cbt2/4/Suppl. 1

[Cancer Biology & Therapy 2:4:Suppl. 1, S169-S177; July/August 2003]; ©2003 Landes Bioscience

Models of Anti-Cancer Therapy

The Molecular Target of Rapamycin (mTOR) as a Therapeutic Target Against Cancer ABSTRACT

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Previously published online as a CB&T E-publication at: http://www.landesbioscience.com/journals/cbt/toc.php?volume=2&issue=0

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*Correspondence to: Eric K. Rowinsky, M.D.; Department of Clinical Research; Institute for Drug Development; Cancer Therapy and Research Center; 7979 Wurzbach Road; 4th Floor Zeller Building; San Antonio, Texas 78229 USA; Tel.: 210.616.5945; Fax: 210.616.5865; Email: [email protected]; or Monica M. Mita, M.D., Pager: 210.746.1038; Email: [email protected]

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Institute for Drug Development; Cancer Therapy and Research Center; San Antonio, Texas USA

The molecular target of rapamycin (mTOR), which is a member of the phosphoinositide 3-kinase related kinase (PIKK) family and a central modulator of cell growth, is a prime strategic target for anti-cancer therapeutic development. mTOR plays a critical role in transducing proliferative signals mediated through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway, principally by activating downstream protein kinases that are required for both ribosomal biosynthesis and translation of key mRNAs of proteins required for G1 to S phase traverse. By targeting mTOR, the immunsuppressant and antiproliferative agent rapamycin (RAP) inhibits signals required for cell cycle progression, cell growth, and proliferation. RAP, a complex macrolide and highly potent fungicide, immunosuppressant, and anti-cancer agent, is a highly specific inhibitor of mTOR. In essence, RAP gains function by binding to the immunophilin FK506 binding protein 12 (FKBP12) and the resultant complex inhibits the activity of mTOR. Since mTOR activates both the 40S ribosomal protein S6 kinase (p70s6k) and the eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), RAP blocks activation of these downstream signaling elements, which results in cell cycle arrest in the G1 arrest. RAP also prevents cyclin-dependent kinase (cdk) activation, inhibits retinoblastoma protein (pRb) phosphorylation, and accelerates the turnover of cyclin D1 that leads to a deficienciy of active cdk4/cyclin D1 complexes, all of which potentially contribute to the prominent inhibitory effects of RAP at the G1/S phase transition. Both RAP and several RAP analogs with more favorable pharmaceutical properties have demonstrated prominent growth inhibitory effects against a broad range of human cancers in both preclinical and early clinical evaluations. This review will summarize the principal mechanisms of action of RAP and RAP derivatives and their potential utility of these agents as anti-cancer therapeutics. The preliminary results of early clinical evaluations with RAP analogs and the unique developmental challenges that lie ahead will also be discussed.

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Monica M. Mita Alain Mita Eric K. Rowinsky

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KEY WORDS

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Eric K. Rowinsky, M.D.

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Rapamycin, CCI-779, mTOR, PTEN

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INTRODUCTION

Unlike normal cells, which cannot proliferate in the absence of exogenous stimulation, cancer cells generate many of their own growth signals. This lack of dependence on stimulation from the physiological microenvironment disrupts a critical homeostatic mechanism that regulates the growth of various types of cells in any given tissue. In fact, growth signaling pathways may be constitutively overactive and/or disregulated in most human malignancies. Since such aberrant signaling pathways distinguish malignant from normal cells, they represent possible strategic targets for therapeutic development against cancer.1,2 One such potential target, the mammalian target of rapamycin (mTOR), also known as FRAP, RAFT1, and RAPT1, is a central modulator of proliferative signals generated by many physiological processes, as well as by aberrant signaling pathways and mutated genes. mTOR relays proliferative and anabolic signals downstream to transcriptional and translational apparatus that regulate the utilization of energy and G1 to S phase traverse.3-6 mTOR is a member of the recently identified phosphoinositide 3-kinase related kinase (PIKK) family of protein kinases. PIKK family members regulate cell cycle progression and cell cycle checkpoints that govern cellular responses to DNA damage and DNA recombination.7 The inhibition of mTOR results in a profound decrement in the transmission of proliferative signals through several critical elements in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) transduction pathway.7

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Figure 1. The structure of RAP and the RAP ester CCI-779.

HISTORY AND DEVELOPMENT Similar to the identification of several well validated targets for cancer therapeutic development, the identification of mTOR and its subsequent elucidation as a potential strategic target for anti-cancer therapeutics occurred somewhat serendipitously after the natural product RAP demonstrated unique antineoplastic properties. The lipophilic macrolide RAP (sirolimus; Rapamune®; Wyeth-Ayerst, Philadelphia, PA, USA) was identified more than 20 years ago during antibiotic screening. RAP was isolated from a strain of Streptomyces hygroscopicus found in the soil of the Vai Atore region of Easter Island (Rapa Nui). The agent is a white crystalline solid that is virtually insoluble in aqueous solutions, but readily soluble in organic solvents.8,9 Upon structural characterization, the RAP molecule was found to consist of a mixture of two conformational isomers due to cis-trans rotation about an amidic bond in its 31-membered macrolide ring.10 Its proposed chemical structure, as shown in Figure 1, was later confirmed by total organic synthesis. Although lacking antibacterial activity, RAP was demonstrated to be a potent fungicide, particularly against Candida albicans and other filamentous fungi.11 However, its antiproliferative effects were subsequently found to be more generalized beyond yeast after RAP demonstrated highly potent immunosuppressant and antineoplastic properties against mammalian cells.12 RAP received more serious consideration as an immunosuppressant nearly a decade later, coincident with the discovery of another Streptomyces derivative, FK506, at the Fujisawa Pharmaceutical Laboratories (Ibaraki, Japan).13 FK506 demonstrated 100-fold greater potency as an immunosupressant than cyclosporin A.14 The discovery that the macrobactam rings of both FK506 and RAP contain a unique hemiketal-masked α, β-diketopipecolic acid amidic component provoked a resurgence of interest in RAP as a possible immunosuppressive agent.15,16 RAP was subsequently determined to be substantially more potent than FK506 at suppressing organ allograft rejection and prolonging the survival of both skin and organ allografts.17 Furthermore, RAP was found to be capable of reversing acute active allograft rejection and enhancing long-term donorspecific allograft tolerance.18 Because of its high potency as an immunosuppressant, favorable therapeutic index compared with cyclosporin A, and negligible renal and hematopoietic toxicities, RAP received regulatory approval for use in preventing allograft rejection following organ transplantation. Although both RAP and www.landesbioscience.com

FK506 bind to the same family of intracellular immunophilin proteins, termed FK506 binding proteins (FKBP), these agents inhibit vastly different aspects of T-cell activation.19,20 Early labeling studies in Candida albicans indicated that RAP strongly inhibits [32P]phosphate incorporation into both DNA and RNA.11 Furthermore, RAP inhibits the growth and function of many other cell types besides lymphocytes and yeast, which led to interest in evaluating its antiproliferative effects in malignant neoplasms.21,22 Although FKBP family is quite large, there is both biochemical and genetic evidence that FKB12 is the most important binding protein with respect to RAP-sensitive signal transduction pathways. In late 1995, several groups of investigators identified the principal protein kinase targeted by the corresponding FKBP12-RAP complex, which was named the molecular target of rapamycin (mTOR), as well as other terms including the FKBP-RAP associated protein (FRAP), RAP FKBP12 target (RAFT1), and RAP target (RAPT1).3-6 mTOR is a 290 kd member of the PIKK family, which are involved in many critical regulatory cellular functions related to cell cycle progression, DNA damage, DNA repair, and DNA recombination. In essence, the binding of RAP to FKBP-12 leads to a “gain of function” as the resultant complex is substantially more efficient than each component alone at inhibiting mTOR. Although the precise function of mTOR is not completely understood, it appears that the protein kinase plays a major role in modulating cellular transition between energy-rich and -depleted states.23 The antiproliferative actions of RAP have been principally attributed to its ability to modulate the synthesis of critical proteins required for ribosome biosynthesis, protein translation, and G1 to S cell cycle phase traverse.24 Furthermore, impressive and unique antiproliferative actions were demonstrated following treatment of a diverse range experimental tumors with RAP.12,22,25 However, RAP’s poor aqueous solubility and chemical stability precluded its clinical development as an anti-cancer agent, which resulted in efforts directed at synthesizing RAP analogs with more favorable pharmaceutical characteristics. The most notable of the RAP analogs currently in clinical development as anti-cancer agents include the cell cycle inhibitor-779 (CCI-779), RAD 001 (Novartis Pharmaceuticals, AG, Basel, Switzerland), and AP23573 (Ariad Pharmaceuticals, Cambridge, MA). These agents have demonstrated impressive antiproliferative activity against a diverse range of malignancies in preclinical studies, and early clinical evaluations have been encouraging thus far.26-30


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Figure 2. The RAP-sensitive signal transduction pathway. Both RAP and RAP analogs bind to the immunophilin FK506 binding protein-12 (FKBP12). The RAP-FKBP12 complex binds to mTOR, inhibiting its kinase activity, which, in turn, inhibits the phosphorylation and activation of the downstream translational regulators, 4E-BP1/ PHAS-1 and p70s6k. These downstream effects decrease the translation of mRNA of specific proteins essential for G1 to S phase traverse.

MECHANISM OF ACTION OF RAPAMCYIN AND RAPAMYCIN ANALOGS RAP interferes with critical elements of signal transduction pathways, particularly those that link mitogenic stimuli to the synthesis of specific proteins required for G1 to S phase traverse. The end result is cell cycle arrest in the mid to late G1 phase, prior to the “restriction point” that modulates S phase initiation. As previously discussed, RAP gains function by binding to FKBP12 and the resultant FKBP12-RAP complex inhibits the mTOR kinase activity, which, in turn, blocks the activation of two critical downstream signaling elements, as shown in Figure 2. By inhibiting the phosphorylation of mTOR, the activation of the 40S ribosomal protein S6 kinase (p70s6k) is blocked, leading to reduced translation of 5´-terminal oligopyrimidine (5’TOP) mRNAs that encode for essential components of the protein synthesis machinery.31 The inhibition of mTOR also blocks phosphorylation of the eukaryotic initiation factor 4E binding protein-1 (4E-BP1), which is also known as PHAS-1 (phosphorylated heat- and acid-stable protein 1). In its dephosphorylated state, 4E-BP1 binds tightly to eIF-4E, thereby inhibiting the translation of mRNAs with regulatory elements in their 5´-untranslated regions (5’UTR) that encode for critical regulatory proteins such as growth factors, oncoproteins, and other cell cycle regulators.32 By inhibiting the translation of proteins that are essential for cell cycle traverse, cell growth, survival, and cell division, RAP and RAP analogs produce profound antiproliferative and immunosuppressive effects. Upstream and mTOR RAP and FK506 bind to similar intracellular proteins. The first to be discovered, FKBP12, a low molecular weight (12kDa) cytosolic protein, binds to FK506 and RAP with equal affinities.33,34 FKBP12 is a peptidyl-prolyl-cis-trans isomerase (PPIase) that catalyzes the cis to trans isomerization of Xaa-proline peptide bonds in short synthetic peptides.35-38 In addition to FKB12, several other related proteins, which possess PPIase activity and variable binding S171

affinities for RAP and FK506, have been identified.35-38 The disparate effects of RAP and FK506 are likely due to the different structural elements on the surfaces of the unique complexes formed between FKBP and these agents.39,40 In yeast, for example, the complex formed between FKBP12 and FK-506 preferentially binds to calcineurin, whereas the FKBP12-RAP complex preferentially binds to TOR proteins. In fact, the first important targets of the FKBP12-RAP complex to be identified, TOR1 and TOR2, were discovered in yeast by genetic screening for mutants resistant to the antiproliferative effects of RAP, and the cDNA encoding for the mammalian ortholog, mTOR, was isolated shortly thereafter.3-7, 36 In mammalian cells, the complex formed between FKBP12 and RAP binds to and inhibits the activity of the large (290 kd) polypepetide kinase mTOR, which is a member of the PIKK family of kinases. PIKK family members possess a common carboxy-terminal catalytic domain that bears sequence homology to the lipid kinase domains of PI3K family members. Among the most important functions of PIKKs relate to the regulation of cell-cycle progression, specifically the regulation of cell-cycle checkpoints that govern cellular responses to DNA damage, DNA repair, and DNA recombination.41,42 Structurally, mTOR contains three basic domains including the FKBP-RAP binding (FRB), catalytic, and HEAT domains. From an evolutionary standpoint, TOR is highly conserved, particularly with regard to the FRB domain, which translates into similar effects in yeast and mammalian cells following RAP treatment. More recently, yeast and mammalian TOR proteins have been shown to be integral in modulating the initiation of mRNA transcription and protein translation in response to intracellular levels of amino acids and other essential nutrients, organization of the actin cytoskeleton, membrane trafficing protein degradation, protein kinase C signaling, and ribosome biogenesis. Although the precise mechanisms by which TOR regulates these activities have not yet been defined, both RAP and RAP-resistant TOR yeast mutants have been invaluable tools for elucidating the essential elements of TOR signaling.44-46 In this context, it is also important to note that TOR


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Figure 3. RAP and RAP analogs inhibit phosphorylation of 4E-BP1/PHAS-1, preventing the release of the eIF-4E and the activation of the eIF4F complex.

mutants with altered FKBP-RAP binding affinities and RAP resistance have been identified.44-46 Although the precise means by which upstream growth factor and receptor interactions and other factors influence the phosphorylation state of mTOR are not completely understood, PI3K/protein kinase B (PI3K/Akt) appears to be the key modulatory factors in the upstream pathway (Fig. 2).47,48 PI3K is a heterodimeric lipid enzyme that play a central role in cellular proliferation, motility, neovascularization, viability, and senescence, and has been demonstrated to be upregulated in many types of malignant cells. Its principal physiological function is to phosphorylate the D3 portion of membrane phosphoinositols. The best characterized phosphorylation target of PI3K is the pleckstrin-homology (PH) domain of Akt, which is the cellular homologue of the retroviral oncogene v-Akt, a serine/threonine kinase activated by a dual regulatory mechanism that requires both translocation to the plasma membrane and phosphorylation. Although other pathways are activated downstream of PI3K, the Akt pathway is of particular interest because of its role in inhibiting apoptosis and promoting cell proliferation by affecting the phosphorylation status of cell-survival and anti-apoptotic proteins like BAD.49 Both PI3K and Akt are considered proto-oncogenes as they possess cell transforming properties, and their activation and downstream signaling are inhibited by the tumor suppressor gene PTEN, which is commonly mutated in solid malignancies.50-55 mTOR mediates many of the signals arising from PI3K- and/or Akt- driven mitogen stimulation, including signals associated with tumorigenesis.56 As such, it has been proposed that RAP and other therapeutics targeting mTOR may be particularly active against malignancies driven by activated PI3K, Akt, or both, as well as by PTEN mutations. Therefore, the characterization of the specific pathways and kinases that mediate these cell transforming effects may lead to the development of both therapeutic and preventative strategies against cancer. For example, the flavonoid derivative, LY294002 (Eli Lilly, Indianapolis, IN), a competitive, reversible inhibitor of the ATP binding site of PI3K, produces G1 arrest in proliferating cancer cells, leading to prominent anti-apoptotic effects and almost complete www.landesbioscience.com

inhibition of the growth of melanoma, osteosarcoma, ovarian carcinoma and other malignanices in preclinical studies.57 The agent also completely inhibits retinoblastoma protein (pRp) hyperphosphorylation that occurs in the G1 phase and upregulates the cyclin-dependent kinase (cdk) inhibitor p27. Downstream of mTOR There is ample experimental data indicating that mTOR functions downstream of the PI3K/Akt pathways and is phosphorylated in response to stimuli that activate the pathway. Following activation via phosphorylation, mTOR plays a central role in modulating two separate downstream signaling elements that control the translation of specific subsets of mRNAs including p70s6k and 4E-BP1/ PHAS-1.58-60 There is ample evidence indicating that the activation of either PI3K or Akt, and/or PTEN mutations, is sufficient to induce the phosphorylation of both 4E-BP1/PHAS-1 and p70s6k, which is mediated through the activation of mTOR.59,61-63 Furthermore, RAP treatment of activated PI3K- or Akt-expressing cancer cells, or cells lacking PTEN suppressor function blocks the phosphorylation of both p70s6k and 4E-BP1/PHAS-1, which suggests that mTOR is required for the mediation of these activities.59,61-63 The processes by which mTOR transmits signals downstream are further modulated by raptor, a 150 kDa evolutionarily conserved regulatory protein that forms a complex with mTOR and also binds to both 4E-BP1/PHAS-1 and p70s6k. The binding of raptor to mTOR is required for the downstream phosphorylation of 4E-BP1/PHAS-1 and facilitates the phosphorylation of p70s6k.64,65 4E-BP1/PHAS-1. 4E-BP1/PHAS-1, which is a low molecular weight protein that represses the initiation of protein translation through its association with eIF-4E, the mRNA cap-binding subunit of the eukaryotic initiation factor-4 (eIF-4F) complex (Fig. 3), is one of two principal downstream elements modulated by the phosphorylation state of mTOR.66,67,68 The importance of the 4E-BP1/ PHAS-1 complex in the regulation of cell growth is underscored by the observation that overexpression of eIF-4E alone is sufficient enough to induce cell transformation.69,70 The binding of 4E-BPs to eIF-4E is dependent on the phosphorylation status of 4E-BP/


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PHAS-1. In its unphosphorylated state that predominates in relatively quiescent cells and under growth factor deprived conditions, 4E-BP1/ PHAS-1 binds tightly to eIF-4E, which inhibits its activity and the initiation of protein translation.68 In response to proliferative stimuli initiated by hormones, growth factors, mitogens, cytokines and G-protein-coupled agonists, 4E-BP1/PHAS-1 is phosphorylated at five serine and threonine sites through the action of mTOR and other kinases, which decrease its binding affinity for eIF-4E. These actions promote the dissociation of the 4E-BP1/PHAS-1 complex, thereby increasing the availability of eIF-4E, which can then bind to eIF-4G (a large scaffolding protein), -4A (an ATP- dependent RNA helicase), and -4B, forming the multisubunit eIF-4F complex and facilitating cap-dependent protein translation (Fig. 3).71-73 These interactions result in an increase in the translation of mRNAs with regulatory elements in the 5-UTR like those encoding for c-myc, cyclin D1, and ornithine decarboxylase. In contrast, growth factor deprivation or treatment with RAP results in the dephosphorylation of 4E-BP1/PHAS-1, an increase in eIF-4E binding, and a concomitant impairment of the initiation of the translation of mRNAs with 5’UTRs that are required for G1 to S phase traverse. There is abundant experimental evidence indicating that mTOR is directly responsible for 4E-BP1/PHAS-1 phosphorylation and the activation of eIF-4E induced by various mitogenic stimuli. For example, the phosphorylation of 4E-BP1/PHAS-1 in insulin-treated cells has been shown to be effectively blocked by RAP, suggesting that mTOR serves as a upstream kinase in the 4E-BP1/PHAS-1 pathway.74-77 In fact, the ratio of 4E-BP1/PHAS-1 to eIF4E may be a determinant of RAP resistance as both intrinsic and acquired resistance in malignant cells have been related to low cellular ratios.78 Furthermore, the five 4E-BP1/PHAS-1 sites that are phosphorylated by mTOR in vitro have been demonstrated to be the identical sites of 4E-BP1/PHAS-1 phosphorylation following insulin treatment, as well as the sites that are rapidly dephosphorylated following RAP treatment.79,80 There is also experimental evidence indicating that mTOR must phosphorylate 4E-BP1/ PHAS-1 on Thr45 before Serine64 and Threonine69 are phosphorylated through an PI3K-independent pathway.81 It has also been speculated that mTOR acts indirectly as an inhibitor of a protein serine-threonine phosphatase, which functions to dephosphorylate 4E-BP1/PHAS-1 when conditions are appropriate for G1 to S phase traverse.82,83 p70s6k. Another principal target downstream of mTOR is the serine-threonine kinase p70s6k. Following activation in response to a broad range of mitogenic stimuli mediated by the PI3K/Akt pathway, mTOR phosphorylates (activates) p70s6k, which, in turn, phosphorylate the 40S ribosomal protein S6.54,84 The phosphorylation of S6 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 for ribosomal proteins, elongation factors, and insulin growth factor-II. Following RAP treatment, p70s6k undergoes rapid and profound degree of phosphorylation, which, in turn, results in a rapid and impressive decrease in protein synthesis.22,85,86 It is also important to note that p70s6k may also be activated by TOR-insensitive signaling pathways involving 3 phosphoinositidedependent kinase-1 (PDK1), MAPK, and SAPK. Further studies are warranted to better understand the role of each pathway in malignant transformation. There has also been experimental evidence suggesting that p70s6k phosphorylates the eIF-4G and eIF-4B units of the eIF-4F complex.68 S173

There appears to be at least three phosphorylation sites on p70s6k, all of which are blocked by RAP. The phosphorylation of Thr389 is particularly important since substitution of this residue with Ala blocks the activation of the kinase domain.87 There is also evidence indicating that the Thr389 is directly phosphorylated by mTOR.87 Alternatively, it has been proposed that mTOR represses a serine–threonine phosphatase that dephosphorylates RAP-sensitive sites onp70s6k.85,86 The derepression of this phosphatase by the binding of the FKBP12-RAP complex to mTOR may explain why p70s6k undergoes rapid dephosphorylation when cells are treated with RAP following stimulation with insulin and other growth factors.81,82,88 Other Effectors of mTOR and Targets of RAP In addition to its well characterized inhibitory effects on the activation of p70s6k and 4E-BP1/PHAS-1, RAP interferes with several other critical intracellular processes that are involved in cell-cycle traverse, which likely contribute to its antiproliferative activities. These pharmacological effects are particularly important in exponentially growing cells that do not undergo G1 arrest in response to the inhibition of p70s6k phosphorylation by RAP.89 RAP increases the turnover of cyclin D1 at both the mRNA and protein levels.90 In addition to a resultant decrease in the translation of cyclin D1 mRNA due to inhibition of 4E-BP1/PHAS-1, this effect results in a deficiency of active cdk4/cyclin D1 complexes required for pRb phosphorylation. RAP also blocks the elimination of the cdk inhibitor p27 and facilitates the formation of cyclin/cdks-p27 complexes.89,91-93 Additionally, RAP upregulates p27 at both the mRNA and protein levels and inhibits cyclin-A-dependent kinase activity in exponentially growing cells.91-93 These actions, along with the inhibition of translation of other mRNAs, may in part explain the profound inhibition of G1 to S phase traverse observed following treatment of cells with RAP. Interestingly, cells derived from p27 knockout mice are only partially resistant to RAP, suggesting that RAP may also block cell-cycle progression by p27-independent mechanisms.93-95 There is evidence indicating that TOR may also regulate the transcription of both rRNAs and tRNAs, which consumes 70–80% of the cell’s transcriptional capacity. Therefore, it has been speculated that TOR functions to regulate protein synthesis at both the transcriptional and translational levels. Among the experimental evidence supporting this hypothesis are studies demonstrating that RAP inhibits the function of Pol I and Pol III in yeast and mammalian cells, thereby reducing the transcription of rRNAs and tRNAs, respectively.96,97 The inhibition of rRNA synthesis by RAP may involve the tumor suppressor pRb, which represses both Pol I and Pol III.98 RAP may inhibit pRb phosphorylation by modulating the stability and expression of cyclin D1 and p27, which regulate cdks upstream of pRb.

RAPAMYCIN ANALOGS—ANTITUMOR ACTIVITY By inhibiting the translation of critical mRNAs involved in G1 to S cell-cycle phase traverse in response to proliferative stimuli, and by interfering with the balance of cyclins/cdks/cdk inhibitors in the early phases of the cell cycle, RAP induces profound inhibition of tumor growth. In early studies, RAP was shown to exert concentrationdependent inhibition of cell proliferation and tumor growth in a wide variety of murine and human cancers growing in both tissue culture and xenograft models including B16 melanoma, P388 leukemia, MiaPaCa-2 and Panc-1 human pancreatic carcinoma, and



TARGETING mTOR

tumors derived from B-cell lymphoma, small cell lung cancer carcinoma, and childhood rhabdomyosarcoma.12,22,24 RAP was also demonstrated to induce p53-independent apoptosis in childhood rhabdomyosarcoma and enhance the apoptosis-inducing effects of cisplatin in murine T-cell and human HL-60 promyelocytic leukemia, and human ovarian SKOV3 carcinoma in vitro.99,100 CCI-779 Collaborative efforts between investigators Wyeth-Ayerst Laboratories and the National Cancer Institute (NCI) to identify RAP analogs with favorable growth inhibitory and pharmaceutical properties led to the selection of the water soluble RAP ester CCI-779 for clinical development. In the NCI’s 60 tumor-type specific cell line screening panel, CCI-779 and RAP were demonstrated to possess nearly identical growth inhibitory profiles (Pearson correlation coefficient, 0.86) and IC50 values that were frequently less than 10-8 M.26 Cultured cell lines of prostate, breast, and small cell lung carcinomas, glioblastoma, melanoma, and T-cell leukemia were among the most sensitive cancers to CCI-779, with IC50 values in the nanomolar range.26 Also, significant growth inhibition was observed in a wide variety of human tumor xenografts, but the preponderance of tumor growth inhibition, in contrast to overt tumor regression, has inferred that disease-directed screening evaluations in the clinic should be designed to assess endpoints indicative of tumor growth delay, as well as overt tumor regression.26 Furthermore, several intermittent CCI-779 dose-schedules were demonstrated to be effective in human tumor xenograft studies, which is important in view of the concern that immunosuppression may result from protracted treatment with RAP analogs. In preclinical studies of CCI-779 administered on intermittent schedules, immunosuppressive effects resolved approximately 24 hours after treatment.26 Studies in PTEN knockout mice, which are prone to developing several types of dysplastic lesions leading to overt tumor formation, have demonstrated that PTEN-deficient tumor cells are extraordinarily sensitive the growth inhibitory effects of RAP and RAP analogs. This may be explained by the fact that PIP3 is constitutively activated in PTEN-deficient tumor cells, raising the possibility that sustained activation of signaling elements downstream of PIP3 renders these cells much more dependent on this pathway for growth and therefore more sensitive to the antiproliferative effects of mTOR inhibitors than wild-type cells. For example, PTEN-deficient tumor cells have been demonstrated to be hypersensitive to the growth inhibitory effects of low concentrations CCI-779, which may have important clinical ramifications.101 RAD 001 RAD 001 is an orally bioavailable hydroxyethyl ether of RAP produced by Novartis Pharma AG (Basel, Switzerland). The agent has demonstrated impressive antiproliferative activity against both human tumor cell lines and a broad range of human tumor xenografts. Furthermore, RAD 001 appears to possess prominent antiangiogenic properties. 29,30 AP23573 AP23573 (Ariad Pharmaceuticals; Cambridge, Massachusetts), a RAP prodrug, has demonstrated prominent antiproliferative activity against several cancers in vitro and in vivo.102 The agent has the same affinity for FKBP12 and mTOR when compared to RAP, but has much more favorable pharmaceutical and pharmacological characteristics. Studies in human xenograft models have shown a potent inhibition of tumor growth with a five daily oral administration schedule of AP23573.102 www.landesbioscience.com

CLINICAL DEVELOPMENT OF MTOR INHIBITORS Two intermittent schedules were selected for initial development of CCI-779 because of the prospect for protracted immunosuppression with continuous drug administration. Initial phase I studies were designed to evaluate the feasibility of administering CCI-779 as a weekly 30-minute intravenous (IV) infusion and a 30-minute IV infusion daily for 5 days every 2 weeks.27,28 Similar to the traditional paradigm used to develop nonspecific cytotoxic agents, initial phase I studies sought to recommend doses of CCI-779 for subsequent disease-directed studies based solely on the maximal tolerable rate of dose-limiting toxicities (e.g., maximum tolerated dose [MTD]). The principal side effects of CCI-779 on both schedules included dermatological toxicity, myelosuppression, reversible elevations in liver function tests, and asymptomatic hypocalcemia. The dermatological manifestations consisted of aseptic folliculitis, erythematous maculopapular rashes, eczcematoid reactions, dry skin, vesicular lesions, and nail disorders. Thrombocytopenia was the principal hematological toxicity, whereas anemia, leukopenia, and neutropenia were noted infrequently. Other drug-induced effects, including mucositis, hypertriglyceridemia, hypercholesterolemia, and reversible decrements in serum testosterone, were typically mild to moderate in severity, reversible, and noted over wide dosing ranges. The MTDs of CCI-779 on the daily-for-5-day-every-2-week schedule were 15 and 24 mg/m2/day for patients who had received extensive and minimal prior myelotoxic therapy, respectively. In contrast, the MTD was not determined for CCI-779 administered on the weekly schedule since antitumor activity was noted over a wide range of tolerable doses. The preliminary results of pharmacokinetic studies indicated that the pharmacokinetic behavior of CCI-779 is dose-dependent; the elimination half-life is approximately 15 to 17 hours; and the agent preferentially partitions in red blood cells.27,28 Unexpectedly, based on the results of preclinical studies, in which the predominant therapeutic effect of CCI-779 was delayed tumor growth, major tumor regressions were documented in previouslytreated patients with renal cell carcinoma, non-small cell lung carcinoma, and soft tissue sarcoma, and minor tumor regressions were observed in previously-treated patients with serous papillary carcinoma of the endometrium, breast carcinoma, squamous cell carcinoma of the skin, and non-Hodgkin’s lymphoma. The observation that CCI-779 consistently induced tumor regressions at relatively nontoxic doses suggests that the optimal therapeutic dose is lower than the MTD. In phase II studies of CCI-779 in patients with advanced renal cell and breast carcinomas, objective evidence of antitumor activity has been demonstrated consistently.103,104 In a phase II study, in which patients with advanced renal cell carcinoma were randomized to treatment with CCI-779 at doses of either 25, 75, or 250 mg/m2 IV weekly, although the overall rates of major regressions (at least 50% reduction in tumor size) were low on all treatment arms (3%, 5%, 6% partial response rates at doses of 25, 75 and 250 mg/m2, respectively) an appreciable proportion of patients in all groups (69–78%) experienced either major tumor regression, minor response, or prolonged stable disease.103 The median time to tumor progression (5.8 months) and overall survival (12 months) were also notable, particularly for heavily pretreated patients. Interestingly, there were no differences in antitumor activity between the three treatment groups. Toxicities included skin rash, mucositis, asthenia, nausea, acne, thrombocytopenia, hypertriglyceridemia, and anemia. A phase II study of CCI-779 in previously treated patients with metastatic breast cancer who were randomized to receive treatment


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with either 75 or 250 mg/m2 IV on a weekly schedule was also performed.104 Two major responses, as well as several minor responses, were observed in the first 16 patients treated in each of the two dose cohort, with regressions of liver, lung, and chest wall metastases. Treatment-related toxicities, including myelosuppression, mucositis, diarrhea, hyperglycemia, hypokalemia, and depression, were generally mild to moderate in severity.104 Based on the favorable results of experimental studies of RAP analogs in models of malignant glioma, in which there is a high incidence of PTEN deletions and other aberrations resulting in constitutive activation of signaling elements comprising the PI3K/Akt pathway, evaluations of the antitumor activity of CCI-779 in patients with glioma have been initiated. Since CCI-779 is principally metabolized by cytochrome P450 mixed function oxidases, which are induced by many types of anticonvulsant agents, the toxicities, pharmacokinetics, and optimal doseschedule of CCI-779 are also being evaluated in glioma patients who are concurrently receiving treatment with P450 mixed function oxidase-inducing anticonvulsant agents. In addition, phase I studies evaluating the feasibility of administering CCI-779 in combination with various nonspecific cytotoxic chemotherapeutics, such as 5-fluorouracil and gemcitabine, are underway, and an oral formulation of CCI-779, which would increase the feasibility of protracted drug administration, is in early clinical development.

NEW TARGETS AND NEW CHALLENGES For RAP analogs and other rationally-derived target-based therapeutics, in which tumor growth delay is anticipated to be the predominant therapeutic effect based on the results of preclinical studies, traditional nonrandomized phase II tumor screening paradigms that focus solely on tumor regression are likely to be suboptimal since clinically significant inhibitory effects on tumor growth, which may translate into real clinical benefit, may not be readily appreciated.105 In addition to evaluating the rate of major tumor regressions, which has largely been used to gauge the potential of nonspecific cytotoxic therapeutics in small phase II trials and for making “go or no go” decisions about the subsequent development of anti-cancer therapeutics, screening trials must also be designed to appreciate relevant inhibitory effects on tumor growth. Clinical endpoints that have been proposed for the evaluation of these antiproliferative agents include time to progression, the proportion of patients without early progressive disease, improvement in the quality of life and/or disease-related symptoms, and reduction of relevant biomarkers that reflect tumor growth. Potential surrogate endpoints include biological evidence of target inhibition, particularly if this endpoint can be validated a priori to reflect tumor growth delay and/or tumor regression in preclinical models, and assessment of cell proliferation using functional imaging modalities (e.g., positron emission tomography, nuclear magnetic resonance scanning). In contrast to the principal therapeutic expectations of RAP analogs based on the results of preclinical studies, regressions of several types of advanced malignancies have been observed in early clinical trials.106,107 However, the overall rates of tumor regression that have been noted with RAP analogs and other rationally-derived targeted therapeutics have been lower than those that can be readily appreciated in small phase II clinical trials. Therefore, if tumor regression is a potential construct based on the agent’s specific mechanism of action and preclinical results, nonrandomized screening studies must be sufficiently sized and powered to enable detection of relatively low regression rates (e.g. 5–10%) since tumor regression may predict for a more sizeable S175

proportion of patients with clinically relevant degrees of tumor growth delay. However, since delayed tumor growth may be the sole therapeutic effect of these agents, randomized clinical trials may be the optimal means to gauge the agent’s potential to ultimately produce a relevant degree of therapeutic benefit since an agent that is capable of inducing profound inhibitory effects on tumor growth without overt tumor regression may still have profound clinical relevance. Another important issue pertaining to the development of RAP and other rationally-designed, target-based therapeutics is the need to assess relevant target effects in order to guide dose selection in phase I trials and facilitate the assessment of clinical benefit in phase II studies. The selection of an appropriate dose of CCI-779 for phase II trials is especially challenging since objective antitumor activity has been observed in patients treated with a wide range of doses, whereas toxicity has been more directly related to dose. Unlike the situation with nonspecific cytotoxic agents, in which the relationship between dose and response is roughly linear and the MTD is ultimately sought, most preclinical data suggest that therapeutic activity generally plateaus above a “threshold” dose. However, since the relationship between dose and toxicity is still likely to be somewhat linear, the selection of an optimal biological dose for clinical trials is desirable. The intracellular target of RAP and its downstream signaling pathways have been well-characterized, and the development of biological or biochemical assays that measure the phosphorylation status of either 4E-BP1/PHAS-1 and/or p70s6k may assist in determining whether patients are receiving biologically relevant doses.108 Nevertheless, a critical issue is whether these downstream effects relate to antitumor activity and can be validated, particularly since malignant cells can traverse the cell cycle and proliferate despite inactivation of 4E-BP1/PHAS-1 and p70s6k by RAP.90,108 These observations suggest that either these pathways are not the only mechanisms by which cell cycle progression is regulated or mTOR-4E-BP1/PHAS-1 and mTOR-p70s6k pathways are redundant. Although such assays may facilitate the determination of whether a delivered dose is pharmacologically relevant, they may not be gauging drug efficacy since the assays may reflect targets that are not related to the effects of the agent on tumor proliferation. Alternatively, downstream factors may render tumor cells resistant to the antiproliferative effects of the agent. Therefore, optimal assessments of target inhibition in the tumor or surrogate tissues may require additional information about the relevance of target inhibition in terms of tumor cell proliferation, cell cycle arrest, apoptosis, and/or angiogenesis. Such assays will ultimately be validated following the acquisition of late-stage clinical outcome data, particularly information pertaining to tumor regression, time to progression, clinical benefit, and survival in relatively large numbers of patients. At this juncture, the utility and feasibility of assays measuring the inactivation of p70s6k in peripheral blood mononuclear cells are being assessed in the course of early phase II studies of RAP analogs.109 Another important issue regarding the optimal development of RAP analogs is whether tumors with specific molecular abnormalities may be “hypersensitive” or alternatively “hyperresistant.” Malignancies that are principally driven by paracrine or autocrine stimulation of receptors that constitutively stimulate PI3k/Akt/mTOR-related pathways or tumors with aberrations that activate PI3K/Akt-related elements (e.g., PTEN mutations) may be particularly dependent on RAP-sensitive pathways for growth and therefore may be especially sensitive to RAP analogs.99,110,112 In addition, aberrations of G1 checkpoint regulators, such as pRb, p16, p27, and cyclin D1, may also portend hypersensitivity to RAP and may be markers that enable prediction of drug efficacy.112



TARGETING mTOR

CONCLUSION The natural product RAP and various water-soluble RAP analogs possess potent and unique immunosuppressant and antiproliferative properties, most likely due to their ability to modulate signal transduction pathways that link growth stimuli to the synthesis of specific proteins required for G1 to S phase traverse. RAP and its analogs inhibit the activity of mTOR, which, in turn results in decrements in the activation of two critical downstream translational regulators, p70s6k and 4E-BP1. These actions result in a profound inhibition of protein synthesis and G1 to S phase traverse. By virtue of these effects, RAP and RAP analogs inhibit the proliferation of a broad range of human malignancies in vitro and in vivo, which has served as the rationale for their development. In early clinical studies, RAP analogs appear to be well tolerated at doses that have resulted in impressive antitumor activity against several types of refractory malignancies. However, many developmental challenges, such as defining an optimal dosing range for clinical evaluations, detecting relevant drug effects on tumor growth delay in nonrandomized trials, and identifying patients who are most likely to benefit from treatment, must be overcome. References 1. Besson A, Robbins SM, Yong VW. PTEN/MMAC1/TEP1 in signal transduction andtumorigenesis. Eur J Biochem 1999; 263:605-11. 2. Sherr CJ. The Pezcoller Lecture: Cancer Cell Cycle revisited. Cancer Res 2000; 60:3689-95. 3. Sabatini DM, Erdjument-Bromage H, Lui M, et al. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin- dependent fashion and is homologous to yeast TORs. Cell 1994; 78:35-43. 4. Sabers CJ, Martin MM, Brunn GJ, Williams JM, Dumont FJ, Wiederrecht G, et al. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem 1995; 270:815-22. 5. Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 1994; 369:756-8. 6. Chiu MI, Katz H, Berlin V. RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex. Proc Natl Acad Sci U S A 1994; 91:12574-8. 7. Sarkaria JN, Tibbetts RS, Busby EC, Kennedy AP, Hill DE, Abraham RT. Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res 1998; 58:4375-82. 8. Vezina C, Kudelski A, Sehgal SN. Rapamycin (AY-22,989), a new antifungal antibiotic I. Taxonomy of the producing streptomycete and isolation of the active principle. J Antibiot 1975; 28:721-6. 9. Baker A, Sidorowicz A, Sehgal SN, Vezina C. Rapamycin (AY-22,989), a new anti-fungal antibiotic. III. In vitro and in vivo evaluation. J Antibiot 1978; 31:539-45. 10. Finlay JA, Radics L. On the chemistry and high field nuclear magnetic resonance spectroscopy of rapamycin. Can J Chem 1980; 58:579-90. 11. Singh K, Sun S, Vezina C. Rapamycin (AY-22,989), a new antifungal antibiotic IV. Mechanism of action. J Antibiot.1979; 32:630-45. 12. Eng CP, Sehgal SN, Vezina C. Activity of rapamycin (AY-22,989) against transplanted tumors. J Antibiot (Tokyo) 1984; 37:1231-7. 13. Kino R, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J. Antibiot. 1987 (Tokyo); 40:1249-55. 14. Kino R, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J Antibiot 1987 (Tokyo); 40:1256-65. 15. Tanaka H, Kuroda A, Marusawa H, Hatanaka H, Kino T, Goto T, et al. Structure of FK-506: a novel immunosuppressant isolated from Streptomyces. J Am Chem Soc 1987; 109:5031-3. 16. Morris RE, Meisser BM. Identification of a new pharmacologic action for an old compound. Med Sci Res 1989; 71:609-10. 17. Morris RE, Wu J, Shorthouse R. A study of the contrasting effects of cyclosporine, FK 506, and rapamycin on the suppression of allograft rejection. Transplant Proc 1990; 22:1638-41. 18. Morris RE, Meiser BM, Wu J, et al. Use of rapamycin for the suppression of alloimmune reactions in vivo: schedule dependence, tolerance induction, synergy with cyclosporin, and FK-506 and effect of host-versus-graft and graft-versus-host reactions. Transplant Proc 1991; 23:521-4. 19. Dumont F, Melino M, Staruch M, et al. The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells. J Immunol 1990; 144:1418-24. 20. Dumont F, Staruch M, Koprak S, et al. Distinct mechanism of suppression of murine T cell activation by the related macrolides FK-506 and Rapamycin. J Immunol 1990; 144:251-8.

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