Temsirolimus, an Inhibitor of Mammalian Target of RapamycinBrian I. Rini

Temsirolimus (Torisel, Wyeth Pharmaceuticals) is an inhibitorof mammalian target of rapamycin (mTOR), a molecule impli-cated in multiple tumor-promoting intracellular signaling path-ways. Temsirolimus recently became the first Food and DrugAdministration–approved mTOR-targeted agent based on aphase III trial showing an overall survival advantage over IFN-amonotherapy in advanced renal cell carcinoma (RCC) patientswith multiple adverse risk features (1).Temsirolimus is a soluble ester of rapamycin, a natural pro-

duct that was initially developed as an antifungal drug and thenas an immunosuppressive agent, with anticancer activity notedmore than 20 years ago. Rapamycin (sirolimus, Rapamune)was isolated from the soil bacteria Streptomyces hygroscopicusfound on Rapa Nui (commonly known as Easter Island) in theSouth Pacific in 1975, but its development for cancer ther-apeutics was not prioritized. The immunosuppressant effectsof rapamycin were pursued and resulted in Food and DrugAdministration approval in 1999 for prevention of renal allo-graft rejection. Laboratory studies of rapamycin starting in theearly 1980s showed antitumor effects in several solid tumors.Cell cycle inhibitor-779 (now known as temsirolimus), a deri-vative of rapamycin, was identified in the 1990s and subse-quently developed as an anticancer agent (2).mTOR, also known as rapamycin-associated protein, rapa-

mycin target, or sirolimus effector protein, is a molecule im-plicated in multiple tumor-promoting intracellular signalingpathways (Fig. 1). mTOR is a 289-kDa serine/threonine-specifickinase with highly conserved structure. It exists in cytoplasm ina complex with three peptides: regulatory-associated protein ofmTOR (raptor), mLST8, and GhL. Regulation of mTOR path-way activation is mediated through a series of complex sig-naling interactions linking growth factor receptor signalingand other cell stimuli, phosphatidylinositol 3-kinase activation,and activation of the Akt/protein kinase B pathway. Two dis-tinct pathways have effects on cell cycle regulation downstreamof mTOR. mTOR phosphorylates and activates p70 S6 kinase,leading to enhanced translation of certain ribosomal proteinsand elongation factors. This process leads, among other effects,to the production of hypoxia-inducible factor-1a, which regu-lates the transcription of genes that stimulate cell growth andangiogenesis, including VEGF . Indeed, it has been shown thatmTOR inhibition leads to RCC cell line and xenograft tumor

growth inhibition, and this effect is mediated by hypoxia-inducible factor (3). The second major mTOR effect is on the4E-binding protein-1 and eukaryotic initiation factor-4 subunitE complex. Activated mTOR phosphorylates 4E-binding pro-tein-1, promoting dissociation of this complex and allowingeukaryotic initiation factor-4 subunit E to stimulate an increasein the translation of mRNAs that encode cell cycle regulators,such as c-myc, cyclin D1, and ornithine decarboxylase. mTORinhibition results in a block of 4E-binding protein-1 phospho-rylation, sequestration of eukaryotic initiation factor-4 subunitE, and failure to form the complex required for translation.Inhibition of mTOR by temsirolimus requires a specific bind-ing complex. Temsirolimus forms this complex with theFK506-binding protein and prohibits the activation of mTOR.This mechanism of action is similar for sirolimus, a temsir-olimus metabolite, and both are likely responsible forinhibition of mTOR and antitumor effects after administrationof temsirolimus. Temsirolimus/FK506-binding protein affectsonly one subpopulation of mTOR proteins, which reside in amultiprotein complex termed mTORC1. An additional com-plex, mTORC2, holds mTOR in a form that cannot be inhibitedby temsirolimus and could have downstream signaling impli-cations despite temsirolimus administration. The biology ofthe mTOR signaling pathway and relevance to cancer has beenextensively reviewed (4, 5). Abnormalities in various compo-nents of the mTOR pathway have been described for a widevariety of sporadic malignancies. In addition, mutations thatdisrupt the tuberous sclerosis complex and other pathwaysassociated with inherited hamartoma syndromes also result inunregulated activation of mTOR (6).Temsirolimus showed antitumor effects across a wide

variety of tumor histotypes in preclinical models, particularlythose with defective PTEN (7–9). An initial dose-escalationphase I trial administered temsirolimus i.v. weekly at dosesranging from 7.5 to 220 mg/m2 (10). Although thrombocy-topenia was dose limiting and reversible maculopapular rashand stomatitis were observed, the formal definition of amaximum tolerated dose was not met. In addition, objectivepartial and minor responses were observed at lower doselevels. Based on these observations and pharmacokinetic dataindicating comparable area under the curve variabilitybetween body surface area–normalized and flat dosing,subsequent phase II testing investigated one or more of thefollowing dose levels: 25, 75, and/or 250 mg i.v. weekly.Phase II trials were conducted in glioblastoma multiforme(11, 12), with well-documented PTEN alterations, mantle celllymphoma (13), with potential mTOR-mediated cyclin D1regulation, as well as melanoma (14), neuroendocrine tumors(15), breast cancer (16), and lung cancer (17). Modest single-agent activity was observed in these trials of pretreatedpatients, most notably in mantle cell lymphoma and breastcancer (Table 1). No evidence of a dose-response relationshipwas evident in any trial, and higher dose levels generallyresulted in greater toxicity.

CCRDrug Update

Author’s Affiliation:Department of SolidTumorOncology andUrology, ClevelandClinicTaussig Cancer Center, Cleveland, OhioReceived11/19/07; revised12/5/07; accepted12/13/07.Requests for reprints: Brian I. Rini, Department of Solid Tumor Oncologyand Urology, Cleveland ClinicTaussig Cancer Center, 9500 Euclid Avenue/DeskR35, Cleveland, OH 44195. Phone: 216-444-9567; Fax: 216-636-1937; E-mail:rinib2@ccf.org.

F2008 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-07-4719

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Temsirolimus was also tested in treatment-refractory, meta-static RCC in a phase II trial that randomized 111 patients to25, 75, or 250 mg i.v. weekly (18). The overall response ratewas 7%, with an additional 26% of patients showing minorresponses (Table 1). Retrospective assignment of risk criteria topatients in this study identified a poor-prognosis group withthree or more adverse risk features as established for metastaticRCC patients receiving IFN-based initial systemic therapy (19).These risk factors consisted of Karnofsky performance status<80%, lactate dehydrogenase >1.5� laboratory upper limit ofnormal, hemoglobin < laboratory lower limit of normal, serumcalcium corrected for albumin >10 mg/dL, and time from firstdiagnosis of RCC to start of therapy of <1 year. Temsirolimus-

treated patients in this poor-prognosis group had a medianoverall survival of 8.2 months compared with 4.9 months forhistorical control IFN-a–treated patients (19). No evidence ofa dose response was seen in this trial.

A subsequent randomized phase III trial was conducted inpatients with metastatic RCC and three or more adverse riskfeatures as defined by existing prognostic schema [the fivenoted above plus a sixth factor of three or more metastatic sitesidentified as prognostic in a separate analysis (20)]. A totalof 626 patients were randomized with equal probability to25 mg temsirolimus i.v. weekly (n = 209) versus 18 millionunits IFN-a thrice weekly (n = 207) versus 15 mg temsirolimusi.v. weekly + 6 million units IFN-a thrice weekly (n = 210).

Fig. 1. mTOR signaling and temsirolimus.Cell stimulation through various means initiatesan intracellular cascade that leads to the activationof the kinase mTOR, which has a key role in cellgrowth and proliferation, regulation of apoptosis,and angiogenesis.Temsirolimus binds toFK506-binding protein (FKBP), and the resultantprotein-drug complex inhibits the kinase activity ofthe mTORC1complex. PI3-K, phosphatidylinositol3-kinase; p70S6K, p70 S6 kinase; 4E-BP1,4E-binding protein-1; eIF-4E, eukaryotic initiationfactor-4 subunit E; HIFa, hypoxia-inducible factor a.

Temsirolimus, an Inhibitor ofmTOR

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A separate phase I trial had established the safety maximumtolerated dose of combination therapy with temsirolimus (21).The primary study end point of the phase III study was overallsurvival and the study was powered to compare each of thetemsirolimus-containing arms with the IFN-a arm. Patientstreated with temsirolimus had a statistically longer overallsurvival than IFN-a monotherapy patients (10.9 versus 7.3months; P = 0.0069; Table 1; ref. 1). There was also aprogression-free survival benefit from temsirolimus monother-apy versus IFN (median, 3.8 versus 1.9 months; P < 0.0001).The objective response rate was 9% in the temsirolimusmonotherapy arm. There was no survival advantage to thecombination therapy arm over IFN-a monotherapy, perhapsdue to the lower dose of temsirolimus delivered, which mayhave been inadequate for mTOR inhibition. These data lead toFood and Drug Administration approval of temsirolimus foradvanced RCC on May 31, 2007. Results from these studiesvalidated mTOR as a relevant therapeutic target in RCC, at leastin the subset of patients with multiple adverse risk features.Indeed, the study population was truly poor risk, with a third ofpatients without prior nephrectomy and 80% with a Karnofsky

performance status V70%. Recent subset analysis from this trialsupports a hypothesis of the greatest benefit of temsirolimus inthe poorest-risk patients and in patients with non–clear cellhistologies (22). These results must be interpreted cautiouslygiven small numbers and the retrospective nature but mayidentify the clinical phenotype associated with response to thisagent. The tumor biology of poor-risk and/or non–clear cellRCC patients that may account for a greater effect of mTORinhibition is unclear at present.Toxicity of this agent is best evaluated from the prospective

randomized RCC trial. Grade 3 adverse events occurred in 67%of the temsirolimus monotherapy group (lower than the othertwo treatment arms). The most frequently occurring temsiroli-mus-related grade 3 or 4 hematologic toxicities included anemiaand thrombocytopenia. Hypercholesterolemia, hyperlipidemiaand hyperglycemia were also more common in the temsi-rolimus arm, reflecting inhibition of mTOR-mediated lipid andglucose metabolism, and generally manageable with dietary ormedical management. The most frequently occurring grade 3adverse events in the temsirolimus arm were asthenia (11%),anemia (20%), and dyspnea (9%). Dyspnea may be due in part

Table 1. Select clinical trial results of temsirolimus

Disease Trial design Clinical outcome

RCC (1) Randomized phase III trial: temsirolimus ORR: 9%monotherapy vs IFN-a vs combinationtemsirolimus/IFN-a in poor-risk metastatic

PFS: 3.7 mo (vs 1.9 mo for IFN-amonotherapy arm; P = 0.0001)

RCC patients (n = 626) OS: 10.9 mo (vs. 7.3 mo for IFN-amonotherapy arm; P = 0.0069)

RCC (18) Randomized phase II: 25, 75, or 250 mg i.v. ORR: 7%weekly in treatment-refractory patients(n = 111)

PFS: 5.8 mo

Mantle cell lymphoma (13) Phase II in relapsed/refractory patients: ORR: 38%250 mg i.v. weekly (n = 35) PFS: 6.5 mo

Duration of response: 6.9 mo

Breast cancer (16) Randomized phase II: 75 or 250 mg i.v. ORR: 9%weekly in treatment-refractory patients PFS: 12 wk(1-2 prior regimens; n = 109) Duration of response: 6 mo

Extensive-stage small cell lung Randomized phase II: 25 or 250 mg i.v. ORR: 1.2%cancer (17) weekly in stable/responding patients after

induction chemotherapy (n = 87)PFS: 2.2 mo

Melanoma (14) Phase II trial of 25 mg i.v. weekly in treatment- ORR: 3%refractory patients (1-2 prior biotherapy/chemotherapy regimens; n = 33)

PFS: 10 wk

Neuroendocrine carcinoma (15) Phase II trial of 25 mg i.v. weekly(0-4 prior regimens; n = 37)

ORR: 5%; tumor burden reductionin 57% of patients

PFS: 6 mo

Glioblastoma (12) Phase II trial of 25 mg i.v. weekly(0-1 prior regimens; n = 65)

ORR: 0%; 36% of patients withregression*

PFS: 2.3 mo

Glioblastoma (11) Phase II trial of 250 mg i.v. weekly ORR: 5%(radiation-refractory and 0-3 prior chemotherapyregimens; n = 43)

PFS: 9 wk

Abbreviations: ORR, objective response rate; PFS, progression-free survival; OS, overall survival.*Regression defined as unequivocal reduction in size of contrast enhancement or decrease in mass effect.

CCRDrug Update

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to an underlying treatment-induced pneumonitis. Pulmonarytoxicity with sirolimus has been reported in the transplantpopulation as patchy or diffuse infiltrates accompanied bydyspnea/dry cough and either a restrictive pattern or reduceddiffusing capacity for carbon monoxide on pulmonary functiontesting (23). The phase III RCC trial reported six patients (5%)with nonspecific pneumonitis not related to dose. One retro-spective review of 22 patients with endometrial cancer or neu-roendocrine tumors enrolled on phase II trials of 25 mgtemsirolimus i.v. weekly identified 8 patients (36%) withradiographic changes on routine computed tomography scans(either ground glass opacification or parenchymal consolida-tion) consistent with possible drug-induced pneumonitis (24).Dyspnea and dry cough were observed in the 50% of patientswho had clinical symptoms. No specific treatment was given,and temsirolimus treatment was continued in four patientswithout reported worsening of pulmonary abnormalities. Theexact incidence of radiographic changes consistent with pneu-monitis, percentage of patients with clinically relevant symp-toms, and the mechanism of this toxicity is poorly defined atpresent. Immunosuppression is an additional potential toxicityof temsirolimus given the known immunosuppressive effects ofsirolimus. The phase III RCC trial, however, showed nosignificant difference in the incidence of neutropenia, lympho-penia, or infection versus the IFN control arm (1). In addition, alimited analysis of lymphocyte subsets and proliferativeresponses to nonspecific antigens did not show any significanteffect in temsirolimus-treated patients on a phase I trial (25).The weekly schedule of temsirolimus, in contrast to the dailyschedule of sirolimus, may have implications for effect ofimmune function. Additional experience in a greater number ofpatients and with longer-term treatment is required to fullycharacterize the effect of temsirolimus on immune variables andclinically relevant immune-mediated toxicity.As with any targeted agent, the presence of a viable target,

measurement of target inhibition, and defining the molecularphenotype of susceptible versus resistant tumors are criticalgoals to allow for rational drug development. Some initialefforts in this regard relevant to the mTOR pathway andtemsirolimus have been made. Evidence of mTOR pathwayactivation has been investigated through immunohistochemi-

cal staining of paraffin-embedded RCC samples with demon-stration of increased expression of phosphorylated Akt, mTOR,and S6 in the majority of samples (26–28). Target inhibitionhas been shown after temsirolimus administration by decreasedp70 S6 kinase activity as assayed in peripheral blood mono-nuclear cells (2) and inhibition of S6 phosphorylation asmeasured by immunohistochemistry of posttreatment neuro-endocrine tumors (15). Molecular determinants of responsehave also been investigated. Baseline expression of phosphor-ylated S6 (15, 29) or phosphorylated p70 S6 kinase (12) wasidentified as associated with objective response to temsirolimusin small populations of patients. Further, a decrease in p70 S6kinase activity in paired pre- and post-temsirolimus peripheralblood mononuclear cells was associated with a longer time totreatment failure (2). These translational efforts require expan-sion and validation in large prospective sample sets but sup-port target inhibition by temsirolimus and generate hypothesesabout prediction of response.

In summary, the mTOR pathway is likely critical across abroad spectrum of tumor types. Temsirolimus has shown anti-tumor activity, most notably in poor-risk advanced RCC where ademonstration of overall survival benefit has been observed. Arandomized trial of RAD001 versus placebo in treatment-refractory RCC has completed accrual and results are pending.The lack of significant antitumor effect of temsirolimus-mediated mTOR inhibition in some tumors, especially thosewith predicted sensitivity based on alterations such as PTENmutation, underscores the complex interplay of multiple signal-ing pathways within a single tumor. Other rapamycin analogueinhibitors of mTOR have also begun clinical testing. Everolimus(RAD001, Novartis) and AP23573 (Ariad Pharmaceuticals)have shown tolerability and preliminary antitumor activity insmall trials (30, 31). Potential differences in antitumor effect ortolerability among these agents due to route/schedule of admi-nistration, potency against mTOR, or other drug effects are notwell characterized at present. More potent or complete mTORinhibition (e.g., through agents that inhibit both mTORC1 andmTORC2), inhibition of multiple signaling pathways simul-taneously, and/or more precise molecular phenotyping oftumors to define mTOR pathway reliance are needed to buildon the clinical benefits of temsirolimus observed to date.

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References1. Hudes G, Carducci M,Tomczak P, et al. Temsirolimus,interferon alfa, or both for advanced renal-cell carcino-ma. NEngl JMed 2007;356:2271^81.

2. PeralbaJM,DeGraffenried L, FriedrichsW, et al.Phar-macodynamic evaluation of CCI-779, an inhibitor ofmTOR, in cancer patients. Clin Cancer Res 2003;9:2887^92.

3. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors ofmTOR in kidney cancer. Nat Med 2006;12:122^7.

4. Abraham RT, Gibbons JJ. The mammalian target ofrapamycin signaling pathway: twists and turns in theroad to cancer therapy. Clin Cancer Res 2007;13:3109^14.

5. Sabatini DM. mTOR and cancer: insights into a com-plex relationship. Nat Rev Cancer 2006;6:729^34.

6.BrugarolasJB,Vazquez F, ReddyA, SellersWR, KaelinWG, Jr. TSC2 regulates VEGF through mTOR-depen-dent and -independent pathways. Cancer Cell 2003;4:147^58.

7. Neshat MS, Mellinghoff IK, Tran C, et al. Enhancedsensitivity of PTEN-deficient tumors to inhibition of

FRAP/mTOR. Proc Natl Acad Sci U S A 2001;98:10314^9.

8. Podsypanina K, Lee RT, Politis C, et al. An inhibitorof mTOR reduces neoplasia andnormalizes p70/S6ki-nase activity in Pten+/- mice. Proc Natl Acad SciUS A 2001;98:10320^5.

9.Yu K,Toral-Barza L, Discafani C, et al. mTOR, a noveltarget in breast cancer: the effect of CCI-779, anmTOR inhibitor, in preclinical models of breast cancer.Endocr Relat Cancer 2001;8:249^58.

10. Raymond E, Alexandre J, Faivre S, et al. Safetyand pharmacokinetics of escalated doses of weeklyintravenous infusion of CCI-779, a novel mTOR inhibi-tor, in patients with cancer. J Clin Oncol 2004;22:2336^47.

11. Chang SM,Wen P, CloughesyT, et al. Phase II studyof CCI-779 in patients with recurrent glioblastomamultiforme. Invest New Drugs 2005;23:357^61.

12.Galanis E, BucknerJC,MaurerMJ, et al. Phase II trialof temsirolimus (CCI-779) in recurrent glioblastomamultiforme: a North Central CancerTreatment GroupStudy. JClin Oncol 2005;23:5294^304.

13.WitzigTE, Geyer SM, Ghobrial I, et al. Phase II trial ofsingle-agent temsirolimus (CCI-779) for relapsedman-tle cell lymphoma. JClinOncol 2005;23:5347^56.

14. Margolin K, Longmate J, BarattaT, et al. CCI-779 inmetastatic melanoma: a phase II trial of the CaliforniaCancer Consortium. Cancer 2005;104:1045^8.

15.Duran I, KortmanskyJ, SinghD, et al. A phase II clin-ical and pharmacodynamic study of temsirolimus inadvanced neuroendocrine carcinomas. Br J Cancer2006;95:1148^54.

16. Chan S, Scheulen ME, Johnston S, et al. Phase IIstudy of temsirolimus (CCI-779), a novel inhibitor ofmTOR, in heavily pretreated patients with locally ad-vanced or metastatic breast cancer. J Clin Oncol2005;23:5314^22.

17. Pandya KJ, Dahlberg S, HidalgoM, et al. A random-ized, phase II trial of two dose levels of temsirolimus(CCI-779) in patients with extensive-stage small-celllung cancer who have responding or stable diseaseafter induction chemotherapy: a trial of the EasternCooperative Oncology Group (E1500). J ThoracOncol 2007;2:1036^41.

CCRDrug Update

www.aacrjournals.orgClin Cancer Res 2008;14(5)March1, 2008 1290

18. Atkins MB, Hidalgo M, StadlerWM, et al. Random-ized phase II study of multiple dose levels of CCI-779,a novel mammalian target of rapamycin kinase inhibi-tor, in patients with advanced refractory renal cellcarcinoma. JClin Oncol 2004;22:909^18.

19. Motzer RJ, Bacik J, Murphy BA, Russo P, Mazum-dar M. Interferon-alfa as a comparative treatment forclinical trials of new therapies against advanced renalcell carcinoma. JClin Oncol 2002;20:289^96.

20. Mekhail TM, Abou-Jawde RM, Boumerhi G, et al.Validation and extension of the Memorial Sloan-Kettering prognostic factors model for survival inpatientswith previously untreatedmetastatic renal cellcarcinoma. JClin Oncol 2005;23:832^41.

21.Motzer RJ, Hudes GR, Curti BD, et al. Phase I/II trialof temsirolimus combined with interferon alfa for ad-vanced renal cell carcinoma. J Clin Oncol 2007;25:3958^64.

22. DutcherJP, Szczylik C,Tannir N, et al. Correlation ofsurvival with tumor histology, age, and prognostic risk

group for previously untreated patients with advancedrenal cell carcinoma (adv RCC) receiving temsirolimus(TEMSR) or interferon-a (IFN). Proc Am Soc ClinOncol 2007;25:5033.

23. PhamPT, PhamPC, Danovitch GM, et al. Sirolimus-associated pulmonary toxicity. Transplantation 2004;77:1215^20.

24. Duran I, Siu LL, Oza AM, et al. Characterisation ofthe lung toxicity of the cell cycle inhibitor temsirolimus.EurJCancer 2006;42:1875^80.

25.HidalgoM, BucknerJC, Erlichman C, et al. A phase Iand pharmacokinetic study of temsirolimus (CCI-779)administered intravenously daily for 5 days every 2weeks to patients with advanced cancer. Clin CancerRes 2006;12:5755^63.

26. Lin F, Zhang PL, Yang XJ, Prichard JW, Lun M,Brown RE. Morphoproteomic and molecular con-comitants of an overexpressed and activated mTORpathway in renal cell carcinomas. Ann Clin Lab Sci2006;36:283^93.

27. Pantuck AJ, Seligson DB, KlatteT, et al. Prognosticrelevance of the mTOR pathway in renal cell carcino-ma: implications for molecular patient selection fortargeted therapy. Cancer 2007;109:2257^67.

28. Robb VA, Karbowniczek M, Klein-Szanto AJ,Henske EP. Activation of the mTOR signaling path-way in renal clear cell carcinoma. J Urol 2007;177:346^52.

29. Cho D, Signoretti S, Dabora S, et al. Potential histo-logic andmolecular predictors of response to temsiro-limus in patients with advanced renal cell carcinoma.Clin Genitourin Cancer 2007;5:379^85.

30. Amato RJ, Misellati A, Khan M, Chiang S. A phaseII trial of RAD001in patients (Pts) with metastatic re-nal cell carcinoma (MRCC) [abstract 4530]. Proc AmSoc Clin Oncol 2006;24:4530.

31. Metcalf C, Bohacek R, Rozamus L, et al. Structure-based design of AP23573, a phosphorus-containinganalog of rapamycin for anti-tumor therapy [abstract2476]. Proc Am Assoc Cancer Res 2004;45:2476.