9 Combinations of Cytotoxic Drugs, Ionizing Radiation, and Mammalian Target of Rapamycin (mTOR) Inhibitors

9 Combinations of Cytotoxic Drugs, Ionizing Radiation, and

Mammalian Target of Rapamycin (mTOR) Inhibitors

Jann N. Sarkaria

J. N. Sarkaria, MD

Assistant Professor, Department of Oncology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA

9.1

Introduction

Rapamycin and its analogs are novel, molecularly targeted drugs that are being developed as anti- cancer agents. The parent compound, rapamycin (Sirolimus, Rapamune, Wyeth, Madison NJ, USA) is approved by the Food and Drug Administration (FDA) for the prevention of allograft rejection fol- lowing renal transplantation, and for incorpora- tion into drug-eluting stents to prevent re-stenosis following coronary angioplasty. Experience in the transplant setting suggests that long-term use of this agent is safe and well tolerated. Rapamycin analogs with more favorable pharmacokinetic properties are currently being developed as anti-cancer drugs (Fig. 9.1). CCI-779 (Temsirolimus, Wyeth Pharma- ceuticals) is an ester of rapamycin, with superior

oral bioavailability compared with the parent com- pound rapamycin. This drug is available in oral and intravenous formulations, and clinical development of this drug is well underway with several phase- II and phase-III trials being conducted. RAD001 (Everolimus, Novartis International AG, Basel, Swit- zerland) is an orally available hydroxyethyl deriva- tive of rapamycin developed for applications in the transplant, cardiovascular, and oncological settings, and clinical testing for all these indications is ongo- ing. The newest mammalian target of rapamycin (mTOR) inhibitor to be developed for clinical use is AP23573 (Ariad Pharmaceuticals, Inc., Cambridge, Massachusetts, USA). Early phase-I clinical trials with this agent, which is also an analog of rapamy- cin, are now underway.

Rapamycin and its analogs inhibit the signal- ing activity of the serine-threonine protein kinase mTOR. mTOR functions downstream from mul- tiple growth factor receptor tyrosine kinases to promote cell growth and proliferation. Key down- stream targets of mTOR include p70S6 kinase and eukaryotic initiation factor 4E-binding protein (4EBP1), which modulate the translation of select mRNA transcripts that ultimately impact on cell growth and cell cycle progression. More recent data have linked mTOR signaling with the cellular response to hypoxia and the expression of vascular endothelial growth factor (VEGF), which suggests that mTOR may be an important mediator of tumor angiogenesis. In tumors that are reliant on mTOR signaling, disruption of these key signaling path- ways by rapamycin results in cell cycle arrest and inhibition of angiogenesis, and these effects may account for the anti-neoplastic activities of mTOR inhibitors seen in multiple tumor types. Based on promising preclinical studies, rapamycin and its analogs currently are being tested as anti-neoplas- tic agents, both given alone or in combination with conventional cancer therapies. In this chapter, the biology of mTOR signaling and the cellular phar- macology of mTOR inhibitor monotherapy and combination therapy are reviewed.

CONTENTS

9.1 Introduction 127 9.2 Biology of mTOR 128

9.3 mTOR-Dependent Signaling Pathways 129 9.3.1 p70S6K 129

9.3.2 4EBP1 130 9.3.3 HIF-1 130

9.4 Anti-Tumor Effects of mTOR Inhibition 131 9.5 Clinical Experience with mTOR-Inhibitor Monotherapy 132

9.6 Combination Therapies with mTOR Inhibitors 132 9.7 Conclusion 134

References 134

9.2

Biology of mTOR

mTOR is a serine-threonine-directed kinase that belongs to the family of phosphatidyl-inosi- tol 3-kinase-related kinases (PIKK). Members of this PIKK family all contain a C-terminal kinase domain that shares significant homology with that of the phosphatidyl-inositol 3-kinase (PI3K). Other members of this family include ataxia telangiecta- sia mutated (ATM), ATM and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK;

Abraham 2001; Durocher and Jackson 2001).

These latter three kinases play key roles in orches- trating DNA damage checkpoint responses and DNA repair (Zhou and Elledge 2000). In contrast, mTOR monitors intracellular nutrient and energy availability and promotes cell growth and prolifera- tion following mitogenic stimuli, dependent upon the availability of requisite nutrients (Raught et al. 2001).

Rapamycin is a highly specific inhibitor of mTOR function. Rapamycin is unable to bind directly to mTOR but forms a complex with the immunophilin, 12 kDa FK506-binding protein (FKBP12; FK-506 is an unrelated immunosuppressant). It is this drug- protein complex that binds to mTOR through an FKBP12-rapamycin binding (FRB) domain (Cutler et al. 1999). The FRB domain is adjacent to the kinase domain in mTOR and formation of this tri-molecu- lar complex markedly attenuates downstream sig- naling from mTOR. Interestingly, rapamycin treat-

ment does not inhibit mTOR catalytic kinase activity directly, since autophosphorylation of mTOR is unaf- fected by rapamycin treatment. Instead, binding of the FKBP12/rapamycin complex is thought to pre- vent interaction of mTOR with its kinase substrates and thus prevent downstream signaling (Edinger et al. 2003). The interaction of the rapamycin/FKBP12 complex with mTOR is highly specific and is so stable that inhibition of mTOR by rapamycin is essentially irreversible. The cellular and biochemical effects of rapamycin are generally believed to result exclu- sively from inhibition of mTOR signaling (Brown et al. 1994; Sabatini et al. 1994).

The mTOR signaling network (Fig. 9.2) is impor- tant for driving cell growth and proliferation in mul- tiple tumor types. Several receptor tyrosine kinases (RTKs), including the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor receptor (IGFR), can activate PI-3 kinase activity, which, in turn, phosphorylates phosphatidylinosi- tol (PI) on the D-3 position (Grant et al. 2002). The resulting accumulation of phosphatidylinositol-3, 4, 5-triphosphate on the cytoplasmic surface of the plasma membrane leads to activation of a number of kinase-signaling pathways including that regulated by protein kinase B (PKB, Akt). Akt stimulates mTOR function both through direct phosphorylation of a negative regulatory domain within mTOR (Sekulic et al. 2000) as well as through its effects on the tuber- ous sclerosis complex-2 (TSC2) protein (Dan et al.

2002; Inoki et al. 2002; Potter et al. 2002; Tee et

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Fig. 9.1. Structure of rapamycin and rapamycin derivatives

al. 2002). TSC2, in a complex with tuberous sclerosis complex-1 (TSC1) protein, functions as a GTPase- activating protein towards the Rheb1 GTPase.

Akt-mediated phosphorylation of TSC2 disrupts the TSC1/TSC2 complex and relieves inhibition of Rheb1 activity; activated Rheb1 then can stimulate mTOR phosphorylation and signaling (see Fig. 9.2;

Castro et al. 2003; Garami et al. 2003; Inoki et al.

2003; Saucedo et al. 2003). The inhibitory effects of TSC2 on mTOR activity are stimulated in nutrient- deprived conditions by activation of LKB-1, which signals through AMP-activated protein kinase (AMPK) to enhance TSC2 activity (Corradetti et al. 2004; Shaw et al. 2004). Signaling from mTOR also is regulated by association with either Rictor or Raptor, which function as scaffolding proteins to promote selective association and phosphorylation of downstream targets (Hara et al. 2002; Kim Do et al. 2002). Collectively, these data highlight the idea that mTOR functions within a molecular complex of multiple proteins that regulate its activity.

PI3K-mediated activation of Akt is normally opposed by the lipid phosphatase PTEN (phospha- tase and tensin analog), which dephosphorylates phosphatidylinositol at the D-3 position. Deletion or mutation of the gene encoding this tumor sup- pressor protein commonly occurs in multiple tumor types and results in constitutive activation of PI3K- dependent signaling pathways that include Akt and

activated Ras as signaling mediators. Consistent with the potential role of the Akt/mTOR signaling pathway in tumorigenesis, overexpression of acti- vated Akt and activated Ras in glial progenitor cells leads to formation of glioblastoma-like tumors in a transgenic mouse model (Holland et al. 2000), and these tumors are exquisitely sensitive to treatment with rapamycin. Germ-line inherited deficiencies in PTEN, TSC, or LKB-1 result in Cowden’s disease, tuberous sclerosis, and Peutz-Jeghers syndrome, respectively, which are all characterized by the development of multiple benign hamartomas. Loss of function in any one of these three proteins results in hyperactivation of mTOR signaling, and this pre- sumably accounts for the development of the char- acteristic hamartomatous lesions; thus, constitutive activation of mTOR signaling can be an important contributor to tumorigenesis in both benign and malignant tumors.

9.3

mTOR-Dependent Signaling Pathways

mTOR signals downstream to multiple protein targets including but not limited to p70 S6 kinase (p70S6K), initiation factor 4E binding protein (4E- BP1), and the hypoxia-inducible transcription factor, HIF-1D. These three well-characterized downstream signaling targets have been implicated in control of hypoxia- and mitogen-induced tumor proliferation, and disruption of these pathways may play an impor- tant role in the anti-tumor effects of rapamycin. In the sections below, we describe the potential links between the anti-tumor effects of mTOR inhibitors and disruption of downstream signaling to p70S6K, 4EBP1, and HIF1 in more detail.

9.3.1 p70S6K

mTOR regulates translation of select mRNA tran- scripts containing 5’-terminal oligopyrimidine (5’TOP) tracts through phosphorylation of p70S6K.

Following PI3K-dependent phosphorylation of resi- dues within an auto-inhibitory domain, mTOR reg- ulates the phosphorylation of Thr-389 (Volarevic and Thomas 2001). Modification of this residue is essential for subsequent phosphorylation of other residues within the activation loop of the kinase domain, which allows for full catalytic activity.

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Fig. 9.2. The mTOR signaling network

After mitogen stimulation, activated p70S6K phos- phorylates the S6 component of the 40S ribosomal subunit, and this promotes translation of mRNA containing 5’TOP (Jefferies et al. 1997). Because transcripts for many ribosomal proteins and trans- lation elongation factors contain this 5’TOP motif, rapamycin-mediated suppression of p70S6K activity may inhibit cell growth and proliferation by lim- iting ribosomal biogenesis and restricting protein synthesis capacity.

9.3.2 4EBP1

mTOR modulates protein translation initiation by regulating the assembly of the eukaryotic initiation factor 4F (eIF4F) complex on the 5’-methyl-GTP cap of mRNA transcripts. The eIF4F complex is a heterotrimer composed of the mRNA cap-binding protein eIF4E, a scaffolding protein eIF4G, and a helicase eIF4A (reviewed by Raught et al. 2001).

This tripartite complex regulates the rate of cap- dependent protein translation by mediating the rate-limiting step of mRNA loading onto the small 40S ribosomal subunit. Formation of a functional eIF4F complex is controlled by the phosphorylation status of an eIF4E binding protein (4E-BP1). In nutri- ent- or growth-factor-deprived cells, the association of hypophosphorylated 4E-BP1 with eIF4E blocks binding of eIF4G to the cap structure and inhibits cap-dependent translation. In contrast, nutrient- or mitogen-induced phosphorylation of 4E-BP1 dis- rupts association with eIF4E and allows formation of a functional eIF4F complex (Gingras et al. 1998;

Herbert et al. 2002). 4E-BP1 is phosphorylated on at least five serine or threonine sites, and these mitogen-induced modifications are regulated, in part, by mTOR. Several studies have suggested that mTOR directly phosphorylates all five sites on 4E- BP1 (Brunn et al. 1997; Mothe-Satney et al. 2000), whereas others have argued that direct mTOR phos- phorylation of two of the sites (Thr-37 and Thr-46) serves as a priming event that allows subsequent phosphorylation of the other sites (Ser-65, Thr-70, and Ser-83) by other signaling pathways (Gingras et al. 1999). Rapamycin diminishes 4E-BP1 phosphory- lation, prevents dissociation of 4E-BP1 from eIF4E, and results in inhibition of cap-dependent transla- tion. Because translation is less efficient for tran- scripts with complex secondary structures, rapa- mycin preferentially inhibits translation of mRNA transcripts containing complex 5’ untranslated

regions (UTR; De Benedetti and Harris 1999).

Transcripts with complex 5’UTRs whose transla- tion is inhibited by rapamycin include key proteins involved in cell proliferation and angiogenesis, such as cyclin D1, ornithine decarboxylase, and VEGF.

9.3.3 HIF-1α

Hypoxia induces the expression of multiple genes containing hypoxia response elements (HREs), and transcription from this response element is regu- lated primarily by the HIF-1 transcription factor.

HIF-1 is a heterodimer composed of HIF-1D and HIF-1E. Under normoxic conditions, HIF-1 het- erodimer levels are undetectable due to rapid deg- radation of HIF-1D subunit. The stability of HIF-1D is regulated through post-translational modifica- tion of an oxygen-dependent degradation (ODD) domain. In the presence of oxygen, prolyl hydroxy- lases modify two conserved proline residues (Pro- 402 and Pro-564) within the ODD (Jaakkola et al.

2001). Hydroxylation of these residues promotes HIF-1D association with the von Hippel Lindau- containing ubiquitin ligase complex and subsequent (Yu et al. 2001) ubiquitin-mediated proteosomal degradation. Because molecular oxygen is required for catalysis of this reaction, hypoxic conditions prevent hydroxylation of the ODD, which results in stabilization of HIF-1D. Other transactivating post-translational modifications and dimerization with the HIF-1E subunit result in formation of an active HIF-1 transcriptional complex. HIF-1 drives expression of genes, such as VEGF, which contain HREs within their promoter region. The repertoire of hypoxia-inducible genes enables tumor or normal tissues to adapt to low oxygen environments and include genes involved in oxygen and glucose trans- port, glycolysis, growth-factor signaling, immortal- ization, genetic instability, invasion and metastasis, apoptosis, and pH regulation (Harris 2002).

Signaling through the PI3K/mTOR pathway reg-

ulates HIF-1D expression and activity (Abraham

2004). The link between PI3K signaling and HIF-1D

activity was first established in Ras-transformed

cells, where hypoxia-induced signaling to HIF-1 was

blocked by genetic or pharmacological inhibition of

PI3K activity (Mazure et al. 1997). Subsequent stud-

ies have demonstrated that restoration of wild-type

PTEN function, expression of a dominant-negative

Akt construct, or treatment with rapamycin blocks

hypoxia and mitogen-induced HIF-1 signaling

(Zhong et al. 2000; Zundel et al. 2000; Laughner et al. 2001). Moreover, a recent study demonstrated that rapamycin blocks both hypoxia-induced HIF- 1D accumulation and transactivation, and that this effect of rapamycin is specifically due to pharma- cological inhibition of mTOR (Hudson et al. 2002).

Collectively, these data suggest the existence of a PI3K/Akt/mTOR signaling pathway that regulates HIF-1D expression, stability, or activation.

9.4

Anti-Tumor Effects of mTOR Inhibition

The central role for mTOR in modulating cell pro- liferation in both tumor and normal cells and the importance of mTOR signaling for the hypoxic response suggests that rapamycin-based therapies may exert anti-tumor effects primarily through either inhibition of tumor cell proliferation or sup- pression of angiogenesis. Preclinical and early clini- cal results demonstrate that only a subset of tumors respond to rapamycin-based therapies. In the fol- lowing sections, we review the preclinical data and the evidence supporting the anti-angiogenic and cytostatic properties of rapamycin.

Preclinical studies have demonstrated efficacy of rapamycin analogs and the parent compound in mul- tiple tumor types. In the National Cancer Institute (NCI) 60 tumor cell line panel, both rapamycin (NSC 226080) and CCI-779 (NSC 683864) demonstrated growth inhibitory activity against a broad spectrum of tumors with a subset of leukemia, lung, brain, prostate, breast, as well as renal and melanoma tumor cell lines being inhibited at low nanomolar concen- trations (http://dtp.nci.nih.gov/). Early animal stud- ies at the NCI and at Ayerst Research Laboratories demonstrated modest growth inhibitory properties of rapamycin in murine tumor models of B16 mela- noma, P388 lymphocytic leukemia, EM ependymo- blastoma, CD8F1 breast carcinoma, Colon 38, CX-1, and 11/A colon cancer models (Eng et al. 1984). Sub- sequently published studies have documented sig- nificant tumor growth inhibition with rapamycin or CCI-779 treatment of DAOY medulloblastoma, U251 or SF295 glioma, PC-3, DU-145, LAPC4, or LAPC9 prostate carcinoma, and Rh-18 rhabdomyosarcoma human tumor xenografts (Houchens et al. 1983;

Dudkin et al. 2001; Geoerger et al. 2001; Neshat et al. 2001). These data have provided the impetus for development of mTOR inhibitor therapy in a variety of tumor types.

Treatment of tumor cells in vitro with rapamycin results in an accumulation of cells in the G

phase of the cell cycle. Similarly, rapamycin treatment in tumor-bearing animals results in decreased tumor cell proliferation as indicated by bromodeoxyuridine (BrdU) labeling index (Eshleman et al. 2002). At the molecular level, this drug-induced cell cycle arrest is associated with an accumulation of the cyclin-depen- dent kinase inhibitor, p27

, decreased expression of cyclinD1, and a corresponding decrease in phos- phorylation of the retinoblastoma protein. CyclinD1 mRNA contains a complex 5’UTR, and reduction in cyclinD1 levels presumably results from rapamy- cin-mediated inhibition of 4EBP1 phosphorylation and the resulting inhibition of eIF4F function. Like- wise, the mRNA encoding for multiple oncogenes or proteins involved in DNA metabolism and S-phase progression contain complex 5’UTRs and are regu- lated by mTOR (Kozak 1991). Although rapamycin can induce apoptosis in select tumor models, rapa- mycin treatment typically slows tumor growth but does not induce tumor regression, suggesting that increased tumor cell loss through apoptosis or other mechanisms of cell death likely are not major con- tributors to drug effect in most tumors. Collectively, these data support the idea that rapamycin exerts a cytostatic effect on tumor cell growth.

Rapamycin also has cytostatic effects on normal and tumor vasculature. As discussed previously, mTOR plays an important role in modulating the cellular response to hypoxia through stabilization and activation of HIF-1D±HIF-1 and drives expres- sion of hypoxia-responsive genes, and these genes include those such as VEGF that are important for tumor angiogenesis. Translation of VEGF mRNA also is regulated in an mTOR-dependent manner via its complex 5’UTR. Consistent with these mechanisms of regulation, treatment of tumor-bearing animals with rapamycin results in decreased expression of VEGF mRNA in tumors (Luan et al. 2003) and decreased circulating levels of VEGF protein (Guba et al. 2002).

The VEGF drives endothelial proliferation through interaction with its cognate receptor tyrosine kinases, VEGF receptors 1 and 2, and these receptors can signal downstream through the PI3K/Akt pathway to mTOR;

thus, proliferation of smooth muscle and endothelial cells is inhibited by mTOR inhibition (Vinals et al.

1999; Mayerhofer et al. 2002), and this effect is likely

related to the efficacy of rapamycin-eluting stents in

preventing vascular re-occlusion. In animal models,

rapamycin inhibits tumor growth and neo-vascular-

ization of CT-26 colon tumors grown in a dorsal skin-

fold model and suppresses serum levels of VEGF in

tumor-bearing animals (Guba et al. 2002); thus, the anti-angiogenic effects may contribute to the efficacy of mTOR inhibitors in cancer therapy.

9.5

Clinical Experience with mTOR-Inhibitor Monotherapy

Clinical efficacy of mTOR inhibitors has been eval- uated in patients with a variety of tumor types.

Although three distinct mTOR inhibitors (CCI-779, RAD001, and AP23573) are being evaluated in clini- cal trials, mature published phase-II study results are only available for CCI-779. In renal cell cancer, 111 patients were randomized to receive weekly IV infusions of 25, 75, or 250 mg (Atkins et al. 2004).

One patient achieved a complete response, and 7 patients had partial responses with >50% reduc- tion in tumor size. Approximately half of patients had at least stabilization of disease for >24 weeks.

Based on these data, a phase-III study in renal cell cancer is now underway. Promising results also have been seen in mantle cell lymphoma, where 13 of 34 patients had either a complete (1 patient) or partial response (12 patients) with single agent weekly treat- ment and modest hematological toxicities (Witzig et al. 2005). Two clinical trials have evaluated the efficacy of CCI-779 in recurrent malignant glioma.

In a study by the North American Brain Tumor Con- sortium, partial responses were observed in 2 of 43 patients and disease stabilization was observed in another 50% of patients (Chang et al. 2005). Similar results were observed in a clinical trial run by the North Central Cancer Treatment Group (NCCTG), where 20 of 65 patients had evidence of reduced T2 signal abnormality on magnetic resonance imaging (MRI) and this response correlated with a time to progression of 5.4 months relative to 1.9 months for non-responders (Galanis et al. 2005). In contrast, monotherapy with CCI-779 in patients with meta- static melanoma did not significantly affect disease progression (Margolin et al. 2005). In addition to the trials discussed here, several phase-II clinical trials using CCI-779 and RAD001 are currently ongo- ing in a variety of tumor types (reviewed by Rao et al. 2004). Collectively, this early clinical experience suggests that mTOR inhibitors may be useful thera- peutic agents for specific tumor types.

The anti-tumor effects of rapamycin therapies are likely secondary to both inhibition of tumor cell proliferation and inhibition of tumor angiogenesis.

The relative contribution of these two effects in any given tumor may be difficult to delineate; however, preclinical and clinical data suggest that only a subset of tumors respond to rapamycin. This obser- vation has spurred investigations into the combi- nations of mTOR inhibitors with conventional and molecular targeted chemotherapeutic agents.

9.6

Combination Therapies with mTOR Inhibitors

Angiogenesis and tumor cell proliferation have been implicated as important mediators that can influence the efficacy of traditional cytotoxic cancer therapies;

therefore, much research effort has focused on the

evaluation of combinations of mTOR inhibitors with

standard cancer therapies. Rapamycin potentiates

cisplatin-induced apoptosis in multiple cell lines

including HL-60 leukemia cells and SKOV3 ovarian

cancer cells (Shi et al. 1995). Likewise, IGF-induced

resistance to cisplatin in Rh30 rhabdomyosarcoma

cells can be reversed by rapamycin treatment (Wan

and Helman 2002). Geoerger et al. (2001) dem-

onstrated that combinations of rapamycin with

either cisplatin or camptothecin provides additive

growth inhibition in the rapamycin-sensitive DAOY

medulloblastoma cell line but not in the rapamycin-

resistant D283 cell line. Consistent with this in vitro

data, CCI-779 therapy provides for additive tumor

growth inhibition in animals when combined with

doxorubicin, mitoxantrone, docetaxel, or cisplatin

in prostate cancer and medulloblastoma xenografts

(Geoerger et al. 2001; Wu et al. 2005). Several other

investigators have confirmed the in vitro effect of

rapamycin on sensitizing cancer cells to chemo-

therapeutic agents such as adriamycin, VP-16, and

cisplatin. While the mechanism(s) of these effects

are not fully understood, in one study inhibition of

mTOR signaling blocked the cytoprotective effects

of IGF-II overexpression in rhabdomyosarcoma

cells (Wan and Helman 2002). Similarly, in another

study, RAD001-dependent inhibition of translation

blocked p53-induced accumulation of p21

in

response to cisplatin treatment, and the failure of

this anti-apoptotic molecule to accumulate resulted

in increased apoptosis (Beuvink et al. 2005); thus,

the increased efficacy of chemotherapies with con-

comitant mTOR inhibition is likely due to disrup-

tion of key survival signaling pathways leading to

increased cell killing.

PI3K/Akt/mTOR-dependent signaling is impor- tant in the development of hormonally responsive cancers involving breast and prostate, and pre- clinical studies suggest that combinations of mTOR inhibitors with hormonal therapies may be worth evaluating in the clinic. In keeping with the impor- tance of the PI3K/Akt/mTOR signaling axis, trans- genic overexpression of Akt in the ventral lobe of the prostate results in development of prostatic intra- epithelial neoplasia (PIN), and this PIN phenotype is completely reversed with RAD001 treatment (Majumder et al. 2004). These effects were associ- ated with an increase in apoptosis and suppression of HIF-1D signaling. In tumor models of prostate cancer progression, hormone-refractory states are associated with activation of p70S6 kinase down- stream from mTOR, upregulation of genes known to be regulated by the Akt/mTOR pathway, and increased sensitivity to mTOR inhibition (Mousses et al. 2001; Ghosh et al. 2005). These observations have prompted the evaluation of mTOR inhibitors in hormone-refractory prostate cancer, and it would be interesting to evaluate whether mTOR inhibitor therapy would prevent or delay the development of a hormone-refractory state in up-front therapy of metastatic prostate cancer.

Combinations of mTOR inhibitors with hormonal therapies are also promising in breast cancer models.

Hyper-activation of Akt signaling is associated with an adverse prognosis in breast cancer and has been implicated in the development of a hormone-refrac- tory state (Kirkegaard et al. 2005). In cell culture, tamoxifen resistance can be induced by over-expres- sion of constitutively active Akt, and sensitivity to tamoxifen can be restored by concurrent treatment with rapamycin or CCI-779 (De Graffenried et al. 2004). Similarly, others have demonstrated that the combination of RAD001 with letrazole, an aro- matase inhibitor, results in synergistic anti-tumor effects (Boulay et al. 2005). Collectively, these stud- ies support the concept of combining conventional hormonal therapies with mTOR inhibitors in breast and prostate cancers.

Rapamycin also can enhance the efficacy of radiation therapy. Based on significant preclinical and clinical data demonstrating that tumor pro- liferation during fractionated radiotherapy con- tributes to clinical radiation resistance (Fowler 2001; Bentzen 2003), we hypothesized that rapa- mycin-mediated inhibition of tumor proliferation during radiotherapy would enhance the efficacy of radiation. Consistent with the idea that mTOR is not involved in DNA damage responses, unlike

the related ATM, ATR, and DNA-PKcs kinases, rapamycin had no effect on the in vitro radiation sensitivity of several glioma cell lines including U87 cells. In contrast, intermittent dosing with rapamy- cin throughout a fractionated course of radiation significantly enhanced the efficacy of treatment in radioresistant U87 xenografts (Fig. 9.3). Although the mechanism of this effect is not entirely under- stood, a recent study demonstrated that the mTOR inhibitor RAD001 can sensitize endothelial cells to radiation and that GL261 xenografts treated with both radiation and RAD001 have delayed tumor regrowth associated with reduced tumor blood flow (Shinohara et al. 2005). While the molecu- lar mechanisms involved in increased endothelial cell death are unclear, these two studies suggest that novel combinations of mTOR inhibitors with radiation would be interesting to evaluate in clini- cal trials. Given the promising responses seen in the NCCTG trial in recurrent glioma, we are now initiating a clinical trial evaluating the addition of CCI-779 to standard therapy with high-dose radia- tion and temozolomide in newly diagnosed glio- blastoma multiforme.

A network of parallel signaling pathways often contributes to resistance to mTOR inhibitors or other signal transduction inhibitors, and this realization has led to increasing interest in simultaneous inhi-

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Fig. 9.3. Rapamycin enhances the effi cacy of radiation in U87 xenografts. Nude mice with established U87 fl ank xenografts were randomized into four treatment groups: (a) placebo; (b) radiation only (4 Gy u 4); (c) rapamycin only (1 mg/kg); or (d) radiation and rapamycin. The tumor regrowth for each treat- ment group is shown. Data points represent the mean relative tumor volumerSE. Treatment was initiated on day 0 with the fi rst injection of rapamycin (Rap). The schedule for Rap and radiation (RT) treatments is depicted below the x-axis. Highly similar results were obtained in two independent experiments.

(From Eshleman et al. 2002)

bition of multiple targets within the mTOR signaling pathway. This approach is best highlighted by the example of chronic myelogenous leukemia (CML).

The CML is caused by a chromosomal translocation which leads to an oncogenic chimeric BCR-Abl gene product. The resulting constitutively active kinase drives leukemia proliferation and survival and can be selectively inhibited by the kinase inhibitor ima- tinib. Imatinib treatment is highly effective in CML, but chronic therapy invariably leads to imatinib resistance through mutation of the BCR-Abl kinase domain, amplification of the BCR-Abl gene product, or other mechanisms. Imatinib treatment in naive CML leads to compensatory hyper-activation of Akt/

mTOR signaling (Burchert et al. 2005), and several groups have demonstrated that combined inhibition of mTOR and BCR-Abl leads to synergistic cell kill- ing (Ly et al. 2003; Mohi et al. 2004; Dengler et al.

2005), which suggests that the hyperactive mTOR signaling is an important survival mechanism in this disease. Moreover, chronic Akt/mTOR signaling has been implicated in the ultimate development of imatinib resistance, since combined treatment with rapamycin and imatinib blocks development of ima- tinib resistance (Burchert et al. 2005). These data suggest that simultaneous mTOR/BCR-Abl inhibi- tion in CML may improve the anti-tumor effects of therapy and also delay the induction of resistance to BCR-Abl inhibitors.

Compensatory feedback mechanisms also can lead to relative resistance to molecularly targeted therapeutics. As mentioned previously, mTOR can be targeted to specific substrates by association with either Rictor or Raptor, and rapamycins spe- cifically inhibit the mTOR/raptor complex. This leads to a shift in signaling to the mTOR/Rictor complex, and one substrate for this rapamycin- insensitive mTOR/Rictor is a key regulatory resi- due (Ser-473) on Akt (Sarbassov et al. 2005); thus, mTOR inhibitor-induced up-regulation of Akt signaling (Sun et al. 2005) may actually promote survival, and conversely, simultaneous inhibition of mTOR and Akt could lead to enhanced cell kill- ing. In support of this hypothesis, a recent study demonstrated that combined inhibition of mTOR with either PI3K or Akt inhibitors leads to syner- gistic anti-tumor effects (Takeuchi et al. 2005).

Similarly, we have demonstrated that EGFR inhibi- tor therapy also can block rapamycin-mediated upregulation of Akt phosphorylation, and that combined EGFR/mTOR inhibition is significantly more effective than either therapy alone in glioma cell lines (Rao et al. 2005). Similar results have

been reported by others in a glioma (Goudar et al. 2005) and renal cell cancer models (Gemmill et al. 2005). The Ras signaling pathway also is impor- tant for promoting survival, and in leukemia cells, combined inhibition of mTOR and MEK resulted in significant synergistic interactions (Bertrand et al. 2005). Combinations of mTOR inhibitors with relatively non-specific kinase inhibitors, such as 7-hydroxystaurosporine, also have provided interesting results (Hahn et al. 2005; Takeuchi et al. 2005). With the numerous signal transduc- tion inhibitors currently in development for clini- cal use, the results summarized here represent just the beginning of what are likely to be a multitude of studies evaluating mTOR inhibitors in combina- tion with other targeted therapies.

9.7

Conclusion

Rapamycin and its analogs are versatile drugs with proven efficacy in cardiovascular and transplant medicine and with promising results in early cancer clinical trials. In specific tumor types, a select minor- ity of patients likely will benefit from monotherapy.

The challenge for the future will be to dissect fur- ther the molecular signaling pathways modulated by rapamycin in order to appreciate fully the molecular mechanisms underpinning sensitivity or resistance to mTOR inhibition. This understanding will pro- vide insight into rational combinations of mTOR inhibitors with classic cytotoxic agents, radiation, and other molecularly targeted therapies.

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