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ahlg,Uiest fCmrde il od abig B X,UK. 0XZ, CB2 Cambridge Road, Hills Cambridge, of University Pathology, Teeatoscnrbtdeulyt h project. the to equally contributed authors *These UK. 4TG, SK10 Cheshire K04G UK. 4TG, SK10 UK. 3AT, CB22 Cambridge lieL Cope L. to Claire eIF4E of translation amplification by cap-dependent inhibitors maintain kinase mTOR to Adaptation ARTICLE RESEARCH ß 788 2013 November 24 Accepted 2013; June 28 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This { 3 nutrient factors, growth 1 senses as rapamycin) known of formerly target (mTOR, mammalian rapamycin of target mechanistic The INTRODUCTION eIF4E, mTOR resistance, amplification, Drug Gene proliferation, eIF4E Cell 4EBP1, of WORDS: KEY role pathway. key MEK1/2–ERK1/2 also the around They and based the strategies mTOR proliferation. therapeutic and cell for implications maintaining important eIF4E, selumetinib have in mTOR at ERK1/2 inhibitor of signalling of downstream convergence MEK1/2 mTOR the the exemplify and were an results cells is to These (AZD6244). SW620:8055R inhibitors to the cross-resistant combination, mTOR although sensitivity actually drug with Finally, reduce rational inhibitors system. to attractive MEK1/2 increased cell of sufficient heterologous furthermore, combination a was cells; in eIF4E AZD8055 SW620:8055R of in cells. expression SW620:8055R AZD8055 resistance acquired in reversed to and eIF4E AZD8055 of CCND1 to cap-dependent knockdown RNAi-mediated of and refractory expression eIF4E-sensitive was by the transcripts, encoded result, cap-dependent proteins a increased MCL1, As and eukaryotic translation. eIF4E the of mRNA factor expression in in initiation increase translation a an translation exhibited signalling, cells S6K (IRES)-dependent SW620:8055R of contrast, loss site In to cells. cap-dependent SW620 entry and parental from switch ribosome mTORC1 a internal caused inhibited with and signalling cells AZD8055 mTORC2 mTORC2 inhibitor cancer (SW620:8055R). and kinase colon mTOR ATP-competitive AZD8055 (mTORC)1 SW620 the to complex generated resistance acquired and have mTOR respond we will chronic an inhibition, cells kinase is how to and anticipate protein factors To adapt (mTOR) growth target. and drug rapamycin nutrients anti-cancer to of responses target coordinates mechanistic The ABSTRACT alD Smith D. Paul uhrfrcrepnec ([email protected]) correspondence for Author oeua igotc,Ataeea lelyPr,Mclsil,Cheshire Macclesfield, Park, Alderley AstraZeneca, Diagnostics, Molecular Campus, Research Babraham Institute, Babraham The Programme, Signalling 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,7880doi:10.1242/jcs.137588 788–800 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 4 nooyie,Ataeea lelyPr,Macclesfield, Park, Alderley AstraZeneca, iMed, Oncology 4 1, yveM Guichard M. Sylvie , ,RbcaGilley Rebecca *, 2 ucio-R eerhCnr n eatetof Department and Centre Research Hutchison-MRC 1, ,KtrnBalmanno Kathryn *, 4 n io .Cook J. Simon and 1, ,MthwJ Sale J. Matthew *, 1, { h 7 RSK1 uui fS iae(6)adthe and (S6K) kinase S6 repress of to phosphorylating subunit rapamycin, by (ULK1) (RPS6KB1) synthesis p70 immune-suppressant 1 protein the promote the kinase and the autophagy mTORC1, unc-51-like of mTORC2. phosphorylates target and multi-protein discrete mTORC1 molecular two the complexes, possess of Cells activation mTOR to RHEB. leading GTPase TSC2) and small 1 (TSC1 sclerosis tuberous heterodimer (PKB)- the 2 B inhibit and to kinase converge that protein RAS, signals and driving Sabatini, dependent and by (PI3K) and mTOR 3-kinase catabolic Laplante activate phosphoinositide 2011; receptors cellular factor al., coordinate Growth et 2012). to (Zoncu status processes anabolic energy and levels TR2 n lctptn n rlne niiino mTOR AZD2014 the of OSI027, CC223, inhibition TORKi prompted prolonged INK128, include and have they and potent targets; mTORC1 elicit inhibit others site, and inhibitors catalytic and mTORC2, mTOR kinase the presence target mTOR results cell directly the ATP-competitive that These new in of of persist drug. driver development to al., the 2010), a et al., (Choo of translation, et cause 4EBPs (Dowling rapalogues cap-dependent the al., proliferation addition, of allowing et In de-phosphorylation S6K-dependent 2008) (Shi 2008). partial a al., and signalling only selective et PI3K–PKB intact: mTORC1- Tamburini because increases signalling the 2005; be loops survival of might feedback PKB suppression This leaves indeed, activity 2011). inhibition anti-cancer al., broad specific mTORC1 exhibited in et not success have (Benjamin some but shown types have the Treatments tumour ‘rapalogues’ complex, 2008). in mTORC1 related the Manning, of and progression inhibitor and allosteric an and (Menon rapamycin, using cancers development of tumour essential majority an is of signalling PTEN, mTOR and component PI3K, mTOR 2012), and al., RAS, 2012) et Sabatini, (Gerlinger in and itself (Laplante mutations LKB1 TSC2, to and TSC1 owing 2011). al., syndromes et to growth Yu phosphorylation Grb10 2011; phosphorylate al., direct et can (Hsu by and signalling RTK 2006) IRS1 inhibit Kandror, inhibits and al., et also (Tzatsos Um 2004; mTOR al., PI3K–PKB et decreasing 2004); (Harrington activity and mTOR degradation and its signalling promoting feedback substrate (IRS1), receptor negative insulin 1 Sabatini, by phosphorylates S6K and regulated to example, tightly Laplante for loops; is Ser473 2005; activity al., on mTOR et 2012). kinases PKB (Sarbassov protein other SGK phosphorylates activates including and activity mTORC2 enzymatic its 1998). Gingras increase 1997; al., al., et Brunn 4EBPs 1994; cap-dependent et al., initiates et that of (Pause complex (4EBPs) translation eIF4F which mRNA the eIF4E, Phosphorylation of factor proteins part forms initiation 2012). then translation 4E-binding eukaryotic Sabatini, the releases factor and (Laplante initiation eukaryotic TRi ye-ciae nmn acr rfmla over- familial or cancers many in hyper-activated is mTOR 1 ae .Howarth D. Karen , 2 akHampson Mark , 3 ,

Journal of Cell Science ehnsso nrni rint eitnet TRkinase mTOR to resistance innate and/or or addiction’ intrinsic ‘mTOR they PKB) little of Rather exhibit mechanisms S1A). cells (P-Ser473 resistant CO115 Fig. and that material (COLO205) mTORC2 reflect suggest sensitive (supplementary both cells not and in inhibited (CO115) nM AZD8055 did 100 P-S6) at that signalling sensitivity given (P-S6K, inhibition in mTORC1 target differences in differences These 1A). (Fig. yl ret ihn vdneo edcls(u-1DNA (sub-G1 cells dead of cell evidence G1 a no and 1B) with (Fig. 2009; expression arrest, al., (CCND1) caused et D1 cycle cells that cyclin Feldman SW620 of of 2009; phosphorylation loss treatment reports al., a AZD8055 4EBP1 2010). et previous al., P-S6 Thoreen of et 2008; Chresta with inhibition al., and et little consistent (Choo P-S6K cause S1B), rapalogues Fig. inhibit failed an (supplementary rapamycin Thr37/Thr46 being material at did Notably, phosphorylation de- it 4EBP1 S1B). inhibit with to Fig. but the (consistent material PKB (supplementary inhibitor) detects P-Ser473 rapamycin inhibit mTORC1 comparison, antibody to In avidly. failed non-phospho-4EBP1 more because 4EBP1 possibility the phosphorylated the out 4EBP1; rule cannot we that AZD8055 although of that 4EBP1, suggest hours results stabilize 48 these may 2008) de-phosphorylation proteasome-dependent al., least et from (Elia degradation rapid 4EBP1 at commensurate protects increased, for also dephosphorylation the 4EBP1 persisted of with levels effect Total 1B). this and (Fig. 4EBP1 P-Thr37/Thr46 PKB; P-S6K, of P-Ser473 loss causing hour, signalling 1 mTORC2 within and mTORC1 inhibited AZD8055 treatment, lines. cell some in operate inhibition e eerltvl eitn,sc sC15(IC CO115 as such resistant, relatively were few n Z85 Toene l,20;Flmne l,2009; al., (IC et potently Feldman AZD8055 2009; 2011). al., al., et et (Thoreen Benjamin AZD8055 and ARTICLE RESEARCH eysniie uha W2 iha IC were an Most with assays. SW620 their as proliferation for such cell lines sensitive, in cell very AZD8055 cancer colorectal to human sensitivity seven cells screened cancer We colorectal AZD8055-sensitive of Identification RESULTS eut rvd motn e nihsit o uorcells tumour inhibition. how mTOR into chronic to insights adapt These new and selumetinib. respond important inhibitor are provide MEK1/2 eIF4E increased the results and through to AZD8055 mTOR cross-resistant to also that resistant the eukaryotic find are we that the Notably, and cells eIF4E, pathway, on eIF4E. converge signalling pathways factor MEK1/2–ERK1/2 mTORC1 initiation the just translation of of (IRES)- upregulation arm site selective chronic one through to entry arises resistance exposure ribosome acquired AZD8055 contrast, internal In translation. switching to dependent by show AZD8055 cap-dependent We with treatment cells. from acute to cancer to the colorectal respond resistance studied cells human all have that in acquire in we AZD8055 and this, of and, Anticipating effects adapt 2013) inhibitors. will al., kinase mTOR cells et BRAF tumour and Little BCR-ABL likelihood, 2012; as (Rosenzweig, such agents, inhibitors targeted successful MEK1/2 most (Holt the studies with well pre-clinical 2012). combines in al., (Chresta it et (AZD6244) death addition, proliferation, and selumetinib cell In cases, cell inhibitor S6K) 2010). some of al., in and and, et inhibition de- autophagy (4EBP1 of substrates, sustained induction mTORC1 strong (PKB) both in mTORC2 of resulting mTOR, phosphorylation inhibits selectively hnS60clswr ujce oatm oreo drug of course time a to subjected were cells SW620 When curddu eitnehslmtdteipc fee the even of impact the limited has resistance drug Acquired 50 f3–0n,bta but nM, 30–50 of 50 , 50 M and nM) 1 . 10 m M) eecmltl oti h W2:05 el.Ti a o due not was This signals cells. 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Technologies/Stratagene performed number (Agilent 4316831) were catalogue system Products, assays PCR number Real-Time All Mx3000P (catalogue Biosystems. the Applied RNaseP from and purchased (Hs01094626_cn) (catalogue TERT 4403315), 4EBP1 genes control the number (Hs01689624_cn), and 4400291) eIF4E number catalogue (both for assays qPCR Q-PCR Genomic rn,G . usn .C,Sekulic C., N. C. Hudson, M. J., G. Hall, Brunn, and C. Moroni, M., Colombi, D., Benjamin, G. C. Proud, and D. B. Fonseca, A. J., P. Averous, Edwards, and E. A. Teschendorff, M., E., Bhat, A. N., Alsop, Siddiqui, A., Yanagiya, D., B. Fonseca, M., Morita, T., Alain, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.137588/-/DC1 online available material Supplementary release. material immediate for Supplementary PMC in receives Deposited which BBSRC. S.J.C.), to and the R.G. C1023/A14545 from K.B., number funding (to [grant Institute strategic UK Babraham M.J.S. The Research (to by Cancer studentship and from BBSRC K.D.H.]; a grant S.J.C.); a and S.J.C.); C.L.C. and awarded (to and Institute Research AstraZeneca Babraham Science to the assigned Biological to studentship and PhD Biotechnology CASE a (BBSRC) by Council supported was work This Funding the authors. wrote other S.J.C. all authors and from All R.G. input data. K.B., with the C.L.C., manuscript analysed interpretation. M.S., and data K.B., experiments to R.G., the contributed C.L.C., performed M.S. experiments. M.H. K.B., the and R.G., designed K.D.H. C.L.C., and project. conceived the S.J.C. conceived and S.G.M. and P.D.S. S.J.C., C.L.C., contributions Author AstraZeneca. of employees paid are S.M.G. and P.D.S. M.H., interests Competing pRL-IRES-FL; 4EBP1 for of Bitterman provision Peter for HA-eIF4E; Proud for Chris Sonenberg Nahum thank We Acknowledgements AAATTCCTGATGG-3 GGCTTCAA-3 iel owr,5 forward, Firefly ees,5 reverse, AGGCCATGATAA-3 TTGCAGCATATCTTGAACCAT-3 9 -CCCGCTTATCTTCTGGGCTA-3 asstetrh e eeaino TRinhibitors. mTOR of generation new 10 a torch: the passes 4E-binding factor initiation 1. eukaryotic protein requires signaling mTORC1 by expression carcinoma pancreatic and colorectal, breast, lines. in cell 18 chromosome on breakpoints therapies. targeted mTOR al. of et 6476. efficacy A. the L. Voyer, predicts P., ratio Metrakos, V., Marcus, D., Zammit, .J,Lwec,J . radArhm .T. R. Abraham, and Jr C., J. Lawrence, J., P. 868-880. , 9 -TCTGGCATGCGAGAATCTGA-3 9 ora fCl cec 21)17 8–0 doi:10.1242/jcs.137588 788–800 127, (2014) Science Cell of Journal -ATTAGATTCCGTTTTCTCCTCTTCTG-3 acrGnt Cytogenet. Genet. Cancer Oncogene 9 WA ees,5 reverse, YWHAZ ; 9 -CCTCTGGATCTACTGGGTTACCTAAG-3 27 9 . 9 1106-1113. , WA owr,5 forward, YWHAZ ; 9 -ATGGCGACTGTCGAACCG-3 AA ´ . ilas .M,Hsi . Houghton, H., Hosoi, M., J. Williams, A., , n sfldsusosadSuet-Feung and discussions useful and 9 9 164 -CCGCCAGGACAAACCAGTAT-3 9 ; ;4EBP1reverse,5 Renilla 97-109. , 20) euaino ylnD1 cyclin of Regulation (2008). 9 ees,5 reverse, 9 19) hshrlto fthe of Phosphorylation (1997). ; -ACTTTTGGTACATTGT- Renilla acrRes. Cancer a.Rv rgDiscov. Drug Rev. Nat. 20) itiuinof Distribution (2006). 9 21) eIF4E/4E-BP (2012). owr,5 forward, 9 9 EP forward, 4EBP1 ; -CTATGACCGG- -GGATTTCACG- 21) Rapamycin (2011). 9 9 72 9 ;Firefly -GAAT- eIF4E ; 6468- , 9 ;

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