Current development of mtor inhibitors as anticancer agents

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1 Current development of mtor inhibitors as anticancer agents Sandrine Faivre*, Guido Kroemer and Eric Raymond* Abstract Mammalian target of rapamycin (mtor) is a kinase that functions as a master switch between catabolic and anabolic metabolism and as such is a target for the design of anticancer agents. The most established mtor inhibitors rapamycin and its derivatives showed long-lasting objective tumour responses in clinical trials, with CCI-779 being a firstin-class mtor inhibitor that improved the survival of patients with advanced renal cell carcinoma. This heralded the beginning of extensive clinical programmes to further evaluate mtor inhibitors in several tumour types. Here we review the clinical development of this drug class and look at future prospects for incorporating these agents into multitarget or multimodality strategies against cancer. Receptor tyrosine kinases (RTKs). A family of transmembrane receptors that are physiologically activated by the extracellular binding of growth factor(s) and which initiate intracellular signalling, ultimately leading to many cellular responses such as proliferation. Abnormalities of these receptors are often reported in human malignancies. Translational research Research aimed at using biological tools for clinical applications. *Service Inter Hospitalier de Cancérologie (SIHC), Beaujon University Hospital, 100 Boulevard du General Leclerc, Clichy Cedex, France. CNRS-UMR 8125, Institute Gustave-Roussy, 39 rue Camille-Desmoulins Villejuif Cedex, France. Correspondence to E.R. eric.raymond@bjn.aphp.fr doi: /nrd2062 Signal transduction in cancer cells frequently involves the conditional or constitutive activation of receptor tyrosine kinases (RTKs) that trigger multiple cytoplasmic kinases, which are often serine/threonine kinases. Such cellular signalling pathways can operate independently, in parallel and/or through interconnections to promote cancer development. Three major signalling pathways that have been identified as important in cancer include the phosphatidylinositol 3-kinase (PI3K)/AKT kinase cascade 1,2, the protein kinase C (PKC) family 3,4 and the mitogen-activated protein kinase (MAPK)/Ras signalling cascades 5. Mammalian target of rapamycin (mtor, also known as FRAP, RAFT1 and RAP1) has been identified as a key kinase acting downstream of the activation of PI3K 6. Cumulative evidence supports the hypothesis that mtor acts as a master switch of cellular catabolism and anabolism, thereby determining whether cells and in particular tumour cells grow and proliferate. In addition, mtor has been found to have profound effects on the regulation of apoptotic cell death, which is mainly dictated by the cellular context and downstream targets including p53, B-cell lymphoma 2 (BCL2), BCL2-antagonist of cell death (BAD), p21, p27 and c-myc 7. Rapamycin and rapamycin derivatives that specifically block mtor have been developed during the past 5 years as potential anticancer agents. In this review we focus on the role of the PI3K/AKT/mTOR signalling pathway in cancer, and the effects of drugs that inhibit mtor on the function of cancer cells and tumour angiogenesis. We further emphasize the role of translational research on the determination of the biologically active dose and the identification of tumour types that are likely to respond, or be resistant, to mtor inhibition. The PI3K/AKT/mTOR signalling pathway The PI3K/AKT/mTOR pathway has a cardinal role in cancer cell metabolism (for a review, see REF. 8). Activation of various RTKs leads to autophosphorylation of the intracellular portion of these receptors, which in turn serves as a docking station for selected intracellular proteins. In particular, phosphorylated tyrosine residues of the RTK interact with p85, the regulatory subunit of PI3K. PI3K is a heterodimer consisting of the p85 regulatory subunit and a p110 catalytic subunit, which can transfer the γ-phosphate group from ATP to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ), thereby generating phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and ADP. The binding of p85 to phosphotyrosine residues of the RTK serves to bring PI3K into proximity to its substrate PtdIns(4,5)P 2 in the plasma membrane and probably also induces the allosteric activation of PI3K. RTKs can also indirectly activate PI3K by causing activation of Ras, which in turn binds to and activates the p110 subunit of PI3K. To negatively regulate PI3K, cells contain phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and other phosphatases that dephosphorylate PtdIns(3,4,5)P 3 back to PtdIns(4,5)P 2. A reduction in PTEN expression indirectly stimulates PI3K activity (and PtdIns(3,4,5)P 3 concentrations), thereby contributing to oncogenesis in humans. PtdIns(3,4,5)P 3 serves as a ligand to recruit AKT to the plasma membrane through direct interactions with the pleckstrin homology (PH) domain of AKT. Once at the inner leaflet of the plasma membrane, AKT becomes phosphorylated by the serine/threonine kinase phosphatidylinositol 3-dependent kinase 1 (PDK1), resulting in AKT activation. Activated ATK, itself a NATURE REVIEWS DRUG DISCOVERY VOLUME 5 AUGUST

2 serine/threonine kinase, promotes cell proliferation, growth and survival and other processes involved in cancer development by phosphorylating various intracellular proteins. Of particular interest among the AKT targets is the downstream effector mtor. Interestingly, several genetic syndromes with well-characterized somatic gene mutations affecting PTEN or the PI3K/AKT/mTOR pathway have enhanced our understanding of human carcinogenesis In such genetic syndromes, the use of mtor inhibitors could control disease progression and/or exert antitumour effects (TABLE 1). General functions of mtor Activation of mtor. The TOR family of proteins has pleiotropic functions, and participates in the regulation of the initiation of mrna transcription and protein translation in response to intracellular concentrations of amino acids and other essential nutrients, in the organization of the actin cytoskeleton, membrane trafficking, protein degradation, PKC signalling and ribosome biogenesis 14,15. Of note, there are two mtor-containing complexes: a rapamycin-sensitive complex (also called mtor complex 1, mtorc1), which is defined by its interaction with the accessory protein Raptor (regulatory-associated protein of mtor); and a rapamycin-insensitive complex (also called mtor complex 2, mtorc2), which is defined by its interaction with RICTOR (rapamycin-insensitive companion of mtor). In contrast to mtorc1, which phosphorylates the well-characterized mtor effectors S6 kinase 1 (S6K1, also known as p70 S6K ) and eukaryotic initiation factor 4E (eif4e)-binding protein 1 (4EBP1, which is encoded by the gene phosphorylated heat- and acid-stable protein regulated insulin 1 (PHAS1)), mtorc2 controls the actin cytoskeleton as well as AKT/PKB 16,17. mtor regulates essential signal transduction pathways and is involved in coupling growth stimuli to cell-cycle progression. In response to growth-inducing signals, quiescent cells increase the translation of a subset of mrnas, the protein products of which are required for progression through the G1 phase of the cell cycle. PI3K and AKT are the key elements of the upstream pathway that links the ligation of growth factor receptors to the phosphorylation and activation state of mtor 18,19. With regard to the role of the PI3K/ AKT/mTOR pathway in the genesis and proliferation of cancer cells, elements of the PI3K/AKT/mTOR pathway have been demonstrated to be activated by the erythroblastic leukaemia viral oncogene homologue (ERB) family of surface receptors, the insulinlike growth factor receptors (IGFRs), and oncogenic Ras Overexpression of insulin-like growth-factor 1 receptor (IGF1R) and its ligand, insulin growthfactor 1 (IGF1), commonly occurs in several cancers Additionally, several elements of the PI3K/AKT/mTOR pathway have been demonstrated to be constitutionally activated in malignancies The hyperactivation of PI3K/AKT/mTOR signalling elements in PTENdeficient malignancies suggests that cancers often depend on this pathway for growth and sustenance 32. Downstream effects of mtor. Following phosphorylation, mtor modulates two distinct downstream signalling pathways that control the translation of specific subsets of mrnas including S6K1 and 4EBP1. Activation of either PI3K and/or AKT, and/or loss of PTEN suppressor function, is necessary and sufficient to induce the phosphorylation of both S6K1 and 4EBP1 through mtor Thus, rapamycin-derivatives block the phosphorylation of S6K1 and 4EBP1 in cells expressing activated PI3K or AKT or lacking PTEN 36,37. The process by which mtor transmits signals depends on its interaction with Raptor, an evolutionarily conserved protein of 150 kda that forms a complex with mtor and also binds to both 4EBP1 and S6K1. Although Raptor itself is not a kinase, it is required for the mtor-mediated phosphorylation of 4EBP1 and S6K1 (REFS 38,39). 4EBP1. 4EBP1 is a small protein that represses the initiation of protein translation through its association with eif4e, the mrna cap-binding subunit of the eif4f complex Overexpression of eif4e alone is sufficient to induce cell transformation 43,44. The binding of 4EBPs to eif4e depends on the phosphorylation status of 4EBP1. A recent review by Houghton et al. 45 has summarized the interaction of mtor with translation proteins. In quiescent cells and under growth-factor-deprived conditions, unphosphorylated 4EBP1 binds tightly to eif4e, inhibiting initiation of protein translation. In response to proliferative stimuli triggered by hormones, growth factors, mitogens, cytokines and G-protein-coupled agonists, 4EBP1 becomes phosphorylated at several serine/threonine sites through the action of mtor and other kinases, promoting the dissociation of eif4e from 4EBP1. Free eif4e can then bind to eif4g (a large scaffolding protein), eif4a (an ATP-dependent RNA helicase), and eif4b, forming the multisubunit eif4f complex and facilitating cap-dependent protein translation This cascade of events induces an increase in translation of mrnas with regulatory elements in the 5 -untranslated terminal regions (5 -UTR), including mrnas that encode c-myc, cyclin D1 and ornithine decarboxylase. By contrast, growth-factor deprivation or treatment with rapamycin results in dephosphorylation of 4EBP1, increased eif4e binding and a concomitant impairment of the initiation of the translation of mrnas with 5 UTRs that is required for the G1-to-S phase transition of the cell cycle. There is abundant experimental evidence indicating that mtor is directly responsible for 4EBP1 phosphorylation and the activation of eif4e induced by various mitogenic stimuli. For example, the phosphorylation of 4EBP1 in insulin-treated cells has been shown to be effectively blocked by mtor inhibitors In fact, a low cellular ratio of 4EBP1 to eif4e can cause resistance to mtor inhibitors 53. Furthermore, sites of 4EBP1 that are phosphorylated by mtor are identical to those induced by insulin treatment, and are rapidly dephosphorylated following exposure to mtor inhibitors Some observations indicate that mtor might also act indirectly as an inhibitor of a protein serine/threonine phosphatase, which functions to dephosphorylate 4EBP1 when conditions are appropriate for the G1-to-S phase transition 57, AUGUST 2006 VOLUME 5

3 Table 1 Tumour-prone syndromes that might benefit from treatment with mtor inhibitors Gene Clinical presentation Syndrome Aetiology PTEN TSC1 TSC2 NF1 Hamartomatous tumour syndromes Malignancies Hamartomas in multiple organs Abnormal proliferation of smooth muscle-like cells in the lung Benign and malignant peripheral nerve sheath tumours Cowden disease Cowden syndrome Cowden syndrome-like phenotype Bannayan Zonana syndrome Bannayan Riley Ruvalcaba syndrome Lhermitte Duclos disease Endometrial carcinoma Prostate carcinoma Malignant melanoma Tuberous sclerosis complex Lymphangioleiomyomatosis Neurofibromatosis 1 PTEN is a negative regulator of PI3K TSC1 is part of a heterodimer (with TSC2) that negatively regulates mtor TSC2 is part of a heterodimer (with TSC1) that negatively regulates mtor Loss of NF1 cause AKT activation via PI3K and Ras AMPK Cardiomyopathy Familial hypertrophic cardiomyopathy On ATP depletion, AMPK can activate TSC2 LKB1 Gastrointestinal hamartomas Peutz Jeghers syndrome LKB1 (STK11) phosphorylated and activated AMPK on ATP depletion AMPK, AMP-dependent protein kinase; STK11, serine/threonine kinase 11; mtor, mammalian target of rapamycin; NF1, neurofibromin 1; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; TSC1, tuberous sclerosis 1. S6K1. Another important downstream target of mtor is the serine/threonine kinase S6K1. Activated mtor phosphorylates S6K1 on Thr389, which, in turn, phosphorylates the 40S ribosomal protein S6K1 59. mtor might therefore function as a rapamycin-activated phosphatase. The phosphorylation of S6K1 leads to the recruitment of the 40S ribosomal subunit into actively translating polysomes, thereby enhancing the translation of mrnas with a 5 -terminal oligopolypyrimidine (5 -TOP), including mrnas that encode ribosomal proteins, elongation factors and insulin growth factor 2 (REFS 60 62). Importantly, S6K1 might also be activated by TOR-insensitive signalling pathways involving PDK1, MAPK and stress-activated protein kinase (SAPK). Reportedly, S6K1 can also phosphorylate the eif4g and eif4b units of the eif4f complex. At least three phosphorylation sites have been identified in S6K1, and all of them are blocked by mtor inhibitors. The phosphorylation of Thr389 is particularly important because substitution of this residue with alanine blocks the activation of the kinase domain 63. There is also evidence indicating that Thr389 is directly phosphorylated by mtor. Alternatively, or in addition, mtor might repress a serine/threonine phosphatase that dephosphorylates rapamycin-sensitive sites on S6K1. If this is correct, the de-repression of this phosphatase by the binding of the FKBP12-RAP complex to mtor might explain why S6K1 undergoes rapid dephosphorylation when cells are treated with mtor inhibitors following stimulation with insulin and other growth factors 64. Other molecular interactions of mtor. In addition to its well-characterized inhibitory effects on the activation of S6K1 and 4EBP1, mtor inhibitors block cell-cycle progression and therefore mediate antiproliferative effects 65. mtor inhibitors increase the turnover of cyclin D1 at both the mrna and protein levels 66. A decrease in the translation of cyclin D1 mrna due to inhibition of 4EBP1 results in a deficiency of active cyclindependent kinase 4 (CDK4) cyclin D1 complexes, which are required for phosphorylation of the protein retinoblastoma (prb). mtor inhibition also blocks the elimination of the CDK inhibitor p27, thereby prolonging its half-life and facilitating the formation of complexes between p27 and CDK/cyclin complexes mtor inhibition also results in the upregulation of p27 at the mrna and protein levels, and inhibits cyclin-adependent kinase activity in exponentially growing cells. Combined together, these cellular effects, along with the translational inhibition of other mrnas, might explain the profound inhibition of the G1-to-S transition that mtor inhibitors cause in sensitive cells. Of note, however, is that cells derived from p27-knockout mice are only partially resistant to mtor inhibitors, suggesting that mtor is also involved in cell-cycle progression through p27-independent mechanisms 70,71. Recent evidence indicates that mtor also modulates the transcription of DNA to RNA. Inhibition of mtor might therefore block the function of polymerases (Pol) I and III in yeast and mammalian cells, thereby reducing the transcription of rrnas and trnas, respectively 72,73. Inhibition of mtor activity results in the inhibition of rrna synthesis and might involve the tumour suppressor prb, which represses both Pol I and Pol III 74. In addition, mtor inhibition might also inhibit prb phosphorylation by modulating the stability and expression of cyclin D1 and p27, which regulate CDKs upstream of prb. mtor might therefore regulate protein synthesis at both transcriptional and translational levels. NATURE REVIEWS DRUG DISCOVERY VOLUME 5 AUGUST

4 TSC1/2 4EBP1 and eif4e S6K1 Ras PI3K S6K1 Activation p110 p85 AKT mtor Protein Dysfunction/effect K-Ras Mutation resulting in activation Receptor tyrosine kinases Receptor activation p110 Gene amplification Gene mutation p85 Gene mutation PTEN Gene mutation, deletion or promoter methylation (loss of function) AKT Gene amplification Protein overexpression Glioblastoma High-grade brain malignancy arising from astrocytes with abnormal cellular proliferation and increased tumour angiogenesis. This cancer is usually refractory to chemotherapy and has a very poor prognosis. Gene mutation Gene amplification Protein overexpression Gene amplification Receptor tyrosine kinase (ErbR, PDGFR/KIT, IGFR) eif4e PTEN TSC1/2 RHEB 4EBP1 Tumour type Pancreatic, gastric, colon Many tumour types Head and neck, ovarian Gastrointestinal, brain Colon, ovarian Endometrial, glioblastoma, thyroid, HCC, Cowden syndrome Breast, ovarian, colon Ovarian, breast TSC syndrome Breast Squamous cell, adenocarcinoma Breast, ovarian Figure 1 Dysregulation of the PI3K/AKT/mTOR signalling pathway in human cancer. Dysregulation of the PI3K/AKT/mTOR pathway can result from exogenous or endogenous activation. Exogenous factors include activation by Ras, mostly restricted to gastrointestinal malignancies, whereas receptor tyrosine kinase activation has been reported in a broad variety of haematological and solid tumours. Endogenous factors include either kinase activation resulting from gene mutation/amplification or PTEN loss of function. The tumour types that are most frequently affected are shown in the table. eif4e, eukaryotic initiation factor 4E; 4EBP1, eif4e-binding protein 1; HCC, hepatocellular carcinoma; IGFR, insulin-like growth factor receptor; mtor, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; RHEB, Ras homologue enriched in brain; TSC, tuberous sclerosis 1. The PI3K/AKT/mTOR pathway in human cancer A plethora of distinct mechanisms can result in the constitutive activation of the PI3K/AKT/mTOR pathway in cancer cells (FIG. 1). Cell-intrinsic processes resulting in mtor activation involve loss of PTEN function, mutation or amplification of the PI3K p110 catalytic unit, mutation of the PI3K p85 regulatory unit, amplification of either of the AKT isoenzymes AKT1 and 2, and inactivation or mutations of AKT-associated mtor-regulatory proteins such as tuberous sclerosis 1 (TSC1) or TSC2. The mtor pathway can also be activated via exogenous oncogenes, including overexpressed or mutated tyrosine kinase receptors such as human epidermal growth factor receptors 1 4 (HER1 4), platelet-derived growth factor receptor (PDGFR)/KIT and IGFR, and Ras directly binds the p110 subunit of PI3K 75, the latter interaction being important for membrane anchoring. Downstream of mtor, the overexpression and/or amplification of S6K1 or eif4e could also contribute to oncogenesis. Interestingly, no mutation of mtor itself has been described. However, there is another rationale for mtor activation in cancer. Activated p53 acts as a negative regulator of mtor for instance, in conditions of glucose deprivation 76. p53 function is often lost in cancer, and so this might favour the constitutive activation of mtor. The role of these signal transduction proteins in carcinogenesis has been extensively studied in a number of cellular and animal models, suggesting that activation of the PI3K/AKT/mTOR pathway alone is not sufficient to induce cancer, but rather requires a secondary oncogenic event to induce cellular transformation. PI3K/AKT/mTOR activation affects many tumour types, as specified in FIG. 1. RTK activation is frequent in malignancies. For instance, activation of PI3K is mediated by K-Ras mutations in certain gastrointestinal cancers, and more particularly in pancreatic, gastric and colon cancer. Loss of PTEN function via gene mutation, deletion or promoter methylation has been reported in a more selected subset of tumours, including endometrial cancer, glioblastoma, prostate, ovarian, thyroid carcinoma, and less frequently in hepatocellular carcinoma, breast, lung, renal cell carcinoma and melanoma. Tumours associated with PTEN inactivation are particularly susceptible to the therapeutic effects of mtor inhibitors. In addition to rare genetic syndromes associated with PTEN mutation, such as Cowden syndrome, PTEN inactivation corresponding to genetic or epigenetic alterations can result in the loss of protein expression in several sporadic tumours, such as endometrial carcinoma, glioblastoma and thyroid carcinoma. In TABLE 2, we attempt to classify major dysfunctions in selected tumour types that might be relevant for therapeutic intervention using mtor inhibitors. Genetic alterations of PTEN. PTEN is found mutated in as many as 36 66% of endometrial carcinomas, which is one of the highest incidences observed among analysed tumours 77,78. This feature is associated with high levels of phospho-akt (p-akt represents the active form of AKT) and BAD (p-bad loses its apoptogenic activity), which improves tumour cell survival 69. Among 99 patients with advanced endometrial carcinoma analysed for PTEN, p-akt and Ki-67 expression in tissue specimens, patients with PTEN-positive and p-akt-negative expression had a higher survival rate than patients in all other groups. Multivariate analysis revealed that the combination of PTEN/ and AKT expression was an independent prognostic factor for survival. Negative p-akt expression was related to a decrease in Ki-67 that could at least in part explain the better prognosis. Interestingly, these molecular events are observed 674 AUGUST 2006 VOLUME 5

5 Table 2 Molecular events resulting in the activation of the PI3K/AKT/mTOR pathway in human tumours Tumour types Molecular events Comments Endometrial carcinoma PTEN PI3K AKT Early mutation (30 50%) Increased PI3K activity Early activation (66% in hyperplasia) Glioblastoma Loss of expression (30 40%) Increased PI3K activity Increased expression or activation Head and neck cancer Colon cancer Pancreatic cancer Gastric cancer Hepatocarcinoma No alteration in most cases Mutation uncommon in sporadic colon cancer; methylation of PTEN promoter in MSI-H sporadic colon cancer Infrequent loss of function (PTEN mutation in sporadic endocrine pancreatic tumours) Loss of function infrequent (11%) Mutation Promoter inactivation Overexpression associated with lymph node metastasis: EGFR-independent Involved in angiogenic switch Involved in cisplatin resistance Activity not altered in most cases Low incidence of PI3KCA mutation (13.6%) Increased activity caused by Ras mutation Infrequent upregulation or mutation ( %) of PI3KCA Concurrent K-Ras mutation Increased PI3K activity Ovarian cancer Rare cases of mutation Gene amplification Somatic mutation (12%) Thyroid carcinoma Breast cancer Prostate cancer Lung cancer Increased activation (57 81%) Increased expression or activation Increased expression or activation Increased expression or activation (28.9%) Increased expression or activation Increased expression or activation of AKT2 (gene amplification) S6K1 gene amplification Deletion (20 60%) Increased PI3K activity Increased expression or activation Loss of PTEN in 30% of sporadic tumours of which 60% have a methylated promoter Frequent in Cowden disease Low expression in advanced tumours Mutation rare Reduced protein expression (24 74%), presumably by promoter silencing PI3KCA mutation (18 26%), mostly if PTEN functional Increased PI3K activity Increased PI3K activity Increased expression or activation of AKT S6K1 gene amplification Increased expression or activation of AKT Increased expression or activation of AKT, activation of S6K1 Rapamycin analogues for the treatment of platinum-refractory endometrial carcinoma EGFR has a role in PI3K activation Early genetic alteration of PI3Kα Role of PI3K in transition of dysplasia to carcinoma Bad prognostic value of PI3Kα Early genetic alteration induced by K-Ras mutations Increase tumorigenic potential Early event in carcinogenesis p-akt might have a role in cisplatin resistance PI3K pathway might contribute to the angiogenic switch Higher percentage of PTEN loss in aggressive phenotypes Role of EGR1 transcription factor in silencing PTEN PTEN loss is a bad prognosis in high-grade tumour with distant metastases and poorer diseasefree survival Correlated with nodal involvement and resistance to tamoxifen Long-term androgen deprivation reinforces the PI3K/AKT pathway Role of EGFR in PTEN silencing PI3K/AKT activated by MAPK EGFR, epidermal growth factor receptor; EGR1, early growth response 1; MAPK, mitogen-activated protein kinase; MSI-H, microsatellite instability-high; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10. already in pre-malignant endometrial hyperplasia, which can be successfully treated with oral progesterone in 30 50% of cases. Among non-responders to progesterone, increased or persistent p-mtor expression was observed. Although mtor phosphorylation does not imply its activation, studies are ongoing to evaluate the role of mtor inhibitors in progesterone-refractory endometrial hyperplasia 79. In 30 40% of glioblastomas, PTEN has been found to be inactivated by mutation, homozygous deletion or loss of expression. In previously untreated patients, loss of PTEN was associated with significant increase in p-akt, p-mtor and p-s6k1. Expression of the mutant epidermal growth factor receptor VIII was also tightly correlated with phosphorylation of these effectors, demonstrating an additional route to PI3K pathway activation in glioblastomas in vivo 80. Among 92 patients with glioma, the levels of p-pi3k, p-akt and p-s6k1 all correlated inversely with the levels of cleaved caspase 3, indicating that PI3K pathway activation suppresses apoptosis. Importantly, activation of the PI3K/AKT/ mtor axis was associated with increasing tumour grade and reduced patient survival 81. PTEN gene inactivation or loss has been correlated with poor prognosis in NATURE REVIEWS DRUG DISCOVERY VOLUME 5 AUGUST

6 Box 1 Protective use of mtor inhibitors in neurodegeneration Inhibitors of mammalian target of rapamycin (mtor) might have other roles than controlling proliferation of cancer cells. As part of multiple effects of mtor inhibition, there is genetic evidence that invalidation of the mtor pathway can increase the lifespan of nematodes and fruit flies, presumably by mimicking the effects of caloric restriction 121. Whether chronic mtor inhibition prolongs lifespan in mammals, however, has yet to be tested. Interestingly, inhibition of mtor has been proposed as a strategy for stimulating the autophagic removal of neurotoxic proteins carrying polyglutamine extensions, such as mutated huntingtin. Accordingly, CCI-779 improved performance in behavioural tasks and decreased aggregate formation in a mouse model of Huntington s disease 122. Furthermore, Alzheimer s disease is accompanied by a marked activation of the mtor pathway, correlating with the levels of the protein tau, which participates in the formation of neurofibrillary tangles 123. In addition, enhanced expression of mtor has been found in multi-nucleated giant cells (also called syncytia) that develop in human immunodeficiency virus (HIV)-associated encephalitis 124. In an in vitro model of HIV1- elicited syncytium formation, mtor was able to participate in pathological cell death by phosphorylating p53 on serine 15, thereby causing activation of this lethal transcription factor 125. Inhibition of mtor can therefore inhibit syncytial cell death in vitro, and it might also be possible to use mtor inhibitors to palliate HIV-associated encephalitis. Further research is underway to better characterize the role of mtor in diseases associated with cell loss and the effects of mtor to prevent neurodegeneration. high-grade astrocytomas. In particular, the PI3K/AKT pathway might be involved in glioma cell migration and stimulated by the urokinase-type plasminogen activator in glioma cells 82. Cowden disease (also known as multiple harmartoma syndrome) is an autosomal-dominant cancer syndrome associated with a high risk of breast and thyroid cancer caused by germline mutations in PTEN. Although somatic mutations of the PTEN gene are rare in sporadic thyroid carcinoma, loss of heterozygosity at 10q23 (which includes the PTEN gene) is present in 20 60% of thyroid malignancies, with a higher percentage in the more aggressive histotypes. For example, a screening for PTEN mrna expression in 87 sporadic thyroid tumours including 14 anaplastic carcinomas, 37 follicular carcinomas, 21 atypical adenomas and 15 ordinary adenomas showed a complete loss of PTEN mrna expression in six of the tumours, including four anaplastic carcinomas. PTEN transcriptional silencing is therefore probably involved in the carcinogenesis of highly malignant or late-stage thyroid cancers, whereas it seems to be of minor importance in differentiated follicular thyroid tumours 83. Epigenetic alteration of PTEN. It has previously been demonstrated that PTEN promoter activity is increased by overexpression of the transcription factor early growth response protein 1 (EGR1) 84. Preclinical studies using thyroid tumour cell lines revealed that the absence of EGR1 mrna is associated with the silencing of PTEN gene expression that occurs during thyroid cell transformation 85. A similar scenario applies to nonsmall-cell lung cancer (NSCLC). Most NSCLC cell lines demonstrate PI3K/AKT/mTOR activation yet harbour a wild-type PTEN gene 86. In tumour specimens from patients, about 24 74% of cases showed no PTEN protein expression 87, presumably because of the silencing of the PTEN promoter gene by the transcription factor EGR1. A recent study suggests that EGR1 gene expression is predictive of PTEN concentrations and that low levels of EGR1 are associated with an increased risk of recurrence and poor survival after surgical resection of tumours in patients with early stage NSCLC 88. In cervical cancer, PTEN mutations seem uncommon 89 at early stages but might increase in frequency in advanced disease, particularly in tumours exposed to radiation therapy 90. Conversely, loss of function of PTEN, either by loss of heterozygosity or promoter methylation, was more frequently reported from the early stages of carcinogenesis, including dysplasia to invasive carcinoma 91. Loss of PTEN function in patients with cervical carcinoma is associated with a poor outcome. PI3K/AKT pathway abnormalities. Inactivation of PTEN, as measured by immunohistochemistry, was found in about one-third (28%) of 236 breast cancer specimens 92. The reduced expression of PTEN protein correlated with lymph-node metastases and poor prognosis. In another study including 100 breast cancers, downregulation of PTEN expression was predictive of the failure of tamoxifen treatment 93. In addition to loss of PTEN, activating PI3K mutations on the catalytic subunit were identified in 26% of 342 breast cancers. Interestingly, PI3K mutations mostly occurred when PTEN function was retained. In addition, PI3K mutations were associated with expression of oestrogen (ER) and progesterone receptors (PR), lymph-node metastasis, and HER2/neu overexpression. Although PI3K mutations correlate with ER/PR-overexpression, PTEN loss correlates with the loss of either/or ER/PR, suggesting that breast cancer might follow two distinct patterns of carcinogenesis 94. In yet another study, among 402 ERα-positive breast carcinomas taken from patients treated with tamoxifen, high p-akt levels were predictive of reduced overall survival 95. These data support in vitro evidence that AKT mediates tamoxifen resistance. Allelic imbalance and mutations of the PTEN gene have only been found in 9% of ovarian cancers 96. However, somatic missense mutations affecting the catalytic unit of PI3K have been reported in 24 of 198 (12%) of ovarian cancers 97. Increased expression or activation of AKT2 due to gene amplification, together with S6K1 gene amplification, has also been observed 24. In prostate cancer, the probability of PTEN loss and PI3K/AKT pathway activation increases in advanced stages 98,99. Androgen independence of prostate cancer has been associated with the activation of either the PI3K or the MAPK pathways 100, suggesting a rationale for novel therapeutic approaches. Conflicting data have been published on the role of PTEN loss in squamous head and neck carcinoma. Although one study reported PTEN loss of heterozygosity in 41% of the cases 101, another analysis of 21 cases found no PTEN loss or mutation 102. The most frequent abnormality is the overexpression or amplification of the catalytic unit of PI3K, which can be found both in the early stage of carcinogenesis (dysplasia) and in advanced head and neck cancer. Activation of the PI3K pathway is correlated with lymph node metastases, enhanced angiogenesis and resistance to cisplatin AUGUST 2006 VOLUME 5

7 G1 S cell-cycle transition 4EBP1 Cyclin D1 p27 Rapamycin derivatives mtor p34 cdc2 Cyclin E p53 Effects on apoptosis BCL2 Figure 2 Effect of mtor inhibitors on cancer cells. Cellular effects of mtor activation include the facilitation of G1 S cell cycle transition and inhibition of apoptosis through the interaction with key molecules of cell-cycle control (4EBP1, cyclin D1 and p27) and apoptosis (BAD, BCL2 and p53). These processes are reversed by mtor inhibitors. 4EBP1, eukaryotic initiation factor 4E-binding protein 1; BAD, BCL2-antagonist of cell death; BCL2, B-cell lymphoma 2; mtor, mammalian target of rapamycin. BAD In gastrointestinal tumours, the role of PTEN seems to be limited, with infrequent mutations. Sporadic PTEN loss of function has been described in colon, gastric and pancreatic carcinoma In colon and pancreatic tumours, K-Ras-induced PI3K/AKT activation was reported as an early event in carcinogenesis. In hepatocellular carcinoma, studies were mainly performed using human cancer cell lines, with no available data from patient specimens. These preclinical studies indicated that the function of the PTEN gene might be impaired by point mutations or promoter inactivation 110,111. Rapamycin: a prototype for mtor inhibition So far, four mtor inhibitors are available for clinical trials: the prototype rapamycin and three rapamycin derivatives, CCI-779 (temsirolimus), RAD001 (everolimus) and AP Each of these inhibitors forms a complex with the intracellular immunophilin FKBP12; the resulting complex interacts with and inhibits mtor. No other proteins have been identified as rapamycin targets, and the requirement for a cofactor makes the mtor rapamycin interaction very specific. Indeed, treatment of mice embryos with rapamycin induces exactly the same developmental defect as the mtor knockout. This accurate phenocopy suggests that rapamycin is a truly monospecific mtor inhibitor. Rapamycin (also named sirolimus) is a macrocyclic lactone produced by Streptomyces hygroscopicus. Rapamycin was initially developed as an antifungal drug directed against Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus 112. It is a white crystalline solid that is insoluble in aqueous solutions, but soluble in organic solvents. When rapamycin was evaluated by the Developmental Therapeutic Branch of the National Cancer Institute (NCI), it was identified as a non-cytotoxic agent that had cytostatic activity against several human cancers in vitro and in vivo. However, the development programme of rapamycin as an anticancer agent was given a low priority and was halted in 1982, resuming in 1988 to be postponed again until the end of the 1990s following the discovery of CCI-779, a novel soluble rapamycin derivative formulated for intravenous infusion that had a safe toxicological profile in animals. In the meantime, rapamycin became an immunosuppressive agent, which stimulated the exploration of the mechanism of action of this agent. Rapamycin inhibits T-cell proliferation induced by cross-linking of the T-cell receptor or antigenic peptides presented by major histocompatibility complex (MHC) molecules. Rapamycin also inhibits proliferative responses induced by several cytokines, including interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-6, IGF, PDGF and colony-stimulating factors (CSFs). The preclinical development of rapamycin as an immunosuppressor has been extensively reviewed 113. Rapamycin can exert its immunosuppressive effect in synergy with cyclosporine 114. The combination of rapamycin and cyclosporine A enhanced rejection prevention in renal transplantation, and enabled the use of lower doses of cyclosporine, minimizing its toxicity 115,116. Rapamycin was approved in the USA in 1999 (and 2000 in Europe) for the prevention of acute rejection in combination with cyclosporine and steroids. Interestingly, rapamycin, unlike cyclosporine, does not increase the risk of malignancy but rather decreases the risk of post-transplant lymphoproliferative disorders 117. Apart from its immunosuppressive capacity, rapamycin was also recently shown to be capable of preventing coronary artery re-stenosis 118. High doses of rapamycin block the proliferative responses to cytokines in vascular smooth-muscle cells after mechanical injury, such as balloon angioplasty 119. Growth, migration and differentiation of vascular smooth-muscle cells are also two major features of neo-intimal proliferation after vascular injury. The proposed mechanisms of inhibition of vascular smooth-muscle cell proliferation were related to cell-cycle blockage and/or the inhibition of PDGFinduced migration 120. Recent studies have suggested applications of rapamycin derivatives in the treatment of neurodegenerative diseases (BOX 1). Recently, rapamycin has been shown to inhibit the growth of several murine and human cancer cell lines in a concentration-dependent manner, both in tissue culture and in xenograft models including B16 melanoma, P388 leukaemia, MiaPaCa-2 and Panc-1 human pancreatic carcinomas 126,127. In the 60-tumour cell-line screen by the US NCI, the spectrum of activity of rapamycin was different from that of other anticancer agents in leukaemia, ovarian, breast, central nervous system, and small-cell lung cancer cell lines. Furthermore, rapamycin alone induces p53-independent apoptosis in childhood rhabdomyosarcoma and sensitized cells to apoptosis induced by cisplatin in HL-60 promyelocytic leukaemias and ovarian SKOV3 carcinoma cell lines 128,129. Conversely, rapamycin inhibits taxol-induced apoptosis in human B-cell lines, probably through prevention of BCL2 inactivation 130. Rapamycin also inhibits hybridoma cell death in bioreactors, thereby increasing the production of monoclonal antibody 131. In addition, rapamycin NATURE REVIEWS DRUG DISCOVERY VOLUME 5 AUGUST

8 VEGFR PDGFR HIF1α Endothelial cells Rapamycin derivatives PI3K/AKT/mTOR Anabolism Proliferation Migration PDGFR KIT Tumour cells IGFR Figure 3 The effect of modulation of the PI3K/AKT/mTOR pathway with rapamycin derivatives on endothelial and tumour cells. Key molecular factors that activate the PI3K/AKT/mTOR pathway in either endothelial or cancer cells are shown. In addition to effects reported in cancer cells, mtor inhibitors might also act as anti-angiogenic agents by intercepting the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) signalling cascades. ERB, erythroblastic leukaemia viral oncogene homologue; HIF1α, hypoxia-inducible factor-α; IGFR, insulin growth factor receptor; mtor, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase. ERB inhibits the oncogenic transformation of human cells induced by either PI3K or AKT, and inhibits metastatic tumour growth and tumour vascularization in vivo in suitable mouse models 132. On the basis of these preclinical results, studies with rapamycin as an anticancer drug have been launched, and rapamycin derivatives with improved pharmacokinetics and reduced immunosuppressive effects have been developed. Unlike rapamycin, the three rapamycin derivatives do not mediate any manifest immunosuppression when administered intermittently in clinical settings 1. CCI-779 is administered either orally or intravenously, RAD001 is available as an oral formulation and AP23573 is either given orally or intravenously. Properties of tumour growth inhibition Direct exposure of cancer cells to rapamycin and rapamycin derivatives results in several effects that depend on specific cellular characteristics and drug concentration. In addition, mtor inhibitors can target tumour growth indirectly, by interacting with the maintenance of endothelial cells and pericytes that are required for tumour angiogenesis. Effects of mtor inhibitors in cancer cells. In cancer cells possessing an activated PI3K/AKT/mTOR pathway, rapamycin and its derivatives block the binding of raptor to mtor, which is required for downstream phosphorylation of 4EBP1 and S6K1 (FIG. 2). This effect restores the proper control of the activated PI3K/AKT/mTOR signalling pathway and can be viewed as a gain of function rather than a trivial inhibitory effect on protein function. Interestingly, molecular interactions between rapamycin, FKBP12 and mtor can persist for about 72 hours even after a short exposure to rapamycin, blocking mtor function for several days. As a result, S6K1 undergoes dephosphorylation, which results in a reduction of protein synthesis, linked to reduced cell size and motility. The rapamycin-triggered dephosphorylation of 4EBP1 results in a reduced translation capacity of mrnas encoding c-myc, cyclin D1 and ornithine decarboxylase, increased eif4e binding, and a concomitant impairment of the initiation of the translation of mrnas with 5 -UTRs that are required for the transition in the G1/S phase of the cell cycle. Overall, effects of rapamycin consistently induce a decrease in cyclin D1 expression and lead to an increase in p27 that will lead to late G1/S cell-cycle blockage. These rapamycin-mediated metabolic and cell-cycle effects slow down the proliferation of several human cancer cell lines in a dose-dependent manner. In a relatively limited number of tumour models, rapamycin was shown to induce cancer cell death either by inducing apoptosis or autophagy. The molecular mechanisms leading to apoptosis in cancer cells have not yet been fully deciphered. One link between mtor inhibition and apoptosis induction might be provided by the downstream target S6K1, which can phosphorylate the pro-apoptotic molecule BAD on Ser136, a reaction that disrupts BAD s binding to the mitochondrial death inhibitors BCL-XL and BCL2, thereby inactivating BAD 133,134. In this scenario, rapamycin-mediated S6K1 inactivation would indirectly cause BAD activation. In addition, two recent studies have suggested that members of the BCL2 family could be downstream mediators of the IGF1-stimulated, PI3K-dependent survival of cells 135,136. Furthermore, several growth factors that activate the PI3K and S6K1 pathways were recently shown to increase expression of BCL2, thereby promoting cell survival in myeloid progenitor cells 123. The contribution of BCL2 to chemotherapy resistance has been investigated extensively in cultured cells, animal models and clinical studies. In studies with patients suffering from ovarian carcinoma, high levels and/or aberrant patterns of BCL2 expression have been correlated with resistance to commonly used anticancer agents 137. This also applies to rapamycin-like agents. Ovarian cancer cells that are resistant to mtor inhibitors overexpress BCL2 and an antisense BCL2 oligonucleotide can sensitize ovarian cancer cells to rapamycin and RAD Furthermore, one of the most potent mtor activators is a high level of nutrients, and in particular amino acids. As mtor acts as an endogenous suppressor of autophagy, nutrient depletion (which inhibits mtor) results in the activation of autophagy, allowing the cell to catabolize macromolecules and meet its energy demands with low molecular mass metabolites 139. Rapamycin activates autophagic processes that might, at least in some instances, participate in the cytostatic or cytotoxic effects of rapamycin on tumour cells 140. Surprisingly, cell death induced by rapamycin and its derivatives does not seem 678 AUGUST 2006 VOLUME 5

9 Table 3 Current status of mtor inhibitors in clinical development Compounds Development status Dose and schedule (number of patients) CCI-779 (temsirolimus) intravenous and oral formulation RAD001 (everolimus) oral formulation AP23573 intravenous and oral formulation Phase I Phase II*: endometrial carcinoma Phase II: renal cell carcinoma mg per m 2 per week (n = 24) mg per m 2 per day x 5 days every 2 weeks (n = 24) Main clinical results Recommended doses <220 mg per m 2 Dose-limiting toxicity: mucositis, depression, thrombocytopaenia and hyperlipaemia Maximum tolerated dose: 19.1 mg per m² Dose-limiting toxicity: thrombocytopaenia and mucositis 250 mg per week (n = 16) Response rate: 31% Rate of stable disease: 63% 25 versus 75 versus 250 mg per week (n = 111) Response rate: 7% with a 2 3 fold survival improvement for intermediate/poor prognosis patients versus historical series Phase III: renal cell carcinoma 25 mg per week (n = 626) Significantly longer survival in CCI-779 arm (10.9 months) compared with interferon-α (7.3 months) Phase II: breast cancer Phase II: glioblastoma 75 versus 250 mg per week (n = 109) 250 mg per week (total n = 65) Response rate: 9.2% (recommended dose: 75 mg per week) No clear dose effect relationship Time to progression: 2.3 months Partial response reported Phase II: mantle cell lymphoma 250 mg per week (n = 43) Response rate: 38% Time to progression: 6.5 months Phase I Phase I/II in patients with Gleevec-refractory gastrointestinal stromal tumours Single agent: 5 20 mg per week Combination: mg per day with imatinib at 600 mg per day Recommended dose: 20 mg per week Dose-limiting toxicity: thrombocytopaenia and gastritis Recommended dose: 2.5 mg per day Partial response in 2 patients Tumour stabilization in 8 patients Refs ,154 Phase I mg per day weekly Antitumour activity in sarcoma 151 Phase II in advanced sarcomas 3 28 mg per day x 5 days every 2 weeks 12.5 mg per day x 5 days every 2 weeks Objective responses in 4 patients, decrease in PET uptake in 8 patients and symptomatic improvement in 13 patients 164 *On the basis of pharmacokinetic data, all Phase II trials were conducted using flat dosing. PET, positron emission tomography to be dose-dependent. Altogether, it seems that rapamycin can induce growth arrest and death of tumour cells through various pathways. Effects of mtor inhibitors on tumour angiogenesis. Tumour angiogenesis relies on an intricate interplay between tumour cells, endothelial cells and surrounding mesenchymal cells (pericytes in microvessels and vascular smooth-muscle cells in large vessels) to activate endothelial cell proliferation, to recruit migrating endothelial cells and pericytes and to form new vessels/ capillaries through vascular remodelling and maturation 141. At the molecular level, tumour angiogenesis depends on shear stress and coordinated interactions between endothelial vascular growth factors such as vascular endothelial growth factor (VEGF), angiopoietin 1 (ANG1), ANG2, basic fibroblast growth factor (bfgf), PDGF-B, ephrin-b2 and members of the tumour growth factor-β (TGFβ) superfamily; intracellular signalling molecules including NOTCH1 and COUP-TFII; and intercellular contacts via connexins and vascular cell-adhesion molecule 1 (VCAM1) 130. Interestingly, all the above factors can activate the PI3K/AKT/mTOR pathway in cancer cells, endothelial cells or pericytes 142. Cellular proliferation, survival and migration required for vascular sprouting, and endothelial cell differentiation leading to tubule formation are primarily driven by VEGF/VEGFR activation that can in turn trigger the PI3K/AKT/mTOR pathway. One of the major stimuli of cancer angiogenesis is hypoxia, which activates hypoxia-inducible transcription factors (HIFs), which in turn induce the expression of VEGF, VEGFR, bfgf, PDGF and ANG2 (REF. 143). mtor can facilitate the translation of HIF1α mrna, thereby enhancing vascular growth factor expression 144, a process that is influenced by PTEN. In normal vessels, HIF1α is transiently expressed as a result of the action of the HIF-prolyl hydroxylase that targets HIF1α to a ubiquitin ligase complex containing von Hippel Lindau (pvhl), which marks it for destruction by the proteasome. In tumour cells, a number of factors stabilize HIF1α and translocate HIF1α into the nucleus. For instance, BCL2 and hypoxia synergize to induce HIF1α and VEGF expression in melanoma cells 145. In addition, loss-offunction mutations of VHL, as they frequently occur in renal cell carcinoma, can cause HIF1α stabilization, thereby inducing PDGF and VEGF overexpression and sustained tumour angiogenesis in humans 133. NATURE REVIEWS DRUG DISCOVERY VOLUME 5 AUGUST

10 Box 2 Chemical structure of rapamycin and rapamycin derivatives The rapamycin derivatives used as inhibitors of mammalian target of rapamycin (mtor) share the same molecular scaffold but with substitution of the lactonic macrocycle, making the compound either suitable for intravenous (CCI-779 and AP23573) or oral (RAD001) formulation. Rapamycin derivatives have the same binding sites Mucositis Inflammation and/or ulceration of mouth mucosa. Thrombocytopaenia Low level of platelets in circulating blood. Objective response Shrinkage of at least 50% of malignant target lesions (according to World Health Organization criteria) after administration of anticancer treatment as compared with baseline measurement. Binding site to FKBP12 O HO N O O O O OCH 3 O OH OCH 3 H 3 CO OH O Binding site to mtor for FK506-binding protein 12 (FKBP12) and mtor. The semi-synthetic modifications of rapamycin derivatives produced only few structural changes, mostly located at the C40 hydroxyl group outside FKBP12- and mtor-binding domains. All these derivatives are therefore likely to share similar properties to rapamycin. Modifications in the rapamycin biosynthetic gene cluster using conjugate methods for DNA transfer to the rapamycinproducing organism Streptomyces hygroscopicus NRRL5491 could provide access to novel rapalogues. CCI-779 can be considered as a prodrug that is bioconverted into sirolimus, whereas RAD001 and AP23573 are not metabolized in this way. It is possible that the anticancer effects of mtor inhibitors involve anti-angiogenic processes mediated by effects on pericytes and endothelial cells rather than on cancer cells themselves 131. As outlined above, mtor inhibition can block angiogenesis by inhibition of HIF1α translation as well as by intercepting the VEGF/VEGFR and/or PDGF/PDGFR signalling cascades (FIG. 3). Chemical inactivation of mtor in hypoxia-activated endothelial cells and pericytes can induce a G0 G1 cell-cycle block (associated with reduced cyclin D1 expression and p27 accumulation) rather than apoptosis 146. In mice bearing human tumour xenografts, RAD001 significantly reduced tumour angiogenesis by blocking tumour growth. If these data can be extrapolated to the human system, then mtor inhibitors should be particularly efficient in inhibiting the angiogenesis of tumours that bear VHL mutations and/or constitutively activate the VEGFR and PDGFR tyrosine kinase signalling pathways, such as renal cell carcinoma. The assumption that VHL has a role in sensitivity/resistance to mtor inhibitors has been supported by recent work showing that CCI-779 preferentially inhibits VHLnull renal cell carcinomas, which provides a rationale for prospective biomarker-driven clinical trials in patients with kidney cancer 147. It remains to be seen whether mtor inhibitors are particularly efficient when combined with anti-angiogenic drugs targeting VEGF (bevacizumab (Avastin; Genentech)), PDGFR (imatinib mesylate (Gleevec; Novartis)) or VEGFR (sunitinib malate (Sutent; Pfizer) and sorafenib (Nexavar; Bayer)). Clinical development of rapamycin-derivatives So far, most data exist for the clinical development of the rapamycin derivatives CCI-779 (temsirolimus), RAD001 (everolimus) and AP23573 (TABLE 3, BOX 2). These compounds were studied in the clinic using three main oral and intravenous schedules: five-times daily dosing every two weeks, once-weekly dosing or daily oral continuous dosing until tumour progression Doselimiting toxicities were consistent between the three compounds and consisted of reversible mucositis, asthenia (weakness and fatigue) and thrombocytopaenia, and were mainly observed with the five-times daily dosing schedule. Severe psychiatric disorders at very high doses were also reported in the weekly schedule of CCI-779 (REF. 146). No significant immunosuppression was observed for CCI-779, RAD001 and AP23573, and at the recommended dose the most prevalent toxic effects were reversible cutaneous side effects, such as herpes lesions, aseptic acne-like rash, maculopapular rash and nail disorders, and were observed in about 75% of the patients. The longer-term side effects of rapamycin derivatives are not yet known, but should be carefully assessed because it is speculated that long-term treatment with weekly CCI-779 might cause nonspecific pneumonia. At the recommended doses, these compounds displayed a side-effect profile that renders them potentially amenable to combination with cytotoxic agents or other targeted therapies. The weekly administration schedule that showed the lowest toxic side effects was selected for further clinical evaluation of rapamycin derivatives in Phase II and III trials. Early on in Phase I studies of rapamycin-derivatives, antitumour activity included an objective response in renal (FIG. 4), breast and non-small cell lung cancer, as well as several cases of minor responses and prolonged disease stabilization. These responses were observed over a broad range of dose levels. Phase II trials were continued using weekly oral and intravenous schedules in tumours types that had shown promising results in Phase I trials or in tumours likely to be driven by the PI3K/AKT/mTOR pathway and/or the loss of PTEN. One such example is a recent Phase II trial investigating CCI-779 that was conducted in recurrent or metastatic endometrial cancer, based on the fact that this type of tumour frequently loses PTEN. In the first part of the study, 18 patients received CCI-779 at the dose of 250 mg per week for a median duration of treatment of 6 months. Five out of the 16 patients who were suitable for evaluating efficacy showed partial responses (31%) and ten out of 16 patients (63%) had stable disease, with only one patient showing progressive disease. These preliminary results suggest that monotherapy with CCI-779 could be an option for the treatment of endometrial carcinoma, a disease for which no standard of care currently exists 152. Two Phase II studies have been performed on patients with recurrent glioblastoma treated with CCI-779 at the initial dose of 250 mg per week 153,154. The results of both studies are consistent, showing limited antitumour activity as measured by a computerized tomography (CT) scan and/or magnetic resonance imaging (MRI) with a median time to progression of disease of 2.3 months. Reports of objective responses in this disease were anecdotal and mainly based on non-validated functional neuroimaging methods. So, therapy with CCI-779 as a single-agent for recurrent glioblastoma seems to have only limited activity. However, it might be possible to combine CCI-779 with other treatment modalities such as radiotherapy and temozolomide chemotherapy. It is 680 AUGUST 2006 VOLUME 5

11 a Before treatment b After treatment Sarcoma Malignant tumour arising from the bone (osteosarcoma) or the soft tissues with high risk of lung metastasis. Patients with soft tissue sarcomas are frequently poor responders to classical chemotherapy. Lung metastases Tumour size reduction Modification of tumour density Figure 4 Antitumour effects of mtor inhibitors in patients with cancer. Antitumour activity, including objective response and tumour stabilization, has been reported consistently in patients with renal cell carcinoma in several trials. The computerized tomography scans in this figure show lung metastases in a patient with advanced renal cell carcinoma before (a) and after (b) treatment with an mtor inhibitor. The reduction in tumour size and modification of tumour density might reflect a direct effect on cancer cells and on tumour angiogenesis, respectively. mtor, mammalian target of rapamycin. significant that patients receiving concomitant cytochrome P450 (CYP450) enzyme-inducing anti-epileptic drugs such as phenobarbital and carbamazepine tolerated higher doses of CCI-779 than patients who did not receive such a co-treatment and who required dose reduction to 170 mg per week. CCI-779 was also investigated in a large dose-randomized Phase II study of patients with advanced renal cell carcinomas that were classified in three groups according to Motzer s criteria (good, intermediate and poor prognosis) 155. As a single agent, CCI-779 displayed a relatively low objective response rate of 7%, with 26% additional minor responses, and was associated with better survival in patients with intermediate or poor prognosis who experienced a two- or threefold increase in survival (22.5 months for the intermediate group, 8.2 months for the group with poor prognosis) compared with historical controls treated with interferon-α (IFNα) (13.8 and 4.9 months for the intermediate and poor prognosis groups, respectively). Those promising results subsequently led to the initiation of a large multicentre randomized Phase III trial comparing IFNα given either alone or with CCI- 779, or single agent weekly intravenous administration of 25 mg CCI-779 as a first-line treatment in a total of 626 high-risk patients with advanced/metastatic renal cell carcinoma. This trial was presented in June 2006 at the annual meeting of the American Society of Clinical Oncology 156, and showed that patients treated with CCI- 779 had a statistically significant longer median survival (10.9 months) than those receiving IFNα (7.3 months). The combination of CCI-779 with IFNα did not improve survival in those patients. On the basis of these data, requests for registration for CCI-779 have been filed in the USA and Europe for first-line treatment of patients with advanced/metastatic renal cell carcinoma. Promising preliminary results showing antitumour activity have also been reported with RAD001 in patients with advanced renal cell carcinoma 157. Although no translational research was associated with those trials, consistent results showing antitumour effects of rapamycin derivatives in patients with renal cell carcinoma suggest an important role for mtor in either renal cell carcinogenesis and/or tumour-associated angiogenesis. CCI-779 has also shown some efficacy in breast cancer. In a study of 109 patients with advanced or metastatic breast cancer and extensive treatment histories, patients were randomized to receive 75 or 250 mg of CCI-779 per week 158. Efficacy was similar for both doses with a rate of objective response of only 9.2%. However, given the favourable safety profile, it might be worthwhile to combine CCI-779 with other agents in future clinical studies of advanced breast cancer. Preclinical studies revealed that mtor inhibitors could downregulate cyclin D1 in mantle cell lymphoma, a disease driven by a chromosomal translocation t(11;14)(q13;q32) that places the cyclin D1 gene under the influence of the immunoglobulin heavy chain enhancer region, resulting in cyclin D1 overexpression. A Phase II trial in 35 patients with mantle cell lymphoma that had relapsed after chemotherapy and rituximab treatment indicated that CCI-779 treatment resulted in a remarkable overall response rate of 38%, with a median duration of responses of 6 months. This suggests that mtor inhibitors can mediate sustained antilymphoma effects 159. Interestingly, the effect seems to be similar in those patients who received 250 mg and 25 mg weekly, suggesting no dose activity relationships in this disease 160. The most promising results with mtor inhibitors, so far, have been obtained in renal cell carcinoma, mantle cell lymphoma and endometrial cancers. This is particularly significant because these patients had relapsed after therapy or had not responded to standard therapies. In other tumour types, including glioblastoma, breast cancer and neuroendocrine carcinoma 161,162, objective response rates with CCI-779 were consistently lower than 10%. Preliminary results with RAD001 and AP23573 given as single agents in patients with gastrointestinal sarcoma and different pathological subtypes of advanced sarcoma (including osteosarcoma, leiomyosarcoma and liposarcoma) also yielded a low rate of objective responses 163,164. This low response rate in many tumour types remains a major hurdle to the development of rapamycin derivatives. Biomarkers for mtor inhibitor development Since 2000, major efforts have been undertaken to identify surrogate markers for assessing the efficacy of mtor inhibitors, either in skin, in peripheral blood mononuclear cells (PBMCs) and/or in the tumour itself (FIG. 5). Progress in this field has been disappointing, but this is at least in part because there is great variation in the reproducibility and robustness of using NATURE REVIEWS DRUG DISCOVERY VOLUME 5 AUGUST