cell lung cancer (Buck et al., 2006).everal of these studies extended the efficacy of these com-inations to xenograft experiments (Birle and Hedley, 2006;uck et al., 2006; Jimeno et al., 2007). Buck et al. noted

e-sensitization and synergistic growth inhibition with theombination of rapamycin and erlotinib in cells lines thatere previously resistant to erlotinib (Buck et al., 2006). Li

t al. noted significant regression of lung tumors in trans-enic mice that possessed the secondary resistance mutation790M when treated with the combination of rapamycinnd the irreversible EGFR TKI, HKI-272 (Li et al., 2007).n human glioma cell lines with mutant PTEN, additionf the dual PI3K/mTOR inhibitor PI-103 to erlotinib wasecessary to induce growth arrest, suggesting that activa-ion of the PI3K/Akt/mTOR pathway by EGFR-independent

echanisms confers resistance to EGFR inhibitors, which
an nonetheless be overcome by the addition of pathwaynhibitors. Collectively, these data suggest that the use ofGFR antagonists with pathway inhibitors may be espe-ially beneficial in patients whose tumors harbor mutations in

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GFR and/or PTEN, as well as patients who have developedesistance to EGFR TKIs.

.2.1.2. Combinations with erbB2 antagonists. Anotherotentially useful combination is proximal inhibition ofrbB2, also known as her-2/neu, with distal inhibition of Aktr mTOR. Inhibition of Akt phosphorylation is a requirementor the anti-proliferative effects of the her-2/neu antago-ist, trastuzumab, and trastuzumab-resistant cells exhibitustained activation of the PI3K/Akt/mTOR pathway (C.T.han et al., 2005; Nagata et al., 2004; Yakes et al., 2002).preclinical study was recently reported combining tricirib-

ne with trastuzumab in an effort to circumvent trastuzumabesistance due to loss of PTEN (Lu et al., 2007). In breast can-er cell lines and xenografts, triciribine restored sensitivityo trastuzumab, concomitant with induction of apoptosis andnhibition of tumor growth. In the same study, RAD-001 waslso able to re-sensitize trastuzumab-resistant cells to apop-osis in vitro and in vivo. Similar results have been observedith rapamycin (Liu et al., 2005; Wang et al., 2007), and clas-

ical PI3K inhibitors have also been successfully combinedith trastuzumab in vitro (C.T. Chan et al., 2005; Nagata et

l., 2004).

.2.1.3. Combinations with IGF-IR antagonists. Mono-lonal antibodies directed against the IGF-IR, a transmem-rane RTK, have been used extensively in preclinical studies.hen bound by IGF-I or IGF-II, IGF-IR is autophosphory-

ated and activates PI3K. Additionally, feedback activationf Akt induced by mTOR inhibition is partially mediatedia upregulation of insulin receptor substrate 1 (IRS1), andubsequent signaling through IGF-IR, suggesting that dualnhibition of IGF-IR and mTOR may be more effective than

TOR inhibition alone. For example, combining rapamycinith a small molecule inhibitor of IGF-IR abrogated feedback

ctivation of Akt and enhanced cytotoxicity of rapamycin inlioma cells (Steinbach et al., 2004). Similarly, combinationf a neutralizing antibody directed against IGF-IR with RAD-01 reversed Akt phosphorylation induced by RAD-001, andesulted in additive anti-proliferative effects in leukemic cellsTamburini et al., 2007). These data demonstrate that proxi-al inhibition of IGF-IR combined with inhibition of distal

athway components, such as Akt and mTOR, may abrogateeedback activation that results from mTOR inhibition alone.

.2.2. Pathway inhibitors and other targeted agentsIn addition to combining PI3K/Akt/mTOR inhibitors with

gents that inhibit either the same or parallel pro-survival sig-aling pathways, PI3K/Akt/mTOR inhibitors have also beenombined with targeted agents that defy easy categorizationuch as imatinib and those that do not directly affecting sig-aling pathways, e.g., histone deacetylase (HDAC) inhibitors
nd proteasome inhibitors (Landis-Piwowar et al., 2006)Fig. 2, right panel). Although the mechanisms behind thefficacy of these combinations are not completely under-tood, they represent potentially useful combinations for
ccei

e Updates 11 (2008) 32–50

atients whose tumors do not respond to more conventionalherapy regimens.

.2.2.1. PI3 kinase and Akt inhibitors. PI3K inhibitors haveeen successfully combined with imatinib in leukemic cellsKlejman et al., 2002), as well as sulindac, a non-steroidalnti-inflammatory drug that inhibits COX-2 (Yip-Schneidert al., 2003). LY294002 and wortmannin sensitize cancerells to HDAC inhibitor-induced apoptosis in vitro and inivo (Denlinger et al., 2005; Rahmani et al., 2003) (Wang etl., 2002). Rahmani et al. (2005) found that treatment withY294002 inhibited ERK phosphorylation and p21 induc-ion, both of which normally protect leukemic cells fromDAC inhibitor-induced apoptosis. They concluded that the

atter mechanisms, rather than inhibition of Akt signalinged to increased cell death. In contrast, sensitization of A549SCLC xenografts by LY294002 to HDAC inhibitor-induced

poptosis resulted from Akt-dependent regulation of nuclearactor kappa B transcription (Denlinger et al., 2005). Com-ined treatment with an HDAC inhibitor and LY294002nhibited tumor growth concurrently with inhibition of Aktn vivo. In addition to PI3K inhibitors, the Akt inhibitor peri-osine has been combined with a handful of other targetedherapies in vitro. Perifosine treatment of PTEN-deficientreast and prostate cancer cells enhanced growth inhibitionnduced by cetuximab (Li et al., 2006), as well as apoptosisnduced by HDAC inhibitors in leukemic cells (Rahmani etl., 2005).

.2.2.2. mTOR inhibitors. mTOR inhibitors have also beenuccessfully combined preclinically with other targeted ther-pies. In chronic myelogenous leukemia cells with moderateesistance to imatinib, treatment with imatinib and rapamycinr its analogue, RAD-001, resulted in synergistic inhibi-ion of leukemic cell growth. Rapamycin has also beenffectively combined in breast cancer models with targetedgents such as herceptin (Wang et al., 2007), cotylenin AKasukabe et al., 2005), and luteolin (Chiang et al., 2007).n MM, rapamycin sensitizes MM cells to apoptosis inducedy hsp90 inhibitors (Francis et al., 2006), dexamethasone,nd thalidomide analogs (Raje et al., 2004; Stromberg et al.,004). In addition, rapamycin acts cooperatively with smallolecule inhibitors of c-met and VEGF, where in the latter

tudy, combination therapy inhibited primary and metastaticrowth of orthotopic pancreatic cancer tumors, as well asiver metastasis (Ma et al., 2005; Stephan et al., 2004). mTORnhibition can be combined with other types of therapeuticpproaches. For example, rapamycin and RAD-001 enhancehe efficacy of oncolytic viruses that target tumor cells in

edulloblastoma and colon cancer xenografts (Lun et al.,007; Homicsko et al., 2005). Recently, it was shown thatnfection with a herpes simplex viral vector activates Akt in
ancer cells, and that concurrent treatment with viral parti-les and LY294002 enhanced the efficacy of the virus (Liut al., 2007). This may be a common feature of pathwaynhibition, whereby oncolytic viral infection activates the

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. Clinical trials with PI3K/Akt/mTOR pathwaynhibitors as single agents and in combination withther therapies

Although combinations of pathway inhibitors with vari-us types of chemotherapy have been investigated extensivelyn preclinical studies, only a few clinical trials with Aktnhibitors and mTOR inhibitors have been reported upo now, while no clinical trials using PI3K inhibitorsave been published. These data will be discussed belowTable 1).

.1. Akt inhibitors

.1.1. PerifosineA number of phase I and II clinical trials investigating per-

fosine monotherapy in a variety of tumor types have beenompleted. In initial phase I trials employing high daily dosesf perifosine, gastrointestinal toxicity led to frequent treat-ent discontinuations (Crul et al., 2002). More recent phasetrials utilized a loading dose followed by smaller dailyaintenance doses of perifosine, allowing for rapid achieve-ent of steady state plasma concentrations, and reduced

astrointestinal toxicity during the maintenance phase (Vanmmersen et al., 2004). The loading dose followed by main-

enance perifosine has been used in phase II clinical trialsn breast cancer, pancreatic cancer, prostate cancer, head andeck cancer, melanoma, and sarcoma (Argiris et al., 2006;ailey et al., 2006; Ernst et al., 2005; Knowling et al., 2006;eighl et al., 2007; Marsh Rde et al., 2007; Posadas et al.,005). In these trials, the activity of single agent perifosinen solid tumors has been disappointing, with few objectiveesponses noted. Gastrointestinal and constitutional toxic-ties were problematic, particularly in a trial in advancedancreatic cancer (Marsh Rde et al., 2007). A phase II trial oferifosine in 23 patients with soft tissue sarcomas yieldedpartial response of 9 months duration in 1 patient with

hondrosarcoma, as well as stabilization of disease in 5atients at 8 weeks. Despite not meeting their objectives forrogression-free survival (>40% at 6 months) in this trial, thenvestigators plan to conduct further studies of perifosine inatients with selected soft tissue sarcoma histologies, perhapsnriching their patient population with patients whose tumorsear high expression of p-Akt, and attempting to achieveigh steady state plasma levels of perifosine (>6 �g/ml)hat appears to correlate with clinical benefit (Bailey et al.,006).

Given the limited efficacy of perifosine monotherapy in a
ariety of solid malignancies in phase II trials, an alternativetrategy would be to combine perifosine with chemother-py, radiation therapy and targeted agents in an attempt tonhance cytotoxicity and overcome chemotherapeutic resis-
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e Updates 11 (2008) 32–50 39

ance through inhibition of the Akt pathway. A phase I trialombining perifosine and radiotherapy in advanced solidumors demonstrated that perifosine could be safely utilizeds a radiation sensitizer, and phase II trials with this strat-gy are in development (Vink et al., 2006b). In addition,reliminary reports from a number of phase I trials investigat-ng the combination of perifosine with traditional cytotoxichemotherapeutic agents such as taxanes and gemcitabinendicate that these combinations can be safely administeredCervera et al., 2006; Ebrahimi et al., 2006; Goggins et al.,006; Weiss et al., 2006).

MM is a rational target for perifosine combination ther-py based upon in vitro data in which perifosine inducesytotoxicity in MM cell lines and patient MM cells resistanto conventional therapy (Hideshima et al., 2006). Perifos-ne also shows antitumor activity in a human plasmacytoma

ouse model (Catley et al., 2007). Preliminary results fromphase II study of perifosine alone or in combination withexamethasone for patients with relapsed or refractory MMevealed that single agent perifosine induced stabilizationf disease in 6 of 25 evaluable patients. The addition ofexamethasone to perifosine in patients who progressed onerifosine monotherapy conferred a minor response in threef nine evaluable patients and stabilization of disease in twof nine patients (Richardson et al., 2006). Based upon theromising activity of perifosine as a single agent and in com-ination with dexamethasone, further studies of perifosinen MM utilizing different dosing schedules as well as inombination with the proteosome inhibitor bortezomib arelanned.

.1.2. Triciribine (API-2)In the 1980s and 1990s, a number of phase I and II

linical trials were performed utilizing triciribine as a cyto-oxic agent in various advanced malignancies at differentosing schedules (Feun et al., 1984, 1993; Hoffman etl., 1996; Mittelman et al., 1983; O’Connell et al., 1987;chilcher et al., 1986). Minimal efficacy was observed withew objective responses, and triciribine at high doses caused

number of serious toxicities, including hepatotoxicity,yperglycemia and hypertriglyceridemia. Whether the hyper-lycemia and hypertriglyceridemia were related to inhibitionf Akt 2 is unknown. In these trials, pharmacokinetic anal-sis revealed erratic drug levels of triciribine, particularlyith a 5-day continuous infusion dosing schedule. With the

ecent discovery of triciribine as a bona fide Akt inhibitor,hase I clinical trials are currently underway utilizing loweroses of triciribine-phosphate by weekly IV infusions inatients with metastatic solid tumors whose tumors bearigh expression of phospho-AKT as well as in patientsith myeloid malignancies. In addition, trials combining

riciribine-phosphate with tyrosine kinase inhibitors such asrlotinib and lapatinib to overcome primary and secondaryesistance mechanisms to ErbB family inhibitors are cur-ently in development.

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Table 1Clinical trials combining PI3K/Akt/mTOR pathway inhibitors with other anticancer agents

Pathway inhibitor Combination agent Trial phase/tumor type Toxicity (CTC grade 3–5) ≥10% ofpatients

Response rates Comments Reference

Perifosine Radiation Phase I (inoperable solidtumors)

Nausea/vomiting (10%), dysphagia(10%)

6/21 (29%) CR, 5/21(24%) PR

Perifosine as radiosensitizer is safe andtolerable

Vink et al.(2006a,b)

Rapamycin Radiation/cisplatin Phase I (stage III NSCLC) Dysphagia (14%), MTD not defined Not reported Rapamycin as radiosensitizer is safeand tolerable

Sarkaria et al.(2007)

Gefitinib Phase I (recurrent malignantglioma)

Mucositis (68%), diarrhea (42%),fatigue (37%), anemia (32%),leukopenia (32%), infection (26%),hypercholesterolemia (21%),hypertriglyceridemia (21%),thrombocytopenia (21%), AST/ALT(16%), nausea/vomiting (16%)

2/34 (6%) PR, 13/34(38%) SD

Increased grade 3–4 toxicity due tohigh doses in attempt to crossblood–brain barrier. Combination ofEGFR TKI and mTOR inhibitor inGBM currently in phase II

Reardon et al.(2006)

CCI-779 5-FU/leucovorin Phase I (advanced solidtumors)

Asthenia (19%), mucositis (19%),hyperglycemia (15%), diarrhea (15%),anemia (15%), nausea/vomiting (11%)

3/26 (12%) PR,11/26 (42%) SD

Discontinued due to twotreatment-related deaths. Combinationof agents at this dosing schedule notrecommended

Punt et al. (2003)

IFN-� Phase III (advanced RCC) Asthenia (28%), anemia (38%),dyspnea (10%), infection (11%),neutropenia (15%)

8.1% CR/PR, 28.1%SD

No difference OS or PFS forcombination vs. IFN-� alone. Singleagent CCI-779 superior OS and PFS toIFN-� and combination. Larger samplesize (∼210 patients per group)

Hudes et al.(2007)

RAD-001 Gefitinib Phase I (advanced NSCLC) Hypotension (11%), acidosis (11%),azotemia (11%), lymphopenia (11%),stomatitis (11%)

2/8 (25%) PR 2/2 responses seen in former smokers.Currently in phase II trials

Riely et al.(2007)

Bevacizumab Phase I (solid tumors) Pain (71%), mucositis (64%), anorexia(57%), bleeding (50%), rash (50%),hyperlipidemia (43%), fatigue (43%)

2/16 (13%) PR, 8/16(50%) SD

Presented only in abstract form.Currently in phase II trials

Zafar et al.(2006)

Imatinib Phase I/II (GI stromaltumors)

Fatigue, diarrhea, vomiting, nausea,anemia, edema, headache, and rash

2/31 (6%) PR, 8/31(26%) SD

Presented only in abstract form Van Oosterom etal. (2005)

CR: complete response; PR: partial response; SD: stable disease; MTD: maximally tolerated dose; OS: overall survival; PFS: progression-free survival; TKI: tyrosine kinase inhibitor.

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.2. mTOR inhibitors: rapamycin, RAD-001, andCI-779

.2.1. Clinical trials of mTOR inhibitors as single agentsThe mTOR inhibitors, CCI-779 and RAD-001, have been

ested as single agents in phase II trials in a variety ofumor types, and objective responses and stabilization ofisease have been noted in breast cancer (S. Chan et al.,005), glioblastoma (Chang et al., 2005; Galanis et al., 2005),euroendocrine carcinoma (Duran et al., 2006), renal cell car-inoma (Atkins et al., 2004), mantle cell lymphoma (Witzigt al., 2005), and myeloid malignancies (Yee et al., 2006). Aecent phase III trial of CCI-779 in poor risk metastatic renalell carcinoma randomized patients to CCI-779, interferon-lpha (IFN-�) or a combination of the two. Single agentCI-779 showed statistically significant improvement inrogression-free survival (5.5 months vs. 3.1 months) andverall survival (10.9 months vs. 7.3 months) as compared toFN-�, while the combination of CCI-779 and IFN-� showedo statistically significant differences in progression-free sur-ival (4.7 months) or overall survival (8.4 months) (Hudes etl., 2007). This trial resulted in regulatory approval of singlegent CCI-779 as front-line therapy in advanced renal cellarcinoma.

.2.2. Clinical trials combining mTOR inhibitors withonventional chemotherapy and radiation

Although responses have been observed with single agentapamycin analogues in a variety of tumor types, in mostases activity is modest and of short duration. This may note unexpected, as preclinical data have shown not only thatapamycin and its analogues are predominantly cytostatic initro, but also that feedback activation of Akt after mTORnhibition may limit the efficacy of mTOR inhibitors as sin-le agents. Therefore, a number of clinical trials are utilizingTOR inhibitors in combination with chemotherapy and

adiation to overcome resistance mechanisms and enhanceesponse. A phase I trial added rapamycin to concomitantadiation and cisplatin for patients with unresectable stageII non-small cell lung cancer (Sarkaria et al., 2007). Unfor-unately, this trial was terminated prematurely due to lack ofurther funding. Despite not reaching the maximal toleratedose of rapamycin with combination chemo-radiation, theeasibility of utilizing mTOR inhibitors as radiosensitizinggents was established.

Other trials that combined mTOR inhibitors with con-entional cytotoxic chemotherapy have revealed somenexpected toxicities. For example, a phase I trial combiningCI-779 with 5-FU and leucovorin in patients with advanced

olid tumors was discontinued due to two treatment-relatedeaths associated with bowel perforation. Based on the over-apping mucocutaneous toxicities of CCI-779 with 5-FU,
he combination of these agents at this schedule was notecommended for further development (Punt et al., 2003).reliminary results of a phase I trial in advanced cancersith weekly gemcitabine at 600 mg/m2 (a dose lower than
tam2

e Updates 11 (2008) 32–50 41

sually administered) and weekly RAD-001 revealed that theombination was not tolerated in a majority of patients dueo myelosuppression (Pacey et al., 2004). Pharmacokineticnalysis of these trials did not suggest an interaction betweenhe mTOR inhibitor and the cytotoxic agent. Clearly, basedn the unexpected toxicities observed in these trials, investi-ators should be attentive to potential overlapping toxicitiesetween mTOR inhibitors and conventional chemotherapy.

.2.3. Clinical trials combining mTOR inhibitors withGFR antagonists

Because preclinical studies showed that PI3K/Akt/mTORnhibitors can augment the efficacy and overcome resistanceo EGFR TKIs, phase I and II clinical trials are underwayesting the combination of EGFR TKI and mTOR inhibitors.

phase I trial in patients with malignant glioma combiningefitinib with rapamycin revealed that daily administrationf these agents is feasible, and that rapamycin does not sig-ificantly affect gefitinib drug levels. Out of 34 pretreatedatients with refractory disease, 2 attained a partial radio-raphic response and 13 achieved stable disease (Reardon etl., 2006). Based on these results, a number of phase II tri-ls utilizing various combinations of EGFR TKIs and mTORnhibitors in malignant glioma are underway. A phase I trialombining gefitinib and RAD-001 in patients with advancedSCLC patients who had not previously been treated with anGFR TKI yielded partial responses in two out of eight evalu-ble patients (Milton et al., 2007). The investigators havenitiated a phase II clinical trial to further assess the efficacyf this combination.

mTOR inhibitors are also being studied for their abilityo overcome secondary resistance to EGFR TKI therapy inSCLC. In NSCLC patients who progressed after initially

esponding to EGFR TKI therapy and were continued onhe EGFR TKI with subsequent addition of RAD-001, nobjective responses were seen 3 weeks after the addition ofAD-001 (Riely et al., 2007). In spite of these negative pre-

iminary findings, the addition of mTOR inhibitors to EGFRnhibitors as a means of overcoming mechanisms of sec-ndary resistance is not associated with undue toxicity andould be further investigated in clinical trials.

.2.4. Clinical trials combining mTOR inhibitors withther targeted therapies

A number of clinical trials are investigating the combina-ion of mTOR inhibitors with multi-targeted tyrosine kinasenhibitors other than EGFR TKIs, such as imatinib, suni-inib and sorafenib in a variety of malignancies. Preliminaryata from a phase I/II clinical trial combining RAD-001 withmatinib in 31 patients with GI stromal tumors refractoryo imatinib resulted in stabilization of disease for greater
han 4 months in eight patients. Two patients subsequentlychieved partial responses, suggesting that mTOR inhibitionay re-sensitize tumors to imatinib (Van Oosterom et al.,
005).

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Given that mTOR inhibitors have direct anti-angiogenicffects through regulation of HIF-1�, dual angiogenic inhi-ition may be a rational approach. Encouraging efficacyata have been reported from a phase I trial, which com-ined RAD-001 with the anti-VEGF monoclonal antibodyevacizumab in various solid tumors. In a preliminary anal-sis, the investigators reported partial responses in 2 outf 16 evaluable patients, with an additional 8 out of 16atients attaining minor responses or stability of disease. Theombination appeared well tolerated with minimal overlap-ing toxicities and no dose-limiting toxicities (Zafar et al.,006).

Based on strong preclinical in vivo data, a number of phaseI and III randomized, controlled clinical trials are underwayo determine the efficacy and safety of aromatase inhibitorsnd mTOR inhibitors in hormone receptor positive breastancer. Despite promising preliminary phase II data from aandomized trial of CCI-779 in combination with letrozolen postmenopausal women with hormone receptor metastaticreast cancer (Carpenter et al., 2005), a phase III trial inves-igating this combination in the same patient population waserminated after an interim analysis determined that the com-ination yielded no benefit over letrozole alone (Leary andowsett, 2006). Despite this negative trial, the combinationf mTOR inhibitors with other molecularly targeted agentsemains a promising approach to enhance cytotoxicity, over-ome resistance and limit toxicity.

.3. Prediction of response to PI3K/Akt inhibitors andathway modulation

.3.1. Phospho-specific antibodies in IHC andmmunoblotting

The clinical use of PI3K/Akt/mTOR pathway inhibitorsill be optimized by identifying biomarkers to predict

esponse to therapy and to assess target inhibition inivo. Traditional methods of assessing pathway activationnclude immunohistochemistry (IHC) and immunoblottingsing phospho-specific antibodies that recognize pathwayomponents when phosphorylated at specific residues. Phos-horylation at these specific sites is indicative of activation.he advantage of IHC is the ability to localize pathwayroteins intracellularly, including the plasma membrane,ytoplasm and nucleus. A potential disadvantage is that IHCs not easy to quantify objectively. Several clinical trialsave measured pathway components by IHC before andfter drug treatment. For example, in a study of CCI-779n neuroendocrine carcinomas, paired tumor biopsies werebtained at baseline and 2 weeks following treatment. Thenly pre-treatment marker evaluated (including PTEN, p53,hospho-S6, phospho-mTOR, phospho-Akt) that correlatedith improved tumor response was an elevated baseline level
f phospho-mTOR. After 2 weeks of therapy, CCI-779 effec-ively decreased levels of phospho-S6, validating that therug inhibited its intended target. Increased levels of p-AKTxpression and decreased levels of p-mTOR expression after
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e Updates 11 (2008) 32–50

weeks of treatment were associated with a statistically sig-ificant delayed time to progression (Duran et al., 2006). Innother phase II study with CCI-779 in recurrent glioblas-oma multiforme, increased levels of phospho-p70S6 kinasen baseline tumor specimens were shown to correlate withadiographic response (Galanis et al., 2005). From thesemall trials, measurement of pathway components such ashospho-Akt, phospho-mTOR and its downstream substratesay serve as predictive biomarkers for patients most likely to

espond to PI3K/Akt/mTOR inhibitors, either as monother-py or in combination with other agents. Future trials shouldake every effort to incorporate analysis of pathway activa-

ion and target modulation in pre- and post-treatment tumorissue.

Depending on the tumor site, this may, however, requireeveral invasive procedures, which are either not feasible orot safe. Therefore, immunoblotting can be used to assessiomarkers in readily accessible surrogate tissues, such aseripheral blood mononuclear cells (PBMCs). Several trialsith mTOR inhibitors have incorporated analysis of down-

tream substrates of mTOR in PBMCs as a correlate forlinical response. Yee et al. analyzed PBMCs in patientsreated with RAD-001 for relapsed or refractory hematologic

alignancies. In six out of nine samples studied, RAD-001ecreased phosphorylation of mTOR substrates, includingn three patients who demonstrated evidence of a clinicalesponse. Interestingly, treatment with RAD-001 led to inhi-ition of p-AKT in up to two thirds of specimens analyzed,ncluding in all samples where inhibition of mTOR was noted,uggesting that feedback activation of Akt may not be clini-ally relevant in hematologic malignancies (Yee et al., 2006).lthough less invasive than serial tumor biopsies, it is notnown whether PBMCs are a valid surrogate tissue in whicho measure target inhibition in non-hematologic malignan-ies.

.3.2. Imaging modalities

.3.2.1. Molecular imaging. Ideally, the least invasive andost sensitive way to measure inhibition of pathway compo-

ents would be to quantify kinase activity of tumor tissue initu. This has been achieved preclinically, where a reporterystem was developed to quantitatively measure Akt kinasectivity via bioluminescence of an Akt reporter molecule,hereby an increase in luminescence was indicative of inhi-ition of Akt (Zhang et al., 2007). They showed a dose- andime-dependent inhibition of Akt in several human xenograftsollowing administration of perifosine and API-2 (tricirib-ne) to nude mice. Use of this technology in humans wouldequire stable integration of the reporter construct into tumorells, which is not currently feasible. However, monitor-ng Akt activity within live tumor cells provides a dynamicreclinical tool with which to assess target modulation in
ivo.
.3.2.2. FDG-PET. The use of [18F] fluorodeoxyglucoseFDG)-positron emission tomography (PET) as a non-

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nvasive means to assess early response to signal transductionnhibitors has been established for gastrointestinal stromalumor patients treated with imatinib (Gayed et al., 2004).tudying FDG-PET as a pharmacodynamic marker of bio-hemical modulation by PI3K/Akt/mTOR inhibitors is basedn evidence that activation of the pathway controls hexok-nase activity and glycolysis (Majewski et al., 2004) andhat FDG accumulation within tumors depends on hexoki-ase activity. Xenografts of (VHL null) renal cell carcinomaumors showed a twofold increase in FDG-PET uptake rel-tive to parental tumor cells. This FDG-PET uptake waseduced to baseline levels 24 h after administration of CCI-79 (Thomas et al., 2006). These data raise the possibility thatDG-PET can be used to detect modulation of the mTORathway in patients treated with rapamycin analogues. Inprospective assessment of the addition of RAD-001 to

efitinib or erlotinib in NSCLC, Reily et al. showed that 3eeks following the addition of the mTOR inhibitor, 5 of0 patients had a decrease in Standard Uptake Value (SUV)f >15%, with a median reduction in SUV of 18% (Riely etl., 2007). These results need to be confirmed in additionallinical trials, where FDG-PET could be further investigateds a surrogate marker for inhibition of the PI3K/Akt/mTORathway.

. Some clinical considerations for the combinationf pathway inhibitors with other chemotherapies

There is substantial preclinical evidence that PI3K/kt/mTOR pathway inhibitors can be effectively combinedith chemotherapy, radiotherapy and targeted agents to

nhance efficacy and overcome mechanisms of resistance.he early clinical trials suggest that pathway inhibitors maye beneficial when added to other anticancer therapies, partic-larly other targeted therapies such as EGFR TKIs, imatinib,nd bevacizumab, although there remains a paucity of phaseI and phase III data to corroborate these findings. Two majorssues that will determine whether pathway inhibitors can beuccessfully used in combination with other anticancer agentsre toxicity considerations and patient selection, which wille discussed below in more detail.

.1. Toxicity concerns

The toxicity of pathway inhibitors will vary with the par-icular drug as well as with the class of inhibitor. For example,ithin the class of Akt inhibitors, lipid-based compounds

uch as perifosine or the PIAs may induce more gastrointesti-al toxicity than a nucleoside analogue such as triciribine.ertain toxicities, however, may be class specific to all path-ay inhibitors, such as de-regulation of glucose and lipid
etabolism, which have been clinically observed with Akt
nhibitors such as triciribine (dose-limiting hyperglycemiand hypertriglyceridemia in phase I and II trials) as well asith mTOR inhibitors such as rapamycin and its analogues.

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e Updates 11 (2008) 32–50 43

hether a therapeutic index can be achieved with pathwaynhibitors is presently unknown, as normal cells also rely onhe activation of the PI3K/AKT/mTOR pathway. However,ancer cells may have greater reliance on pathway activationor survival than normal cells because they are selectivelyxposed to stressors such as hypoxia or aneuploidy, whichncrease activation of PI3k/Akt/mTOR. Thus, pathway inhi-ition may result in selective cytotoxicity of cancer cells. Itas yet to be determined whether the development of severeyperglycemia and/or hypercholesterolemia would correlateith patient response, in a manner analogous to rash inatients responding to EGFR inhibitors. In support of thisossibility in a study of CCI-779 in glioblastoma, develop-ent of grade 2 or greater hyperlipidemia was associatedith a higher rate of radiographic response (Galanis et al.,005).

A significant concern with mTOR inhibitors is immuneuppression, given that rapamycin is FDA approved for therevention of allograft rejection and blocks IL-2-induced Tell proliferation. However, there is little evidence in theiterature to suggest that the mTOR inhibitors cause sig-ificant immune compromise when used as single agents.idalgo et al., in a phase I trial of CCI-779 in advancedalignancy, found no changes in lymphocyte cell surface

henotypic markers and lymphocyte subsets. Furthermore,here was no significant change in lymphocyte proliferationssays nor was there clinical evidence of immune compro-ise (Hidalgo et al., 2006). In a different study, Yee et al.

oted a high frequency of infectious episodes in patientsith hematologic malignancies treated with RAD-001, buto opportunistic infections were observed. The investigatorsoted that this increased frequency could be due to underlyingmmune-compromised states associated with hematologic

alignancies (Yee et al., 2006).In trials combining mTOR inhibitors with conventional

hemotherapy, unexpected toxicities in two trials lead to earlyiscontinuation of the studies (Punt et al., 2003; Pacey et al.,004). However, overlapping toxicities were not observed inreliminary data from trials combining perifosine with con-entional chemotherapy (Cervera et al., 2006; Ebrahimi etl., 2006; Goggins et al., 2006; Weiss et al., 2006). Nev-rtheless, combining pathway inhibitors with conventionalytotoxic chemotherapy could result in more toxicity thanhen combining inhibitors with molecularly targeted agents.

f overlapping toxicities with combination agents are a con-ern, phase I trials should be designed utilizing doses lowerhan established single agent doses, even if it resulted inlower achievement of biologically effective pathway inhi-ition in vivo.

.2. Patient selection

In designing clinical trials for pathway inhibitors inombination with other agents, particularly phase II tri-ls, investigators should stratify patients by relative strengthf pathway activation, or alternatively exclude patients

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hose tumors do not demonstrate pathway activation. If theI3K/Akt/mTOR pathway is not activated in tumor cells,

hen pathway inhibitors would not be expected to have effi-acy, assuming that these agents’ clinical activities will not beue to off-target effects. Of the pathway inhibitors discussedn this review, rapamycin is exquisitely specific for mTOR,nd has no described off-target effects. One could arguehat patients whose tumors did not exhibit mTOR activationould not be expected to benefit from an mTOR inhibitor.ertainly, any changes in design of early phase clinical tri-ls that results in exclusion of patients based on molecularriteria must be accompanied by the development of vali-ated assays that can reliably measure activation of pathwayomponents.

In addition to utilizing activation state specific antibodiesn IHC or immunoblotting, other methods for measur-ng pathway activation are in development. Recently, Saalt al. developed a gene expression signature for PTENoss which correlated with adverse outcomes in breast,rostate, and bladder cancer (Saal et al., 2007). Future tri-ls could prospectively assess cancer cell gene expressionignatures of key components of the pathway. Compar-sons of pathway component gene expression at baselinend after therapy may be a means by which to deter-ine if an inhibitor is altering gene expression of pathway

omponents and to evaluate if a given gene signature isredictive of response to a pathway inhibitor. An alter-ative strategy to validate target modulation by pathwaynhibitors early in their clinical development would beo test these agents in a patient population with uniformctivation of the PI3K/Akt/mTOR pathway and accessi-le tissues. Such populations might include patients withTEN hamartomatous tumor syndromes (PHTSs) such asowden Syndrome. These are rare syndromes in whichatients possess germline mutations of PTEN, leading toonstitutive activation of the PI3K/Akt/mTOR pathway inenign and malignant tumors. Patients with this syndromere at increased risk for developing certain malignancies,ncluding thyroid, breast and endometrial cancer. Agentshat effectively modulate the pathway in tissues such asBMCs, gastrointestinal hamartomas, and skin trichilemmo-as might have promise as anticancer therapeutics. Those

gents that demonstrated modulation of the pathway inatients with PHTS could subsequently be tested in the gen-ral population of cancer patients whose tumors bear pathwayctivation.

In conclusion, the proper selection of patients for clini-al trials and reliable demonstration of target inhibition inivo will be critical to the development of PI3K/Akt pathwaynhibitors as anticancer therapeutics.

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