Ontario Institute for Cancer Research, MaRS Centre, South Tower, 101 College Street, Suite 800, Toronto, ON M5G 0A3, Canada. janet.dancey@ oicr.on.ca
mToR signaling and drug development in cancer Janet Dancey
abstract | Mammalian target of rapamycin (mTOR) is a protein kinase of the PI3K/Akt signaling pathway. Activation of mTOR in response to growth, nutrient and energy signals leads to an increase in protein synthesis, which is required for tumor development. This feature makes mTOR an attractive target for cancer therapy. First-generation mTOR inhibitors are sirolimus derivatives (rapalogs), which have been evaluated extensively in cancer patients. Everolimus and temsirolimus are already approved for the treatment of renal-cell carcinoma. Temsirolimus is also approved for the treatment of mantle-cell lymphoma. These drugs, in addition to ridaforolimus (formerly deforolimus) and sirolimus, are currently being evaluated in clinical trials of various cancers. Second-generation mTOR inhibitors are small molecules that target the kinase domain, and have also entered clinical development. Clinical trials are underway to identify additional malignancies that respond to mTOR inhibitors, either alone or in combination with other therapies. Future research should evaluate the optimal drug regimens, schedules, patient populations, and combination strategies for this novel class of agents.
Dancey, J. Nat. Rev. Clin. Oncol. 7, 209–219 (2010); published online 16 March 2010; doi:10.1038/nrclinonc.2010.21
Introductionmammalian target of rapamycin (mtor) is a protein kinase ubiquitously expressed within cells and a vali-dated target in the treatment of cancer. sirolimus (also known as rapamycin) was originally used as an anti-fungal agent, but it also has immunosuppressive and anti proliferative properties. sirolimus, the first identi-fied inhibitor of mtor, and three derivatives (termed rapalogs)—temsirolimus, everolimus and ridaforolimus (formerly deforolimus)—are currently being developed as anti cancer agents. these first-generation mtor inhibitors form a complex with the intracellular recep-tor FK506 binding protein 12 (FKBP12), which interferes with mtor activity. second-generation mtor inhibi-tors are small-molecule mimetics of atP that target the mtor kinase domain and have also entered clinical trials (table 1). these second-generation kinase inhibi-tors might have some degree of specificity for mtor, or could inhibit mtor, phosphatidylinositol 3-kinase (Pi3K) and other Pi3K-related protein kinases.
as monotherapy, rapalogs have antitumor activity with mild toxic effects. temsirolimus and everolimus are already approved for the treatment of patients with metastatic renal-cell carcinoma (rCC), and temsiro-limus is also approved for mantle-cell lymphoma (mCl). multiple trials of single agents and combination regimens involving mtor inhibitors are currently underway to identify and improve the use of these drugs. this review addresses the mechanism of action and clinical experience of mtor inhibitors.
The PI3K/Akt/mTOR signaling pathwaythe Pi3K/akt/mtor signaling pathway regulates cell proliferation, survival and angiogenesis. an important role has also been identified in the response of cells to hypoxia and energy depletion.1 noncancerous cells, such as lymphocytes, endothelial cells and fibroblasts, as well as cancer cells depend on this signaling pathway. aberrant activation of this pathway has been linked to the development of cancer.1
mtor is a member of the Pi3K-related protein kinase (PiKK) family, which includes atm, atr, Dna-dependent protein kinase catalytic subunit, and smG1.2,3 PiKKs share a number of conserved regions, including the kinase catalytic domain. mtor also has a binding domain that sirolimus binds to in a complex with FKBP12. while other PiKK family members are involved in Dna and mrna surveillance and repair pathways, mtor integrates signals from growth factors and nutrients to promote cell growth, proliferation, and survival. atP, amino acids and growth factor signals from the Pi3K/akt pathway modulate mtor function. mtor signaling is upregulated in benign and malignant neoplastic dis-orders and drugs targeting mtor activity are, therefore, anticipated to be useful for treating these conditions.
mtor activities are mediated by its binding to different proteins to produce two notable complexes—mtorC1 and mtorC2—which have distinct functions.4,5 mtorC1 contains raptor (regulatory- associated protein of mtor). activation of Pi3K and akt inhibits hamartin and tuberin repression of rheb (ras homolog enriched in brain), which leads to mtorC1 activation and phos-phorylation of ribosomal protein s6 kinase 1 (s6K1) and eukaryotic translation initiation factor 4e-binding-protein-1 (4e-BP1). negative regulators of mtor include
competing interestsThe author declares an association with the following company: Wyeth. See the article online for full details of the relationship.
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FKBP8, which prevents rheb from activating mtorC1, and proline-rich akt1 substrate 1, which competes with raptor for binding to s6K1 and 4e-BP1. when intra-cellular atP is depleted relative to amP, amP-activated kinase and its upstream regulator serine/ threonine-protein kinase 11 (stK11) phosphorylate tuberin, which inactivates rheb and mtorC1 signaling. Hypoxia and low amino acid levels also negatively regulate mtor. mtorC1 modulates several downstream signaling effectors and transcription factors, which influences cell proliferation, survival and angiogenesis. mtorC1 also regulates translation, ribosome biogenesis, metabolism, and cellular response to hypoxia and autophagy.
By contrast, the mtorC2 complex contains rictor (rapamycin-insensitive companion of mtor) and phos-phorylates akt at serine 473 (ser473), increasing the degree of akt activation. akt is pivotal in mtor signal-ing, as it is both an upstream activator of mtorC1 and downstream effector of mtorC2. in addition to aug-menting akt activation, mtorC2 signaling is involved in the organization of the actin cytoskeleton. mtor is phos-phorylated differentially when associated with mtorC1 and mtorC2.6 additional details of the mtor pathway are provided elsewhere and in Figure 1.7,8
Feedback loops exist within the mtor signaling pathway. For example, s6K1 activation exerts negative
Key points
Mammalian target of rapamycin (mTOR) is a central regulator of cell ■proliferation in response to growth factors and energy stimuli
In some tumor types, such as renal-cell carcinoma and certain lymphomas, ■mTOR has a key role in tumor cell proliferation and angiogenesis
Temsirolimus and everolimus administered as single agents are associated ■with substantial improvements in patients with advanced renal-cell carcinoma, and are approved for use in this indication
Temsirolimus is also approved for use in mantle-cell lymphoma, where it has ■shown a notable improvement in progression-free survival
Biomarkers that assess pathway activation are being explored to identify tumor ■types that are sensitive to mTOR inhibition
Combinations of targeted therapies could improve outcomes by augmenting ■anti-tumor activity and overcoming mechanisms of resistance
feedback to restrict insulin and insulin-like growth factor 1 (iGF-1) signaling.9 insulin receptor sub-strates 1 and 2 (irs-1 and irs-2) link insulin and iGF-1 signaling to activate Pi3K, akt and mtor. s6K1 phos-phorylates irs-1 and irs-2, which destabilizes these proteins and prevents iGF-1/insulin signaling to Pi3K.9 loss of this negative feedback and induction of other pathways, such as mitogen-activated protein kinase (maPK),10 occur in cells and tumors exposed to rapa-logs. this response might limit the antitumor effects of mtor inhibition with these agents, and a number of potential drug combinations are currently being investi gated (Figure 2).11
the biochemical effects of mtor signaling are complex, incompletely understood and potentially context specific.12,13 rapalogs associate with FKBP12 and preferentially disrupt mtorC1 whereas small-molecule mtor kinase inhibitors target both mtor complexes. inhibition with rapalogs and mtor kinase inhibitors is likely to differentially interfere with these processes, and the activity and toxic effects of these drugs will, therefore, probably differ.
The mTOR pathway and canceralthough mutations have not been reported in mtor in human cancers, deregulation of upstream pathway effec-tors can lead to hyperactivation of the mtor protein (Box 1). activation of Pi3K, akt and growth factor recep-tors that occur as a result of mutations, amplification or overexpression of relevant genes, are found in different tumor histologies. similarly, the loss of tumor suppres-sors that regulate the Pi3K/akt/mtor pathway, such as phosphatase and tensin homolog (Pten), hamartin and tuberin, and stK11, are linked to the development of hamartoma syndromes, including tuberous sclerosis, Peutz–Jeghers syndrome, and Cowden disease.14 in a number of in vitro cell-line and in vivo murine xenograft models, aberrant pathway activation through oncogene stimulation or loss of tumor suppressors contributes to tumor growth, angiogenesis, metastasis, and resistance to standard cancer therapy.14 these features are relevant for the development of cancer therapeutics as aberrant pathway activation could increase sensitivity to agents that target mtor.
Mechanisms of mTOR inhibitionin addition to sirolimus, three rapalogs are now being used in humans. temsirolimus, everolimus and rida-forolimus differ from the parent structure of sirolimus at position C-42, which is modified to increase solubi lity and bioavailability by the addition of an ester, ether, or phosphonate group, respectively.15 all rapalogs bind to FKBP12 and preferentially inhibit mtorC1 functions (Figure 1). Few preclinical studies have assessed rapalogs under the same experimental conditions, but initial results suggest that the spectra of clinical activity and toxic effects of these drugs are similar under comparable conditions of exposure.16–19 the mtor kinase inhibitors seem to inhibit both mtorC1 and mtorC2 functions and are active in rapamycin-insensitive cell lines (Figure 1).20,21
Table 1 | mTOR inhibitors in clinical development
agent company Target Status
Rapalogs (first-generation inhibitors)
Temsirolimus Wyeth mTORC1 Approved
Sirolimus Wyeth mTORC1 Approved
Everolimus Novartis mTORC1 Approved
Ridaforolimus Ariad Pharamceuticals/ Merck mTORC1 Phase III
Phosphorylation effectsrapalogs and mtor kinase inhibitors share certain biochemical and cellular effects. Both classes of agents reduce downstream mtor functions in diverse carci-noma, lymphoma and glioblastoma cell lines.14 they inhibit phosphorylation of s6K1 and 4e-BP1 by mtorC1, which alters the translation of certain mrnas, especially those involved in the regulation of cell-cycle progression. these agents also inhibit protein transla-tion and cancer cell proliferation, induce G1 cell-cycle arrest and apoptosis in some cell lines, and reduce angio genesis.22–25 studies conducted with in vitro and in vivo models show that rapalog- mediated inhibition of mtorC1 leads to increased rates of mtorC2 phospho-rylation of akt ser473 in some models.26,27 By contrast, small-molecule kinase inhibitors compete with atP in the kinase catalytic site and inhibit the kinase-dependent functions of both mtorC1 and mtorC2,25 particularly the phosphorylation of akt ser473. since mtor acti-vities are mediated by effects on its kinase and binding actions,26 the different ways rapalogs and kinase inhibi-tors interact with mtor will determine the antitumor activities and toxicities of these drugs.
the inhibition of mtorC1 and subsequent phos-phorylation of akt ser473 by mtorC2 was thought to be a potential mechanism of resistance to rapalogs and was one reason for developing mtor kinase inhibitors. one small, single-arm, open-label clinical trial of siro-limus in 15 patients with glioblastoma has suggested that enhanced phosphorylation of akt, which was identified in seven patients, may be associated with shorter time to progression.28 the effects of rapalogs on mtor com-plexes, however, are not so clear-cut. rapalogs inhibit both mtorC1 and mtorC2 under certain experimental conditions,26,27 and the enhanced phosphorylation of akt ser473 seen in the presence of rapalogs does not correlate with the antiproliferative effects of these drugs.21 there are a number of possible explanations for these discordant reports. First, mtorC1 contains mtor phosphorylated predominantly on ser2448, whereas mtorC2 contains mtor phosphorylated predominantly on ser2481. when the latter was used as a marker, mtorC2 formation was shown to be sensitive to sirolimus in several cancer cell lines that had been previously reported to be sirolimus insensitive.27 second, in a large panel of cell lines from different tumors, the antiproliferative response to evero-limus correlated with the basal phosphorylation of akt ser473 and some akt targets.21 By contrast, everolimus-induced akt ser473 phosphorylation did not correlate with anti proliferative effects.21 these data suggest that the divergent sensiti vities of cell lines to rapalogs and kinase inhibitors might not be entirely due to rapalog-induced akt ser473 phosphorylation.
concentration-dependent effectsthe inhibition of mtorC1 and mtorC2 by rapalogs may be concentration dependent. while sirolimus can suppress both mtorC1 and mtorC2 in certain cancer cell line models,27,29 it does so at different concentrations: mtorC1 is suppressed by low nanomolar concentrations
of sirolimus, whereas mtorC2 generally requires low micromolar concentrations. the low-dose inhibitory effect on mtor signaling is likely to be mediated by binding of rapalogs to FKBP12 and the effects on s6K1 and 4e-BP1 activation. at clinically relevant concentra-tions (iC50 >1.0 μmol/l), temsirolimus suppressed prolif-eration of a broad panel of tumor cells.29 this high-dose drug effect did not require binding of temsirolimus to FKBP12 and correlated with an FKBP12-independent suppression of mtor signaling, leading to profound translational repression in a number of cancer cell lines, such as lung, colon, prostate and breast.29
Phosphatidic acidthe concentration-dependent efficacy of rapalogs could relate to the intracellular level of phosphatidic acid, which is required for the assembly of both mtorC1 and mtorC2.30 Phosphatidic acid is generated through hydrolysis of phosphatidylcholine by phospho lipase D. Phosphatidic acid interacts with mtor in a manner that is competitive with sirolimus, and elevated phospholipase D
Figure 1 | mTOR signaling pathways. mTOR forms complexes with other proteins, including Raptor (forming mTORC1) or Rictor (forming mTORC2). ATP, amino acids and signals from the PI3K/Akt pathway modulate mTOR function. Activation of PI3K and Akt inhibits hamartin and tuberin repression of Rheb, which leads to mTORC1 activation and phosphorylation of S6K1 and 4E-BP1. Akt is pivotal in mTOR signaling, as it is both an upstream activator of mTORC1 and downstream effector of mTORC2. Negative regulators of mTOR include FKBP8, which prevents Rheb from activating mTORC1, and PRAS40, which competes with Raptor for binding to S6K1 and 4E-BP1. When intracellular ATP is depleted relative to AMP, AMPK and its upstream regulator STK11 phosphorylate tuberin, which inactivates Rheb and mTORC1 signaling. Hypoxia and low amino acid levels also negatively regulate mTOR. Rapalogs associate with FKBP12 and preferentially disrupt mTORC1 whereas small-molecule mTOR kinase inhibitors target both mTOR complexes. Abbreviations: AMPK, AMP-activated kinase; 4E-BP1, eIF4E-binding protein 1; FKBP12, FK506 binding protein 12; GBL, G protein beta subunit-like; mTORC, mammalian target of rapamycin complex; PI3K, phosphatidylinositol 3-kinase; PRAS40, proline-rich Akt1 substrate 1; PTEN, phosphatase and tensin homolog deleted on chromosome 10; S6K1, p70 S6 kinase 1; Sin1, stress-activated protein kinase interaction protein 1; STK11, serine/threonine-protein kinase 11; TSC, tuberous sclerosis complex.
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activity confers resistance. if phospha tidic acid levels are suppressed, mtorC2 becomes sensi tive to low micro-molar concentrations of sirolimus that can be achieved clinically. these results suggest that phosphatidic acid is a determinant of rapalog sensitivity.
Rapalogs in clinical trialsrapalogs have proven clinical benefit as immuno-suppressants and are used to prevent rejection in organ transplantation, in eluting stents to prevent coronary artery reocclusion, and as inhibitors of cancer growth. the ability of rapalogs to affect tumor and stromal cells might contribute to their clinical antitumor activity and toxic effects. rapalogs are currently being evaluated across a broad range of human neoplasia in addition to the approved indications.
Dose, schedule and pharmacologyalthough rapalogs have different formulations, recom-mended doses, schedules and pharmacology (table 2), they have similar biochemical activities in vitro at com-parable concentrations and exposures.1,2,14 sirolimus and everolimus are oral formulations. temsirolimus
and ridaforolimus were initially tested with intravenous formulations; oral formulations subsequently entered clinical development. intermittent and continuous daily dosing schedules have been evaluated. results show that intermittent dosing limits immunosuppression, whereas long-term, continuous schedules maximize target inhibi-tion. toxic and other pharmacodynamic effects, such as inhibition of the phosphorylation of downsteam proteins s6K1 and 4e-BP1, are similar for rapalogs and mtor kinase inhibitors used at different schedules.
the recommended phase ii doses of the agents are suf-ficient for target inhibition based on extrapola tion from preclinical models, pharmaco dynamic effects measured in normal and tumor tissue, and toxic effects. the recom-mended doses of temsiro limus and everolimus are below the maximum tolerated doses, whereas for ridaforolimus, the recommended dose is the maximum tolerated dose (table 2). all the agents inhibit phosphorylation of s6K1 and/or 4e-BP1 in skin, blood mononuclear cells, and tumor tissue.31–33 whether higher doses lead to greater antitumor activity or whether differences in outcomes are unrelated to the drug’s ability to induce toxic effects, requires further evaluation. emerging data from phase ii and iii trials suggest, however, that antitumor effects will be dependent on schedule and dose for some agents.34,35
sirolimus and the rapalogs share certain pharmaco-logic properties. these include high blood-to-plasma ratios, increased total clearance as the dose increases, and, for oral agents, peak concentrations and exposure (area under the curve for plasma concentration versus time) that increase less than proportionally with dose.31–33 the terminal plasma half-lives of these agents are long, ranging from 20–50 h. In vitro, both temsirolimus and sirolimus bind to FKBP12 and exert similar anti-proliferative effects. when temsirolimus is administered to patients, the ester moiety is hydrolyzed to yield siro-limus, which has a longer terminal half-life (12–15 h for temsirolimus versus 40–50 h for sirolimus).36 rapalogs easily cross the blood–brain barrier owing to their high lipophilic indices and could, therefore, be of value for the treatment of primary and secondary malignancies of the central nervous system.37 sirolimus, temsirolimus and everolimus are metabolized by the cytochrome P450 enzyme CYP3a4, and metabolites are mainly excreted through the gastrointestinal tract. the bioavailability of oral sirolimus and everolimus is low, approximately 14% and 15–30%, respectively.38–40 the efficiency of absorption of orally administered drugs is modulated by p- glycoprotein.41 the coadministration of siro-limus, temsiro limus or everolimus with strong inhibi-tors or inducers of CYP3a4 and p-glycoprotein (for oral agents), should be avoided owing to the risk of drug–drug interactions that may alter the metabolism of one or both drugs.
Toxic effectsall the rapalogs in clinical development are well- tolerated. Common toxic effects include skin reactions, stomatitis, thrombocytopenia, diarrhea, fatigue, hyperlipidemia and hyperglycemia. less common effects include renal
Figure 2 | mTOR pathway feedback loops. A negative feedback loop from the mTORC1/S6K1 pathway inhibits growth factor signaling to PI3K. IRS-1 links the insulin receptor and IGF-1 signaling to PI3K, which leads to activation of Akt and mTOR. S6K1 phosphorylates IRS-1, which destabilizes this protein and uncouples IGF–insulin signaling to PI3K. Thus, mTOR/S6K1 activation exerts negative feedback to restrict insulin and IGF-1 signaling. Loss of this negative feedback mechanism has been shown to occur in cells and tumors exposed to rapamycin. Rapalogs preferentially inhibit mTORC1, which leads to mTORC2 assembly and an increase in phosphorylation of Akt Ser473. mTOR inhibition also leads to an increase in Ras pathway activation.105 Abbreviations: 4E-BP1, eIF4E-binding protein; FKBP12, FK506 binding protein 12; GBL, G protein beta subunit-like; IGF-1, insulin-like growth factor 1; IRS-1, insulin receptor substrate 1; mTORC, mammalian target of rapamycin complex; PI3K, phosphatidylinositol 3-kinase; PRAS40, proline-rich Akt1 substrate 1; PTEN, phosphatase and tensin homolog deleted on chromosome 10; S6K1, p70 S6 kinase 1; Sin1, stress-activated protein kinase interaction protein 1; TSC, tuberous sclerosis complex.
insufficiency, peripheral edema, interstitial pneumoni-tis and infections.42 these adverse effects are generally mild to moderate in severity (grade 1–2) and reversible with interruption of dosing or with specific treatment for hyperlipidemia and hyperglycemia
results from laboratory and clinical studies suggest that the occurrence of infections and pneumonitis could be related to the drug, dose, schedule and clinical setting. in preclinical models, intermittent dosing of sirolimus reduced immunosuppression while maintaining effec-tive delay in tumor growth.43,44 results from phase iii trials, however, indicate that the overall risk of infection is twofold higher with chronic daily administration of everolimus and with weekly administration of tem siro-limus than with controls of placebo or interferon, respec-tively (table 3);34,45,46 however, severe and opportunistic infections are rare. Clinical trials evaluating dose and schedule suggest that the risk of pneumonitis could be greater with higher trough drug concentrations and with prolonged exposures achieved with chronic daily dosing versus intermittent weekly dosing. in a randomized study of everolimus in patients with breast carcinoma, toxic effects—in particular, pulmonary effects—seemed to be dose and schedule-related; 41% patients had pulmo-nary toxic effects on 10 mg daily versus 13% of patients on 70 mg weekly.35 objective response rates were 10% and 0%, respectively, for the daily and weekly schedules. interstitial lung disease resolved upon drug discontinu-ation or dose reduction, or with corticosteroid treatment in most cases. Differences in drug dose, schedule, and pharmaco kinetics, or unidentified differences in uptake and effects on target, could explain the toxic effects of the agents seen in clinical trials.
Post-transplant malignancyPost-transplant malignancy, morbidity and mortality are important limitations in organ transplantation, and risk of occurrence could be reduced with rapalogs.47 Clinical studies have shown a lower incidence of new malignan-cies in organ transplant recipients who were given siro-limus.47 Post-transplant lymphoproliferative disorders, Kaposi sarcoma and nonmelanotic skin malignancies might regress after patients are treated with sirolimus, especially if lesions are small and low grade.48 treatment with mtor rapalogs could prevent immunological rejection and reduce the risk of secondary cancer in this population.
Hamartoma syndromesHamartomas are benign, tumor-like nodules usually found on the skin and mucous membranes. evidence from clini-cal trials demonstrates that rapalogs successfully prevent or delay morbidity associated with hamar toma syndromes, which are linked to aberrant mtor pathway activation. inactivation of the tumor suppressors Pten, the tuberous sclerosis proteins hamar tin, tuberin, and stK11, results in the development of Cowden syndrome, tuberous sclerosis, and Peutz-Jeghers syndrome, respectively.49
Deregulation of mtor seems to be critical to the pathogenesis of tuberous sclerosis, the related lung disease
lymphangioleiomyomatosis,50 and in neoplasias that arise from germline PTEN loss. mtor inhibition seems to have therapeutic effects in these conditions. Phase i and ii clinical trials of rapalogs have demonstrated reductions in renal angiomyolipomas associated with tuberous sclero-sis and lymphangioleiomyomatosis, and improvement in lung function.51,52 a patient with Proteus syndrome, a hamartoma syndrome associated with PTEN mutations, experienced sustained normalization of respiratory and gastrointestinal function, and regression of mediastinal and mesenteric tumors after starting treatment.53 larger trials of mtor inhibitors for treatment of renal, lung and brain manifestations of disease associated with hamartin and tuberin are underway.50
mTOR inhibitors and cancerrCC was the first cancer for which an mtor inhibi-tor was approved. Clear-cell rCC, the most common subtype, is associated with loss of function of the von Hippel–lindau gene (VHL) and overproduction of hypoxia-inducible factor (HiF), deregulation of which correlates with high levels of veGF in rCC. the bene-ficial effects of mtor inhibitors are probably partly because of their ability to downregulate HiF, as the expression of HiF is under mtor regulation.54 Both
Box 1 | Proto-oncogenes and tumor suppressor genes in the mTOR pathway
Proto-oncogenesMutations and amplifications of growth factor receptor proteins, such as ■EGFR, HER2, FGFR and c-kit have been described in multiple tumors including breast, lung, endometrial, glioblastoma, gastrointestinal stromal tumors and melanoma
Aberrantly high PI3K ■ activity is implicated in cell transformation, tumor progression and treatment resistance
PIK3CA ■ amplification has been observed in a variety of human cancers, including breast, cervical, uterine, ovarian, gastric, thyroid, lung, oral and HNSCC
PIK3R1 ■ is mutated in glioblastoma, colon cancer and ovarian cancer
Amplification of the ■ Akt genes are observed in gastric cancer, glioblastomas and gliosarcomas (Akt1), and breast, ovarian, pancreatic, gastric and HNSCC (Akt2). Akt1 mutation leads to breast, ovarian, HNSCC and pancreatic cancer
eIF4E ■ amplification is found in breast and head and neck cancer
S6K1 ■ amplification is found in breast cancers
Cyclin D ( ■ CCND1) amplification and translocation is seen in breast cancer and mantle-cell lymphoma
Tumor suppressorsGermline ■ PTEN loss is found in hamartoma tumor syndromes (Cowden disease, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Duclos disease)
PTEN ■ loss through mutation, deletion or hypermethylation is found in a large fraction of advanced human cancers, such as breast, gastric, glioblastoma, HNSCC, lung, renal, prostate, ovarian, uterine, endometrial, cervical, melanoma, thyroid, hepatocellular and astrocytoma
Germline ■ TSC1 or TSC2 loss leads to tuberous sclerosis complex characterized by the formation of hamartomas in many organs. Somatic mutations of these genes have been reported in endometrial carcinoma
Germline ■ STK11 loss leads to Peutz-Jeghers syndrome, characterized by hamartomas in the gastrointestinal tract. Somatic mutations of STK11 have been reported in lung, pancreatic and biliary cancers and melanomas
Abbreviations: FGFR, fibroblast growth factor receptor; HNSCC, head and neck squamous cell carcinoma; PI3K, phospatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog.
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temsirolimus and evero limus have demonstrated clini-cal benefit for patients with rCC (table 4). in first-line treatment of rCC patients with poor prognosis, temsiro-limus improved overall survival compared with inter-feron (hazard ratio for death 0.73, 95% Ci 0.58–0.92, P = 0.008).46 everolimus improved progression-free survival compared with placebo in rCC patients with tumors that had progressed after sunitinib and/or sorafenib therapy.45 For the optimal clinical applica-tion of mtor inhibitors, further evaluation of the dose, schedule and sequence of administration of agents alone and in combination is needed.
temsirolimus has demonstrated clinical benefit in mCl. mCl is characterized by constitutive activation of akt and overexpression of cyclin D1 due to t(11;14)(q13;q32) translocation. in preclinical studies, inhibition of mtor signaling by sirolimus resulted in cyclin D1 downregulation.55 a phase iii study assigned 162 patients with relapsed or refractory mCl to receive one of two temsirolimus regimens: 175 mg weekly for 3 weeks fol-lowed by either 75 mg (175/75 mg) or 25 mg (175/25 mg) weekly, or the investigator’s choice therapy.34 Progression-free survival, response rate and overall survival favored the higher-dose regimen compared with the control. of particular note, patients treated with higher-dose temsiro limus had significantly longer progression-free survival than those treated with investigator’s choice of
standard chemotherapy (hazard ratio 0.44, P = 0.0009). Patients treated with lower-dose temsirolimus showed a non significant trend towards longer progression-free sur-vival than controls (hazard ratio 0.65, P = 0.0618). median progression-free survival was 4.8, 3.4, and 1.9 months for the temsirolimus 175/75 mg, 175/25 mg, and standard chemotherapy, respectively. the results from this trial not only demonstrate the positive effect of temsirolimus on mCl, but they also show a dose–response relationship for temsirolimus in this disease setting that could be relevant in other diseases.
everolimus (10 mg orally, daily)56 and temsirolimus (25 mg intravenously, weekly)57 have since been evalu-ated in other lymphomas with promising results. in a single-arm study of 145 patients with non-Hodgkin lym-phomas and Hodgkin disease, the everolimus-induced objective response rate was 33% (48 of 145 patients). the median time to progression for all 145 patients was 4.3 months (95% Ci 3.6–5.9 months). the median dura-tion of response for the 48 responders was 6.8 months (95% Ci 5.4–11 months). a similar objective response rate of 35% (26 of 74 patients) was seen in a study of patients with B-cell non-Hodgkin lymphoma treated with temsirolimus.57 median progression-free survival was 4 months and the median duration of response was 3.8 months. a phase iii study has been initiated to assess the efficacy of everolimus in patients with diffuse large B-cell lymphoma who have achieved a complete remis-sion with the combination of first-line rituximab and chemotherapy but who are at high risk of recurrence. temsirolimus is under evaluation in combination with standard agents in patients with lymphoma.
antitumor activity among patients with a variety of malignancies has been reported with all rapalogs used as single agents (table 5). the highest reported objec-tive response rates have been seen in lymphomas56,58,59 and previously untreated endometrial carcinoma.60 in other disease settings, rates of response have been low, although rates of stable disease, time to progres-sion and survival have been promising compared with historical clinical trial data in soft-tissue sarcoma,61,62
Table 2 | Features of rapalogs
Rapalog alternative name and manufacturer
c-42 substitution Formula Molecular weight
Formulation Maximum administered dose
Recommended dose/schedule
Dose-limiting toxic effects(grade >3)
Sirolimus Rapamycin, Wyeth
Not applicable C51H79NO13 913.5 Oral Not reported 2–5 mg per day Stomatitis
neuro endocrine tumors,63,64 and previously treated endo-metrial carcinoma.65,66 Phase iii trials testing everolimus and ridaforo limus in neuroendocrine and soft-tissue sarcoma, respectively, are underway. minimal activity has been seen in trials of patients with lung cancer,67–69 pancreatic carcinoma,70,71 melanoma,72,73 glioma74,75 or leukemia,76,77 which suggests that only a subset of patients are likely to have tumors sensitive to single mtor inhibit ing agents. thus, the identification of sensiti-vity or resistance markers and development of effective combination regimens will be important for improving patient outcomes.
Improvement of clinical outcomeswhile it is clear that certain tumor types are sensitive to mtor inhibition, only a minority of patients benefit from treatment and none have been cured. Four strate-gies could improve outcomes achieved with mtor inhib-itors: optimization of drug administration, identifi cation of patients who are most likely to benefit, identifi cation of drug combinations that increase activity or overcome mechanisms of resistance, and development of more effective mtor inhibitors.
Drug administrationthe rapalogs differ in formulation, administration, dose and schedule, which results in different drug exposures. in addition, genetic and phenotypic variations between patients might lead to differences in bioavailability and metabolism of mtor agents mediated through CYP3a4 and p-glycoprotein, especially for oral agents. whether these differences are clinically meaningful remains an important question to be addressed in clinical trials.
there are compelling scientific and economic reasons to evaluate drugs, doses and schedules. sirolimus might be the least expensive agent to utilize as its patent has expired, but the poor bioavailability of this drug and lack of patent are frequently cited as reasons why it is not being widely studied as an anticancer agent. the modi-fied oral rapalogs might have better bio availability, and
for temsirolimus,2,28 higher concentrations are clinically achievable compared with sirolimus. Clinical data also strongly suggest a dose-related response to temsirolimus favoring high intermittent dosing in mCl.34 Continuous administration of low-dose oral everolimus, however, seems more effective than high-dose inter mittent schedules, which may in part be related to reduced bio-availability and exposures achieved with higher doses, but also greater toxicity.47
it is important to establish the most-efficacious dose and schedule for each drug, but the most cost- effective treatment must also be determined. newer rapalogs should be favored over sirolimus because of their pharma-cology and activity rather than simply for their patent protection. optimal dosing of individual patients for a given rapalog could be determined through assessment of blood concentrations, functional assays or imaging to evaluate target modulation. the question of preferred drug, dose and schedule might only be answered through randomized, comparative trials.
Markers of sensitivity and resistanceinhibition of a target is necessary for drug acti vity, although not always sufficient for clinical benefit. assessment of the inhibition of s6K1 or 4e-BP1 phos-phorylation in white blood cell samples from patients treated with rapalogs demonstrated that inhibition of mtor correlates with blood levels of the agents.31,78–80 in addition, imaging with fluorodeoxyglucose Pet could be a useful method to assess pharmacodynamic features of inhibition, as glucose uptake and Glut1 levels are affected by mtor inhibition. although phosphory-lation of s6K1 or 4e-BP1 is not consistently predictive of response to mtor inhibition, these proteins could act as potential biomarkers to determine inhibition in indivi dual patients.81,82
sensitivity to mtor inhibitors is thought to be related to deregulation of critical elements of the pathway. the molecular processes involved in mtor activation, however, are probably tumor specific, such as vHl loss
Table 4 | Clinical trials of mTOR inhibitors in RCC
Reference Phase Treatment number of patients
Setting objective response rate (%)
Progression free survival (months)
Hazard ratio (ci 95%)
overall survival (months)
Temsirolimus
Atkins et al. (2004)106
IIb Temsirolimus 111 Second-line 7 5.8 NA 15
Hudes et al. (2007)46
III IFN-αTemsirolimusTemsirolimus + IFN-α
626 First-line, poor prognosis
59, NS8, NS
3.15.54.7
0.66 (0.5, 0.8),P <0.001NS
7.310.98.4
Everolimus
Amato et al.(2009)107
II Everolimus 41 First-line, second-line
14 11.2 NA 22.1
Motzer et al.(2008)45
III EverolimusPlacebo
362 Second-line, third-line
30
41.9
0.30(0.2–0.4), P <0.0001
Not reached8.8
Abbreviations: IFN, interferon; NA, not applicable; NS, not significant; RCC, renal-cell carcinoma.
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of function in rCC, overexpression of cyclin D1 in mCl and loss of Pten function in endometrial carcinoma and in mCl. results from preclinical models suggest that genetic alterations and aberrant protein expression resulting in pathway activation correlate with sensiti-vity to mtor inhibition.1,14 Germline mutations in or methylation of the tumor suppressor genes PTEN, TSC and STK11 probably contribute to pathway activation and subsequent sensiti vity to sirolimus in patients with neoplasms associ ated with hamartoma syndromes.49 Despite extensive clinical evaluation, no biomarkers have been identified in patients with sporadic cancers. this outcome could be due to numerous genetic abnor-malities, feedback loops and crosstalk between signal-ing pathways involved in malignant transformation, which might determine activity of mtor inhibitors. a broader and more systematic interrogation of mutations and amplification of oncogenes, loss of tumor suppressor
genes, and gene-expression profiling are required to identify clinically relevant markers to select patients for treatment.83
combinations of targeted agentssynergistic antitumor effects have been observed when rapalogs are combined with conventional therapeutic agents, radiation and other targeted agents.1,2,84–86 ideally, targeted combinations should augment inhibition within a pathway, across pathways (circumventing resistance to mtor inhibition), or target several tumor processes. in addition, these combination regimens should use agents with differing mechanisms of action and adverse effect profiles, which would allow dosing for maximum target inhibition and interaction. since signaling pathways and their interactions might be context specific, differ-ent combination strategies might be required depen-dent on the setting. to date, strategies have focused on
Table 5 | Summary of phase II trials with rapalogs
Reference Tumor type number of patients
Dose (mg), schedule
objective response rate (%)
Median progression-free survival (months)
Chan et al. (2005)108 MBC 109 75 weekly250 weekly
9.2 3
Witzig et al. (2005)58 MCL 35 250 weekly 38 6.5
Ansell et al. (2006)59 MCL 29 25 weekly 41 6
Oza et al. (2006)60 EC (no prior treatment) 19 25 weekly 25 NR
Oza et al. (2008)65 EC (prior treatment) 27 25 weekly 7.4 NR
Pandya et al. (2007)67 SCLC 87 25 weekly250 weekly
improving inhibition within and between pathways, and of the mtor and veGFr pathways.
a substantial amount of preclinical data suggest that lack of response to eGFr and Her2-directed thera-peutics is associated with deregulation of downstream signaling elements and mtor activation.87,88 evidence also suggests that resistance to eGFr or Her2 inhibitors could be circum vented by rapalogs.89,90 unfortunately, acti vity was not seen in early trials of eGFr inhibitors combined with rapalogs in glioblastoma patients.91,92 also, lung cancer patients resistant to eGFr inhibitors experi enced mucocutaneous and gastro intestinal toxic effects that required discontinuation or dose reductions in some patients.91,92 Phase i and ii trials of mtor inhibi-tors in combination with erlotinib, gefitinib, cetuximab or trastuzumab are underway.
the ability of mtor inhibitors to downregulate HiF and veGF when used as single agents, and thus con-tribute to an antitumor and enhanced antiangiogenic effect, makes the combination of these drugs and veGFr inhibitors particularly interesting.93,94 Preliminary results from trials of temsirolimus or everolimus with bevaci-zumab, sorafenib or sunitinib have been reported.95–99 Combinations of mtor inhibitors with bevacizumab seem to be better tolerated and more active than com-binations using small molecules targeting veGFr. in a phase i study of temsirolimus and bevacizumab in rCC patients who had not previously received mtor or veGFr inhibitors, each agent was administered at its full single-agent dose. toxic effects were within an accept-able range. eight partial responses were observed in 14 patients.97 in the phase ii trial conducted in patients pre-viously treated with veGFr tyrosine kinase inhibitors, an 18% objective response rate and 68% stable disease rate were observed.100 By contrast, the combination of temsirolimus with sorafenib, which targets raf-1 and other kinases in addition to veGFr, required a 50% reduction of the single-agent dose of sorafenib to achieve an acceptable adverse event profile.98 in a similar patient population, intravenous temsirolimus 15 mg weekly and oral sunitinib (which also inhibits veGFr and other kinases) 25 mg daily caused excessive toxic effects.95
the relative lack of tolerability of rapalogs with suni-tinib and sorafenib could be due to pharmaco logical inter actions or unanticipated intracellular effects caused by inhibition of mtor, veGFr and other targets of sorafenib and sunitinib. two temsirolimus combinations— temsirolimus combined with bevaci-zumab or temsirolimus combined with sorafenib—are currently being evaluated and compared with bevaci-zumab alone in a randomized phase ii study of untreated patients with metastatic rCC (nCt00378703). one ongoing phase iii trial is evaluating the combination of bevacizumab and temsirolimus as second-line therapy for rCC (nCt00631371).
other drug combinations of interest are those that counter the feedback loops triggered by mtorC1 inhibi-tion. as rapalog inhibition leads to akt activation through iGF-1–irs-1 signaling,101 mtorC2 phosphorylation of akt ser473, or activation of the maPK pathway,10 it is
unsurprising that combinations of inhibitors target-ing these pathways have already undergone preclinical evalu ations. studies of rapalogs with iGF-1 inhibitors or maPK2 inhibitors reported synergistic effects.102–104 Phase i clinical trials are now underway to evaluate the safety and tolerability of these combination therapies.
Development of more-effective mToR inhibitorsDespite success with rapalogs in certain cancers, it is ques-tionable whether they are the optimum means of mtor inhibition. small molecules designed to compete with atP in the catalytic site of mtor would be expected to inhibit all the kinase-dependent functions of mtorC1 and mtorC2. thus, if mtorC2 is an important target either through its phosphorylation of akt or through other potential mtorC2 functions, then mtor kinase inhibi-tors may be superior to rapalogs. most, if not all of these small-molecule mtor kinase inhibitors also suppress the actions of other enzymes, especially class i Pi3Ks. as Pi3K regulates mtor activity, inhibitors that target both these proteins could have a therapeutic advantage over single-target inhibitors in certain disease settings. initial trials are underway to determine the true therapeutic index and clinical value of mtor kinase inhibitors.
Conclusionsmtor is a validated target for the treatment of cancer. Clinical trials show that rapalogs are generally well tol-erated and could induce stable disease and even tumor regression in a subset of patients. trials in rCC and mCl suggest that certain genetic abnormalities will lead to pathway activation and predispose some tumor types to respond to treatment. Drug, dose and administration schedule could influence clinical activity as well as toxic effects profiles. analysis of the role of mtor in signal-ing pathways that drive cancer has provided insights into determinants of sensitivity and resistance. translational research to identify markers of sensitivity and define rational drug combinations should continue. whether the new class of mtor kinase inhibitors will have greater clinical activity at tolerable doses and schedules than rapa logs and other cancer drugs, remains to be seen. the determination of optimal doses, schedules, patient selec-tion, and combination strategies for this novel class of agents requires continued basic, translational and clinical scientific exploration.
Review criteria
The information for this Review was compiled by searching PubMed for articles published between 1 January 2005 and 1 July 2009 including electronic publications available ahead of print. Search terms included “temsirolimus”, “everolimus”, “ridaforolimus”, “deforolimus”, “sirolimus”, “mTOR”, and “cancer”. Full articles were obtained and references were checked for additional material and primary references when appropriate. Abstracts submitted to ASCO were searched by use of a similar strategy. Listings of ongoing clinical trials of mTOR inhibitors in cancer were retrieved from www.clinicaltrials.gov on 15 July 2009.
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