Huang and Houghton
`Mechanisms of resistance
`to rapamycins
`
`Shile Huang, Peter J. Houghton
`
`Department of Molecular Pharmacology, St. Jude Children’s Research Hospital,
`Memphis, TN 38105-2794
`
`Abstract Rapamycins represent a novel family of anticancer
`agents, currently including rapamycin and its derivatives,
`CCI-779 and RAD001. Rapamycins inhibit the function of the
`mammalian target of rapamycin (mTOR), and potently suppress
`tumor cell growth by arresting cells in G1 phase or potentially
`inducing apoptosis of cells, in culture or in xenograft tumor
`models. However, recent data indicate that genetic mutations or
`compensatory changes in tumor cells influence the sensitivity of
`rapamycins. First, mutations of mTOR or FKBP12 prevent
`rapamycin from binding to mTOR, conferring rapamycin
`resistance. Second, mutations or defects of mTOR-regulated
`proteins, including S6K1, 4E-BP1, PP2A-related phosphatases,
`and p27Kip1 also render rapamycin insensitivity. In addition, the
`status of ATM, p53, PTEN/Akt and 14-3-3 are also associated
`with rapamycin sensitivity. To better explore the role of
`rapamycins against tumors, this review will summarize the
`current knowledge of the mechanism of action of rapamycins,
`and progress in understanding mechanisms of acquired or
`intrinsic resistance. C(cid:176) 2002 Elsevier Science Ltd.
`
`Key words: Rapamycin, mTOR, signaling pathways, p27kip1, drug
`resistance
`
`INTRODUCTION
`
`R apamycin, a macrocyclic lactone (Fig. 1), is produced
`
`by the soil bacteria Streptomyces hygroscopicus that
`was first found on Easter Island in the South Pacific.
`A group led by Dr. Suren Sehgal, then senior scientist at
`Ayerst Research Laboratories in Montreal, Canada, firstly iso-
`lated rapamycin from the bacteria and identified it as an
`antifungal agent.1–3 Soon rapamycin (sirolimus), as a struc-
`tural analogue of the macrolide antibiotic FK506 (tacrolimus,
`r
`Prograf
`) (Fig. 1), was also found to potently suppress the
`immune system.4–7 When rapamycin was sent to the Na-
`tional Cancer Institute (NCI) for testing, surprisingly, the drug
`also demonstrated potent inhibitory activity against numer-
`ous solid tumors.8–10 Whereas the NCI quickly designated ra-
`pamycin as a priority antitumor drug Ayerst abandoned it,
`because at that time company researchers failed to develop
`a satisfactory intravenous formulation for use in clinical tri-
`als. Also at that time, little was known about the mechanism
`of action of rapamycin in blocking signal transduction. Not
`until 1988, after Wyeth and Ayerst merged, did studies of ra-
`pamycin resume. While solid data convinced Wyeth-Ayerst to
`develop rapamycin as an immunosuppressant, the NCI and
`many other laboratories continued to study the antitumor ac-
`r
`tivity of rapamycin. Rapamycin (Rapamune
`), as an immuno-
`suppressive drug, was finally approved by the Food and Drug
`
`......................................................................................................................................................................................................................
`
`Administration (FDA) in the USA in September, 1999, and the
`European Commission in March, 2000, respectively. So far,
`results from many laboratories have demonstrated that ra-
`pamycin, in contrast to FK506, is not only a potent immuno-
`suppressant, but also a potential antitumor agent. Rapamycin
`can act as a cytostatic, arresting cells in G1 phase or potentially
`inducing apoptosis in many malignant cells in culture. To date,
`studies have revealed that rapamycin potently arrests growth
`of cells derived from rhabdomyosarcoma, neuroblastoma and
`glioblastoma, small cell lung cancer,11–17 osteoscarcoma,18
`pancreatic cancer,19,20 breast and prostate cancer,21–23 murine
`melanoma and leukemia, and B-cell lymphoma.9,24–26
`However, direct use of rapamycin as an anticancer
`drug is clinically impractical, because of its poor water-
`solubility and stability in solution. Recently,
`two ra-
`pamycin ester analogues (Fig. 1), CCI-779 [rapamycin-42, 2,
`2-bis(hydroxymethyl)-propionic acid] (Wyeth-Ayerst, PA, USA)
`and RAD001 [everolimus, 40-O-(2-hydroxyethyl)-rapamycin]
`(Novartis, Basel, Switzerland), with improved pharmaceuti-
`cal properties have been synthesized and evaluated. CCI-779
`is designed for intravenous injection, whereas RAD001 for
`oral administration. Both have similar antitumor effects as
`rapamycin,17,21–23,27–30 and are currently being developed as
`antitumor agents and undergoing phase I/II clinical trials. So
`far, preclinical results have revealed that rapamycin and its
`derivatives (designated here as rapamycins) suppress growth
`of numerous human tumor cells in vitro, and in some hu-
`man and murine tumor models in vivo.11–30 When combined
`with other chemotherapeutic agents, rapamycins generally
`show at least additive antitumor activity.10,12,17,31 Preliminary
`data from clinical trials have indicated that rapamycins are
`well tolerated and successfully suppress growth of various hu-
`man tumors.32–34 However, increasing evidence has suggested
`that genetic mutations or compensatory changes in tumor
`cells may affect the sensitivity of rapamycins. For instance,
`mutations of the mammalian target of rapamycin (mTOR)
`or FKBP12 prevent rapamycin from binding to mTOR and
`confer rapamycin resistance. Mutations or defects of mTOR-
`controlled downstream effector molecules, such as S6K1, 4E-
`BP1, PP2A-related phosphatases, and p27Kip1, also render ra-
`pamycin insensitivity. At least in some systems the status of
`ATM, p53, PTEN/Akt and 14-3-3 also determines rapamycin
`sensitivity. This review will summarize the current knowl-
`edge of action mechanism of rapamycins, and resistance
`mechanisms.
`
`MECHANISM OF ACTION OF RAPAMYCINS
`
`Rapamycins represent a novel family of anticancer agents, cur-
`rently including rapamycin and its derivatives, CCI-779 and
`RAD001. Rapamycins share a common mechanism of antitu-
`mor action. Simply, they inhibit the function of mTOR that
`links mitogen stimulation to protein synthesis and cell cycle
`progression, and potently suppress tumor cell growth by ar-
`resting cells in G1 phase, potentially inducing apoptosis of
`cells.
`
`mTOR and its inhibition by rapamycin
`The mammalian target of rapamycin, mTOR [also designated
`FRAP (FKBP12 and rapamycin-associated protein), RAFT1
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`c(cid:176) 2002 Elsevier Science Ltd. Drug Resistance Updates (2001) 4, 378–391
`doi: 10.1054/drup.2002.0227, available online at http://www.idealibrary.com on
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`Fig. 1 Structures of rapamycin, FK506, and two rapamycin analogues in clinical trials, CCI-779 and RAD001.
`
`(rapamycin and FKBP12 target 1), RAPT1 (rapamycin target 1)
`or SEP (sirolimus effector protein)], was identified as a 289 kDa
`serine/threonine kinase from mammalian cells.35–38 Accord-
`ing to Genebank database, TOR proteins are evolutionarily
`conserved from yeast to human beings in the catalytic do-
`main. In the yeasts, Saccharomyces cerevisiae and Schizosac-
`charomyces pombe, two TOR genes, designated TOR1 and
`TOR2, have been cloned, both sharing 67% homology and en-
`coding »280 kDa proteins.39–41 In the fruit fly, Drosophila
`melanogaster, a single TOR orthologue, termed dTOR, has
`been characterized, sharing 38% identity with TOR2 from Sac-
`
`......................................
`
`charomyces cerevisiae.42,43 Mammalian TOR (mTOR) shares
`»45% identity with TOR1 and TOR2 from the yeast Saccha-
`romyces cerevisiae, and 56% identity with dTOR in overall
`sequence.44,45 Human, mouse and rat mTOR proteins share
`95% identity at the amino acid level.46,47 Structurally, mTOR is
`composed of a catalytic kinase domain, FRB (FKBP-rapamycin
`binding) domain and a putative auto-inhibitory domain (“re-
`pressor domain”) near C-terminus, and up to 20 tandemly re-
`peated HEAT (Huntingtin, EF3, A subunit of PP2A and TOR)
`motifs at the N-terminus, as well as FAT (FRAP-ATM-TRAPP)
`and FATC (FAT C-terminus) domains (Fig. 2).47,48 Since the
`
`Fig. 2 Schematic representation of the domains of mTOR. Structural domains of mTOR. HEAT: (huntingtin elongation A subunit
`TOR) repeats (positions 71–522 and 628–1147); FAT: (FRAP-ATM-TRAPP) domain, which is unique to PIK-related kinases located
`N-terminal to the FKBP12-rapamycin binding domain (FRB); the role of FAT sequences is less clear, but they are associated with
`C-terminal FAT (FATC) sequences in mTOR Interaction between FAT and FATC domains may facilitate protein binding or act as a
`structural scaffold; CD: Catalytic domain; RD: regulatory domain.
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`Fig. 3 Scheme of the mTOR signaling pathway. Arrows represent activation, whereas bars represent inhibition. IRS, insulin receptor
`substrates; PI3K, phosphatidylinositol 30 kinase; PIP2, phosphatidylinositide (4,5)-P2; PIP3, phosphatidylinositide (3,4,5)-P3; PTEN,
`phosphatase and tensin homologue deleted on chromosome ten; PDK1, phosphoinositide-dependent protein kinase 1; Akt/PKB,
`protein kinase B; rapamycin-FKBP12, rapamycin-FK506-binding protein 12 complex; mTOR, mammalian target of rapamycin; pRb,
`retinoblastoma protein; Pol I/II/III, RNA polymerase I/II/III; 4E-BP1, eIF-4E-binding protein 1; eIF-4A/4E/4F/4G/3, eukaryotic initiation
`factor-4A/4E/4F/4G/3; S6K1, p70 S6 kinase; S6, 40S ribosomal protein; 50TOP, 50-terminal oligopyrimidine.
`
`C-terminus of mTOR is highly homologous to the catalytic
`domain of phosphatidylinositol 3 kinase (PI3K), mTOR is
`considered a member of PI3K-related kinase family (desig-
`nated PIKK), which also includes MEC1, TEL1, RAD3, MEI-41,
`DNA-PK, ATM, ATR, and TRRAP.47,49 Both PI3K and, poten-
`tially, Akt/PKB lie upstream of mTOR, whereas two trans-
`lational components, ribosomal p70S6 kinase (S6K1) and
`eukaryotic translation initiation factor-4E (eIF4E) binding
`protein 1 (4E-BP1), are the best characterized downstream
`effector molecules of mTOR (Fig. 3). However, the full spec-
`trum of cellular events controlled by mTOR extends beyond
`these pathways. Increasing evidence has implicated mTOR as
`a sensor that integrates extracellular and intracellular events,
`coordinating 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.47 Recent results suggest that mTOR may also
`sense cellular ATP levels, suppressing protein synthesis when
`ATP levels decrease.50
`Rapamycins are specific inhibitors of mTOR. Although
`rapamycin and FK506 are both potent immunosuppressive
`agents, their mechanisms of action are quite different. Both
`rapamycin and FK506 competitively binds to a Mr 12,000
`cytosolic protein termed FK-binding protein (FKBP-12). The
`FKBP-FK506 complex inhibits calcineurin, preventing dephos-
`phorylation, nuclear translocation of NF-ATp, and activation of
`interleukin 2 transcription.46 The FKBP-rapamycin complex
`binds to the FRB domain of mTOR, resulting in inhibition of
`
`........................................................................................................
`
`the function of mTOR. The specific binding of rapamycin has
`been confirmed by studies of genetic mutations of mTOR and
`FKBP12 (see review below for details). Currently, a major unre-
`solved issue is how rapamycin inhibits the function of mTOR.
`As we know, many small molecule kinase inhibitors reduce
`the activity of kinases by direct competition for ATP bind-
`ing, thus preventing ligand-induced autophosphorylation and
`signaling. However, whether rapamycin or FKBP-rapamycin
`complex directly inhibits the kinase activity of mTOR is con-
`troversial. FKBP-rapamycin complex inhibited autokinase ac-
`tivity of mTOR in vitro at high concentration (500 nM).51
`Rapamycin in vitro also blocked the modest insulin-induced
`increase of kinase activity of immunoprecipitated mTOR.52
`However, treatment of cells with rapamycin did not alter au-
`tophosphorylation level of Ser2481, and had little or no effect
`on the kinase activity of immunoprecipitated mTOR.42,45,53
`Possibly, mTOR may repress a phosphatase activity associated
`with downstream targets. Binding of FKBP-rapamycin com-
`plex to mTOR may first result in de-repression of this phos-
`phatase, which then dephosphorylates downstream effector
`molecules, e.g. S6K154,55 and p44/42 MAP kinases (our un-
`published data).56 More recently, phosphatidic acid has been
`identified as a critical component of mTOR signaling, and its
`binding to mTOR is necessary for activation of mTOR down-
`stream effector molecules.57 It is also possible that FKBP-
`rapamycin complex may compete with phosphatidic acid to
`bind the FRB domain of mTOR, preventing mTOR from activat-
`ing downstream effectors although without inhibiting mTOR’s
`catalytic activity.57 Alternatively, mTOR may act as a scaffold
`
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`Resistance to rapamycins
`
`and the FKBP-rapamycin complex presumably disrupts higher
`order mTOR-protein complexes. Obviously, more studies are
`required to establish a suitable model for rapamycin action.
`
`Rapamycin-sensitive signaling pathways mediated
`by mTOR
`As mentioned above, 4E-BP1 and S6K1 are the best character-
`ized downstream effector molecules of mTOR (Fig. 3). Both are
`translational components. 4E-BP1 functions as a suppressor of
`eIF4E. In response to mitogens, six sites (Thr37, Thr46, Ser65,
`Thr70, Ser83, and Ser112) of 4E-BP1 (also termed PHAS-I) can
`be phosphorylated.58 So far, only mTOR and ATM have been
`identified to be involved in phosphorylation of 4E-BP1.59–62
`Little is known whether other kinases participate in phospho-
`rylation of 4E-BP1. ATM phosphorylates 4E-BP1 at Ser112,62
`whereas mTOR in vitro selectively phosphorylates 4E-BP1 at
`two and possibly four Ser/Thr residues (Thr37, Thr46, Thr70
`and Ser65) in the N-terminal region.61,63 4E-BP1 phospho-
`rylation is a hierarchical process.61,63–65 Phosphorylation of
`Thr37/Thr46 is followed by Thr70 phosphorylation, and Ser65
`is phosphorylated last.65 Phosphorylation of Ser65 depends on
`phosphorylation of all three Thr/Pro sites,63,64 whereas muta-
`tions of Thr37 and/or Thr46 to alanine(s) prevents phosphory-
`lation of Ser65 and Thr70, suggesting that phosphorylation of
`Thr37 and Thr46 serves as a requisite ‘priming’ event.55 Single
`phosphorylation of above residues is not sufficient to dissoci-
`ate 4E-BP1 from eIF4E, indicating that a combined phosphory-
`lation of at least Thr37, Thr46, Ser65, and Thr70 in 4E-BP1 is
`essential to suppress association with eIF4E.55,66 In the pres-
`ence of rapamycin, 4E-BP1 becomes hypo-phosphorylated and
`associates with eIF4E. This prevents binding of eIF4E to the
`scaffold protein eIF4G and formation of the eIF4F initiation
`complex required for cap-dependent translation of mRNA. As
`a result, rapamycin may downregulate mTOR-controlled syn-
`thesis of essential proteins involved in cell cycle progression,
`such as cyclin D1,67,68 and ornithinine decarboxylase,69 and
`survival (c-MYC).70
`S6K1 is the other well documented downstream target
`of mTOR. To date, two ribosomal p70S6 kinases have been
`identified: S6K1 and S6K2, and both can be inhibited by
`rapamycin.71,72 S6K1 contains a nuclear localization signal
`domain at the N-terminus, followed by an acidic domain, a
`catalytic domain, a regulatory domain, an auto-inhibitory do-
`main and C-terminal domain.73 Activation of S6K1 is a com-
`plex process that involves the interplay between four different
`domains and at least seven specific sites mediated by multi-
`ple upstream kinases.73 It has been reported that at least 12
`sites (Ser17, Thr229, Thr367, Thr371, Thr389, Ser404, Ser411,
`Ser418, Tr421, Ser424, Ser429, and Thr447) can be phospho-
`rylated in response to serum stimulation.58 However, the ki-
`nases responsible for the phosphorylation of these sites are
`not fully characterized. Phosphoinositide-dependent protein
`kinase 1 (PDK1) phosphorylates Thr229 in vitro and in vivo.74
`Atypical PKC isoforms and the Rho family of small G proteins
`(cdc42 and Rac1) may partially contribute to phosphorylation
`of S6K158, but the specific sites regulated by these kinases re-
`main to be determined. In vitro, mTOR phosphorylates only
`Thr389 in the regulatory domain.75–77 However, whether this
`phosphorylation is directly or indirectly regulated by mTOR is
`in question, since recent data suggest that mTOR may regulate
`
`......................................................................................................................................................................................................................
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`S6K1 activation by inhibiting phosphatases rather than directly
`phosphorylating S6K1.54,73 Similar to 4E-BP1, S6K1 also needs
`a hierarchical phosphorylation process to be activated. The
`initial step for S6K1 activation is the phosphorylation of the
`Ser/Thr-Pro sites in the auto-inhibitory domain, which then
`cooperates with the N-terminus to allow phosphorylation of
`Thr389. This presumably disrupts the interaction of the C-
`terminus with the N-terminus, allowing phosphorylation of
`Thr229 and resulting in S6K1 activation.73 As phosphorylation
`of Thr389 is a primary event for phosphorylation of other sites,
`in vivo rapamycin may affect phosphorylation of more sites,
`including Thr229 in the catalytic domain, and Ser404 in the
`regulatory domain.75 S6K1 functions to increase translation of
`0
`0
`mRNA species with 5
`terminal oligopyrimidine (5
`TOP) tracts.
`These mRNAs primarily code for ribosomal proteins and other
`elements of the translational machinary, such as ribosomal pro-
`teins, elongation factors, the poly(A) binding proteins,72 and
`IGF-II.78 Therefore, inhibition of mTOR by rapamycin primar-
`0
`ily downregulates translation of 5
`TOP-containing mRNAs.
`In addition to inhibition of translation of specific mRNAs
`through 4E-BP1 and S6K1 pathways, rapamycin may also sup-
`press RNA polymerase (Pol) I/II/III-mediated transcription and
`translation by decrease of mTOR-controlled phosphorylation
`of retinoblastoma protein (pRb).66 Furthermore, rapamycin
`may also inhibit activation of G1 cyclin-dependent kinases
`(cdks) causing hypophosphorylation of pRb protein, and slow
`or arrest cell cycle transition from G1 to S-phase.79 The mech-
`anism by which rapamycin inhibits activity of cdks may be
`cell type dependent, either by upregulation of cdk inhibitors,
`or downregulation of cyclins or cdks, or inhibition of associa-
`tion of cyclin-cdks. For example, in T lymphocytes, rapamycin
`increases the level of cdk inhibitory protein p27Kip1 by preven-
`tion of its degradation induced by mitogens.80,81 Involvement
`of p27Kip1 being an effector of rapamycin-induced G1 cell cy-
`cle arrest is strengthened by the observation that p27Kip1 de-
`ficient T lymphocytes or fibroblasts are relatively resistant to
`rapamycin inhibition of growth.82 In NIH3T3 cells rapamycin
`may inhibit the G1 to S transition through inhibition of cdks by
`decrease of the cyclin D1 mRNA level and protein stability,68
`or delay of the expression of cyclin A.83 In vascular smooth
`muscle cells, growth factors elevate the levels of cell cycle
`proteins, such as cyclins (D1, E, B) and cdks (cdk1 and cdk2),
`whereas rapamycin blocks the upregulation of these proteins,
`but not mRNA, and arrests the cells before S phase.84 In con-
`trast to findings in other cell types, in vascular smooth muscle
`cells rapamycin does not affect growth factor-induced down-
`regulation of p27Kip1.84 In MG-63 human osteosarcoma cells,
`rapamycin inhibits cdk activity and cyclin D1-cdk association
`during early G1.85 Similarly, in T lymphocytes, rapamycin also
`blocks activation of cdk1 (p34cdc2) and cdk2 (p33cdk2) by in-
`hibition of cyclin A expression, and formation of active cyclin
`A-cdk1/2 complexes and cyclin E-cdk2 complex, resulting in
`late G1 arrest.86
`
`MECHANISMS OF RESISTANCE TO RAPAMYCINS
`
`As observed by Dilling et al.11 various cell
`lines exhibit
`several thousand-fold differences in their intrinsic sensitiv-
`ity to rapamycin under similar growth conditions. Further
`studies indicate that the response to rapamycin is different
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`between cell lines, being either cytostatic or cytotoxic, or
`cytostatic/cytotoxic.14–16 The mechanism for this intrinsic
`resistance is under investigation. However, increasing data
`have implicated that cells may acquire resistance either with
`or without mutagenesis. Obviously, the mechanisms of ra-
`pamycin resistance are complicated and multiple, some of
`which have been identified whereas others remain to be de-
`termined. Reported mechanisms of rapamycin resistance are
`summarized below.
`
`Mutations in FKBP12 and mTOR
`As aforementioned, rapamycin has a specific mode of action. It
`cannot directly bind to mTOR. It first has to bind to FKBP-12 in
`mammalian cells, forming the FKBP-rapamycin complex. This
`complex then interacts with the FRB domain in mTOR (Fig. 2),
`and inhibits the function of mTOR. Therefore, during such se-
`quential interactions, either specific mutations in FKBP12 that
`prevent the formation of FKBP-rapamycin complex, or certain
`mutations in the FRB domain of mTOR that block binding of
`FKBP-rapamycin complex to mTOR would finally abrogate the
`effect of rapamycin on mTOR, causing rapamycin resistance.
`Such mutations were first found in yeast. For example, S. cere-
`visiae treated with rapamycin irreversibly arrested in the G1
`phase. A mutational screen identified rapamycin-resistant al-
`leles with mutations in genes designated TOR1 and TOR2.
`Strains with mutated to TOR1-1 (Ser1972! Arg) and TOR2-1
`(Ser1975! Arg), were completely resistant to the growth-
`inhibitory effect of rapamycin. These resistant alleles encode
`mutant TOR proteins that lack the ability for FKBP-rapamycin
`complex binding.87–92 The results suggest that a conserved ser-
`ine residue (Ser1972 in TOR1; Ser1975 in TOR2) in yeast TOR
`proteins is critical for FKBP-rapamycin binding. In mammalian
`cells, resistance to rapamycin selected after mutagenesis is re-
`lated to a dominant phenotype also consistent with mutation
`in the FRB domain of mTOR,93 that results in decreased affin-
`ity for binding of the FKBP-rapamycin complex. Expression
`of a mutant mTOR (Ser2035! Ile), having reduced affinity
`for binding the FKBP-rapamycin complex, confers high level
`resistance.14,93,94 Alternatively, in the yeast S. cerevisiae, dele-
`tion of the RBP1 gene, a homologue of mammalian FKBP-12, re-
`sulted in a recessive rapamycin resistance, whereas expression
`of RBP1 restored rapamycin sensitivity.87 This observation has
`been further confirmed by RBP1 disruption experiments us-
`ing the pathogenic yeast Candida albicans, in which the wild-
`type RBP1/RBP1 parental strain and the rbp1/RBP1 heterozy-
`gous mutant were sensitive to rapamycin inhibition, whereas
`rbp1/rbp1 homozygous mutant was rapamycin resistant.95 In
`addition, in S. cerevisiae mutation of a specific residue (Tyr89)
`which is conserved in RBP1 or FKBPs, also resulted in de-
`creased binding of rapamycin and conferred a recessive resis-
`tance phenotype.96 In murine mast cells, two distinct point
`mutations in FKBP12, one altering a hydrophobic residue
`within the drug-binding pocket (Trp59! Leu) and the other
`changing a charged surface residue (Arg49 ! Gln), substan-
`tially reduced binding affinity of FKBP12 for rapamycin, ren-
`dered rapamycin resistance.97
`
`Mutations in S6K1
`As described above, S6K1 is a principal downstream effector
`of mTOR. So far, data have revealed that rapamycin primarily
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`inhibits only phosphorylation of Thr389 in the regulatory
`domain.73 However, since phosphorylation of S6K1 is hierar-
`chical with phosphorylation of several other sites dependent
`on phosphorylation of Thr389,73 rapamycin in vivo influences
`phosphorylation of more sites, including Thr229 in the cat-
`alytic domain, and Ser404 in the regulatory domain.75 There-
`fore, site mutation of Thr389! Glu abrogates the ability of ra-
`pamycin to inhibit S6K1 activation.72,75 Similarly, substitution
`of Thr229 by either a neutral amino acid Ala (Thr229! Ala)
`or by an acidic amino acid Glu (Thr229! Glu), renders S6K1
`insensitive to rapamycin.98 In addition, deletion of the 77 N-
`terminal codons (1N77) conferred rapamycin resistance.99 It
`turns out that truncation of the first 54 residues of N-terminus
`(otherwise identical to 1N77 above) blocked the serum-
`induced phosphorylation of three rapamycin-sensitive sites,
`Thr229, Thr389 and Ser404, causing rapamycin insensitivity.75
`Whether this results in resistance to the growth inhibitory ef-
`fect of rapamycin is less clear, and may be cell context specific.
`
`De-regulation of eIF4E
`Besides S6K1, 4E-BP1, the suppressor of eIF4E, has been
`widely recognized as the other primary downstream effector
`of mTOR.55 Recently, our group has found that acquired re-
`sistance to rapamycin was associated with decreased levels of
`4E-BP1 (Dilling et al. submitted).100 Briefly, rapamycin-resistant
`cell lines, Rh30/Rapa10K and C2 clones, were obtained by
`continuously culturing Rh30 parental cells in the presence of
`increasing concentrations of rapamycin, without prior muta-
`genesis. In the absence of selective pressure, resistance was
`unstable. Within 10 weeks after rapamycin was withdrawn
`from the medium, resistant clones reverted to being sensi-
`tive to growth inhibition of rapamycin. The molecular basis
`of rapamycin resistance in this case has been investigated.
`It turns out that in Rh30/Rapa10K and C2 cells, the levels
`of the suppressor protein 4E-BP1 bound to eIF4E were sig-
`nificantly lower (»10-fold), as were total cellular levels of
`4E-BP1. However, mRNA levels of 4E-BP1 were unaltered,
`indicating post-translational regulation. Further studies indi-
`cate that the synthesis of 4E-BP1 did significantly decrease
`in rapamycin-resistant clones, but whether the steady state
`level of 4E-BP is also regulated by increased degradation re-
`mains to be determined. Thus, the changes in 4E-BP levels
`are reminiscent of those reported for p27Kip1 in BC3H cells.82
`In cells (Rh30/Rapa10-revertant) that reverted to be sensitive
`to rapamycin, total levels of 4E-BP1 became similar to those
`in parental cells, and 4E-BP1 bound to eIF4E had similar re-
`sponse to serum starvation and IGF-I stimulation as found in
`parental cells. In contrast, no significant changes were de-
`tected for S6K1 levels or activity between parental and re-
`sistant clones. Activation of S6K1 was equally inhibited in
`parental and rapamycin-resistant clones. Both Rh30/Rapa10K
`cells and Rh30/Rapa10K-revertant cells exhibited elevated
`c-MYC levels, and increased anchorage-independent growth,
`indicating that inhibition of c-MYC translation by rapamycin
`is not critical in determining rapamycin sensitivity. These
`data suggest that decrease of 4E-BP1 expression results in de-
`regulation of eIF4E, conferring rapamycin resistance.
`According to the above findings, rapamycin-regulated
`eIF4E pathway is crucial in inducing growth arrest, and de-
`regulation of eIF4E may facilitate malignant phenotype. This
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`Resistance to rapamycins
`
`is supported by certain clinical observations that de-regulation
`of the eIF4E pathway does promote tumor progression.101
`In addition to decrease of 4E-BP1 expression, as described
`above, increased eIF4E levels may also cause de-regulation
`of eIF4E. In advanced head and neck carcinoma,102 breast
`carcinoma103 and gastrointestinal carcinoma,104 eIF4E levels
`are elevated. However, levels of 4E-BP proteins have not been
`reported in a consistent manner. Potentially, the ratio of 4E-
`BP:eIF4E may determine whether inhibition of mTOR elicits
`a biologically significant tumor response. Certainly, intrinsic
`resistance to rapamycin has been shown in glioblastoma cells
`and colon adenocarcina that have very low 4E-BP:eIF4E ratios
`(our unpublished data). In addition, HCT8 colon carcinoma
`cells are highly resistant to rapamycin (IC50 > 10,000 ng/ml).
`When 4E-BP1 is overexpressed, these cells become sensitive
`(IC50 > 10 ng/ml) to rapamycin (Dilling et al. submitted). How-
`ever, further studies will be necessary to determine if this ratio
`has predictive value for drug sensitivity of tumors.
`
`Mutations of PP2A-related phosphatases
`So far, several Ser/Thre protein phosphatases, such as PP2A,
`PP4 and PP6, have been identified as the components of mTOR
`signaling pathway in mammalian cells.66 Mammalian PP2A is
`composed of a common core dimer of a 39 kDa catalytic C-
`subunit (PP2Ac) and a 65 kDa A-subunit associated with di-
`verse distinct regulatory B-subunits (50»130 kDa). Rapamycin
`resistance caused by mutations of PP2A-related phosphatases
`was first studied in yeast. In S. cerevisiae, PPH21 and PPH22
`encode C-subunits of PP2A (Pph21 and Pph22), whereas TPD3
`and CDC55 respectively encode 64 kDa A-subunit and 60 kDa
`B-subunit. Tap42 is the yeast homologue of mammalian fi4,
`and Sit4 is the yeast homologue of PP6, and the catalytic sub-
`unit of a PP2A-related phosphatase in yeast. Early studies in
`yeast indicate that Tap42 associates with Sit4 and Pph21/22.105
`The function of Tap42 remains to be determined. However, it
`appears to be a gene essential for cell division and survival.
`Strains overexpressing isogenic tap42-11 mutants were almost
`completely resistant to rapamycin.105 In addition, overexpres-
`sion of Sit4, but not Pph21, also resulted in weak rapamycin
`resistance.105 The mechanism of rapamycin resistance in this
`case is still unknown. Rapamycin did not decrease Tap42 pro-
`tein level, but caused dissociation of Tap42 from Sit4 and
`Pph21/22.105 Two possibilities accounting for rapamycin re-
`sistance were discussed by the authors.105 First, if Tap42 func-
`tioned as a positive regulatory subunit for Sit4 and Pph21/22,
`in rapamycin-resistant tap42-11 strains, the mutant protein
`might be stably associated with the phosphatases and main-
`tain a specific phosphatase activity that is insensitive to ra-
`pamycin. Second, if phosphatases regulated a specific Tap42
`function,
`in the rapamycin-resistant Tap42-11 mutant, the
`function of the Tap42-11 protein would be less dependent on
`the association with the phosphatases, also causing rapamycin
`resistance.
`Similarly, mutations or deletion of either TPD3 (encod-
`ing Tpd3, A subunit) or CDC55 (encoding Cdc55, B sub-
`unit), which regulate Pph21/22 activity, conferred rapamycin
`resistance.106 This is because TPD3 or CDC55 mutants
`failed to compete with TOR-phosphorylated Tap42 bind-
`ing to Pph21/22 C-subunit, resulting in increased associa-
`tion of Tap42 with Pph21/22.106 These findings suggest that
`
`......................................................................................................................................................................................................................
`
`Tap42, Sit4 and PP2A might be downstream effectors of TOR
`proteins. Studies of mammalian cells also indicate that as-
`sociation of fi4 with PP2A, PP4, and PP6 is related to ra-
`pamycin sensitivity.107,108 For example, in rapamycin-sensitive
`Jurkat cells, rapamycin dissociated fi4 from PP2Ac, whereas in
`rapamycin-resistant Raji cells, rapamycin did not affect associa-
`tion of fi4 with PP2Ac.108 Transfection of mouse fi4 into Jurkat
`cells conferred rapamycin resistance,108 further demonstrating
`that these PP2A-related phosphatases are novel rapamycin-
`sensitive targets. Surprisingly, rapamycin inhibits cell prolif-
`eration by decreasing PP2A activity through dissociating fi4
`from PP2Ac,108 suggesting that PP2A may positively regulate
`cell proliferation under certain conditions. However, other
`studies109 did not demonstrate rapamycin-induced dissocia-
`tion of fi4 from PP2A or PP6. Thus, at this time the significance
`of fi4 remains controversial.
`
`Defective regulation of p27Kip1
`p27Kip1, a cdk inhibitor, is downregulated in serum stimu-
`lated cells. Prevention of mitogen-stimulated downregulation
`of p27Kip1 level by rapamycin suggests that p27Kip1 is involved
`in the antiproliferative activity of rapamycin.80,81 Rapamycin
`resistance linked to defective regulation of p27Kip1 has been
`described.82 BC3H1 is a rapamycin-sensitive murine myogenic
`cell line. Prolonged culture of BC3H