[Cancer Biology 8& Therapy 2:3, 222-232, May/June 2003]; ©2003 Landes Bioscience
`
`Review
`
`Rapamycins
`Mechanism of Action and Cellular Resistance
`
`Shile Huang
`Mary-AnnBjornsti
`Peter J. Houghton*
`
`Department of Molecular Pharmacology; St Jude Children's Research Hospital; 332
`N. Lauderdale; Memphis; Tennessee USA
`
`*Correspondence to: Peter J. Houghton; Department of Molecular Pharmacalogy; St
`Jude Children’s Research Hospital; 332 N. Lauderdale; Memphis; Tennessee
`38105-2794 USA; Email: peterhoughton@stjude.org
`
`Received 04/24/03; Accepted 05/01/03
`
`Previously published online os 0 CB&T Paperin Press at:
`http://wwwlandesbioscience.com/journals/cbt/toc.php?volume=28issue=3
`
`KEY WORDS
`
`Rapamycins, Translation initiation, Cancer,
`Resistance, Therapy, Ribosomal biogenesis, Yeast
`
`Work reported from these laboratories was supported by PHS awards (A23099,
`(A58755, CA77776, CA96996 and CA21765 (Cancer Center Support Grant) and by
`American, Lebanese, Syrian Associated Charities (ALSAC).
`
`222
`
` ABSTRACT
`
`Rapamycins are macrocyclic lactones that possess immunosuppressive, antifungal and
`antitumor properties. The parent compound, rapamycin, is approved as an immunosup-
`pressive agent for preventing rejection in patients receiving organ transplantation. Two
`analogues, CCI-779 and RADOO1 are currently being investigated as anticancer agents.
`Rapamycinsfirst bind a cyclophilin FKBP12, and this complex binds and inhibits the
`function of mTOR (mammalian target of rapamycin) a serine/threonine (Ser/Thr) kinase
`with homology to phosphatidylinositol 3’ kinase. Currently, as mTORis the only identified
`target,
`this places rapamycins in a unique position of being the most selective kinase
`inhibitor known. Consequently these agents have been powerful fools in elucidating the
`role of mTORin cellular growth, proliferation, survival and tumorigenesis. Increasing
`evidence suggests that mTOR acts as a central controller sensing cellular environment
`(nutritional status or mitogenic stimulation) and regulating translationinitiation through the
`eukaryotic initiation factor 4E, and ribosomal p70 Sé6 kinase pathways. Here we review
`the conserved TOR signaling pathways, conceptual basis for tumor selectivity, and the
`mechanismsofresistance to this class of antitumor agent.
`
`INTRODUCTION
`
`Rapamycin, a macrocyclic lactone product ofthe soil bacteria Streptomyces hygroscopicus,
`was isolated and identified as an antifungal agent in the mid-1970’s.'3 Rapamycin
`(sirolimus),
`is a structural analogue of the macrolide antibiotic FK506 (tacrolimus,
`Prograf®) (Fig. 1), and like FK506 was foundto potently suppress the immunesystem.*7
`The potential for rapamycin to act as an antitumor agent was recognizedearly in its
`development when the drug demonstrated potent inhibitory activity against numerous
`solid tumors in the NCI screening program.®-!9 However, the drug was not developed
`further dueto stability and solubility problems that prevented developmentof a parenteral
`formulation 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 manylaboratories have demonstrated that
`rapamycin, in contrast to FK506,is not only a potent immunosuppressant, but also an
`active antitumor agent. Rapamycin can actas a cytostatic, slowing or arresting cells in G,
`phase. Under specific conditions, or in some tumorcell lines rapamycins may induce
`apoptosis in culture. To date, studies have revealed that rapamycin potently arrests growth
`ofcells derived from rhabdomyosarcoma, neuroblastomaandglioblastoma, small cell lung
`cancer,!!-!7 osteoscarcoma,!® pancreatic cancer,!?2° breast and prostate cancer,2!-74
`murine melanoma and leukemia, and B-cell lymphoma.?2*?6 In addition to broad
`spectrum activity in vitro, rapamycin andits derivatives (designated here as rapamycins)
`suppress growth of some human and murine tumor models in vivo.!!-39 When combined
`with other chemotherapeutic agents, rapamycins generally showatleast additive antitumor
`activity.!0:12:17:31 Preliminary data from clinical trials have indicated that rapamycins are
`well tolerated and successfully suppress growth of various human tumors.3234
`The use of rapamycin as an anticancer drugis clinically impractical, because ofits poor
`water-solubility and stability in solution. Recently, rapamycin ester analogues (Fig. 1),
`CCI-779 [rapamycin-42, 2, 2-bis(hydroxymethy])-propionic acid]
`(Wyeth-Ayerst, PA,
`USA) and RADOO1 [everolimus, 40-O-(2-hydroxyethyl)-rapamycin]
`(Novartis, Basel,
`Switzerland), have entered clinical trials. These analogues have improved pharmaceutical
`properties. CCI-779 is being developed for both intravenous and oral administration,
`whereas RADO01 is only for oral administration. The antitumoractivity of these analogues
`
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`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
`
`is similar to that of rapamycin.!77!-2327-30 RADO01 is
`in Phase I trials whereas the development of CCI-779
`is more advancedwith several PhaseII 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 tumorcells may affect
`sensitivity to rapamycins. At least
`in some systems
`mutations that occur frequently in malignant trans-
`formations such as GLJ amplification, or mutations
`that inactivate p53, and the dual specificity phos-
`phatase PTEN (phosphatase and tensin homolog
`deleted on chromosometen, also known as MMAC1
`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 mTORincellular regulation, the mechanism
`of action of rapamycins, and currently understood
`resistance mechanisms.
`
`THE RAPAMYCIN TARGET (MTOR)
`
`LLLLLSLLLELLPLLLLL
`
` Rapamycin
`
`CCI-779
`
`Figure 1. The structure of rapamycin and its analogues FK506, CCI-779 and RADOO1. The
`FKBP12 binding face is shown by thefilled bars, whereas the mTOR binding face of
`rapamycin is shownby the hatched bar.
`
`The mammaliantarget of rapamycin, [also named
`FKBP12 and rapamycin-associated protein (FRAP),
`rapamycin and FKBP12 target 1 (RAFT 1), rapamycin
`target
`1
`(RAPT1), or sirolimus effector protein
`(SEP)],
`is a 289 kDa Ser/Thr kinase orthologue of
`TORI and TOR2 in Saccharomyces cerevisiae.>>*6
`TORis 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.3°38 Since the C-terminus ofTORis highly homologousto the
`catalytic domain of phosphatidylinositol 3’ kinase (PI3K), mTORis
`considered to belong to the PI3K-related protein kinase (designated
`PIKK) family, which also includes Mecl, Tell, RAD3, MEI-41,
`DNA-PK, ATM, ATR, and TRRAP?°37 Recently, single TOR
`homologshave also been identified in fungi (TOR1 in Cryptococcus
`neoformans), plants
`(AtTOR in Arabidopsis thaliana), worms
`(CeTORin Caenorhabditis elegans), and flies (ATOR in Drosophila
`menalogaster).”
`The domain structure of mTORis 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 PP2A 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.°*® The remarkable conservation of mTORatthe aminoacid
`level suggests that multiple domainsof 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.3”39*? The Tor signaling pathway in yeast
`is depicted in Figure 3, and controls translation initiation, protein
`turnover, transcription, andactin cytoskeleton organization. In yeast
`these pathwayshave been rigorously established,*” andareat least in
`part maintained in mammalian cells. The Torl/2 complex (desig-
`nated TORC1) comprising Kogl
`(the yeast homologue of the
`mammalian protein raptor) and Lst8 controls translation, protein
`stability and transcription,#3“> whereas the TORC2 complex
`controls actin organization. As TORC2is not a rapamycin targetit
`will not be considered further. The evolutionarily conserved TORC1
`complex controls translation initiation probably through activation
`of eIF4E, and transcription of ribosomal genes, stress response
`genes, ribosomal biogenesis and tRNAsynthesis.
`Tor, through the TORC1 complex,controls protein turnover and
`some aspects of transcription through regulation of protein phos-
`
`Figure 2. Schematic representation of the domains of 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 PlK-related kinases located N-terminal to
`the FKBP12-rapamycin binding domain (FRB);
`the role of
`FAT sequencesis less clear, but they are associated with
`C+erminal
`FAT (FATC)
`sequences
`in mTOR.
`Interaction
`between FAT and FATC domains mayfacilitate protein bind-
`ing or act asastructural scaffold; CD: Catalytic domain;
`RD: regulatory domain.
`
`
`|RO]}FATC
`
`2549 aa
`
`1| C
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`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
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`Amino acids/glucose
`Cell membrane
`?
`
`
`Sale
`
`Translation
`
`rr
`
`= GD
`
`Le
`
`Actin
`organization
`
`protein turnover
`
`Transcription
`
`Figure 3. Nutrient signaling in yeast. (Adapted from Jacinto and Hall.37)
`
`phatases. Tor positively regulates Tap42, which binds to and inacti-
`vates the Sit4 and Pphprotein phosphatases.464” Pph21 and Pph22
`are calaytic subunits of PP2A,andSit4 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 someaspects of tran-
`scription appearto be regulated through Tap42 binding andinactivat-
`ing Sit4, whereas pathwaysregulated byrelease ofPph phosphatases are
`less well defined. In the presence ofnutrients Tor signaling represses
`autophagy andleads to stabilization of proteins by suppressing
`ubiquitin-dependent degradation.4® For example, Tor signaling
`prevents ubiquitylation, vacuolar targeting and degradation of the
`tryptophan transporter Tat2 by maintaining phosphorylation and
`inactivation ofNprl a putative Tat2 kinase.4?>° Under conditions of
`starvation Sit4 becomesactivated leading to dephosphorylation of
`Npr1 and degradation ofTat2. The Tap42-Sit4 pathwayalso 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.*94 The pathway(s) downstream of Tap42 involving Pph
`are less clear. Similarly, Tor negatively regulates the heterodimeric
`transcription factor Rtgl-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 ofstress-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 Bmh1 and Bmh2.°°
`Proximal Signaling in Mammalian Cells. In mammaliancells,
`mTORis regulated not only by nutrients but also by growth fac-
`tors.3”39-42 Tt appears that growth factors regulate mTORsignaling
`through both PI3K and Akt pathways, whereas proximal activators
`regulated by nutrients and ATPare less well characterized. In mam-
`malian cells mTORis activated as a consequenceofligand binding
`to various growth factorreceptors that result in activation of PI3K,
`Figure 4. Activated PI3K catalyzes the conversion ofphostidylinosi-
`tol (4,5)-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trispho-
`sphate (PIP3). This pathwayis negatively regulated by a dual-speci-
`ficity protein andlipid phosphatase PTEN. Full activation of Akt,
`downstream of PI3K requires binding of PIP3 to the pleckstrin
`homology domain of Akt, and phosphorylation by phosphoinositide-
`dependent kinases 1/2 (PDK1/2) and other unidentified kinases.
`Pharmacological studies with albeit relatively non-specific inhibitors
`of PI3K (wortmannin and LY294002) indicate that mTORis down-
`stream of PI3K. How mTORisregulated by PI3K or Akt, however,
`is still not well understood. Akt can phosphorylate mTOR
`(Ser2448) directly, although the significance remains to be deter-
`mined.3738 Recent studies have placed the tuberous sclerosis
`(TSC1/2) complex as a modulator between PI3K/Akt and mTOR.>?>4
`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.
`Conversely co-expression of TSC1 and TSC2 inhibits amino
`acid-induced activation of SGK1 in nutrient-deprived cells.>4
`Mitogenic stimuli, such as insulin or serum, activate Akt which can
`directly phosphorylate TSC2 on multiple sites in vitro and in
`vivo.>25556 Phosphorylation of TSC2 at Ser939 and Thr1462is
`PI3K-dependent.°2>° Akt-mediated phosphorylation of TSC2
`destabilizes TSC2 and thereby inhibits the formation of TSC1/2
`complex,
`leading to de-repression of mTOR, and consequently
`increasing the kinase activity of mTOR. By contrast, treatment with
`rapamycin does notinfluence the phosphorylation of TSC2,°? and
`together with other data,>>4 suggests that mTORlies downstream
`of TSC2. Other results?”°* 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 mTORand TSC1/2.
`mTORAssociated Proteins. mTOR forms a scaffold complex
`with other proteins, such as raptor (regulatory associated protein of
`mTOR) and mLST8,°™© which are the mammalian counterparts of
`yeast Kog] (kontroller of growth 1) and Lst8,°! respectively. The
`exactrole ofraptor remainsunclear.>8°?-6! Raptor mayactas a scaffold
`protein linking mTORto SGK1and 4E-BP1.°Alternatively, it may
`have a dual function, inhibiting mTOR under nutrient-deficient
`conditions andstimulating mTORina nutrient-replete environment.>?
`
`Thus TSC1/2, raptor and possibly mLST$8actas potential modulators
`of mTORfunction in responseto availability of nutrients.
`Signaling Distal to mTOR. mTORcontrols translation initiation
`through two pathways, S6K1 and eukaryotic initiation factor 4E
`(eIF4E) binding proteins (4E-BPs). mTOReitherdirectly phospho-
`rylates Thr389 of S6K1, or suppresses a phosphatase responsible for
`maintaining the hypophosphoryaltedstate ofthis residue. Activation
`of S6K1 enhances translation of mRNAs bearing a 5’
`terminal
`oligopyrimidine tract (5’ TOP).Inactivation of S6K1 decreases
`synthesis of ribosomal proteins and translation factors.°%
`Activation of S6K1 is complex. The process involves interplay
`between fourdifferent domainsandat least seven specific sites medi-
`ated by multiple upstream kinases.°4 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.% Atypical
`PKC isoforms and the Rho family of small G proteins (cdc42 and
`Racl) maycontribute to phosphorylation of S6K1,©although the
`specific sites regulated by these kinases remain to be determined. In
`vitro, mTOR phosphorylates only Thr389 in the regulatory
`domain.®”-However, whether this phosphorylation is directly or
`indirectly regulated by mTORis in question, since recent data suggest
`that mTOR may regulate S6K1 activation by inhibiting phos-
`phatases rather than directly phosphorylating S6K1.°7°
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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) of4E-BP1 can be phosphorylated.©
`So far, only mTORand ATM havebeen identified to be involved in
`phosphorylation of 4E-BP1.7!-74 Other kinases that phosphorylate
`4E-BP1 remainto be characterized. ATM phosphorylates 4E-BP1 at
`Serl12,”4 however the physiological significance of this remains to
`be elucidated. In vitro mTOR phosphorylates 4E-BP1 at twosites
`(Thr37, Thr46) and possibly at two additional Ser/Thr residues
`(Thr70 and Ser65) in the N-terminal region.”*7° Phosphorylation
`is a hierarchical process.’3”>’7 Phosphorylation of Thr37/Thr46is
`followed by Thr70 phosphorylation. Ser65 is phosphorylated last”7
`and is dependent on phosphorylation ofall three Thr/Prosites.”>»”°
`Mutation ofThr37 and/or Thr46 toalanine(s) prevents phosphory-
`lation of Ser65 and Thr70,
`indicating that phosphorylation of
`Thr37 and Thr46serves as a requisite “priming” event.>° Single
`phosphorylation of these residues is not sufficient
`to dissociate
`4E-BP1 from elF4E,
`indicating the requirement of combined
`phosphorylation of at least Thr37, Thr46, Ser65, and Thr70 in
`4E-BP1 to suppress association with eIF4E.9%78 Inhibition of
`mTORleads to rapid hypophosphorylation of 4E-BP1 which then
`tightly binds to eIF4E. This prevents formation of elF4F complex
`that contains eIF4E, eIF4G, eIF4A and elIF3, and inhibits
`cap-dependenttranslation initiation.>° Once 4E-BP1 is hyperphos-
`phorylated, it releases eIF4E,facilitating e[F4F complex formation
`and promoting cap-dependentprotein synthesis.>° Overall inhibition
`of mTORby rapamycin leads to a decrease in protein synthesis of
`15 to 20 percent. However, as the elF4E pathway is required for
`translation of mRNA’s encoding cyclin D1,7%°° and ornithine
`decarboxylase®! inhibition of mTORleads to slowing or arrest of
`cells in G, phase ofthe cell cycle. However, the exact mechanism(s)
`by which mTORregulatescell 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 pathwayregulates trans-
`lation initiation of survival
`factors
`such as
`c-MYC®* and
`hypoxia-inducible factor 1a, and consequently vascular endothelial
`growth factor.8384 In addition, mTORis involved in the regulation
`of cyclin A, cyclin dependent kinases (cdk1/2), cdk inhibitors
`(p21C'P! and p27KiP!), retinoblastoma protein, RNA polymerase
`I/II/I1-transcription and translation of rRNA and tRNA,protein
`phosphatases (PP2A, PP4 and PP6), and CLIP-170.337.85-91
`
`TUMORSELECTIVITY OF RAPAMYCINS
`
`To date there are noreports suggesting that activating mutations
`of mTOR,or overexpression occur as primary events in malignant
`transformation. However, activation of signaling pathways both
`proximal anddistal to mTOR appearto occur frequently in human
`cancer. Loss of the phosphatase PTEN by deletion, silencing or
`mutation leads to constitutive activation of Akt,2%3%9? and upregu-
`lation of mTOR-dependent pathways. In PTEN-deficient tumor
`cells or mouse embryofibroblasts (MEFs), activated Akt is associat-
`ed with enhanced activity of S6K1 and hyperphosphorylation of
`4E-BP1,?9° or increased levels of c-MYC.”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 cancercells,
`multiple myelomacells and in MEFs.?39 The association of PTEN
`deficiency andsensitivity to rapamycinis further supported by the
`
`activity of CCI-779 against the growth of human tumors implanted
`in athymic nude mice.2339 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 independentvariable predicting
`for rapamycinsensitivity 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.?> These studies suggest that tumors
`developing in patients with tuberous sclerosis may besensitive to
`rapamycins. Oncogeneexpression mayalso regulate the response to
`rapamycin. For example, in RK3Ecells transformed with c-MYC or
`Ras rapamycin treatmentincreased global protein synthesis. In contrast
`rapamycin inhibited global protein synthesis and turnover in GLI
`transformed isogeniclines leading to inhibition ofproliferation.™4
`Although generally considered to be cytostatic agents, rapamycins
`can induce apoptosis in some cell systems. Rapamycins induce
`apoptosisof B-cells, rhabdomyosarcomacells, renal tubular cells and
`dendritic cells.1%9>°” Rapamycin enhances transforming growth
`factor-B induced cell cycle arrest,98 and through blocking survival
`factor signaling”? rapamycins enhancecell death. Ourresults 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
`p21! 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 inducesa cellular stress response characterized by rapid
`andsustained activation ofASK1 (apoptosis signal-regulating kinase
`1) signaling in p53-mutantcells.!°° This leads to sustained phos-
`phorylation of c-JUN (Ser63) that appears to be responsible for
`inducing apoptosis. Rapamycin-inducedstress appears distinct from
`that inducedbyultra violet radiation in that MEKK1is notactivated,
`and from cytotoxic agents such as DNA damaging agents where
`other stress pathways (p38 or ERK1/2) are also activated.!0!:!0? In
`contrast, cells expressing wild type p53,
`(or constitutive p21@P!
`expression)
`there is only transient activation of ASK1,!°
`Suppression of ASK1 is associated with binding of p21@P!
`in
`rapamycin treated cells, and protection from apoptosis. Since the
`rapamycin-induced apoptosis is specifically prevented by insulin-like
`growthfactors (IGF-I/II) and insulin,®© combination of IGF receptor
`inhibitors with rapamycins maybeselectively cytotoxic and induce
`regression of tumors with p53 mutations. Whether such anticipation
`is justified requires vigoroustesting.
`
`MECHANISMS OF RESISTANCE TO RAPAMYCINS
`
`Intrinsic sensitivity to rapamycins betweencell lines may vary by
`several orders of magnitude. For example, rhabdomyosarcomacells
`in vitro are very sensitive (concentration for 50% inhibition (IC50)
`of proliferation ~ 1 nM) comparedto colon carcinomacells (IC50 >
`5000 nM).!! Mechanismsofintrinsic and acquired resistance may
`have either a genetic or epigenetic basis.
`Mutations in FKBP12 and mTOR.Rapamycinsfirst 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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`Synthetic lethality
`a genetic defect alters cellular response to drug action
`MEFs
`
`
`
`o 1a es
`
`2cc
`
`Se=
`
`1cw
`s ue
`Be
`Ey
`
`p53"+
`
`Rapamycin|
`
`Alive
`
`p53"
`
`Rapamycin|
`
`
`Dead
`
`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 mTORthatblock binding
`of FKBP-rapamycin complex to mTOR. Such
`mutations werefirst identified in budding yeast
`S. cerevisiae in which treatment with rapamycin
`irreversibly arrests cells in the G, phase. In the
`yeast S. cerevisiae, 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.!°4
`This observation has been further confirmed by
`RBP1 disruption experiments using the patho-
`genic
`yeast Candida
`albicans. Wild-type
`RBP1/RBP1 parental strain and the rbp1/RBP1
`heterozygous mutantweresensitive to rapamycin
`inhibition, whereas
`rbp1/rbp1 homozygous
`mutantwas rapamycin resistant.!In addition, in
`S. cerevisiae mutation ofa specific residue (Tyr89)
`which is conserved in RBPJ or FKBPs, also
`resulted in decreased binding of rapamycin and
`conferred a recessive resistance phenotype.!° In
`murine mastcells, two distinct point mutations
`in FKBP12 confer
`resistance. By altering a
`hydrophobic residue within the drug-binding
`pocket (Trp59Leu) or changing a charged. sur-
`face residue (Arg49>Gln), the bindingaffinity for
`rapamycin decreases substantially.!6
`A genetic screen identified rapamycin-resist-
`ant alleles with mutations in genes designated
`TORI and TOR2. Strains with mutatedto tor]-1
`(Ser1972>Arg) and tor2-1 (Ser1975>Arg), were
`completely resistant
`to the growth- inhibitory
`effect ofrapamycin. Theseresistantalleles encode mutantTorproteins
`that do not bind the FKBP-rapamycin complex.!03:107-111 This
`result suggests that a conserved serine residue (Ser1972 in Torl;
`Ser1975 in Tor2) in Tor proteinsis critical for FKBP-rapamycin
`binding. In mammalian cells mutations in the FRB domain confer
`a dominantresistant phenotype consistent with decreased affinity
`for binding of the FKBP- rapamycin complex.'!! Expression of a
`mutant mTOR(Ser2035 lle), having greatly reduced bindingaffin-
`ity for the FKBP-rapamycin complex, confers high level resist-
`ance, 14112,113
`Deregulation of eIF4E. mTORphosphorylates and regulates the
`function of 4E-BP1, the suppressor of e[F4E.2° Recently, our group
`has found that acquired resistance to rapamycin wasassociated with
`decreased levels of 4E-BP1.!?! In the absenceofselective pressure
`(rapamycin), resistance was unstable andcells 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-BP1
`levels increased to thosein wild type cells. Levels of 4E-BP1 transcripts
`were unaltered in rapamycinresistant clones suggesting post-tran-
`scriptional regulation. Further studies indicate that the synthesis of
`4E-BP1 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 p27*P! in
`rapamycin resistant BC3H cells.!!> In contrast, no consistent
`changes were detectedin thelevel or activity of S6K1 between parental
`
`10 1 1"
`10"
`a0" 40"
`me jo"
`Annexin V-FITC Fluorescence
`
`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 p537 MEFs, and p537 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
`quadrantis presented (from Huanget al. 20014).
`
`andresistant clones. Rapamycin also inhibited growth factor activa-
`tion of S6K1 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 HCT8colon carcinomacells
`that are highly resistant to rapamycin (IC50 > 10,000 ng/ml). When
`4E-BP1 is overexpressed, these cells becomesensitive (IC50 < 10
`ng/ml) to rapamycin, Figure 6.'!4 These data suggest that low levels
`of 4E-BP1 results in de-regulation of eIF4E, conferring rapamycin
`resistance.
`These results suggest that rapamycin-regulation of the elF4E
`pathwayis crucial in inducing growth arrest. Further de-regulation
`of eIF4E mayfacilitate a malignant phenotype. Of interest is that
`both rapamycin-resistant and -revertant cells exhibited elevated
`c-MYClevels, and increased anchorage-independent growth. That
`deregulation of the e[F4E pathway is associated with increased
`malignancy is supported bycertain clinical observations that dereg-
`ulation of the e[F4E pathway does promote tumor progression.!1°
`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,!!? and peripheral carcinomaofthe lung!?°
`elF4E levels are elevated. However, levels of 4E-BP1 suppressor
`proteins have not been reported in a consistent manner. Potentially,
`the ratio of 4E-BP:eIF4E may determine whether inhibition of
`mTORelicits 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 & Therapy
`
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`Exhibit 1018
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`RAPAMYCINS: MECHANISM OF ACTION AND CELLULAR RESISTANCE
`
`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 ofseveral other sites dependent on phospho-
`rylation ofThr389,rapamycin in vivo influences phosphorylation
`ofothersites, including Thr229 in thecatalytic domain, and $404
`in the regulatory domain.” Mutation ofThr389>Glu abrogates the
`ability of rapamycin to inhibit S6K1 activation.o”!2! Similarly,
`substitution of Thr229 by either a neutral amino acid Alanine
`(Thr229>Ala) or by an acidic amino acid Glu (Thr229>Glu),
`renders S6K1 insensitive to rapamycin.!?? In addition, deletion of
`the 77 N-terminal codons (AN77) confers rapamycin resistance.!?4
`Ofnote truncationofthe first 54 residues of N-terminus blocks the
`serum-induced phosphorylation of three rapamycin-sensitive sites,
`Thr229, Thr389 and Ser404, causing rapamycin insensitivity.°”
`Whetherthis results in resistance to the growth inhibitory effect of
`rapamycinis less clear, and maybecell context specific.
`Mutations of PP2A-Related Phosphatases. The regulation of
`protein phosphataseactivity is thoug