[GANEER RESEARCH 60,3.504~35n, July 1, 1000]
`
`A Direct Linkage between the Phosphoinositide 3-Kinase-AKT Signaling Pathway
`and the Mammalian Target of Rapamycin in Mitogen-stimulated and
`Transformed Cells1
`
`Aleksandar Sekulic,2 Christine. C. Hudson,2 James L. Homme, Peng Yin, Diane M. Otterness, Larry M. Karnitz, and
`Robert T, Abraham3
`Oepartrmnt .of lrr:tnJnology [A.SJ, Ma\{Q Sc;bool of Medicine [J,L.HJ., and Division of Oncology Re!mrch [LM.K.], Mayo Clinic, Rocftoo:er, Mihneoota .55905, and
`D'ej',attrmnt .of Pharrmcology and Cancer Bi.ol~y. Duke.University Medical Genter, Durt:nim, North carolina .27710 [C. C. H., P. Y., D. M. o,, R. T. A.]
`
`ABSTRACT
`
`The miqpbially derived antqlroUterative agent rapamyi;in inhibits ci:11
`growth by interfering with the .signaling functions . of the mammalian
`target uf rapamycin OnTOR); In this study~ we .demonstrate that inter-(cid:173)
`leukin-3 stimuiatiOn induces 11. wortmanninc~ensitive :iru,rease in mTOR
`kina~ 3:ctivity _ in a myliloid prQgenitor cell . line. The. iuvolve,meut. at
`phosph()inositide 3 1-kinase (PI3K) in the regulation of mTOR actjvi~ was
`further suggested by findings that mTOR was. phosphotylated i h vitro and
`in vivo bythe PI3K-regulated protein kinase, AKT/PKB. Although AKT
`phosphorylated mTOR a,t t:wo COOR-terminal sites (Thr2ll46 and Ser2!!~8)
`Jn vitro, Se..24"8 was the lllajor phosphorylation site iu insulin4;Jtimulate4
`or -actitated. AKT -expressing huiliail enihryonic kidn~ cells. Ttlmsient
`transfection assays with mTOR mutants bearing Ala substitution$ at
`Ser24il8 andlor Thr2446indicated that AKT-depend.ent mTOR phosphoryl(cid:173)
`ation W!fs 11ot ~ential t;or either PHAS-I phosphorylation Qr p70!iliK:
`activation in HE.K cells. .How~er,. a ddeti911 ()f aridtt.il acids 2430-2:450 in
`mTOR, which incl.udes the potentiaIAKT phosphQrylation Sites, signifi(cid:173)
`cantly increased both the basal protein kinase arlivity mid in vivo signal-.
`ing functions of mTOR. These results demonstrate that mTDR is a direct
`target uf the ~l3KcAKT signaling pathway in n1itogen.-stirrttllated cells,
`artd that the identified AKT ph95phorylati1m sites i..re neste,d within a
`''repressor domain" that negatively regnlates the. catalytic a.ctivity -of
`mTOR. Furthermore,.'the activation status of the Pl3K0AKT pathway in
`cancer cells may .be an important detenninant otcllllulal'. sensitivity to th'll
`eytostadc etlect of rapamycin,
`
`lNTRODUCTION
`
`Rapamycin isa-potentimrnunosuppressive drug and investig;ttional
`anticancer agent,the major mechanism of action of which in:volvesthe
`inhibition of lymphoid or tumor c~ll proliferation, through interfer(cid:173)
`ence with an event(s) required for Grio-8 phase progression in
`cycling cells. The block to Gl phase .progression imposed by tapa(cid:173)
`mycm occurs prior to the "restriction point,'1 hased on the observa(cid:173)
`tiPns that rapamycin inhibits the. phosphoryktti<1n of the retinoblas(cid:173)
`toma, protein and that. rapa:mycin-treated cellS; are not fully committed
`to enter S-phase of the. ce11 cycle after relea:s:e from drug-induced G1
`arrest( 1-3). !he sensitivity of certain tumor cell lines to the cytostatic
`effects ofrapamycin.has prompted consideral>le interest u1 the nossi(cid:173)
`bili'ty th.at. this drug might be a useful cancer chemotherapeutic agent
`Indeed, a tapam,ycin analogµe (CCI-779; Wyeth-Ayerst) is now in
`
`.R.lheeived 1/10/0Q;c accepted .4/27/00.
`To.ecbsts 0IpJ1b;licalion bf this .article were defrayed in part hy·the payment ofpag!'
`charges. This article must then;fore be hei:eby m;irked adV~i~ in a~orclante with
`L~ U/S,C. Sec.lion 1734 solely to lndica.te thi$ fact
`1 This.work was lillpported by .USPHS. Grants CA 76193 and CA5'299.'i (to R.T. A)
`from the; National Cancer Institute, by a Collaborative Research Prag,am in Cancer
`Research Grant (to R. :r-. A) from Glax<i-Wellcom,e, and by the Mayo Foundation.
`C G. H. is th\' tecipient of postcloctor:al fellowship PF-99-100-01-TBE.from the.Am~(cid:173)
`ican Cancer Society: RT, A. is a Glaxo-Wel1¢ome prqf,:ssor df Molecular Carie.er
`Biulogy.
`2 .These· authors contributed equally to this work.
`3 To wliom.requests for reprints-should be a,lilressed, at Departme~t of'Pbarmacology
`and Cancer Biology, R.oom C3.3'.3B LSRC, Bo½ '.3813, Duke-University Medical Center,
`Durham, r:lc 277TO. Phone: (919)<,1;3-8650; Fa~; (919)684·$461 E-mail:_ abrah008@
`mc.ctuke;edu.
`
`Phase I. cli111cal trials in cancer patients in. the. United States .and
`Europe.
`The molecular pharmacology underlying the cellular effects of
`rapamycin .is now unperstood in cemsider.able detail. The principal
`r.apamycin ''receptor'; is a widely expr:essed intracellular protein
`tertned FKBP4 -12. In lilatnmalran cells, the l:nteracti:on of rapamycin
`with FKBF12· generates a pharmacologically active compleK that
`billds. with.high affinity to themTOR [Ref. 4; also named FRAP (5),
`RAFTl (6), orRAPTl (7) by otfiers]. This.rapamycin targetpr-otein is
`a member of a.recently described family of protein kinases, termed
`PIKKs. The PIKK family members share a COOH-tertninal catalytic
`domain that bears significant• seqµence homology to the .lipid kinase
`domains of PI3Ks (8). Other Ifiembers of the PIKK family mclude
`TORI p and 10R2p, the budding yeast orfho1ogues of nffOR. The
`finding that rapamycin inter1;tcts with. FPRlp; the budding yea:St or(cid:173)
`thologue of :EKBP12, to ar;rest yeast cell growth in Gj phase (9)
`suggests that the TOR signaling pathway has been at least partially
`conserved dining eukaryotic evolution.
`Toe specificity of rapamycin as an inhibitor of mTOR function
`facilitatt::d th,e identification of the downstream signaling events gov(cid:173)
`erned by mTOR.in initogen--stirnulated t:ells. io date, the rapamycin(cid:173)
`sensitive signaling. activities ascribed to' mTOR impinge primarily on
`the translational machinery. Rapamycill treahnent triggers the raprd
`dephosphorylation and inactivation of p7086K in mitogen-stimulated
`cells (10-14). The •overall :effect of p7os5K activation is lo stimulate
`ribosome biogenesis, and, in turn, ta increase the capacity of the
`translational mac:hinery, which allows cells to m:eet J:he increased
`demand for protein ~nthesis i:mposecl by cell cycle ptogressiQn (15-
`
`17). Although p'.708= activation involves a co~plex series of phos(cid:173)
`
`phorylation events catalyzed by multiple protein ki:nases (t8-21), the
`prompt reversal of p70S6Rt ac;tivation by rapamycin (11_, 14) suggests
`that this prQtein kinase requiie$c continuous signaling threugh ntTOR
`to both achieve and maintain the activated state. The exact nature of
`the. input supplied by mTO.R remains unclear; however, re.cent find(cid:173)
`ings suggest thatmTOR phosphorylates and suppresses the activity of
`a type 2A protein phosphatase bourid directly to p7086K (22} Hence.
`rapatnycin treatment may inactivate p7()SGK by removing.a mTOR.(cid:173)
`dependent inhibitory c.onstl'~t on the activity of a p70stk-targeted
`type. 2A protein phpsphatase EP2A.
`A se.con:d downstream protein targeted by m TOR is the trrutsla.,..
`tional repressor, PID\S-1, also termed 4E-BPL PHAS-I represses
`tr;mslation iru.'tiation thro~h association with el:E-4E, the mRNA
`cap-binding subunit of the eIF-4F complex, The binding of PHAS~I to
`e1F-4E blocks assembly of the eIF-4F complex1ttthe 5'•cap stltLcture
`of the mRNA template, thereby decreasing the efficiency of translac
`tion initiation (23). Stimulation of cells with insulin or growth factors
`
`4 Tlie abbreviations used are: FKBP, FK506-binding prot~in; tnTOR, ;ma(nmalian
`targt;t of rapamy.cin; Pf3K, phosphoinositide 3' -kinase; Pna:'., PI3K-related kinases;
`
`p70S6K, ribosomal_ ro 86 kinase; cll,: eukaryotic .irritiation factor; FBS, fetal bovine
`
`serum; IL, interleukin; HEK, humarr embryonic kidne;Y; HAc hemagglutinin; mAb; mono•
`clonal antjpqdy; PMSF, plfenylajethylsulf6nyl fluoride; PDK, p,hbsphoinositide-depen°
`dent kina$~, PKC, protein kinase c.
`35!)4
`
`Downloaded from eancerres,aacrjournals.QrQ 61') July 21., 2017.@ 2000 Arneritan Associatioh for CatJBAll!AilHr.005161
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`NOVARTIS EXHIBIT 2042
`Breckenridge v. Novartis, IPR 2017-01592
`Page 1 of 11
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`

`

`mTOR R:EGULATIO}! BY THE .PI3K,AKT PATHWAY
`
`suggest that deregulated signhli:n:g tfuough mT(cid:144)R may contribute tb
`the transformed phenotype of :f>TEN-deficient cancer cells.
`
`causes the phosphorylatibn of'PHAS-I at five sites, which leads tb the.
`release of eIF-4E, and, in .turn, a:ri increase in eIF-4f:Cdependent
`translation initiation (24-'29):. The phosphorylatton ofPHAS.-1 in(cid:173)
`duced.by hQnnonal stimuli is strongly :irihipited by rap.unycin (27, 30, MAT:ElUALS AND METHODS
`31 ). Barliettesults suggested that mTORis ditectiy responsible for the
`phosphorylatibn of PHAS.-1 in intact cells (3:2, :3~J, although addi-
`Flasmids_, Reagents, and Antibodies, The expression vectors encoding
`tional proline-ditec.ted kinases appear to be required for :full phospho- AUl-fagged wild-type m:TdR (AmTOR~ and. catalytfoidly inactive '("ldnase0
`dead") mTOR (AmT0Rk<l, were described previously (32). The rapam:ycin-
`ryl:ation ofPBAS..I in :insulin--stimulattid cells (14).
`1c
`resistant versions of mTOR contain an additional Ser (2035)--+Ile mutation
`The signaling pathway that couples growth factor receptor occu-
`and.are usually designated with the suffix "SI'' (ag., AmTOR-SI). PCR-based
`pancy to mTOR activation is only partially understood .. However, mutagenesis W!l!l used to cons1:t"uct single and double mTOR point mutants in
`accJJlllulating evidence places mTOR do}Wstt:eam -of both PBI< and which T!n-2446 'and Se..7448 Wtlfe i::h®ged io alanine (A) tesrdues. The internal
`the Pl3.K-regul&ted protein kina,;e, A.KT (also .tenned PKB); i.n: growth
`deletion mutant, AriiTORLi, which !Mks :amiM acids 2430-2450, was pre(cid:173)
`factbr-s:timulated cells. This model .is based in part on ~etic artd
`pared by the PCR~based SOEing techniqrre .(40). The e;g,ression vectors for
`pharmacologica:l eviden.ce thll,t links.activation of P13l{ and/orAKT to HA-tagged wild4ype AKT (cAKT), catalyticalfy inactive AKT (AKT-kd) and
`the two intracellular events koo-wn to be governed by mTOR, the
`ihe co.nsti1;utively ;ietiv,: mytistylated form of AKT (n1yrAKT), Wetekind gills
`activation of p7Q36.K and the phosphorylation of PHAS-I (35). The
`from P. N. T~ich:lis (F<ix:-dhase Cancer Center, Phil<1-.d<iilphia, PA,). The: cDNA
`notion that mTOR participates. in signal:ing downstream from PI3K
`encoding p10s5K' (kindly provided by Dr. Naohiro Terada, National Jllwi~h
`may be particularly relevant to .the antitumor activity of rapamycin. Medical .and Research Center, Denver; CO) was appended with .nucleotides
`Recent studies have identified a·negativ'e regulator of PI3K-mediated
`encoding an NH2.-termina1 FLAG epitop-e :tag and was cloned info :pcDN:A3
`using EcoRI and Xbairestriotion siies. AliPOR pr.oducts wer.e-subcloned.and
`signaling, PTEN, a:s a tumor suppressor gene product(36).The tumor
`then. Mq_uenced.to etil!ure the fidelity of the amplitication step.
`suppress0r function of PTBN i.s attributed io its activity as a phos-
`Reeombinartt.ifiutine JLs3. was. purchased from R&D Systems, Inc. (Mm(cid:173)
`phoinositide 3-phosphatase, which, effectively tenninates PBK-
`neapolis,.MN). Recombinanthuman insulinfRecombulin) and.G41"& GGeneti(cid:173)
`mediated signaling via dephosphorylation of the second messengers,
`ein} were obtained from Life- Technologies, Inc., (Gaithersburg, MD), and
`phosphatidylinosifol-3;4,5-trisphosphate
`.!tiJ.d phospltatidylinositol~
`FµGene. transfection reagent was purchased frnm Bqehringer Mannheim (In.-
`3,4-bisphospha.te. Thus, loss of PTEN function leads to hyperaefiva-
`to.
`dianapolis, IN). Wortmannin (S,gma) wai;; dissolved in DM:SO .(Meil(cid:144))
`tion of the PI3K signaling cascade, which promotes abnormal cell
`y.ield a 1.2 mM stock solution. The wortm:anrtirr stock solution was aliquo'ted
`growth, survival, and ntig:r:ation,
`an.d stored at -70°C. Rapamycin (Sigma) was, prepared as a lQ µ,M stock
`The importanee of PI3:k signaling du.ring tumorigenesis is under-
`solution in ethanol an4 aliquoted and stored as described /iliove,
`The a-ADI !jlid l~CA5 (a-BA) mAbs wt1re puroha~o from Ba\;co (Rich(cid:173)
`s.cored by observ:atiorts that mutations in the PTEN gene occurs mond, G.A), an.cl the .a-mTOR monoiilonal antibody, :Z6E3, was a generous gift
`frequently in .. a variety nf h1llllan cancers, incl1Jding prostate cancer
`from Dr: Peter Houghton (St Jude Children.'s Research Hospital, Memphis,
`and glioblastoma (37). If mTOR also resides downstream ot"Pt3K
`1N). Peptides corresponding to amino.acid residues 2433~2450. in mT(cid:144)R were
`and/or AKT, then tnTOR activity should also be deregulated in
`synthesized (Re11earch Genetic~, .Huntsville, AL) with or without pho~phate at
`PTEN-deficient tumor.cells, and consequently, PTE];(T status might be
`either orboth of the uruJerlined resiaues in the sequence CDTNAKGNKRSR(cid:173)
`lill importantcpr~ctor of cancer ,celi sensiti~ty to the mTOR inhib-
`TR:'.fD~YS. Polycional antib.odiils directed against the nonpbosphol")'bited pep(cid:173)
`itor, rapamycin. Given these speculative arguments, it beeomes in~
`tide were raised 'by immunizing rabbits with the peptide coupled to keyMle
`creasingly important to defmethe :interactions-camongPI3K,AKT~ and
`limpet hemocyanin, The antiserum '(designated 6'.-mTOR 36"7) was affinity(cid:173)
`purifred over a peptid~coupled Sµlfolink bead eolui;rtn according t.o the mane
`mTOR as fhe nipamycin an~ogue, CQ1-779-, moves.into clinical. tria:l.s
`ufiicturer's ptocedur;e (Pierce, Rockford, IL). Ph0$phospeeific @tibodies were.
`ih patients with different types of cancer.
`prepared in a: si"mihtr Jashion, eJ«iept that a keyhole limpet hemocyanin-
`Atthe ineeptiort of this study, the<most direct evidence for .epistatic
`coupled, dually phosphorylated .peptide ( containing phosphate at both Thr:!446
`rela,tionships among PBK, AKT, and mTOR stems from results
`andSer2448 servedastheimmunugen. Theresultingaatiserumwasfirstpassed
`obucined with a poly clonal antibody, termed mJAbl, whieh recog_-
`over ;i colllf11n consisting of nonphosphQrylated peptici-¢ unmo~ilized ob S.ul(cid:173)
`folink beads, amJ the flow'through traction was t)1err passed through a second
`tii:zes a GOOH-tertninal peptide sequence i:n mTOR (residues 2433".:..
`2450; Ref. 38). The authors noted that cellular stimulation with
`column containingtheitnrhobilized, duallyphoaplu:iryfatedpeptide. The bound
`insulin, or ~pression of mutationally activated Al{T, caused a de-
`antibodies {designated 6'.-tnTORp2) were eluted allow pH and were stored in
`crease in the inun1J.I1.o:reactivity of Ii).TQR in anti-iriTAhl: itmnunoblot
`PBS q{)ntafuing 0.05% a:zide.
`Cell Culture an:d. Tran-sfecti1ms. The .. murine IL-3-dliJlendilnt myelotnQno.
`analyses (39} The loss of mTAbl binding activity was reversed by
`treatment of the immunoprecipitated mTQR with.a protein pbospha-
`,;ytic progenitor cell lin.e, FDC~Pl, was cultured in standard growth m:edinm
`tas€ prior to immunoblot analysis. Collectively, J:hese rey;ults sug-
`[RPMI 164() st,lPP1emented with l0%.(v/v) FBS (Hycl.one, Logan, UT), 2 mM
`gested thatinsulin or AKI stimulation caused the phosphorylation of
`t-glutamine, 50 p,M "2-mercaptoethanol, 10 mM HEPES (pH 7.2), and 100/4
`(vlv} WEHI-3 qell-conditionedmedi)lttl as _a source ofIL-3]. Stablytransfocted
`.FDC-l'l ceils expressing AmTORwt were prepared by suspending r x 107
`mTDR at a site(s) that resulted in a decrease in the recognition of this
`pn'ltein by tlte rnTAbl antibody.
`exponentially growing ~Us in"3,50 µ,l of standard growth medium at 4°C. The
`The gqal o( tlte present study was to furlht!T understand the role of
`cells .were mixed with a total of 45 p,g of plasm,tl DNA suspended intlre same
`the PI3K-AKT signaling pathway in the regulation ofniTOR iunctioh medium. Mock transfeetants received.45 p,g ofpcDNA3 only, whereas mTOR
`by extracellular. stimuli. We demonstrate that stimulation of myeloid
`transfoctants were electroporatedwith 25 p,g <>f mtOR-encoding pla\lmid plus
`progenitor cells with IL-3 triggers a I"apid. :inerease _in_ the protein
`.20 µ,g of pel)N;\.3 as filler. Prior to eiectroporation, J;he cell-ONA niixtu¢~
`kinase aetivity of mTOR l:he ]Lc3-dependent increase in mTOR were incubated fot 10 min at room tempemture, The cellswete electroporated
`activity is blocked by low concelftratibrts of the PI3k inhibitor, with a B'IX =del T820 square-wave electropotator (S@ Diego, CA).. at. a
`wortmanrtin. Furthenn.bre, we provide in ~tro and in Vi'j{() evi.dence
`setting of 350. V (lO•ms pulse duration). The electroporated cells were mixed
`ger1Jly lllfd then.allowed t0 stan4 at r:oom temperat~e {or ati.additionai lo min.
`that AKT phosphory lates rrffOR -at a site( s) located .in. a region Jhat Toe cells were then dilumd intci:.20 ml ofstandard _growth medium and cultured
`represses the catalytic activity of the mTOR kinase domain. Deletion
`for 24 h, at whilih time the .cells wete. transfilrre.d into ftesl:i growth medium
`of this repressor domain generates a mTOR mutant bearing a corrsti-
`containing 800 p,g of 0418/niL S.table clones that expressed Am TOR wt and
`tutively eleva:ted level of protein. kinase activity. These findings out-
`AmTdRk<.i were isolated by limiting dilution, and ei-:pression levels of the
`line a direct lin}cage between the PB;K-AKT pathway and mTQR and
`tra:nsf'ected pro~ins were :assessed by lmmunoblotting with the :A.Ul m:Ab.
`'.3505
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`Downloaded from ca11cerres,aacrJourna1s.org 011 July 21, 2017 .. @2000 American AssooJafioh for CanElA~IHr:005162
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`NOVARTIS EXHIBIT 2042
`Breckenridge v. Novartis, IPR 2017-01592
`Page 2 of 11
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`

`

`mTOR R:EGULATIO}! BY THE .PI3K,AKT PATHWAY
`
`tt:ansfected cells w.:re ttans:fert:ed into serum..-free DMEM and cu\tl\fed for
`24 h. Cellular extra.Gts were: p.repared by removal of the .cult\jte medium,
`followedby&ddition.of400 µl.ofbufferP-petdish [50 m.M Tris-HCI, 100 mM
`NaCl, 50 mM (3-glycetophbsphate {pH 7.4),.containing 10%(wN) i;lycerol, I%
`Triton XolOO, 1 mM DTT, SO U:M microcystin, l mM PMSF, and protease
`inhibitor cocktail]. The: d.etached cells were disrupteq by sonicatio)l, and
`.cleared extracts from the HA-tagged A.KT-expressing cells and the. AmtORwt~
`expressing cells were: m1i¢d at a total protein ratio of 1:9. The epitope-taggea:'.
`in.TOR and AKT proteins were eoirtimunoprecipitated with 1 /J,l ofAU1 mAb
`and 5 {Lg of 12CA5.mAb bX)und to prdtein A~Sepharose beads that had been
`precoupled tlil rabbit a;ntimouse immunoglobillin antibodies'- The immunopre(cid:173)
`cipitates wet.e: washed thtee times in buffer N [25: mM HEPES (pH 7.6), 0. 5 M
`.1 mlv! NiijVO"' @d 0.2% Tween 20] and two.iim,$ in
`NaCl, 10% glycer(cid:144)l,
`kinase buffer F [5:0 mM Tris (pH 7.5), 10 mM MgC12 ,. 50 mM Na3 V04 , and 1
`mM DTT}. The coimmunoprecipitaled proteins were incubated for S:0 min a:t
`30<1() in 20 µ,l o.fkinase buffer F supplemented with 10 /J,M ATP and 20 µ,Ci
`of [y--'32PJATP (spe"Cific activity, 4500 C-i!mmol). The reaction products.were
`separated by SOS-PAGE and transfem:d t(cid:144) an Iinmobilon-P membralle. The
`mcorporation of3Zp into wilu,-type or mutafod forms. of Am TOR was detected
`by autoradiography and quantitated by phosphorimager analysis as described
`above.
`to measure the ac;tivit-y of tt:ansientl-y trans~c;te:d FLAG-p1ds61\ serum(cid:173)
`deprived HEK 29.3 Ce!lls Wete prepared as described above. The: cells wete
`stimulated with l µ,M insulin and then lysed in TNEE buffer [50 mM Tris, HCt
`150 mM NaCl, 2.5 mM EDT A, 2 mM EGTA, 25 mM il,glycerophosphate, 25
`m.M NaF (pH 7.$). 9ontll;ining, 0.5% Trito11 X~lQO., IOd µ,M sodiimt o:tthov!1M(cid:173)
`date,, 2 niM DTT~ a,nd pr"Qtease inhibitor cocktail]. The epitope-tag.ged p7086K
`Wit$ irrimunoprecipitated. froin cellular eKtracts with anti-FLAG Mi affinity
`resin (Sigma Chemical Co., St Louis, MOJ, and protein kinase activity
`WM d~termin;ed with a. p7036k 11ssay kit (Upstate Biotechneflogy, .{Uc.-, Lake.
`Placid, NY),
`
`For ·experiments, exponentially growing FDC~Pl cells (2 X 107 cells/
`sample were wa,she<l twice in PBS, the C,ills wqec resuspendjJ<l i1i W ml of
`starvation medium: [RPMI 1640 co.ntaitting rod µ,g/ml BSA, 2 mM L~gtuta:(cid:173)
`mitre, and 50 p,M 2-mercaptoethanol, buffered to pH 7.2 with ro mM HEPES].
`After 4-6 h i1i culture, the fao:tor-depriYed. cells were trealed.for"30 min with
`the indic;tted pharmacological inhibitors and then were resti;lliulated with either
`30 ng/ml IL-3 ot 20% PBS.
`HEK.293 and 293T cells were maintained in mortoliyer<mltures in DMEM
`(Life Techtmlt\lgies,. Inc,) supplemented with 10% PBS or .5:o/o FBS, respec(cid:173)
`tively. PriortQ fransfectiQfi, 6 X 105 c.ells were seeded intQ a 60-rum tissue
`cult\® dish. The cells w.ete. culwred for .24 h under sta:ndatd condifoms and
`then were trartsfected with a. totitl Qf 5 Jig of plasmid DNA mh,ed with & µ,l of
`FuGene transfection reagettt/dish. The standard amounts of plasinid DNAs
`used for each fransfection wet:e: AKT, 0.25 µ,g, mTOR, 3 µ,g; and p7086K, 2
`µ,g. Wh~n necessary, the total .amount of plasmid DNA was broµghl; to $ µ,g
`by !!ddition of the empty pcDNA3· expression vect<5r. The ttansfucted .c.elfs
`were cultured for 16 h, Washed one time. in PBS, and .arrested for 24 h in
`serum-free DMEM. The serum-deprived cells were pretreated for 30 min with
`wortmannin :or rapamycin and thell stimulated with insulin for the indicated
`times .. The pr<lcedures fortransfe~ion !>f HEK 293T cells were sin,.ilro-, except
`that the 0ells were deprived ofsertmr fbt 2 Ii prior to drug treatment.
`DU 145 alld PC03 p-rrisfate cancer cells were maintained in m.on6layer
`cultures in RPMI 164"0 supplemented with 10% FBS, Prior to assay,2 X 105
`Cl)lls were seeded in 60-mm tissue Clllture dishes. After 24 h, the cells were
`transferred into serum-free RPMI 1640 and were Cultured for an :additional
`20 h. The cells Were washed in. PB.S and lysed in LB buffer [25 inM TriS'-HCL,
`pH 7.4, 50 mivl NaCl, 1.0% (wlv) glycerol, 1 % Triton X-100, 50 mM ff-glyc(cid:173)
`erophlY~phate, .20 nM microt1yst.in- L,R, 1:00 µ,g,iml PM.fill,. l)lld protease inhib(cid:173)
`itor p0cktail .(5 µ,g/ml aprotinin, 5 µ,g/rnl pepstatirr, and 10 p,gXn:il leupertin)L
`The lysates were cleatei.l o:f irtsolubw material, and the cleared extracts were
`assayed fortotal protein to equalize . .sampleloadingpriorto SDS-PAGE.
`Inununoprecipitations. M~k-tr~ected or ,:1.mtQRctransfected FDG,Pl
`cells (2 x 107cells/sl!tnp1C) were growth fa,ctordeprh,ed andrestimµla,ted a.s
`described abo-ye, The cells were washed in PBS and lysed by sotiicatinn in I
`Stimulation ofmTOR CatalyticActivityby Serum ot IL-3. Our
`ml of buffer L [50 mM Tiis,HCI, 50 mM {3-glycero:phosphafo, 100 rriM NaCl
`initial objective was to determine Wh.ether mTOR activity w;,i:s regu(cid:173)
`(pH 7.4), containing 10% glycerol, 0.2% Tween 20, 1 mM DTT, 1 mM
`lated in a Pnt.-dependent fashion by Il.,-3, a cytokine. that pto:m.ote$.
`Na3 V04, l 111M Mt{).l;z, 50 nM mierocystin-LR, 1 tnM PMSF, and proteas¢
`inhibifor cocktail]. Tire lysates were cleared ofillsoltible material b.y i:enttif~
`the proliferation and sutviva1 of m.yeloid lineage proge:ttitor cells. Io
`u~tiort,. and the extracts we.re immunoprecipitated with 1 µ,1 of a-AUl mAb
`fi!ci:litate the E(Mlyses ofmTOR kinase activity, we sta:bly expressed
`for 2 hat 4°0. The immunoprecipitates were washed three times .in buffer W
`AUl-tagged wild,-type or catalytically :inai.:tive ("k:inase,,dea.4"). ver(cid:173)
`[50 mM Tris-HCl, 50 mM /3-glycerophosphate, 100 mM NaCl (pH 7.4); con(cid:173)
`sions of' mTOR tAmTORw:t and AmTORkd, respectively) in 11--3·
`taining 10% glycterot 0.2% Tween 20, :lllld 1 mM DTI] and twice in buffer 11
`dependent FDC-Pl cells. Importantly, the stable cell lines Selected for
`{10 mM HEPES; 5:0 ri1M NaCl, 50 lll1,,j /3-glycetophosphate (pH 7.4), 50 tiM
`these studies were .not overexpressing the recombinant protein, as
`microcy~tin-LR, and the protease inhibitor' cocktail].
`:indj.cated by 1I1)11ll,llloblot amiJ.yses of the transfected clones ftr total
`.Immunoblot Analyses. For immunoblot analyses with a-mTOR 167 or
`mtoR protein le:vels with antibodies that recog;ttize 00th the endog.
`a-mTORp2 antibOtfies, rec6nibinap.t AruTOR was immunoprecipitatl!d with
`th;; tag-specific d'..-AtJl mAb from trartsfe1.;ted FD(;-Pl, HEK 293, or HEK
`enous and transfect.ed proteins (results .not shown). The tagged Am(cid:173)
`TOR proteins therefore serve as a "tracer" subpopulation,. the behavior
`Z93T cells. The irtimurtopte.cipitat¢d proteins were sep&rated by SDS-PAGE
`of which in response to physiological stimuli should. reflect that of the
`through 6% polyaciylamide gels. After transfer to hnmobilon~P, the mem(cid:173)
`branes 'Yere l>l_ocked ifild probed with $ p,g per ml idfinity•.pudfied antibodies
`endogenous mTOR
`in 1ris-buofreted saline-0.2.% Tween 20 (1'B$T) cofita,ining 2%(wiv) BSA (for
`In the initial studies, AmTORwt-expressing cells were deprived of
`a-mTORp21!11tibodfes) ot 5% milk (for a,mT(cid:144)R 367 a;ntibodi:es). Immuno(cid:173)
`serum and IL-3 for 6 h and then were restimulated for 10 min with
`reactive proteins were detected with horseradish peromdase-cimjugated to
`IL-3 prior to the preparation of cellular extracts. The extracts were
`protein A and the Renaissance reagent(New England Nuclear, Boston, MA),
`in11nunopredpitated With a•AUl mAb, and mTOR kinase activities
`Tire blqts were thett stripped andreprobed :with the a-AUi mAb in TBS1-milk:
`were det.ennined With PHAS-I as. the subst:mte. Pllrallel samples were
`solµtioo. The phosphorylation state -0f ertd.ogertous niTOR in DU 145 ot PC-3
`prepared fi:om identii.:al1y treated cells that expressed the .AmTORka
`prostate cancer cells. was aHafyzed by immunoblotting with a;-m.TORp2 as
`mutant. Stimulation of AinTORm.expre~iilg" FDC-Pl cells with IL-3
`described above, followed by reblotting of the same membrane with the
`significantly increased the in vitro kinase activity of the.inrmmopre(cid:173)
`a-mTOR mAb, 26E3, in 113'S1-milk solufron.
`~se AsQys, The protein kinase acthdty of immunopt:ecipitated mtQR
`cipitated Am T0Rwi but did not change ·the amol,lllt of A:mTOR wt in
`was assayed wit}irecombinant: PHAS-1 as the substrate: (Strata.gene, La: J011a,
`th~se immunoprecipitates (Pig. IA fei'f panel) .. The activation of
`CA; Ref 32). The samples were separated by SOS-PAGE, and.radiolabeled.
`mTOR by TI.,.:, was maximal at 5-ld min after cytokine stimulanon:
`PHAS-1 was deteeted by antomdiography: Itreorp@ration of 32P intg PHAS-I
`and then dropped to a lower; but still elevated5 plateauJevel ofactivity
`was quantitat<ld with a Molecular Dynamics Stot:m "840 Phoi,phorimager
`that was sustained for at I.east 4 h after cytolcine addition (Fig. 1 Band
`(Sl\nnyvaJe, CA) and fmageQua:nt sofl;ware,
`data rHit shown}. In. contrast; AUl immunopteC"ipitates fr-om: either
`Phosphorylation of mTORby AKT in vitro :was perfotined by transfe¢tion
`mock-transfected or Am:TORkd_expressirtg, celfa contained fow levels
`of AmTOR""' and HA-tagged myrAKT, c-AKT, or catalytically inactive AKT
`of background protein kinase activity that was not substantially in(cid:173)
`into separate populations of HEK 293 cells. The cells were plated in @-mm
`culture dishes ifild were tt:ansfected as described ab-0"\<e. Af\er Hi h, the
`creased by cellular st:inmlation. With IL-3, Interestingly, ser:um-slarved
`3506
`
`RESULTS
`
`Download19d from cance.rre:s.aacrjo.urtralS.org on July 21, .2017. © 2000 American Association tot CanBA~i~005183
`
`NOVARTIS EXHIBIT 2042
`Breckenridge v. Novartis, IPR 2017-01592
`Page 3 of 11
`
`

`

`mTOR R:EGULATIO}! BY THE .PI3K,AKT PATHWAY
`
`A
`
`mTOR
`A.U1 blot
`
`.32P-PHAS-l
`
`IL-3
`CPM
`
`+
`159
`
`li8
`
`wt wt kd
`
`kd:
`
`.. +
`
`+
`5064 14128 615 914
`
`'"·
`1599
`
`FCS
`
`5046
`
`-:5145
`
`noreactivity of AU1-tagged mTOR isolated from lL-3-stimulated
`FDC~Pl cells(Fig. 3, upper paoel). lhterestingly, the time course of
`the alteration in anti-mTOR antibody reactivity corresponded dosely
`to the changes. in mTOR kinase a<.;tivity indm;ed by IL-3 (Fig. 18).
`The IL-'.l~dependent decrease in mTOR immlllloreactivity, was abro(cid:173)
`gated by pretreatment of the cells with 100 nM wortrnannirt, suggest(cid:173)
`ing that thi:. alteration was mediated through the .activation Qf PI3K.
`As will be descrfbl.:d below, parallel inunµn:crblot ~yses with a
`phospho-mTOR-specific antibody (a-m10Rp2) im:licated that the
`decrease in .a-mTOR 361 reactivity induced by IL-3 stirnulatioh is
`-a:ttributable to the phosphorylation of at least-0.ne mnino acid (Ser2448)
`lo~atedW1tliin the a-mJOR 367 e;:pitope (Fig. 3. rtiddle panel~ see
`below f.or description).
`·
`Examination of the peptide sequenC'e recognized by cx-mTO:R 367
`antibod,ies revealed that this. n::gion cqntajned two eonsensµs pha.s"
`phocylation sites (Thr2445 and Set2'448)for AKT(Fig..4).T-o detennine
`whether mTOR was an in vitro substrate for AKT, we expressed
`AmTORwt, HA-tagged wild-type :AIU (cAKT), . activated AKT
`(niyrAKT), or a catalytfoally inactive AKI (AKTkdJ in .different
`populations ofREK 293 cells.. Cellular extracts were then mix.ed, and
`
`,S
`
`Q
`
`111 15 20 25 30 35 @ 4~ 50 55 60
`'nroe,mln
`Fig. 1. Stimulation of tnTOR kinase a¢1;ivity by l.1-'.3 or serum. FilC-J'l cells were
`stably transfected with wild~type AUi-tagged mTOR or acatalytii;ally il)active vel'Sion of
`Am TOR. Tral)sfected clones were "1!ltured for 6 h .ih mediunuvlthout serum snd ll.-3. 1\.,
`left panel,fa"Ctar-deprived FD<>Pl ®llswere stimlllated for 10 min:with lL-3 or medium.
`only (-J The protein kinase a:ctivities ofwild-ly.pe (wt) AmTOR or the "kinMe-dead•
`(kd) AmTO_R wern det;,rmined in innrtun_e compl,;,t k_inase assays with (y-32P]ATP and
`t.;combinantPHAS-i as; the :substrate {lower panels). ]nco¢oratton o.f. 32l>, 'into ;rHAS-I
`was q\lanhtated with. a Mcllecular Dyn'lmics: Storm /!40 phosphorimaging. system and
`reported as cpm. The.;unouuts of irnmunoprecipitated mTOR were assessed by immuno(cid:173)
`blottingwi:fuAUI ,mAb (upper panels} Righi panel, AmTOR"'-expressing FDC-Pl cells
`were restimulatedcfor m min with ll.03 .or serum,. and 11.rtiTORWI kinase a,;tivities were
`measure,d a-s abovs,. B, !)\TORw'-expressing F.DC-Pl JJells Wer'e restimulated for (qe
`inaicatedtiml\s•with II,-3. The prptein.kina:se act.ivitres in AmTOR.wt.inilliunoprecjpitates
`were determined as d.,scnbed in A Incorporation of 32Pi into PHAS-I wa;; riorrtialized to
`the b.asal level-of phosp-hQrylation in the immunoprecipitate .prepared from-uns-tiinula(ed
`cells.
`
`AU1 blot
`
`s
`C :s
`8
`..,
`0 ,..
`"'i" m
`ct
`X
`Q.
`0..
`~
`
`~
`
`8
`
`7
`
`6
`
`5
`
`4
`
`3
`
`2
`
`1
`
`0
`
`+
`
`IL-3
`+
`+
`+
`+
`+
`Wm,nM
`.30
`10
`1000
`300
`100
`Fig. 2. Inhibition of ti.,...3 d·ependent mTOR activation by cellular treatment with
`wottman.nin. AmTQRwt'tr;tnsfected )WC-Bl celiil' Were. deprived cf growth factors all
`de~qiJied in Fig. l. Am TORwt-expr;,ssiIJg cells were treated for :i!p min With !lie inc!ic.ated
`concentrat;iona of wortmannin (Wn),. 11.fter stimulat;ion of the aells: for 10 milt with )L-3,
`1\.mTtiR wt was immunopreci!)i\ated, and immune complex kinase assays wen; performed
`as described in the Fig. I !~end. The amount of Am TOR wt in egch .immunoprecipitate
`was determined by a-AUi immunoblotting ('upper panel).
`
`FDC-Pl .cells also displayed a dear increase in AmTORwt activity
`after a 10-min exposure to fresh serum (Fig: 1.A, right panel} Toll$,
`ligation of receptors for IL-3, .as well a.s µn:defined serum compancents
`(possibly insulin-like growth factors),. initiates a signaling: pathway
`leading to mTOR activation in FDC-Pl cells.
`8-ole. of P(JKinil'...-3-dependent mTOR Attivaiion, Ear.lier stud(cid:173)
`ies implicated. the PBK J)athway in the activaiiori. of mTOR-depend(cid:173)
`ent signaling events in BEK293. cells and 31:3-L 1 preadipocyles (JS,
`39). Stimulation o.fthe IL-3 receptor also triggers a rapid increase in
`PI3K activity ( 41 ), which, suggested. that PI3K might be re-sponsible.
`for the .activation of mTOR in IL-3•stimulated FDC-Pl cells . .If the
`activation of mTOR by IL-3 is dependent on Pl3K, then this response
`should be :inhibited by pretreatment of tire cells with wortrnannin at
`drug concentrations «-;'; I 00 nM (42). As shown in Fig: 2,. the activ&ffon
`of Am'I'OR"'t by IL-3 was virtually a:brogated by pretreatment of tlie
`FbC-Pi cells with 10 nM wortmannin. ·Thus, the sensitivity dflt-3-
`dependentm'TORactiv:ation tu wortrnannin strongly sl,'.lggests that thj:s
`P1 clohe Co
`response is dependent on the acti