Inhibitors of mammalian target of rapamycin as novel antitumor agents:
`From bench to clinic
`
`Shile Huang & Peter J Houghton*
`Address
`Department of Molecular Phamraoology
`St Jude Children's Research Hospital
`332 N Laudardala
`Memphis
`TN 3B105»2?94
`USA
`Email: peter.houghton@stjude.org
`
`‘To whom correspondence should be addressed
`
`Current Opinion In lnvesligatlonal Drugs 2002 3(2):295-304
`O Phan'naPress Ltd ISSN 1472-44‘i'2
`
`Rapamycin and its derivatives, CCI-779 and RAD-D01, inhibit tlie
`mannnaliau target of
`rapamycin
`(n1'1"OR,l, doronregulating
`translation of specific mRNAs required for cell cycle progression
`from C1 to S phase. Preclinically, mTOR inhibitors potently
`suppress growth and proliferation of numerous tumor cell lines in
`culture or when grown in mice as xenografts. CC!-779 and RAD-
`OOI are being developed as antitumor drugs and are undergoing
`clinical
`trials. Clinically, CCl-779 has
`shown evidence of
`antitumor activity but
`imluce.-:l relatively mild side eflfects
`in
`patients. Here we discuss potential antitumor mechanisms and
`resistance meclmnisnts of mTOR inhibitors, and summarize the
`current status of these compounds as novel antitumor agents.
`
`Keywords Antitumor, cell cycle, mammalian target of
`rapamycin (mTOR), rapamycin
`
`Introduction
`Malignant disease is characterized by genetic mutations or
`compensatory changes in cells that result in unregulated
`population growth due to increased proliferation or decreased
`cell death. Since the early 1950s, extensive chemical synthesis
`and screening programs have resulted in clinical
`trials of
`many potential anticancer agents. In some cases, cytotoxic
`agents have significantly increased survival rates in adult
`diseases and notably for children with hematologic as well as
`solid tumors. However, relatively few cytotoxic agents have
`proven to be useful against a wide spectrum of cancers and
`' most cause significant toxicity. The reasons for the relatively
`poor activity of cytotoxic agents are numerous. Most do not
`target the transforming event, rather they induce forms of
`damage that lead to necrosis or activation of cell-suicide,
`apoptosis. As a consequence, agents are cytotoxic to both
`malignant tumor cells and normal healthy cells, often causing
`severe side effects. Understanding pathways known to be
`critical to the growth and survival of tumors is essential to
`developing potentially selective treatments. Validation of this
`concept has been met with compounds such as imatinib
`(Gleevec, STI—571; Novartis AG). This compound inhibits
`tyrosine kinases such as BCR/ABL in chronic myeloid
`leukemia and c-KIT in gastrointestinal stromal tumors. Less
`success has been met by targeting activated Ras with
`inhibitors of farnesyltransferase.
`
`Wyeth-Ayerst
`Rapamune;
`(sirolimus,
`Rapamycin
`Laboratories; Figure 1), an immunosuppressant, has emerged
`as a potent inhibitor of a signaling pathway that may be
`deregulated in some forms of cancer,
`leading to both
`
`increased growth and malignant characteristics of cells. It is a
`lipophilic
`macrolide,
`that
`selectively
`inhibits
`a
`serine/theronine kinase, specifically, the lammalian target gt
`gapamycin
`(mTOR).
`mTOR
`lies
`downstream of
`phosphatidylinositol 3-kinase (PI3I<)
`in the PI3l( signaling
`pathway. Rapamycin was originally isolated as a fungicide
`from the soil bacteria Streptomyces liygroscopicus, collected
`fron1 Easter Island (known as Rapa Nui to the natives) in the
`South Pacific in 1975 [1,2]. Structurally similar
`to the
`imrnunosuppresive reagent FK-506 ttacrolirnus; Fujisawa
`Pharmaceutical Co Ltd), rapamycin was initially developed
`for transplant rejection [3,4] and was approved by the US
`Food and Drug Administration in September 1999 and the
`European Commission in March 2000. While rapamycin was
`being developed as an immunosuppressant, it was also found
`to exert potent antitumor activity in intro and in viva [5-7].
`However, perhaps because its mechanism of action was
`unknown at that time, rapamycin was not developed as a
`cancer therapeutic.
`
`The potential for rapamycin as a cancer therapeutic was
`refocused, in part, by studies of Dilling et al [8]. These were
`the first studies to demonstrate potent and selective
`inhibition of growth by rapamycin. Rapamycin potently
`inhibited
`the growth of
`rhabdomyosarcoma
`cells
`at
`concentrations of approximately 1 ng/ml, whereas human
`colon cancer cells were inhibited only at micromolar
`concentrations in culture (Table 1). Proliferation of many
`rhabdomyosarcoma cells is regulated by an autocrine loop
`involving secretion of type II insulin-like growth factor {lGF-
`II) and signaling through the type I IGF receptor [9,10].
`Indeed,
`the cell
`lines most sensitive to rapamycin were
`dependent on this autocrine pathway.
`
`Additional findings from various research groups around
`the world support rapamycin as a good candidate for a
`cancer therapeutic agent. In many malignant cells in culture,
`rapamycin can act as a cytostatic agent by arresting cells in
`G1 phase. Another potential mode of its antitumor action is
`via the induction of apoptosis. Rapamycin potently inhibits
`proliferation
`or
`growth
`of
`cells
`derived
`from
`rhabdomyosarcoma,
`neuroblastoma,
`glioblastoma,
`medulloblastoma.
`small
`cell
`lung
`cancer
`[8,11-16],
`osteoscarcoma [17], pancreatic carcinoma [18,19], breast and
`prostate carcinoma [20-2], murine melanoma and leukemia,
`and B-cell lymphoma [6,23-25] (Table 1).
`
`Despite the antiproliferative effects of rapamycin, it has poor
`water-solubility and stability in solution, precluding its
`formulation for parenteral use as an anticancer agent. Two
`rapamycin ester analogs, CCI-7'79 (Wyeth-Ayerst Research;
`Figure 1) and RAD-091 (everolimus; Novartis AG; Figure 1),
`with improved pharmaceutical properties, but
`similar
`cellular effects to rapamycin [16,20—22,26,27,28-,29o], are
`currently undergoing antitumor phase III and phase I
`clinical trials, respectively. This review will discuss potential
`antitumor and resistance mechanisms of rapamycin and its
`derivatives, and summarize the preliminary data about
`these compounds, from the bench to the clinic.
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`296 Current Opinion in Investigations! Drugs 2002 Vol 3 No 2
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`Table 1. sensitivity at different tumor cell lines to rapamycln.
`cell Line
`flhabdomyosaraoma ceiis
`Fth1 (10% serum)
`H111 {serum free)
`H1118
`H1128
`Ft!-I30
`Colon carcinoma cells
`GCJG1
`VFiCJc1
`Ceca
`HCT8
`HOT29
`HCT116
`small cell lung manner cells
`H69
`H345
`H510
`Neurobiastama cells
`NB-SD
`NS-1643
`NB-EB
`NB-1691
`NB-1382.2
`Giiobiastama cells
`SJ-G2
`SJ-G3
`Medullabiastoma ceiis
`DAOY
`Ostoobiaete-like ostaosarcoma coils
`ROS 17.~‘2.B
`Pancreatic cancer cells
`Pane-1
`MiaPaca-2
`Leukemia cells
`FIBL-2H3
`B-col.‘ lymphoma cells
`BKS-2
`L1 .2
`' NFS1.1
`WEHI-279
`Thyrnama coils
`EL4
`
`Fla
`
`In cln C
`tnsfmli
`4630
`3.6
`
`0:80
`iflfiimil
`0.17
`
`
`
`Antitumor mechanism of inhibitors of mTOR
`Rapamycin and its analogs, CCI-779 and RAD-001, are the
`most potent and selective inhibitors of mTOR reported so
`far. The three agents share a common mechanism of
`antitumor action, ie, inhibiting mTOR, which, links mirage,-.
`stimulation to protein synthesis and cell cycle progresgiol-L
`mTOR
`
`antitumor mechanism of
`To better understand the
`rapamycin and its derivatives, we will briefly review the
`emerging cellular role of mTOR. mTOR is referred to by
`various other names, some of which are derived from its
`binding partner Fl(~506-binding protein, FKBP12 (discugsed
`below). These names are FRAP (FKBP12 and rapamycjn-
`associated protein}, RAI-T1 (rapamycin and FKBP12 target
`1), RAPT1 {rapamycin target 1) and SE13 {sirofilnus effector
`protein}.
`In the mid-1990s, mTOR was identified as a
`mammalian serine/threonine kinase of approximately 289
`kDa in humans, mice and rats [30-33]. TOR proteins
`represent a class of evolutionarily conserved kirtases in
`eukaryotes.
`In the yeasts, Saccharomyces
`cerevisiae and
`Scltfzosacclmromyces pombe, two TOR genes, TOR] and TOR2,
`
`have been cloned, which share 67% identity and encode
`proteins of approximately 280 kDa [34-36]. In the fruit fly,
`Drosophila melanogaster, a single TOR ortholog,
`termed
`dTOR, has been characterized, sharing 38% identity with
`TOR2 from Saccharomyces cerevisiae [37,38]. mTOR shares
`approximately 45% identity with TORI and TOR2 from the
`yeast Saccharomyces ccrwisiac, and 56% identity with dTOR in
`overall sequence [39,40]. Human, mouse and rat mTOR
`proteins share 95% identity at the amino acid level [40,41].
`mTOR contains a catalytic kinase domain and a Fl(BP12-
`rapamycin binding (FRB) domain near the C«terminus, and
`up to 2.0 tandemly repeated HEAT {Huntingtin, EF3, A
`subunit of PPZA and TOR) motifs at the N-terminus, as well
`as FAT (FRAP-ATM-TRRAP) and FATC {FAT C-terminus)
`domains (Figure 2}. Since the C-terminus of mTOR shares
`strong homology to the catalytic domain of PI3K, mTOR is
`considered a member of the Pll<-related kinase family,
`which also includes MEC1, TELL RAD3, MEI-41, DNA-PK,
`ATM, ATR and TRRAP [42»]. Both PI3l< and potentially
`protein kinase B (PKB; Akt) lie upstream of mTOR, whereas
`ribosomal p70S6 kinase (p70S6l() and eukaryotic initiation
`factor-4E (eIF—4E) binding protein isoforms (4E-BP1-3) are the
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`Inhibitors of mammalian target of rapamycin as novel antitumor agents: From bench to clinic Huang 8. Houghton 297
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`Figure 1. Molecular structures or raparnycin, col-779 and RAD-001.
`
`ocr-in
`(Wyeth-Ayersii
`
`best characterized downstream mTOR effector molecules.
`
`Figure 2. Schematic representation of mTOR domains.
`
`Increasing evidence has implicated mTOR as a central
`controller of cell growth and proliferation. mTOR may
`directly or indirectly regulate translation initiation, actin
`organization, membrane traffic and protein degradation,
`protein kinase C signaling, ribosome biogenesis and tRNA
`synthesis, as well as transcription [42u]. Recent results also
`suggest
`that mTOR may sense
`cellular ATP levels,
`suppressing protein synthesis when ATP levels decrease
`[43].
`
`Specificity of rapamycin action
`Rapamycin inhibits proliferation and growth of many
`tumor cells, which is clearly a consequence of binding
`mTOR. Whether this action is a consequence of inhibiting
`mTOR kinase activity per se is less clear. Raparnycin cannot
`directly bind to mTOR. It first has to bind to the 12 kDa
`cytosolic immunophilin, FKBP12, found in mammalian
`cells,
`to form the FKBP12-rapamycin complex. The
`complex then interacts with the FRB domain in mTOR
`(Figure 2),
`and inhibits
`function of mTOR. High
`concentrations of rapamycin together with FKBP12 are
`required to inhibit mTOR kinase activity in oiiro and
`mTOR autophosphorylation. However,
`the specificity of
`rapamycin action can be demonstrated in vivo as certain
`mutations in the FRB domain of mTOR affect FI(BP12-
`rapamycin binding, and significantly reduce the cellular
`sensitivity of rapamycin. The first
`rapamycin-resistant
`alleles, TORI-I and TOR2—1, identified in a Saccimromyces
`cereoisiae genetic screen were shown to confer dominant
`resistance. These mutant TOR proteins lost the ability for
`FKBP-rapamycin
`complex
`binding
`[44].
`Similarly,
`mammalian
`cells
`also
`became
`highly
`resistant
`to
`rapamycin when a mutation (Ser’°"—>Ile’“”} occurred in the
`FRB domain of mTOR, which resulted in decreased affinity
`for binding of FKBP12-rapamycin complex [14,45,46]. in
`the yeast. Smtcharonryces cerevisiae, decreased RBP1,
`a
`homolog of mammalian FKBP12, or mutation at Tyr", led
`to decreased binding of
`rapamycin and conferred a
`recessive resistance phenotype [47].
`
`HEAT Huntingtin, EF3, A subunit of PP2A and TOR, FAT FFlAP-
`ATM-TFIFIAP. FFIB FKBP12-rapamycin binding, CD catalytic
`domain. FATC FAT C-tenninus.
`
`Potential models for raparnyc-in inhibition of mTOR
`Small molecule kinase inhibitors act directly,
`regulating
`kinase activity generally by competition for ATP binding.
`However, whether
`the FKBP12-rapamycin complex or
`rapamycin alone directly inhibits the kinase activity of mTOR
`is still controversial. in vitro, rapamycin inhibited the modest
`increase in kinase activity of imrnunopreczipitated mTOR
`induced by insulin [48]. The FKBP12-rapamycin complex also
`inhibited the autokinase activity of mTOR, although a much
`higher concentration of rapamycin was needed in vitro than in
`trim to inhibit
`the activity of mTOR [49]. Conversely,
`treatment of cells with raparnycin did not alter
`the
`autophosphorylation level of Serm, and had little or no effect
`on the kinase activity of immunoprecipitated mTOR [37,49].
`More recently. an alternative model for mTOR function has
`been proposed. Specifically, mTOR may repress phosphatase
`activity associated with downstream targets. The inhibition of
`mTOR induced by bound FKBP12-rapamycin complex, may
`result
`in activation of
`this phosphatase, which then
`dephosphorylates downstream effector molecules such as
`p70S6K [500-,5!-]. Consistent with this model, we [52,
`Houghton 8: Huang, unpublished data] have suggested that
`mTOR regulates the catalytic subunit of PPZA associated with
`p44/42 mitogen-activated protein (MAP) kinases in some
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`293 Current Opinion in lnvestigational Drugs 2002 Vol 3 No 2
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`p44/42
`inhibits
`rapamyciri
`cells,
`these
`In
`cells.
`phosphorylation on Thrm following IGF-I
`stimulation.
`However, more studies are necessary to confirm the
`generality of this phosphatase model. An alternative model is
`for n1TOR to act as a scaffold and for the FKBP12-rapamycin
`complex to disrupt higher order mTOR-protein complexes.
`
`Rapamycin inhibition of mTOFt-controlled signaling
`nathways
`Although specific details of how rapamycin inhibits function of
`mTOR remain to be resolved, it has been widely accepted that
`inhibition of mTOR by rapamycin blocks growth factor
`stimulation of 40S ribosomal p7t}S6 kinase and phcsphorylation
`of 4E-Bl"'l (also designated PHAS-I). This results in a 15 to 20%
`inhibition of overall protein translation and arrests cell cycle
`progression in_ G1. Consistent with this observation, mTOR
`controls the synthesis of essential proteins involved in cell cycle
`progression (cyclin D1 and ornithinine decarboxylase) {53,54]
`
`[55]. A scheme of mTOR-controlled
`(c-Myc)
`and survival
`signaling pathways based on rapamycin effects is shown in
`Figure 3. 4E-BP1, the suppressor of elF-4E, has been reported to
`be a direct substrate for mTOR in cells [56,57]. In oilm, mTOR
`selectively phosphorylates 4E-BP1 at least at two and possibly
`four Ser/Thr residues (Thr", Thr“, 'I‘hr"’ and Set”) in the N-
`terminal region [58--,59]. Phosphorylation of 4E-BP1 appears to
`be an ordered process [53..,59,50]. Phosphorylation of Ser“
`depends
`on
`phosphorylation
`of
`all
`three Ser/Thr
`phosphorylation sites 159,60], whereas mutations of Thr”
`and/or Thr"‘ to Ala(s) prevents phosphorylation of Ser“ and
`Thrw, suggesting that ph0spl'|orylal'it)rt of Th!” and 'I‘hr"’ serves
`as a requisite ‘pr-in-ring‘ event [51I]. lt appears that mTOR also
`plays a critical role in regulating the phosphorylation of Serif
`and Thr”.
`In the presence of raparnycin. 4-IE-BP1 becomes
`hypophosphorylated and associates with elF-4E. This prevents
`formation of the elF-4F initiation complex and cap-dependent
`translation of mRNA.
`
`Figure 3. Rapamyclfl-lflhlbllbd 8l9f|8ll|'|9 pathways controlled by n1'l'OFt.
`
`FKB12-rapamycin
`
`4E—BP1
`
`(PHAS-I)
`
`Arrows represent activation. whereas bars represent inhibition. IGF-IFI type I Insulin-like growth factor, IRS1 insulin receptor substrate 1,
`Plait phosphatldvlinositoi 3-klnase. PTEN phosphatase and tensin tlomolog deleted on chromosome ten. PKBJAICI protein kinase B, FKB12-
`fapamyclrl FK-506-binding protein 12-rapamycin complex, rn'l'OR mammalian target oi rapamycln. alF-4E eukaryotic initiation taotor-4E,
`4E-BP1 (PHA3-I} elF~4E—bincIing protein 1. S6 40S ribosomal protein, prosaic p70S6 kinase.
`
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`Inhibitors at mammalian target at rapamycin as novel antltumor agents: From bench to clinic Huang & Houghton 299
`
`Ribosomal p70S6K represents the other well characterized
`downstream target of mTOR. Two p70S6 kinases have been
`characterized, namely. p70S6K1 and p70S6l(2. The activation
`of both these kinases can be inhibited by rapamycin [61,62].
`rnTOR may directly or indirectly phosphorylate p7{lS6I(1 at
`Thrm or Thr” [50--,63,64,65I--,66o,67]. Phosphorylation of
`these two residues is blocked by rapamycin. Furthermore,
`mutation of either of these residues can abrogate the ability of
`rapamycin to inhibit p'70S6I( activation. p7l}S6l( functions to
`increase translation of 5' terminal oligopyrirnidine (STOP)
`tract
`IHRNAS, primarily Coding
`for
`elements of
`the
`translational machinery,
`such
`as
`ribosomal
`proteins,
`elongation factors, the poIy(A) binding protein [61] and [GF—I[
`[68]. Inhibition of mTOR by rapamycin thus selectively causes
`decreased translation of 5"IlOP-containing mRNAs.
`
`In addition to pathways controlling translation initiation,
`mTOR has been implicated in regulating the retinoblastoma
`protein {pRb), RNA polymerase {Pol} l/ll/III-transcription
`and translation of rRNA and ERNA, and phosphatases (PPZA,
`PP4, PP6) [69]. It seems that these pathways are cell type-
`dependent. For example, in vascular smooth muscle cells,
`rapamycin may act upstream of pRb to slow or arrest cell
`cycle transit ['70]. In this model, rapamycin inhibits activation
`of cyclin—dependent kinases
`{CDl(s), which results
`in
`hypophosphorylation of pRb protein, and inhibits cells
`progressing from C1 to S-phase [70].
`In T—lymphocytes,
`rapamycin induces G1 arrest, in part through inhibition of
`activation of CDK1 (p34'“"’) and the formation of the cyclin B-
`p33"” complex [71,72]. G1 arrest by rapamycin may also be
`due to prevention of the degradation of CDK inhibitory
`protein p27""" that occurs when cells are stimulated by growth
`factors [73,74]. This is further supported by the observation
`that p27""" deficient fibroblasts are somewhat resistant to
`rapamycin as determined by assaying for DNA synthesis [75].
`In NIHST3 cells, rapamycin inhibits the G1 to S transition in
`part through decrease of cyclin D1 mRNA level and protein
`stability ['76], or delay of the expression of cyclin A [77].
`
`Arititumor activity of rapamycin
`As previously mentioned, rapamycin has been approved as an
`irnmunosuppmiive drug for organ transplantation by the
`FDA. So far, rapamycin has been used clinically in organ
`transplantation with great success, particularly in kidney
`transplantation [78,79]. This is because rapamycin can inhibit T-
`cell activation and proliferation. Increasing evidence indicates
`that rapamycin is not only a potent immunosuppressant, but
`also a promising antilurnor agent. As reviewed above (Table 1),
`rapamycin potently inhibits the growth of many tumor cell
`lines in intro, and has demonstrated antiturnor activity in both
`xeno-graft and syngeneic murine tumor models.
`
`However, rather than acting as a cytostatic, rapamycin induces
`cell death under some conditions. Early data show that
`rapamycin induces prograrnrned cell death or apoptosis of B-
`cells [24,25]. Consistent with these findings, recent studies
`indicate that rapamycin alone can also induce apoptosis of
`certain rhabdomyosarcorna cells [14,15] and monocyte- and
`CD34-derived dendritic cells [80]. When combined with other
`chemotherapeutic
`agents
`in wire,
`rapamycin enhanced
`cisplatin-induced apoptosis in human small cell lung cancer cell
`lines
`[11],
`potentiated
`apoptosis
`of
`the murine T-
`lymphoblastoid cell line S49, induced by dexarnethasone [81]
`
`and augmented cisplatin- or camptothecin-induced cytotoxicity
`in DAOY human medulloblastoma cell lines [16]. Raparnycin
`produced
`additive
`cytotoxicity with 5-fluorouracil
`and
`cyclophosphamide in a Colon 38 tumor model [7]. At present,
`little is known about
`the molecular mechanism by which
`rapamycin induces apoptosis of tumor cells. However, Huang
`at al [15] have observed that the responses of malignant and
`normal cells to rapamycin are qualitatively different. When
`treated with rapamycin, cells with wild-type p53 arrest in G1
`phase and maintain viability. In contrast, when grown under
`autocrine conditions
`(ie, serum-free)
`in the presence of
`rapamycin, p53 mutant cells accumulate in G1 phase, but
`progress to S-phase and undergo apoptosis. More than 90% of
`apoptotic Rh:-10 cells {mutant p53 alleles, Argm—>Cysm) were
`BrdU-labeled, suggesting that
`the cells died after initiating
`replication. Thus, rapamycin-induced death appears to be a
`consequence of continued cell cycle progression, suggesting
`that p53 senses inhibition of mTOR and co-operates to reinforce
`a GI arrest. This model has been further tested using Rh30
`infected with adenovirus expressing wild-type p53 (Ad-p53)
`and p53 +/+ or p53 -/- murine embryo fibroblasts (MEFs) [15].
`Restoring the p53-mediated G1 checkpoint by Ad-p53 infection
`causes R1130 rhabdomyosarcorna cells to arrest
`in G1 and
`prevents rapamycin-induced apoptosis (Figure 4). Similarly,
`when exposed to rapamycin, p53 -/- MEF cells continue cell
`cycle progression, and become apoptotic, whereas p53 +/+
`MEF cells arrest in G1 and remain viable. Exactly why cells that
`fail to anest in G1 in the presence of rapamycin die is currently
`unknown.
`
`Mechanisms of resistance to rapamycin
`As shown by Dilling at al [8], under similar conditions of
`growth, various cell lines demonstrated several thousand-
`fold differences in sensitivity to rapamycin. The mechanism
`for this intrinsic resistance is under investigation. Cells may
`also acquire resistance either with or without rnutagenesis.
`
`Mutations in FKBP12, mTOR and p70S6K
`Budding
`yeast
`Saccltaramyces
`cerevisiae
`treated with
`rapamycin irreversibly arrested in the G1 phase. However,
`when yeast TOR1 and TOR2 were genetically mutated to
`TOR1-1 and TOR2-1, these strains were completely resistant
`to the growth-inhibitory effect of rapamycin. These resistant
`alleles encode proteins that have reduced affinity for
`binding the Fl<Bl’12—raparnycin complex [44]. Also in yeast,
`a recessive resistance phenotype was associated with
`decreased RBP1, a homolog of mammalian FKBP12, or a
`mutation altering Tyr”,
`leading to decreased binding of
`rapamycin [47]. In mammalian cells, resistance to rapamycin
`selected after mutagenesis
`is
`related to a dominant
`phenotype consistent with mutation in mTOR [45]. Similar
`to results in yeast, mTOR mutants are associated with
`decreased affinity for binding of the FKBP12-«rapamycin
`complex. High-level resistance to rapamycin is obtained
`when a mutant
`rnTOR (Ser””—)lle’°”), having reduced
`affinity for binding the FKBP12-rapamycin complex,
`is
`expressed [14,46]. mTOR is essential
`for activation of
`ribosornal
`p70S6I<1
`through
`phosphorylation
`of
`the
`rapamycin-sensitive
`sites
`at Thrm or Thr”
`[63,64].
`Substitution of either of these residues can also abrogate the
`ability of rapamycin to inhibit p70S6K activation. Whether
`this results in resistance to the growth inhibitory effect of
`rapamycin is less clear, and may be cell context-specific.
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`Figure 4. Protective effect at the tumor suppressor p53 on repernvclrl-induced epoptosis.
`
`8C
`(D
`
`Ad-Bgal + Flap 100
`
`32o 3t
`
`oE BE
`
`.3
`ED.
`ED.
`
`Ad-p63 + Rap 100
`
`100 101 102 103 104100
`
`to‘
`
`102
`
`103
`
`10‘
`
`Annexin V~FITC fluorescence
`
`Hhao rhabdomyosarooma oells were infected with either adenovirus expressing wild-type p53 (Ad-p53] or Ad-B-galactosidase (|3-gal} at a
`mumpficfly °l i"'°°“°" °' 1' M3’ 2‘? hi "‘°°“'-1m W33 fflnleoecl with serum-tree N2E. and cells were grown for a further 6 days in the absence
`0|’ Pffifieflce 0* 100 n91m|_f§Pam}‘0|n (Fiap 100}. Cells were harvested and apoptosis determined by quantitative FACs analysis [ApoAIert).
`Left panels show dual staining for propidium Iodide uptake and annexin V-FITC. Flight panels show corresponding distribution oi annexin V.
`FITC staining in populations oi cells. (Adapted from Huang 9; a;[-I5”
`
`Decrease of 4E-BP1 protein expression
`Mechanisms of acquired resistance (without use of mutagens)
`or intrinsic resistance have received less attention. Murine
`BC3I-I1 cells selected for acquired resistance demonstrated
`reduced levels of p27""", and consistent with this, embryo
`fibroblasts with disrupted p27'°"" are relatively resistant to
`rapamycin, as determined by inhibition of DNA precursors
`['75]. These data are consistent with G1 arrest, being in part
`due to rapamycin stabilizing this cyclin-dependent kinase
`inhibitor protein. Recently,
`rapamycin-resistant cell
`lines,
`R1130/Rapa1OK and C2, have also been obtained by growing
`
`Rh3C| parental rhabdomyosarcoma cells in the continuous
`presence of increasing concentrations of rapamycin, without
`prior mutagenesis [82, Huang 6: Houghtorb 1-ITIP‘-lblifihed
`data]. When resistant clones were grown without rapamycin
`for 6 or 10 weeks, they reverted to rapamycin sensitivity (in
`terms of ICE by growth inhibition assay). Thus, acquired
`rapamycin resistance was unstable.
`
`Analysis of several clones revealed increased levels of the c-
`Myc protein. Of interest, these rapamycin-resistant clones
`exhibited increased anchorage-independent growth in soft
`
`est-Ward Pharm.
`Exhibit 1017
`Page 006
`
`West-Ward Pharm.
`Exhibit 1017
`Page 006
`
`

`

`2-.
`
`Inhibitors of mammalian target at rapamycin as novel antltumor agents: From bench to clinic Huang 8. Houghton 301
`
`agar. Consistent with increased c-Myc, the levels of the 4E-
`BP suppressor proteins bound to elF—4E were significantly
`lower (approximately 10-fold), as were total cellular levels of
`4E-BP proteins. Steady state levels of £1-E-BP transcripts
`remained unaltered, however, the rate of synthesis appeared
`to be decreased in rapamycin-resistant clones [Huang &:
`Houghton, unpublished data]. In clones that reverted to
`rapamycin sensitivity, total levels of 4E-BP1 became similar
`to those in parental cells. In some cases, intrinsic resistance
`also appears to relate to low 4E-BI’:eIF4E levels. In colon
`carcinoma cells, very low levels of 4-E-BP were detected,
`whereas eIF4E levels were similar to those in sensitive
`
`tumor cell lines. In contrast, no significant changes were
`determined for p70S6 kinase levels or activity between
`parental and resistant clones.
`
`Development of CCl—779 and RAD-001 as
`antitumor agents
`CC!-779
`2,2—
`rapamycin-42,
`inhibitor-779;
`cycle
`(cell
`CCI-7'79
`of
`is
`an
`ester
`bis(hydroxymethyl)-propanoic
`acid)
`rapamycin (Figure 1}, which was developed by Wyeth-
`Ayerst as an antitumor agent. Like rapamycin, CCI-7'79
`acts
`by
`inhibition
`of mTOR,
`preventing
`the
`phosphorylation
`of 4E-BPs
`and p7(}S6I(
`[20-22,26].
`However, in contrast to rapamycin, CCI-779 is stable in
`aqueous
`solution, hence
`it
`can be
`formulated for
`intravenous administration. In preclinical
`tests, CCI-779
`possesses similar antitumor profiles to rapamycin [16,20-
`22,26,28o,290]. CCI-779
`potently inhibits growth
`of
`numerous cultured human tumor cell
`lines including
`human breast, prostate, pancreatic and small cell
`lung
`carcinomas, glioblastoma, medulloblastoma, melanoma,
`rhabdomyosarcoma and T-cell leukemia, with ICE values
`in the nanomolar range ['l6,20-22]. These observations have
`been further supported by the significant inhibitory effect
`of CCI-779 on growth of human tumor xenografts in
`athymic nude mice [16,20,2‘l,24]. When combined with
`cisplatin or carnptothecin, CCI-7'79
`showed additive
`cytotoxicity in subcutaneous implants of human brain
`tumors [16]. Interestingly,
`in viva CCI-7'79 also inhibited
`the growth of human U251 malignant gliorna cells that
`were resistant
`to rapamycin in oitro, although the
`mechanism is unknown [16].
`In addition, more recent
`results have
`revealed
`that
`the
`tumor
`suppressor
`phosphatase and tensin homolog deleted on chromosome
`ten (PTEN)-mutated or -deficient cancer cells are more
`sensitive to CCI-779 [21,28o,29t]. PTEN acts as a major
`negative regulator of the PI3K/Akt signaling pathway [83-
`85]. Loss of PTEN by deletion or mutation occurs in as
`many as 50% of all solid human tumors [86], resulting in
`activation of Akt. Conceptually, this could activate mTOR-
`dependent pathways, hence forming the basis for CCI-779
`hypersensitivity of PTEN deficient cells. However,
`it
`is
`unclear if Akt activates mTOR in situ, as mutation of
`putative Akt phosphorylation sites in the C-terminus of
`mTOR does not abrogate insulin stimulation of p7DS6K
`activation [87]. Of considerable interest, however, is the
`report by Podsypanina at at [280] showing inhibition of
`neoplastic transformation in PTEN +/- mice. These
`animals develop spontaneous multifocal complex atypical
`hyperplasia in the uterine secretory epithelium that
`progresses
`to neoplastic transformation. Tumor cells
`
`demonstrate elevated levels of phosphorylated Akt and
`activated p70S6K. While CCI-7'79 had no effect on Akt
`activation, as anticipated,
`it normalized p’70S6K activity.
`Whether,
`loss of PTEN function consistently sensitizes
`tumor
`cells
`to
`rapamycin analogs
`remains
`to be
`demonstrated.
`
`These preclinical results have revealed that CCI-7'79 exhibits
`impressive cytostatic, and in some instances, cytotoxic
`properties, and may be valuable to delay tumor progression
`and to improve survival when used alone, or in combination
`with other chemotherapeutic agents.
`
`Early data from phase I trials have shown promise of CC!-
`779 in treatment of some cancers [S80]. In European phase 1
`clinical trials [89], CCI-7'79 was administered as a weekly 30-
`min intravenous infusion in 18 patients with different types
`of advanced solid tumors. Doses ranged from 7.5 to 220
`mg/m’/week. After 2 8 weekly doses, significant tumor
`regressions were observed in two patients (receiving 15
`mg/tn’/week) with lung metastasis of renal cell carcinomas
`and in one patient (receiving 22.5 mg/ml/week} with a
`neuroendocrine tumor of
`the lung. Additionally,
`two
`patients experienced tumor stabilization.
`It
`is unclear
`whether the very high dose levels used in this study are
`necessary to elicit antitumor activity. Considering the high
`plasma levels of CCI-7'79 measured in patients (1000-fold
`greater than required for rapamycin activity in vitro), it is
`conceivable that CCI-7'79 is acting through a secondary
`mechanism independent of mTOR.
`
`In the US, phase I clinical trials were also conducted to
`determine the safety and tolerability of CCI-779 [90]. CC]-
`779 was administered as a daily intravenous 30-min infusion
`for 5 days every 2 weeks. Of 45 patients with various types
`of cancer, nine achieved some evidence of tumor response.
`Irnportantly, results from the above two trials indicate that
`CCI-7'79 is well tolerated in patients with only mild Side
`effects,
`such as acneform rash, mild mucositis,
`some
`thrombocytopenia, and elevated triglyceride and cholesterol
`levels. Rapamycin and its analogs potently inhibit T-cell
`activation,
`thus the potential
`for
`immunosuppression in
`patients treated with CCI-779 was anticipated. Interestingly
`CCI-779
`induced
`only
`modest
`evidence
`of
`immunosuppression. Rapamycins
`also
`inhibit
`insulin
`signaling, but in the clinical trials reported there was no
`toxicity consistent with inhibition of insulin signaling. CC]-
`779 is currently in phase III clinical trials. In athymic nude
`mouse xenografts, apart from breast and prostate cancers,
`human glioblastorna
`(U8'7MG)
`tumors and pancreatic
`carcinoma were also very sensitive to CCI-7'79. Thus, with
`an expanded focus,
`it is probable that clinical trials will
`identify other tumor types that are sensitive to CCI-779.
`
`RAD-001
`
`another
`is
`(40-O-(2-hydroxyethyl)-rapamycin)
`RAD-001
`rapamycin analog that is being developed as an antitumor
`agent. Like rapamycin and CC]-7'79, RAD-001 works by
`inhibition of mTOR, downregulating p70S6 kinase activity
`and decreasing the phosphorylation level of 4E-BP1 [27].
`Preliminary data indicate that in wire, RAD-001 potently
`inhibits growth of numerous human tumor cell lines, with
`50% inhibition of growth in the femtomolar range [27].
`
`- st-Ward Pharm.
`Exhibit 1017
`Page 007
`
`West-Ward Pharm.
`Exhibit 1017
`Page 007
`
`

`

`302 Current Opinion in lnvostlgational Drugs 2002 Vol 3 No 2
`
`Unlike CCI-779, RAD-D01 is a hydroxyethyl ether derivative
`of rapamycin, designed for oral administration.
`In viva
`experimental results reveal
`that RAD-001 inhibited the
`growth of human tumor xenografts in nude mice at doses
`ranging from 0.5 to 5.0 mg/kg/day. At these doses, RAD-
`001 was well tolerated, suggesting that it is a very promising
`chemotherapeutic agent. Currently, RAD-001 is entering
`phase I clinical trials for solid tumor.
`
`Summary
`in summary, inhibitors of mTOR, including rapamycin,
`CCI-7'79 and RAD-001, are promising novel anti