[Cancer Biology BC Therapy 2:3, 222-252, May/June 2005] ; ©2003 Landes Bioscience
`
`Review
`
`Rapamycins
`
`Mechanism of Action and Cellular Resistance
`
`ABSTRACT
`
`galand
`' munosup-
`ntation. Two
`ticancer agents.
`5 and inhibits the
`
`
`
`Rapamycins are macrocyclic lactones that possess immunosuppressive,
`antitumor properties. The parent compound, rapamycin, is approved
`pressive agent for preventing rejection in patients receiving orga
`analogues, CCI-779 and RAD001 are currently being investigate
`Rapamycins first bind a cyclophilin FKBPI 2, and this comple
`function of mTOR (mammalian target of rapamycin) a serbt
`with homology to phosphatidylinositol 3’ kinase. Currentl
`target,
`this places rapamycins in a unique position o
`inhibitor known. Consequently these agents haveb
`role of mTOR in cellular growth, proliferation,
`evidence suggests that mTOR acts as a centra
`
`onine (Ser/Thr) kinase
`OR is the only identified
`g the most selective kinase
`owerful tools in elucidating the
`and tumorigenesis. Increasing
`troller sensing cellular environment
`
`(nutritional status or mitogenic stimulation) 0; regulating translation initiation through the
`eukaryotic initiation factor 4E, and rib
`70 S6 kinase pathways. Here we review
`the conserved TOR signaling pathwa
`ceptual basis for tumor selectivity, and the
`mechanisms of resistance to this clfif antitumor agent.
`
`INTRODUCTION
`
`9
`
`Rapamycin, a m
`was isolated an
`
`
`
`ic lactone product of the soil bacteria Strrptomyce: /Jygraxcapiau,
`tified as an antifungal agent in the mid-1970’s.1'3 Rapamycin
`ructural analogue of the macrolide antibiotic FK506 (tacrolimus,
`, and like FK506 was found to potently suppress the immune system.4'7
`rail for rapamycin to act as an antitumor agent was recognized early in its
`nt when the drug demonstrated potent inhibitory activity against numerous
`mars in the NCI screening program.8‘1° However, the drug was not developed
`er due to stability and solubility problems that prevented development of a parenteral
`rmulation for use in clinical trials. Also at that time in the early 1980’s, the mechanism
`of action of rapamycin in blocking signal transduction was not understood.
`Rapamycin (Rapamune®), as an immunosuppressive drug, was finally approved by the
`Food and Drug Administration (FDA)
`in the USA in 1999, and the European
`Commission in 2000, respectively. Results from many laboratories have demonstrated that
`rapamycin, in contrast to FK506, is not only a potent immunosuppressant, but also an
`active antitumor agent. Rapamycin can act as a cytostatic, slowing or arresting cells in G1
`phase. Under specific conditions, or in some tumor cell lines rapamycins may induce
`apoptosis in culture. To date, studies have revealed that rapamycin potently arrests growth
`of cells derived from rhabdomyosarcoma, neuroblastoma and glioblastoma, small cell lung
`cancer,“'17 osteoscarcoma,18 pancreatic cancer,19'2° breast and prostate cancer,21'23
`murine melanoma and leukemia, and B-cell lymphoma.9’24‘26 In addition to broad
`spectrum activity in vitro, rapamycin and its derivatives (designated here as rapamycins)
`suppress growth of some human and murine tumor models in vivo.“'3° When combined
`with other chemotherapeutic agents, rapamycins generally show at least additive antitumor
`activity.1°=12’17’31 Preliminary data from clinical trials have indicated that rapamycins are
`well tolerated and successfully suppress growth of various human tu.mors.52'54
`The use of rapamycin as an anticancer drug is clinically impractical, because of its poor
`water-solubility and stability in solution. Recently, rapamycin 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), have entered clinical trials. These analogues have improved pharmaceutical
`properties. CCl—779 is being developed for both intravenous and oral administration,
`whereas RAD001 is only for oral administration. The antitumor activity of these analogues
`
`
`
`(sirolimus)
`Prograf®)
`The
`devel
`50
`
`
`
`Shile Huang
`
`Mary-Ann Biornsti
`
`Peter J. Houghton*
`
`Department of Molecular Phormacologm St Jude Children's Research Hospital; 332
`II. Lauderdale; Memphis; Tennessee USA
`
`*Carrespnndence to: Peter I. Houghton; Department of Molecular Pharmacology; St
`Jude Children's Research Hospital; 332 N. Lauderdale; Memphis; Tennessee
`30105-2794 USA; Email: peter.houghton@stiude.org
`
`Received 04/24/03; Accepted 05/0l/03
`
`Previously published online as a [It&T Paper in Press at:
`http://VIww.Iundesbioscience.com/iournals/cbt/toc.php?voIume=2&issue=3
`
`KEY WORDS
`
`Rapamycins, Translation initiation, Cancer,
`Resistance, Therapy, Ribosomal biogenesis, Yeast
`
`Work reported from these laboratories was supported by PHS awards (A23099,
`(AS0755, CA77776, (M6996 and (A2l765 (Cancer Center Support Grant) and by
`American, Lebanese, Syrian Associated Charities (AlSA().
`
`$0
`V
`"3
`
`©"'§
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`RAPAMYCTNS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
`
`is similar to that of rapamycin.17’21‘23’27’30 RADOOI is
`in Phase I trials whereas the development of CCI—779
`is more advanced with several Phase II trials completed.
`Why should it be anticipated that
`rapamycins
`could exhibit tumor—selectivity, in a manner analogous
`to the activity of another kinase inhibitor, Gleevec, in
`Bcr—Abl expressing chronic lymphocytic leukemia?
`Accumulating evidence suggests that genetic mutations
`or compensatory changes in tumor cells may affect
`sensitivity to rapamycins. At least
`in some systems
`mutations that occur frequently in malignant trans-
`formations such as GL1 amplification, or mutations
`that inactivate p53, and the dual specificity phos—
`phatase PTEN (phosphatase and tensin homolog
`deleted on chromosome ten, also known as MMACI
`for mutated in multiple advanced cancers) or lead to
`activation of Akt appear to determine rapamycin
`sensitivity. On the other hand there may be multiple
`loci that confer intrinsic or acquired resistance. This
`review will summarize the current knowledge of the
`role of mTOR in cellular regulation, the mechanism
`of action of raparnycins, and currently understood
`resistance mechanisms.
`
`THE RAPAMYCIN TARGET (MTOR)
`
`W;fl’;W fi
`
`Rapamycin
`
`
`
`CCI-T79
`
`Figure l. The structure of rapamycin and its analogues FK506, CCI-779 and RADOOI . The
`FKBPI2 binding Face is shown by the filled bars, whereas the mTOR binding Face of
`rapamycin is shown by the hatched bar.
`
`The mammalian target of rapamycin, [also named
`FKBPI2 and rapamycin—associated protein (FRAP),
`rapamycin and FKBP12 target 1 (RAFT1), rapamycin
`target
`I
`(RAPTI), or sirolimus effector protein
`(SEP)],
`is a 289 kDa Ser/Thr kinase orthologue of
`TORI and TOR2 in Sac:/mromyces cerevz'5z'ae.35’36
`TOR is an atypical serine/threonine kinase highly
`conserved from yeast to mammals. Human, mouse
`and rat mTOR proteins share 95% identity at the amino acid
`level.36‘38 Since the C—terminus ofTOR is highly homologous to the
`catalytic domain of phosphatidylinositol 3' kinase (PI3K), mTOR is
`considered to belong to the PI5K—related protein kinase (designated
`PIKK) family, which also includes Mecl, Tell, RAD3, MEI—4I,
`DNA—PK, ATM, ATR, and TRRAP3637 Recently, single TOR
`homologs have also been identified in fungi (TORI in Cryptococcus
`neofiirmzzm), plants
`(AtTOR in Aralzidopsix t/mliana), worms
`(CeTOR in Caenor/aabziitis elegant), and flies (dTOR in Drosophila
`memz[oga:ter).37
`The domain structure of mTOR is depicted in Figure 2. The
`protein consists of a catalytic kinase domain, an FKBP12—rapamycin
`binding (FRB) domain and a putative auto—inhibitory domain
`(“repressor domain”) near the C—terminus, and up to 20 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. HEAT motifs may serve as
`protein—protein interaction parts, whereas FAT and FATC domains
`
`may participate in modulation of catalytic kinase activity of
`mTOR.36 The remarkable conservation of mTOR at the amino acid
`
`level suggests that multiple domains of this protein are essential for
`its cellular functions.
`
`Tor Signaling in Yeast. In yeast, Tor kinase activity is regulated
`by availability of nutrients (amino acids and glucose), whereas in
`mammalian cells, mTOR is regulated also by phosphatidic acid,
`ATP, and growth factors.37’39'42 The Tar signaling pathway in yeast
`is depicted in Figure 3, and controls translation initiation, protein
`turnover, transcription, and actin cytoskeleton organization. In yeast
`these pathways have been rigorously established,37 and are at least in
`part maintained in mammalian cells. The TorI/2 complex (desig-
`nated TORC1) comprising Kogl
`(the yeast homologue of the
`mammalian protein raptor) and Lst8 controls translation, protein
`stability and transcription,43'45 whereas the TORC2 complex
`controls actin organization. As TORC2 is not a rapamycin target it
`will not be considered further. The evolutionarily conserved TORCI
`complex controls translation initiation probably through activation
`of eIF4E, and transcription of ribosomal genes, stress response
`genes, ribosomal biogenesis and tRNA synthesis.
`Tor, through the TORCI complex, controls protein turnover and
`some aspects of transcription through regulation of protein phos—
`
`2549 aa
`
`Figure 2. Schematic representation at the domains at mTOR. Structural
`domains of mTOR. HEAT :[huntingtin elongation Factor A subunit of PP2A
`and 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 FKBPl2-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 bind-
`ing or act as a structural scatlold; CD: Catalytic domain;
`RD: regulatory domain.
`
`1 I
`
`
`II > II
`ll
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`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
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`Amino acids/glucose
`
`Cell membrane
`
`
`Translation
`
`Actin
`organization
`
`protein turnover
`
`Figure 3. Nutrient signaling in yeast. (Adapted from Jacinio and Hc1||.37)
`
`Transcription
`
`phatases. Tor positively regulates Tap42, which binds to and inacti-
`vates the Sit4 and Pph protein phosphatases.46’47 Pph21 and Pph22
`are calaytic subunits of PPZA, and Sit4 is the catalytic subunit of a
`PP2A—related phosphatase in yeast. Nutrient deficiency or rapamycin
`leads to dissociation of these phosphatases from Tap42, resulting in
`increased phosphatase activity. This leads to dephosphorylation of
`Nprl and Gln3 involved in protein turnover and transcription,
`respectively. Control of protein turnover and some aspects of tran-
`scription appear to be regulated through Tap42 binding and inactivat-
`ing Sit4, whereas pathways regulated by release of Pph phosphatases are
`less well defined. In the presence of nutrients Tor signaling represses
`autophagy and leads to stabilization of proteins by suppressing
`ubiquitin—dependent degradation.“ For example, Tor signaling
`prevents ubiquitylation, vacuolar targeting and degradation of the
`tryptophan transporter Tat2 by maintaining phosphorylation and
`inactivation ofNpr1 a putative Tat2 kinase.49’50 Under conditions of
`starvation Sit4 becomes activated leading to dephosphorylation of
`Nprl and degradation ofTat2. The Tap42—Sit4 pathway also controls
`the Gln3 transcription factor. Under nutrient replete conditions
`Gln3 is phosphorylated and is bound to the Ure2 protein in the
`cytoplasm. Inhibition of Tor by rapamycin or nitrogen starvation
`leads to dephosphorylation of Gln3, its nuclear translocation and
`transcription of genes required for the use of secondary nitrogen
`sources.43’44 The pathway(s) downstream of Tap42 involving Pph
`are less clear. Similarly, Tor negatively regulates the heterodimeric
`transcription factor Rtg1—Rtg3 through an unknown mechanism.“
`The Tor pathway also is important in control of stress—responses
`through modulation of transcription. The TORC1 complex nega-
`
`tively controls transcription of stress-responsive genes through the cyto-
`plasmic sequestration of the general stress transcription factors Msn2
`and Msn4. Although the mechanism is not fully understood this
`may occur through Tor signaling promoting the binding of these
`transcription factors to the 14-3-3 homologues Bmhl and Bmh2.50
`Proximal Signaling in Mammalian Cells. In mammalian cells,
`mTOR is regulated not only by nutrients but also by growth fac-
`tors.37’39‘42 It appears that growth factors regulate mTOR signaling
`through both I’I3K and Akt pathways, whereas proximal activators
`regulated by nutrients and ATP are less well characterized. In mam-
`malian cells mTOR is activated as a consequence of ligand binding
`to various growth factor receptors that result in activation of PI3K,
`Figure 4. Activated PI3K catalyzes the conversion of phostidylinosi—
`tol (4,5)—bisphosphate (PIP2) to phosphatidylinositol—3,4,5—trispho-
`sphate (PIP3). This pathway is negatively regulated by a dual—speci—
`flcity protein and lipid phosphatase PTEN. Full activation of Akt,
`downstream of PI3K requires binding of PIP3 to the pleckstrin
`homology domain ofAkt, and phosphorylation by phosphoinositide—
`dependent kinases 1/2 (PDK1/2) and other unidentified kinases.
`Pharmacological studies with albeit relatively non—specif1c inhibitors
`of PI3K (wortmannin and LY294002) indicate that mTOR is down-
`stream of PI3K. How mTOR is regulated by I’I3K or Akt, however,
`is still not well understood. Akt can phosphorylate mTOR
`(Ser2448) directly, although the significance remains to be deter-
`mined.37’38 Recent studies have placed the tuberous sclerosis
`(TSC1/2) complex as a modulator between PI3K/Akt and mTOR.52‘54
`The TSC1/2 complex comprises harmartin (TSC1) and tuberin
`(TSC2). These proteins form a physical and functional complex in
`
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`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
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`Figure 4. Growth Factor signaling in mammalian cells.
`
`Vivo, which binds and inhibits mTOR.52‘54 Loss of TSC1/2 results
`in mTOR—dependent increase of ribosomal p70 S6 kinase (S6K1)
`activity, and confers resistance of cells to amino acid starvation.53
`Conversely co—expression of TSC1 and TSC2 inhibits amino
`acid—induced activation of S6K1 in nutrient—deprived cells.“
`Mitogenic stimuli, such as insulin or serum, activate Akt which can
`directly phosphorylate TSC2 on multiple sites in vitro and in
`vivo.52’55’56 Phosphorylation of TSC2 at Ser939 and Thr1462 is
`PI3K-dependent.52’56 Akt—mediated phosphorylation of TSC2
`destabilizes TSC2 and thereby inhibits the formation of TSCI/2
`complex,
`leading to de—repression of mTOR, and consequently
`increasing the kinase activity of mTOR. By contrast, treatment with
`rapamycin does not influence the phosphorylation of TSC2,52 and
`together with other data,5354 suggests that mTOR lies downstream
`of TSC2. Other results5758 imply that the TSC1/2 complex may
`mediate S6K1 activation through a pathway parallel
`to mTOR.
`However, further studies will be required to address the relationship
`of mTOR and TSC1/2.
`
`mTOR Associated Proteins. mTOR forms a scaffold complex
`with other proteins, such as raptor (regulatory associated protein of
`mTOR) and mLST8,59'60 which are the mammalian counterparts of
`yeast Kogl (kontroller of growth 1) and Lst8,61 respectively. The
`exact role of raptor remains unclear.38’59‘61 Raptor may act as a scaffold
`protein linking mTOR to S6K1 and 4E—BP1.60 Alternatively, it may
`have a dual function, inhibiting mTOR under nutrient—def1cient
`conditions and stimulating mTOR in a nutrient—replete environment.”
`
`Thus TSC1/2, raptor and possibly mLST8 act as potential modulators
`of mTOR function in response to availability of nutrients.
`Signaling Distal to mTOR. mTOR controls translation initiation
`through two pathways, S6Kl and eukaryotic initiation factor 4E
`(eIF4E) binding proteins (4E-BPS). mTOR either directly phospho-
`rylates Thr389 of S6K1, or suppresses a phosphatase responsible for
`maintaining the hypophosphoryalted state of this residue. Activation
`of S6K1 enhances translation of mRNAs bearing a 5’
`terminal
`oligopyrimidine tract (5’ TOP).62’63 Inactivation of S6K1 decreases
`synthesis of ribosomal proteins and translation factors.62’63
`Activation of S6K1 is complex. The process involves interplay
`between four different domains and at least seven specific sites medi-
`ated by multiple upstream kinases.64 At
`least 12 sites
`(Serl7,
`Thr229, Thr367, Thr371, Thr389, Ser404, Ser411, Ser418, Tr421,
`Ser424, Ser429, and Thr447) can be phosphorylated in response to
`serum stimulation.“ However, the responsible kinases have not been
`fully characterized. Phosphoinositide—dependent protein kinase 1
`(PDK1) phosphorylates Thr229 in vitro and in vivo.66 Atypical
`PKC isoforms and the Rho family of small G proteins (cdc42 and
`Racl) may contribute to phosphorylation of S6K1,65 although the
`specific sites regulated by these kinases remain to be determined. In
`vitro, mTOR phosphorylates only Thr389 in the regulatory
`domain.67'69 However, whether this phosphorylation is directly or
`indirectly regulated by mTOR is in question, since recent data suggest
`that mTOR may regulate S6K1 activation by inhibiting phos-
`phatases rather than directly phosphorylating S6K1.64’70
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`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
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`mTOR phosphorylates and inactivates the 4E—BP suppressor
`proteins causing their dissociation from the RNA cap—binding
`protein eIF4E. In response to mitogens, six sites (Thr37, Thr46,
`Ser65, Thr70, Ser83, and Ser112) of 4E—BP1 can be phosphorylated.“
`So far, only mTOR and ATM have been identified to be involved in
`phosphorylation of 4E—BP1.71‘74 Other kinases that phosphorylate
`4E—BP1 remain to be characterized. ATM phosphorylates 4E—BP1 at
`Ser112,74 however the physiological significance of this remains to
`be elucidated. In vitro mTOR phosphorylates 4E—BP1 at two sites
`(Thr37, Thr46) and possibly at two additional Ser/Thr residues
`(Thr70 and Ser65) in the N—terminal region.73’75 Phosphorylation
`is a hierarchical process.73’75'77 Phosphorylation of Thr37/Thr46 is
`followed by Thr70 phosphorylation. Ser65 is phosphorylated last77
`and is dependent on phosphorylation of all three Thr/Pro sites.75’76
`Mutation of Thr37 and/or Thr46 to alanine(s) prevents phosphory—
`lation of Ser65 and Thr70,
`indicating that phosphorylation of
`Thr37 and Thr46 serves as a requisite “priming” event.36 Single
`phosphorylation of these residues is not sufficient
`to dissociate
`4E—BP1 from eIF4E,
`indicating the requirement of combined
`phosphorylation of at least Thr37, Thr46, Ser65, and Thr70 in
`4E—BP1
`to suppress association with eIF4E.36’78 Inhibition of
`mTOR leads to rapid hypophosphorylation of 4E—BP1 which then
`tightly binds to eIF4E. This prevents formation of eIF4F complex
`that contains eIF4E, eIF4G, eIF4A and eIF3, and inhibits
`cap—dependent translation initiation.36 Once 4E—BP1 is hyperphos—
`phorylated, it releases eIF4E, facilitating eIF4F complex formation
`and promoting cap—dependent protein synthesis.36 Overall inhibition
`of mTOR by rapamycin leads to a decrease in protein synthesis of
`15 to 20 percent. However, as the eIF4E pathway is required for
`translation of mRNA’s encoding cyclin D1,79’80 and ornithine
`decarboxylase81 inhibition of mTOR leads to slowing or arrest of
`cells in G1 phase of the cell cycle. However, the exact mechanism(s)
`by which mTOR regulates cell cycle progression are complex, poor-
`ly understood,
`and potentially context
`specific. Although
`rapamycins are highly specific inhibitors, the TOR pathway regu-
`lates multiple cellular processes. The mTOR pathway regulates trans-
`lation initiation of survival
`factors
`such as
`c—MYC82 and
`hypoxia—inducible factor 10., and consequently vascular endothelial
`growth factor.83’84 In addition, mTOR is involved in the regulation
`of cyclin A, cyclin dependent kinases (cdkl/2), cdk inhibitors
`(p21CiP1 and p27KiP1), retinoblastoma protein, RNA polymerase
`I/II/III—transcription and translation of rRNA and tRNA, protein
`phosphatases (PPZA, PP4 and PP6), and CLIP—170.36’37v85‘91
`
`TUMOR SELECTIVITY OF RAPAMYCINS
`
`To date there are no reports suggesting that activating mutations
`of mTOR, or overexpression occur as primary events in malignant
`transformation. However, activation of signaling pathways both
`proximal and distal to mTOR appear to occur frequently in human
`cancer. Loss of the phosphatase PTEN by deletion, silencing or
`mutation leads to constitutive activation of Akt,29’30’92 and upregu—
`lation of mTOR—dependent pathways. In PTEN—deficient tumor
`cells or mouse embryo fibroblasts (MEFS), activated Akt is associat-
`ed with enhanced activity of S6K1 and hyperphosphorylation of
`4E—BP1,29’30 or increased levels of c—MYC.92 It is speculated that
`Akt—activated cells become dependent on upregulated mTOR
`signaling for proliferation, hence become more susceptible to
`rapamycin or CCI—779. Increased sensitivity to rapamycins has been
`demonstrated in a panel of brain, prostate, and breast cancer cells,
`multiple myeloma cells and in MEFs.25’29 The association of PTEN
`deficiency and sensitivity to rapamycin is further supported by the
`
`activity of CCI—779 against the growth of human tumors implanted
`in athymic nude mice.23’3° There are, however, some exceptions;
`cells with functional PTEN and low constitutive activation of Akt
`
`are equally sensitive to inhibition of proliferation by rapamycins.
`Consequently the role of PTEN as an independent variable predicting
`for rapamycin sensitivity remains to be demonstrated. Disruption of
`the TSC1 or TSC2 gene leads to the development of tumors in
`multiple organs, notably kidney brain heart and lung. Even low
`doses of rapamycin causes apoptosis and necrosis of spontaneous
`renal tumors in Eker rats with germline mutation in TSC2 and
`results in tumor regression.93 These studies suggest that rumors
`developing in patients with tuberous sclerosis may be sensitive to
`rapamycins. Oncogene expression may also regulate the response to
`rapamycin. For example, in RKSE cells transformed with c—MYC or
`Ras rapamycin treatment increased global protein synthesis. In contrast
`rapamycin inhibited global protein synthesis and turnover in GLI
`transformed isogenic lines leading to inhibition of proliferation.”
`Although generally considered to be cytostatic agents, rapamycins
`can induce apoptosis in some cell systems. Rapamycins induce
`apoptosis of B—cells, rhabdomyosarcoma cells, renal tubular cells and
`dendritic cells.16’95'97 Rapamycin enhances transforming growth
`factor—[3 induced cell cycle arrest,98 and through blocking survival
`factor signaling” rapamycins enhance cell death. Our results suggest
`that the functional status of the p53 tumor suppressor may dictate
`the cellular fate of rapamycin treated cells, as depicted in Figure 5.
`For example, under serum free conditions,
`the response to
`rapamycin in cells lacking functional p53 is apoptosis, suggesting
`that only in the absence of p53/p21 inhibition of mTOR becomes
`lethal
`(so—called synthetic lethality). Ectopic expression of p53 or
`p21CiP1 protects cells from rapamycin-induced apoptosis. The
`implication is that rapamycins may have potential tumor—selective
`therapeutic effects.“ Recent results show that inhibition of mTOR
`by rapamycin induces a cellular stress response characterized by rapid
`and sustained activation of ASK1 (apoptosis signal—regulating kinase
`1) signaling in p53—mutant cells.100 This leads to sustained phos-
`phorylation of c—]UN (Ser63) that appears to be responsible for
`inducing apoptosis. Rapamycin—induced stress appears distinct from
`that induced by ultra violet radiation in that MEKK1 is not activated,
`and from cytotoxic agents such as DNA damaging agents where
`other stress pathways (p38 or ERK1/2) are also activated.1°1*1°2 In
`contrast, cells expressing wild type p53,
`(or constitutive p21CiP1
`expression)
`there is only transient activation of ASK1.100
`Suppression of ASK1 is associated with binding of p21CiP1 in
`rapamycin treated cells, and protection from apoptosis. Since the
`rapamycin-induced apoptosis is specifically prevented by insulin—like
`growth factors (IGF—I/II) and insulin,96 combination of IGF receptor
`inhibitors with rapamycins may be selectively cytotoxic and induce
`regression of tumors with p53 mutations. VVhether such anticipation
`is justified requires vigorous testing.
`
`MECHANISMS OF RESISTANCE T0 RAPAMYCINS
`
`Intrinsic sensitivity to rapamycins between cell lines may vary by
`several orders of magnitude. For example, rhabdomyosarcoma cells
`in vitro are very sensitive (concentration for 50% inhibition (ICSO)
`of proliferation ~ 1 nM) compared to colon carcinoma cells (IC50 >
`5000 nM).“ Mechanisms of intrinsic and acquired resistance may
`have either a genetic or epigenetic basis.
`Mutations in FKBPIZ and mTOR. Rapamycins first bind to the
`cyclophilin FKBP12 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.
`
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`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
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`Rapamycin resistance may be conferred by
`mutations in FKBP12 that prevent the forma-
`tion of FKBP—rapamycin complex, or mutations
`in the FRB domain of mTOR that block binding
`of FKBP—rapamycin complex to mTOR. Such
`mutations were first identified in budding yeast
`5. cerevixiae in which treatment with rapamycin
`irreversibly arrests cells in the G1 phase. In the
`yeast 3. caret/iriae, deletion of the RBPI gene, a
`homologue of mammalian FKBP—12, results in a
`recessive rapamycin resistance, whereas expres-
`sion of RBPI restores rapamycin sensitivity.103
`This observation has been further confirmed by
`RBPI disruption experiments using the patho-
`genic
`yeast Czzndida
`at/Means. Wild—type
`RBP1/RBPI parental strain and the rbpl/RBPI
`heterozygous mutant were sensitive to rapamycin
`inhibition, whereas
`rbpl/rbpl homozygous
`mutant was rapamycin resistant.104 In addition, in
`S. cerevisiae mutation of a specific residue (Tyr89)
`which is conserved in RBPI or FKBPs, also
`resulted in decreased binding of rapamycin and
`conferred a recessive resistance pl1eI10type.105 In
`murine mast cells, two distinct point mutations
`in FKBP12 confer
`resistance. By altering a
`hydrophobic residue within the drug—binding
`pocket (Trp599Leu) or changing a charged sur-
`face residue (Arg49-)Gln), the binding aflinity for
`rapamycin decreases substantially_106
`A genetic screen identified rapamycin—resist-
`ant alleles with mutations in genes designated
`TORI and TORZ. Strains with mutated to torl-1
`
`Synthetic lethality
`a genetic defect alters ceilular response to drug action
`
`p53+.l+
`
`p53~‘-
`
`Rapatnytiril
`
`Atiue
`
`Rapamycinl
`
`
`
`PrupidiumIodideFluorescence
` =‘:.5ti..'ii.
`
`+ Rap 11]!)
`
`Ad-p53
`
`W”
`
`in‘
`
`.-_._
`In‘
`
`In’
`10’
`lo‘
`10‘ In“
`11?
`in’
`Annexin V—F|TC Fluorescence
`
`in‘
`
`Figure 5. Loss of p53 function alters cellular response to rapamycin from cytostasis to apoptosis
`in murine embryo fibroblasts (MEFs). Left: schematic representation of synthetic lethality. Right:
`Wild type, and p53’/’MEFs, and p53’/‘ MEFs infected withAd-p53 (MOI of 100) were grown
`without or with rapamycin (100 ng/ml). Cells were harvested after 5 days and apoptosis deter-
`mined by quantitative FACs analysis (ApoAlert) assay. The per cent distribution of cells in each
`quadrant is presented (from Huang et al. 200i '6).
`
`(Ser19729Arg) and t0r2—I (Ser19759Arg), were
`completely resistant
`to the growth— inhibitory
`effect of rapamycin. These resistant alleles encode mutant Tor proteins
`that do not bind the FKBP—rapamycin complex.103’107'1“ This
`result suggests that a conserved serine residue (Ser1972 in Torl;
`Ser1975 in Tor2) in Tor proteins is critical for FKBP—rapamycin
`binding. In mammalian cells mutations in the FRB domain confer
`a dominant resistant phenotype consistent with decreased affinity
`for binding of the FKBP— rapamycin complex.1” Expression of a
`mutant mTOR (Ser2035-)Ile), having greatly reduced binding affin-
`ity for the FKBP—rapamycin complex, confers high level resist-
`anCe_14,112,113
`
`Deregulation of eIF4E. mTOR phosphorylates and regulates the
`function of 4E—BP1, the suppressor of eIF4E.36 Recently, our group
`has found that acquired resistance to rapamycin was associated with
`decreased levels of 4E—BP1.121 In the absence of selective pressure
`(rapamycin), resistance was unstable and cells reverted to being
`sensitive to growth inhibition of rapamycin within ten weeks. In
`resistant cells
`the levels of 4E—BP1 were reduced significantly
`(~10—fold), whereas in rapamycin—sensitive revertants the 4E—BPI
`levels increased to those in wild type cells. Levels of 4E—BP1 transcripts
`were unaltered in rapamycin resistant clones suggesting post—tran—
`scriptional regulation. Further studies indicate that the synthesis of
`4E—BPI
`significantly decreased in rapamycin—resistant clones.
`Whether the steady state level of 4E—BP1 is also regulated by
`increased degradation remains to be determined. Thus, the changes
`in 4E—BP1 levels are reminiscent of those reported for p27KiP1 in
`rapamycin resistant BC5H cells.“5 In contrast, no consistent
`changes were detected in the level or activity of S6K1 between parental
`
`and resistant clones. Rapamycin also inhibited growth factor activa-
`tion of SGKI equally in parental and rapamycin—resistant clones.
`Intrinsic resistance to rapamycin has been shown in glioblastoma
`cells and colon adenocarcinoma that have very low 4E—BP1.“4 For
`example, 4E—BP1 is barely detected in HCT8 colon carcinoma cells
`that are highly resistant to rapamycin (IC50 > 10,000 ng/ml). \X/hen
`4E—BP1 is overexpressed, these cells become sensitive (IC50 < 10
`ng/ml) to rapamycin, Figure 6.114 These data suggest that low levels
`of 4E—BPI results in de—regulation of eIF4E, conferring rapamycin
`resistance.
`
`These results suggest that rapamycin—regulation of the eIF4E
`pathway is crucial in inducing growth arrest. Further de-regulation
`of eIF4E may facilitate a malignant phenotype. Of interest is that
`both rapamycin—resistant and —revertant cells exhibited elevated
`c—MYC levels, and increased anchorage—independent growth. That
`deregulation of the eIF4E pathway is associated with increased
`malignancy is supported by certain clinical observations that dereg-
`ulation of the eIF4E pathway does promote tumor progression.“6
`In addition to decreased 4E—BP1 expression, as described above,
`increased eIF4E levels may also cause de—regulation of eIF4E. In
`advanced head and neck carcinoma,“7 breast carcinoma“8 gas-
`trointestinal carcinoma,119 and peripheral carcinoma of the lung120
`eIF4E levels are elevated. However, levels of 4E—BPI suppressor
`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. Further
`studies will be necessary to determine if this ratio has predictive
`value for drug sensitivity of tumors.
`
`www.landesbioscience.com
`
`Cancer Biology 8: Therapy
`
`227
`
`West-Ward Pharm.
`Exhibit 1018
`Page 006
`
`West-Ward Pharm.
`Exhibit 1018
`Page 006
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`

`

`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
`
`Fiapamycin
`Sensitive
`
`3 7 4- 4E—BP‘t I
`
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`
`B
`
`'03 HCT8-4E-BP1 clones
`[MI
`E
`1
`2
`3
`
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`
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`
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`
`100 -7
`
`Mutations in S6K1. Ribosomal S6K1 is the other principal
`downstream effector of mTOR. Inhibition of mTOR by rapamycin
`primarily inhibits phosphorylation of Thr389 in the regulatory
`domain.“ However, since phosphorylation of S6K1 is hierarchical
`with phosphorylation of several other sites dependent on phospho-
`rylation of Thr389,64 rapamycin in vivo influences phosphorylation
`of other sites, including Thr229 in the catalytic domain, and S404
`in the regulatory domain.67 Mutation ofThr3899Glu abrogates the
`ability of rapamycin to inhibit S6K1 activation.67’121 Similarly,
`substitution of Thr229 by either a neutral amino acid Alanine
`(Thr2299Ala) or by an acidic amino acid Glu (Thr2299Glu),
`renders S6K1 insensitive to rapamycin.122 In addition, deletion of
`the 77 N—terminal codons (AN77) confers rapamycin resistance.123
`Of note truncation of