A role for eukaryotic initiation factor 4B overexpression in the pathogenesis of diffuse large B-cell lymphoma

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1 Leukema (), & Macmllan Publshers Lmted All rghts reserved -/ OPEN ORIGINAL ARTICLE A role for eukaryotc ntaton factor B overexpresson n the pathogeness of dffuse large B-cell lymphoma E Horvlleur, T Sbarrato, K Hll,, RV Sprggs, M Screen, PJ Goodrem, K Sawcka,, LC Chapln, C Tourol, G Packham, KN Potter, S Drnhofer, A Tzankov, MJS Dyer, M Bushell, M MacFarlane and AE Wlls Dysregulated expresson of factors that control proten synthess s assocated wth poor prognoss of many cancers, but the underlyng mechansms are not well defned. Analyss of the dffuse large B-cell lymphoma (DLBCL) translatome revealed selectve upregulaton of mrnas encodng ant-apoptotc and DNA repar protens. We show that enhanced synthess of these protens n DLBCL s medated by the relef of represson that s normally mposed by structure n the -untranslated regons of ther correspondng mrnas. Ths process s drven by sgnalng through mammalan target of rapamycn, resultng n ncreased synthess of eukaryotc ntaton factor (eif) B complex (eifb), a known actvator of the RNA helcase eifa. Reducng eifb expresson alone s suffcent to decrease synthess of protens assocated wth enhanced tumor cell survval, namely, BCL and ERCC. Importantly, eifb-drven expresson of these key survval protens s drectly correlated wth patent outcome, and eifb, and ERCC are dentfed as novel prognostc markers for poor survval n DLBCL. Our work provdes new nsghts nto the mechansms by whch the cancer-promotng translatonal machnery drves lymphomageness. Leukema (), ; do:./leu.. Keywords: lymphoma; eifb; mcroarray analyss; apoptoss; DNA repar; mtor INTRODUCTION It s well establshed that altered regulaton at the level of mrna translaton s mportant n both cancer development and progresson. Rates of proten synthess are controlled both globally and specfcally, and dysregulaton va ether route makes a sgnfcant contrbuton to cell transformaton and tumorgeness. In general, global proten synthess rates are regulated by the avalablty of two complexes, the eukaryotc ntaton factor F complex (eiff) and ternary complex. The eiff complex comprses eife (the cap bndng proten), eifg (a scaffold proten) and eifa (a dead box helcase). Two assocated protens, eifb and the functonally related eifh, anchor the complex to the mrna and ncrease eifa helcase actvty., Dysregulaton of the eiff complex members has a drect role n tumorgeness,, n part due to ncreased synthess of protens that are normally poorly translated, partcularly those encodng growth factors and oncogenes., In dffuse large B-cell lymphoma (DLBCL), the most common lymphoma subtype, transcrptonal proflng has provded mportant nsghts nto lymphoma cell bology and new prognostc nformaton. Ths type of analyss dentfed three dstnct forms: germnal center B-cell-lke DLBCL, actvated B-cell-lke DLBCL and type III DLBCL,,, wth a correlaton between expresson pattern and patent survval., Recent advances n treatment have resulted n a sgnfcant mprovement n outcome for patents wth -year survval rates of B%. However, a sgnfcant proporton of patents stll succumb to the dsease, and new therapes and prognostc markers to nform treatment optons are requred. As transcrptonal changes are not necessarly drectly lnked to proten expresson,, we have analyzed the translatome of DLBCL cells. We show that translatonal dysregulaton n DLBCL results n altered expresson of protens that functon n apoptotc processes and n DNA repar. Ths results from actvaton of mammalan target of rapamycn (mtor) pathway and ncreased eifb expresson, whch overcomes the repressve effects on the translaton of specfc mrnas that s exerted by ther hghly structured -untranslated regons (UTRs). Importantly, we demonstrate that translatonal dysregulaton of these mrnas s assocated wth poor survval of patents wth DLBCL, dentfyng ERCC, and eifb as new prognostc markers for ths dsease. MATERIALS AND METHODS Antbodes Provenance and dlutons of antbodes are descrbed n Supplementary Table. Cell culture All cells were grown n RPMI wth % fetal calf serum, mm L-glutamne. Cell lnes used were GM (control B-cell lne), GM (control B-cell lne) and fve cell lnes derved from GC-DLBCL, that s, DoHH- (secondary DLBCL derved from follcular lymphoma (FL)), (secondary DLBCL derved from FL), SuDHL-, and OCI-LY. Cell suspensons from lymph nodes were from the Faculty of Medcne Tssue Bank, Unversty of Southampton. Approval to use patent tssue was obtaned from the Lecestershre, Northamptonshre and Rutland Research Ethcs Commttee (LREC reference ). Medcal Research Councl Toxcology Unt, Lecester, UK; INSERM UMR CRCT Equpe CHU Ranguel Toulouse, France; Faculty of Medcne, Unversty of Southampton, Southampton General Hosptal, Southampton, UK and Insttute of Pathology, Unversty Hosptal Basel, Unversty of Basel, Basel, Swtzerland. Correspondence: Professor M MacFarlane or Professor AE Wlls, Medcal Research Councl Toxcology Unt, Hodgkn Buldng, Lancaster Road, PO Box, Lecester LE HN, UK. E-mal: mm@le.ac.uk or aew@le.ac.uk Current address: The Babraham Insttute, Cambrdge, UK. Current address: Domnck P. Purpura Department of Neuroscence, Albert Ensten College of Medcne, Bronx, NY, USA. Receved September ; revsed October ; accepted October ; accepted artcle prevew onlne October ; advance onlne publcaton, November

2 Treatments Inhbtors for mtor were purchased from Selleck Chemcals (Houston, TX, USA) for AZD and Sgma Aldrch (St Lous, MO, USA) for PP. Unless stated otherwse, treatments were performed wth nm AZD or mm PP. SDS-polyacrylamde gel electrophoress and western blottng Cell lne extracts were prepared as descrbed prevously. B cells from lymphomas or tonsls were purfed usng Mlteny Botec (Cologne, Germany) CD beads and MS columns followng the suppler s protocol. Proten extracts were subjected to electrophoress on SDS-polyacrylamde gels. Protens were detected wth the relevant antsera usng chemlumnescent reagents. Pulse-labeled mmunoprecptaton For mmunoprecptatons, transfected cells were grown at cells/ml for h. They were ncubated for h n methonne-free medum n the presence of. mc of S Met-Label (Hartmann Analytcs, GmbH, Braunschweg, Germany). Cells were then lysed, and mmunoprecptaton was done usng proten A/G agarose beads (Santa Cruz Botechnology, Dallas, TX, USA). Immunoprecptated protens were boled n SDS contanng buffer and were subjected to electrophoress before Coomasse stanng and autoradography were done. Sucrose densty-gradent centrfugaton and RNA detecton Sucrose densty-gradent centrfugaton was used to separate rbosomes nto polysomal and subpolysomal forms. Gradents were then fractonated wth contnuous montorng at nm and RNA was solated from each fracton as descrbed prevously. RNA analyss Northern analyss of RNA solated from sucrose densty gradents was performed as descrbed prevously on at least three ndependent occasons. Reverse transcrptase-pcr and quanttatve reverse transcrptase-pcr were performed as descrbed prevously. Preparaton of fluorescently labeled cdna for mcroarray hybrdzaton and data analyss Human cdna mcroarrays were used as descrbed prevously. Fluorescently labeled DNA probes were generated from equal proportons of RNAs (B mg) of pooled non-polysomal fractons (fractons, Cy) and pooled polysomal fractons (fractons, Cy). Mcroarray sldes were scanned usng a GenePx A mcroarray scanner and GenePx Pro. software (Axon Instruments, Unon Cty, CA, USA). Analyss of mcroarray data GenePx Pro. was used to quantfy fluorescence ntenstes for ndvdual spots on the mcroarray. The data were processed n the R platform usng the Lmma package., The resultng log-transformed ratos were as follows: M (gene x) ¼ Log[Cy(polysomal-assocated mrna)/cy(non-polysomal mrna)]; ( replcates of experment, ¼ :) The log-transformed ratos of polysomal over subpolysomal sgnal for each condton were then analysed by a non-parametrc rankng approach usng the RankProd package., mrnas were clustered usng Gene Ontology annotatons through DAVID (davd.abcc.ncfcrf.gov) and IPA (Ingenuty Systems, pe charts were created n Mcrosoft Excel and heatmaps were created usng MultExperment Vewer MeV ( Death receptor-nduced apoptoss Cells were grown to cells/ml and treated wth dfferent concentratons of Hs-tagged TRAIL (tumor necross factor-related apoptoss-nducng lgand) or ant-fas antbody. Cells were collected at h post treatment and staned for Annexn V and propdum odde, and then analysed by flow cytometry. Dysregulaton of translaton n DLBCL E Horvlleur et al Tssue mcroarrays The two dfferent tssue mcroarrays have been descrbed prevously. The mmunostanng was performed wth hstostanr plus bulk kt (Invtrogen, Carlsbad, CA, USA) followng the manufacturer s nstructons. eifb stanng showed a presence/absence pattern that was determned for each tumor. As expresson changes were more gradual for the other protens, tssue arrays were scored usng the mmunoreactve score method. Scorng was carred out on two ndependent occasons. The populaton was then dvded nto two groups usng medan stanng ntensty n the DLBCL populaton as a threshold. The survval of these populatons was plotted usng the R package a package for survval analyss and sgnfcance of the changes was assessed by Cox regresson. Plasmds and srna -UTRs of ERCC, BCL and were amplfed by PCR (pfu DNA polymerase) and subcloned between the Spe and Nco restrcton stes of a pgl vector. The psv-rl vector expressng Renlla lucferase was used as an nternal control. The Renlla vectors wth dfferent -UTR szes are descrbed n Supplementary Table. In ths case, pgl was used as an nternal control. eifb small nterferng RNA (srna) and non-targetng control were provded as a SMART-pool of target plus srna (Dharmacon, Thermo Fsher Scentfc, Waltham, MA, USA). Transfecton Transfectons were performed usng a Nucleofector b TM (Lonza, Basel Swtzerland), accordng to the manufacturer s nstructons. All cell lnes were transfected n the soluton L usng the followng programs: GM C-, DoHH-A- and OCI-LY M-. For each transfecton of cells, pmol of srna were used. For lucferase assays, mg of reporter pgl vector and. mg of psv-rl were added. UTR analyss The sequences of the -UTRs were analysed for upstream open readng frames, potental nternal rbosome entry segment and termnal olgopyrmdne tract motfs (n-house Perl scrpts). McroRNA target stes were predcted usng TargetScan ( usng conserved targets of conserved mcrorna famles. Structures were predcted usng RNAfold. RESULTS cdna mcroarray analyss shows that a subset of mrnas are subject to dfferental translatonal regulaton n DLBCL To dentfy selectve changes n the translatome n DLBCL, polysome proflng was carred out. Cytoplasmc extracts prepared from fve DLBCL cell lnes (, DoHH-, SuDHL-, OCI-LY and ) and two control B-cell lnes (GM, GM) derved from dsease-free ndvduals were separated on % sucrose gradents (Supplementary Fgure ) and the RNA solated. RNA derved from fractons (polysomes) was compared wth RNA from fractons (subpolysomes) on mcroarrays to dentfy canddate mrnas whose translatonal behavor dffers sgnfcantly from the translaton pattern observed n the control cells. These transcrpts were then classfed nto dfferent categores based on ther bologcal functon (Fgure, Supplementary Fgure ). In DLBCL cell lnes, mrnas encodng ant-apoptotc protens, transcrpton factors, protens requred for cell mgraton, DNA repar enzymes and subsets of RNA-bndng protens, ncludng eifb (Fgure, Supplementary Fgure ), were translatonally upregulated. In contrast, mrnas encodng pro-apoptotc protens, cell sgnalng and a dfferent subset of RNA-bndng protens were translatonally downregulated (Fgure a). Changes n gene expresson and the synthess of specfc protens n DLBCL valdate cdna mcroarray analyss To assess the changes n expresson of protens that are assocated wth tumorgeness, mrnas whose proten products are nvolved n apoptoss and DNA repar were studed. Cell lysates were & Macmllan Publshers Lmted Leukema ()

3 Dysregulaton of translaton n DLBCL E Horvlleur et al Mgraton/cytoskeleton Apoptoss Immune response DNA repar Transcrpton factors RNA bndng G-coupled sgnalng Other sgnalng pathways Other Sgnalng Transcrpton factors Other Apoptoss Mgraton/cytoskeleton Cell prolferaton Immune response Oxdatve stress response Proten degradaton Ion transport RNA bndng/rbosomal assocated DoHH- SuDHL- OCI-LY Av control Av DLBCL Apoptoss BAK BCL famly member Pro-apoptotc BCL CASP CD IGFR BCL famly member Caspase Multenzyme Transcrptonal repressor IGF receptor Ant-apoptotc Pro-apoptotc sgnal transducton TNF sgnalng pathway IGF sgnalng PEA DED-doman proten FAS/TNF nhbtor RELE NF kappa B sgnalng TNF sgnalng pathway SIAH Ubqutn lgase multple pathways SMARCD SWI/SNF complex member transcrpton regulaton TNF Tumor necross factor TNF sgnalng pathway TNFAIP TNF sgnalng TNF sgnalng pathway TNFRSFC TNF famly member TNF sgnalng pathway TNFSF TNF lgand TNF sgnalng pathway TRAF TNF sgnalng TNF sgnalng pathway TRAIP Ubqutn lgase NFkB nhbtor BIRC BRE CASP CASP FAS FASTK LTBR MAPKIP NGFR TNFRSFD TRADD TRAF Caspase nteractor TNF pathway Caspase Caspase TNF famly member Knase TNF famly member MAP knase nhbtor Nerve growth factor receptor TNF famly member TNF sgnalng TNF sgnalng Ant-apoptotc TNF sgnalng pathway Pro-apoptotc Pro-apoptotc FAS sgnalng pathway FAS sgnalng pathway Death receptor Apoptotc sgnalng nhbtor NGF sgnalng TNF sgnalng pathway TNF sgnalng pathway TNF sgnalng pathway DNA repar BRCA CCNH ERCC FANCG GTFH MAPK MNAT MSH XPA Double strand break repar Cycln dependant knase Damaged DNA excson Member of BRAFT complex Transcrpton factor Map knase knase CDK-actvatng knase Msmatch recognton DNA damage recognton Homologuous recombnaton DNA damage sgnalng Nucleotde excson repar Homologuous recombnaton Nucleotde Excson repar Cell cycle arrest DNA damage sgnalng DNA msmatch repar Nucleotde excson repar NUDT PRG RECQL XRCC Phosphatase Growth regulaton Helcase Helcase Msmatch repar p response DNA repar Non-homologous end jonng Subpolysome Fgure. mrnas nvolved n apoptoss and DNA damage response that dsplayed sgnfcantly dfferent rankng n DLBCL lnes compared wth control cells. (a) Pe charts showng the functonal dstrbuton of protens whose mrna s () more or () less polysome-assocated n DLBCL cells compared wth control. (b and c) A color scale was used to represent the rato of mrna n subpolysome to polysome fractons. Transformed from the log[polysome:subpolysome], where blue ndcates subpolysome-assocated and yellow sgnfes that the mrna s predomnantly polysome-assocated. (b()) mrnas assocated wth apoptotc processes that are translatonally upregulated. Av control represents the average value for the GM and GM cell lnes, av DLBCL the average value for, DoHH-, Sud-HL, OCI-LY and cell lnes. (b()) mrnas assocated wth apoptotc processes that are translatonally downregulated. (c() mrnas assocated wth DNA repar processes that are translatonally upregulated. (c()) mrnas assocated wth DNA repar processes that are translatonally downregulated. polysome separated by SDS-polyacrylamde gel electrophoress and mmunoblotted for the protens shown (Fgures a and b, Supplementary Table ). Dfferences n expresson were observed n all cases, n agreement wth the proflng data (Fgure and Supplementary Fgure ). For protens that functon n apoptoss, the data show that TRADD (tumor necross factor receptor type -assocated death doman proten) and FAS were downregulated, whereas, BCL, TRAIP and c-myc were upregulated, albet to dfferent extents (Fgure a()). There were no correspondng changes n the total amount of mrna present (Fgure a(), Supplementary Fgure ). For example, the level of TRADD mrna was smlar n the control cell lnes and the DLBCL cell lnes, and although FAS mrna levels were more varable, nether of these correlated wth the expresson of the proten. The cell lnes used n the study carry the t(:) translocaton that couples the mmunoglobuln heavy chan locus to the BCL gene resultng n transcrptonal upregulaton of BCL mrna. As expected, the levels of BCL mrna were hgher n four out of fve of the Leukema () & Macmllan Publshers Lmted

4 Dysregulaton of translaton n DLBCL E Horvlleur et al GM GM Proten DoHH- SuDHL OCI-LY TRADD GM GM RNA DoHH- SuDHL OCI-LY TRADD Western blot analyss was carred out on patent tumor samples to examne the levels of BCL, and ERCC (Fgure c). The data show that smlar changes also occur n patent samples wth ncreased expresson of these protens detected when compared wth B cells derved from control tonsls. Proten GM GM DoHH- SuDHL OCI-LY Tonsl B-cells FAS BCL TRAIP c-myc Tubuln ERCC BRCA MNAT Tubuln RNA GM GM DoHH- SuDHL OCI-LY DLBCL-derved cell lnes. However, ths change was nsuffcent to account for the large ncrease n observed proten levels n some of the lymphoma cell lnes. The dfferental expresson of these apoptotc protens was shown to affect cell senstvty to FAS-medated apoptoss (Supplementary Fgure ). The translatonal proflng data also suggested that there would be dfferences n the synthess of protens that functon n DNA repar. Therefore, western analyss was performed to examne the expresson of ERCC, BRCA and MNAT (Fgure b(), Supplementary Table ). The levels of these protens were ncreased wth no correspondng changes n the levels of the mrna, agan strongly suggestng translatonal dysregulaton (Fgure b(), Supplementary Fgure ). Importantly, dysregulated expresson of these protens has not been reported prevously n DLBCL. DLBCL patents BCL- ERCC Actn FAS BCL TRAIP c-myc Tubuln ERCC BRCA MNAT Tubuln Fgure. mrnas dentfed by the translatonal proflng show dfferental proten expresson. (a() and b()) Proten extracts were generated from DLBCL cell lnes or from control B cells (GM and GM). These were subjected to SDS-polyacrylamde gel electrophoress (PAGE), mmunoblotted and probed wth the antbodes ndcated to assess dfferences n expresson n the protens from genes wth a role n apoptoss (a) or DNA repar processes (b). (a() and b()) To measure total RNA levels, samples were taken and RNA was extracted. These samples were subjected to northern analyss usng probes aganst, BCL, FAS and MYC, or reverse transcrptase-pcr usng prmers aganst TRAIP, ERCC, CCNH, BRCA, MNAT, actn and tubuln. Actn/tubuln were used as loadng controls. The data show that there s a large ncrease n the expresson of a number of protens that functon n apoptotc and DNA repar pathways wth no net change n mrna levels, n agreement wth the array data. (c) B cells were purfed from tumors obtaned from ndvduals wth DLBCL or from tonsls. Cell extracts were generated and samples separated by SDS-PAGE and mmunoblotted wth the antbodes shown. The data show that there s ncreased expresson of, ERCC and BCL n patent samples n agreement wth the cell lne and the translatonal proflng data. mrnas wth hghly structured -UTRs are translatonally upregulated n DLBCL The enhanced translaton of subsets of mrnas n DLBCL could be due to unque RNA elements, as the majorty of mammalan mrnas contan regulatory RNA motfs, ncludng upstream open readng frames, nternal rbosome entry segments, termnal olgopyrmdne tracts and mcrorna-bndng stes. Therefore, the UTRs of the mrnas n the up and down lsts were examned to assess whether there were unque features that could contrbute to ther up- or downregulaton. The proporton of mrnas contanng nternal rbosome entry segments, upstream open readng frames, termnal olgopyrmdne tracts or mcrorna target stes remaned unchanged between the gene lsts and the whole array (Supplementary Fgure A). However, the data show a sgnfcant ncrease n the average -UTR length and n the predcted mnmum free energy (DG) of foldng n the lst of genes that were translatonally upregulated n DLBCL, compared wth the complete array (Fgures a( and )). Further examnaton of the -UTR length (Supplementary Fgure B) reveals that the cumulatve dstrbuton correspondng to the translatonally upregulated group s shfted towards the longer lengths compared wth the downregulated and unchanged groups; the DG data shows a smlar pattern wth a shft towards more negatve free energes n the translatonally upregulated group (Supplementary Fgure C()). These data suggest that a populaton of mrnas wth long and/or structured -UTRs s enrched n the translatonally upregulated group. To determne whether the hgher overall DG observed n the translatonally upregulated mrnas was smply a consequence of the longer -UTR lengths, a number of crtera were examned, ncludng the dstrbutons of the number of nucleotdes that were ether pared (Supplementary Fgure C()) or free (Supplementary Fgure C()), and the longest strng of nucleotdes nvolved n a structure (Supplementary Fgure C(v)) or unstructured regons (Supplementary Fgure C(v)). These analyses show that there s a sgnfcant enrchment of mrnas contanng structure n translatonally upregulated mrnas n DLBCL, whereas the number of free nucleotdes and the sze of unstructured regons s smlar n all three groups. Taken together, these data suggest that the degree of structure s more mportant than length n determnng enrchment n the up lst. To nvestgate ths fndng further, the -UTRs of three of the mrnas that were shown to be translatonally upregulated (ERCC, BCL and ) were subcloned nto lucferase reporter vectors (Fgure b()) and transfected nto DLBCL-derved cell lnes (Fgure b()). There was an ncrease n the synthess of the lucferase reporter enzyme from all constructs n the DLBCLderved cell lnes when compared wth the control, suggestng that enhanced translaton of those mrna n DLBCL was dependent upon features n ther -UTRs. Therefore, vector constructs were generated to examne the effect of length/ structure on reporter proten synthess n DLBCL (Fgure c(), Supplementary Table ). These constructs were transfected nto the control and DLBCL-derved cell lnes and lucferase expresson was determned. The data show that constructs wth longer -UTRs were repressed n the control cells, and that ths represson s sgnfcantly less n DLBCL cell lnes (Fgure c()). It has been shown n other systems that modfyng the proten components of the translaton apparatus has the net effect of alterng the dynamc range of translaton effcences across the entre transcrptome, so that those mrnas that are normally poorly & Macmllan Publshers Lmted Leukema ()

5 Dysregulaton of translaton n DLBCL E Horvlleur et al UTR length (bases) Total mcroarray up lst down lst ΔG (kcal/mol) Total mcroarray up lst down lst Nucleotdes n harpn Free nucleotdes Total mcroarray up lst down lst Relatve lucferase actvty ERCC BCL UTR ERCC lucferase BCL SV SV Poly A enhancer GM DoHH- OCI-LY Control Relatve lucferase actvty to bases UTR..... lucferase SV SV Poly A enhancer / / GM DoHH- OCI-LY eifg eifb eifb-p eifa Tonsl B-cells DLBCL patents eifb Tubuln eife eife-p eifh eifα eifα-p Actn hghly expressng tumours (%) CLL eifb FL DLBCL GM GM DoHH- SuDHL OCI-LY Insert length (bases) Fgure. mrnas contanng hghly structured -UTRs are preferentally translated n DLBCL. (a) The cdna mcroarray data were analysed and the average -UTR lengths, and DG values were calculated for those mrnas n the up and down lsts, and the sgnfcance of the dfference to the whole array was assessed by Student s t-tests. The data show that the mrnas n the upregulated lst have longer -UTRs (P ¼.; ), have greater DG values (P ¼.; ) and have more nucleotdes ncorporated nto harpns (P ¼.; ); all three crtera would be expected to decrease the translatablty of mrnas n these groups. (b()) The cdnas correspondng to the -UTRs of, ERCC and BCL were subcloned nto a lucferase reporter vector based on pgl. (b()) The reporter constructs were transfected nto cell lnes derved from DLBCL and control B cell, and lucferase actvty determned. The data show that there s greater expresson of lucferase n the DLBCL-derved cell lnes than n the control cell lnes for all plasmds, suggestng that the -UTRs are less nhbtory n these cell lnes. Sgnfcance was assessed by pared Student s t-tests on three ndependent experments (Po., Po. and Po.). (c()) Unrelated DNA sequences of ncreasng length (Supplementary Table ) were subcloned nto the -UTR of pgl upstream of the lucferase cstron. (c()) These plasmds were transfected nto the control cell lne (GM) and cell lne derved from DLBCL (DoHH-, OCI-LY and ), and lucferase actvty determned. (d) Proten extracts derved from cell lnes representatve of ether DLBCL (Val, DoHH-, SuDHL-, OCI-LY and ) and two control B-cell lnes (GM and GM) were separated by SDS-polyacrylamde gel electrophoress (PAGE). Samples were mmunoblotted wth eukaryotc ntaton factor (eifs)-specfc antbodes. The data show that only eifb levels were ncreased n these cell lnes. (e()) Extracts were generated from B cells purfed from tonsls of tumor-free ndvduals or tumors from patents wth DLBCL, and separated by SDS-PAGE and mmunoblotted to examne eifb levels. Tubuln was used as a loadng control. The data show that eifb levels were elevated n the fve DLBCL samples tested. (e()) Tssue mcroarrays that contaned tssues from lymphomas, ncludng DLBCL, FL and chronc lymphocytc leukemas (CLLs) were probed by an antbody aganst eifb and scored as descrbed n the expermental procedures secton. These data show that there s sgnfcantly hgher expresson of eifb n DLBCL compared wth that n CLL and FL. Sgnfcance between the dfferent categores was assessed by w -test (Po.). translated, for example, those that contan hghly structured -UTRs as shown here, are selectvely upregulated., Therefore, the levels and phosphorylaton status of the translatonal apparatus n DLBCL were assessed (Fgure d). Interestngly, the data show that whereas there was no dfference n the expresson of the eifs that have been shown prevously to be assocated wth a transformed phenotype, namely eife, eifg and eifa,, there was an ncrease n the expresson of eifb and a relatve Leukema () & Macmllan Publshers Lmted

6 GM DoHH- OCI-LY Dysregulaton of translaton n DLBCL E Horvlleur et al Ctrl srna eifb srna Ctrl srna eifb srna Ctrl srna eifb srna... eifb WB ERCC... Tubuln... GM DoHH- OCI-LY ctrl sb ctrl sb ctrl sb eifb BCL IP autoradography coomasse Tubuln IP autoradography coomasse srna Relatve lucferase actvty BCL UTR ERCC UTR control eifb control eifb control eifb control eifb control eifb control eifb GM DoHH- OCI-LY GM DoHH- OCI-LY Relatve lucferase actvty v Relatve lucferase actvty UTR control PGL... srna Control eifb Control eifb Control eifb srna Control eifb Control eifb Control eifb v Relatve lucferase actvty... GM DoHH- OCI-LY GM DoHH- OCI-LY srna ctrl eifb ctrl eifb Insert length Relatve lucferase actvty GM DoHH- OCILY Fgure. eifb modulaton restores represson of translaton on mrnas that s exerted by structured -UTRs. (a) Cell lnes derved from patents wth DLBCL (DoHH- and OCI-LY) and control B cells (GM) were transfected wth srna to eifb. After h, cell extracts were generated, whch were separated by SDS-polyacrylamde gel electrophoress and mmunoblotted for eifb and ERCC levels. Pulse-labeled mmunoprecptaton was used to measure the levels of BCL and after h of eifb srna treatment, as these protens have long halflves. The gels were quantfed usng Image J software. The numbers shown represent expresson normalzed to tubuln (eifb and ERCC) or autoradography normalzed to Coomasse stanng (BCL and ). The data show that n DLBCL-derved cells reducng eifb expresson decreased the expresson of, ERCC and BCL. (b) The levels of eifb were reduced by srna (). The reporter plasmds contanng the -UTRs of BCL, ERCC, or a control pgl ( v) were transfected nto the cell lnes ±srna to eifb. After h, lucferase actvty was determned. The data show that there was a reducton n lucferase actvty when the level of eifb was reduced for all the -UTRs examned. Sgnfcance was assessed by pared Student s t-tests on three ndependent experments (Po., Po., Po.). (c) Plasmds contanng unrelated DNA sequences of or nucleotdes upstream of the lucferease cstron were transfected nto the control cell lne (GM) or cell lnes derved from DLBCL (DoHH-, OCI-LY and ) ±srna to eifb, and lucferase actvty determned. ncrease n eifb-p (Fgure d). eifb stmulates translaton by ncreasng the actvty of the helcase eifa, t s requred for S complex formaton va ts nteracton wth poly(a)-bndng proten and contrbutes to selectve translaton of mrnas wth hghly structured -UTRs.,, Importantly, eifb expresson was also ncreased n DLBCL patent samples, albet to dfferng extents (Fgure e()). To analyze eifb expresson from a larger number of patent samples smultaneously, tssue mcroarrays were used & Macmllan Publshers Lmted Leukema ()

7 Dysregulaton of translaton n DLBCL E Horvlleur et al cell lne data, there was a sgnficant ncrease n eifb expresson n the DLBCL tumors compared wth chronc lymphocytc leukema and FL (Fgure e()). Interestngly, there was a OCI-LY SuDHL- DoHH- GM GM (Supplementary Fgure A, Supplementary Table ). These contaned tssues from lymphomas, ncludng DLBCL, FLs and chronc lymphocytc leukemas. In agreement wth the PIK PIK PKB Total P AKT PKB-P PP AZD P mtor mtor EBP Total EBP E EBP-P P P EBP P E AZD nm AZD nm Gradent fracton AZD nm AZD nm Gradent fracton v AZD nm AZD nm Gradent fracton AZD pp GM ctrl AZD nm AZD nm DoHH- Gradent fracton AZD nm AZD nm Gradent fracton AZD nm AZD nm Gradent fracton AZD nm AZD nm Gradent fracton OCI-LY pp ctrl Gradent fracton RPS mrna (A.U.) eifb mrna (A.U.) pp v eifb mrna (A.U.) eifa mrna (A.U.) RPS mrna (A.U.) AZD nm AZD nm eifa mrna (A.U.) AZD AZD Anm Absorbance (A.U.) ctrl Anm Absorbance (A.U.) Actn EBP eifb eifb-p ERCC Actn Leukema () & Macmllan Publshers Lmted

8 correlaton between eifb overexpresson and the levels of and ERCC n the tumor samples (Fgure c) and n tssue mcroarrays (Supplementary Fgure B). Translatonal dysregulaton of ERCC, and BCL correlates wth enhanced eifb expresson To test the hypothess that ncreased eifb levels drectly elevated the synthess of subsets of protens, ncludng and BCL n Dysregulaton of translaton n DLBCL E Horvlleur et al DLBCL, cells were transfected wth srna drected aganst eifb mrna and potental target protens were analyzed, ether by western blottng (ERCC) or pulse-labelng and mmunoprecptaton (BCL and, due to the long half-lves of these protens, whch would mask changes n net synthess rates). When eifb levels were reduced, there was a decrease n the expresson of ERCC, BCL and (Fgure a). Hghly expressng tumours (%) CLL ERCC FL DLBCL CLL FL DLBCL Hghly expressng tumours (%) Survval rate.... Survval rate Low ERCC Hgh ERCC. p=.. tme (months).... Survval rate..... Low eifb Hgh eifb. p=.. tme (months) Low Hgh p=.. tme (months) Fgure. Tssue mcroarray shows that altered expresson of the protens dentfed by translatonal proflng n tumors from patents wth B-cell lymphoma corresponds wth survval. (a) Tssue mcroarrays that contaned tssues from lymphomas, ncludng DLBCL, FL and chronc lymphocytc leukemas (CLLs) were probed wth antbodes aganst ERCC () and (), and scored as descrbed n the expermental procedures secton. The graphs represent percentage of tumors from each type expressng hgh levels of the scored proten. The data show that there are dfferences n expresson of n the DLBCL patent samples compared wth the other tumor types, and for ERCC a dfference n the expresson n FL and DLBCL when compared wth CLL. Sgnfcance between the dfferent categores was assessed by w -test (Po., Po., Po.). (b) Patents were separated nto groups that have low or hgh expresson of ERCC () or (), or eifb (). Survval curves were generated usng Kaplan Meer approxmaton to compare overall survval n dfferent groups of patents. The sgnfcance of the changes n survval was assessed usng log-rank P-value. The data show that there are sgnfcant decreases n survval n patents that have hgh expresson for ERCC (P ¼.), (P ¼.) and eifb (P ¼.). Fgure. A schematc dagram to represent the changes n DLBCL that occur as a consequence of eifb overexpresson. Taken together, the data suggest a model wheren ncreased sgnalng through mtor n DLBCL ncreases the expresson of eifb, whch drves the translaton of mrnas that contan hghly structured -UTRs and lead to the upregulated synthess of ERCC, and BCL. Increased expresson of these protens negatvely mpacts upon patent survval, provdng three new prognostc markers for ths dsease: eifb, and ERCC. Fgure. Sgnalng through mtor lnks eifb expresson to and ERCC. (a()) Cell lnes derved from DLBCL or control cells, as shown, were lysed, separated by SDS-polyacrylamde gel electrophoress (PAGE), and mmunoblottngs were probed wth antbodes aganst phosphonostde -knase (PIK), proten knase B (PKB), mtor and EBP. The data suggest that the mtor sgnalng pathway s upregulated n these cells when compared wth that n the controls. (a()) Schematc to llustrate sgnalng through the mtor pathway. (b and c) Two DLBCL cell lnes DoHH- (b) and OCI-LY (c) were treated wth the mtor nhbtor AZD at or nm. After h, cells were collected and post-nuclear fractons were appled to sucrose densty gradents to separate the actvely translatng polysomes from the subpolysomal materal (b() and c()). RNA was solated from each fracton and quanttatve reverse transcrptase-pcr was used to assess the proporton of RPS (b() and c()), eifb (b() and c()) and eifa (b(v) and c(v)) assocated wth each fracton. (d) Control cell lne (GM) or DLBCL lnes (DoHH- and OCI-LY) were treated wth PP or AZD, cell extracts were generated, separated by SDS-PAGE and mmunoblotted for EBP-P, eifb, eifb-p, ERCC and. The data show that the reducton n eifb as a result of exposure to mtor nhbtors also reduces the expresson of ts downstream targets ERCC and. & Macmllan Publshers Lmted Leukema ()

9 Dysregulaton of translaton n DLBCL E Horvlleur et al To examne whether the regulaton eifb target mrnas was medated by elements n ther -UTRs, the level of eifb was reduced by srna (Fgure b()) and cells were transfected wth reporter plasmds (Fgure b( v)). There were decreases n the expresson of lucferase from the reporter plasmds that contaned the ERCC, BCL and -UTRs n the lymphoma-derved cell lnes when eifb was reduced (Fgure b( v)). These data suggest that hgh eifb levels could contrbute to the enhanced synthess of subsets of protens, ncludng, BCL and ERCC, by unwndng structured -UTRs. To confrm that the relef of represson on structured mrnas was drectly due to the hgher levels of eifb n these cells and ndependent of sequence, cells were co-transfected wth srnas aganst eifb and constructs contanng structured -UTRs of unrelated sequence (Supplementary Table ). In ths case, the enhanced synthess that was observed n DLBCL-derved cells when compared wth the control lnes was ablated (Fgure c). Sgnalng va the mtor sgnalng pathway upregulates eifb expresson The proflng data (Fgure, Supplementary Fgure ) ndcated that eifb was also dysregulated n DLBCL at the level of translaton. Several lnes of evdence suggest that the translatonal dysregulaton of eifb could result from aberrant sgnalng through the mtor pathway. Actvaton of the mtor pathway has been shown prevously n DLBCL, although not n the cell lnes used heren, and a recent study has also reported actvaton of mtor n a subset of DLCBL patents. Moreover, eifb was present on two lsts of genes whose expresson changed followng mtor nhbton., Therefore, DLBCL and control cells were mmunoblotted to assess the expresson and phosphorylaton status of proten knase B/AKT, mtor and EBP. There was an ncrease n expresson and phosphorylaton of these protens n the DLBCL-derved cell lnes (Fgure a). Although mtor actvaton nduces phosphorylaton of eifb, our data show that there are changes n the total level of eifb proten n DLBCL. To assess whether mtor nhbton decreased the translaton of eifb n DLBCL, cells were ncubated wth the nhbtor AZD. Polysomes were separated by sucrose densty-gradent centrfugaton, and quanttatve reverse transcrptase-pcr was carred out to assess changes n polysomal dstrbuton of mrnas encodng a known mtor target, RPS and eifb (Fgures b( v) and c( v)). eifa was used as a control. The data showed a shft of RPS and eifb mrnas from the polysomes followng ncubaton wth AZD (Fgures b(, ) and c(, c)), but lttle or no effect on eifa mrna (Fgures b(v) and c(v)), whch agan suggests that eifb expresson s regulated va sgnalng through mtor., To assess the effect of mtor nhbtors PP and AZD on eifb proten levels, cells were ncubated wth these compounds and mmunoblottngs carred out. As expected, there were changes n the degree of phosphorylaton of EBP and eifb but, mportantly, also a decrease n expresson of eifb, and two of ts downstream targets, ERCC and, albet to dfferent extents (Fgure d). Dfferences n eifb, ERCC and expresson n cell lnes correlate wth alteratons n B-cell lymphoma tumor samples and patent survval It was then mportant to assess whether the dfferences n proten expresson were also present n patents wth DLBCL and whether ths was assocated wth patent survval. It s well establshed that BCL s upregulated n dfferent non-hodgkn lymphoma types, and ts elevated expresson s assocated wth poor prognoss n DLBCL; hence, the role of ths proten n patent survval was not examned further. However, and ERCC expresson have not been studed prevously n DLBCL n ths regard. Therefore, to determne the expresson of and ERCC n a large number of patent samples smultaneously, tssue mcroarrays were used (Supplementary Fgure, Supplementary Table ). ERCC expresson was sgnfcantly hgher n DLBCL when compared wth that n chronc lymphocytc leukema (Fgure a()). proten expresson n DLBCL was ncreased sgnfcantly when compared wth that n FL and chronc lymphocytc leukema (Fgure a()). Kaplan Meer analyss was performed to determne whether there were dfferences n survval assocated wth the ncreased expresson of, ERCC and eifb (Fgures b( )). Importantly, the data show that hgh, ERCC and eifb expresson correlated wth poor overall survval for all subtypes of DLBCL, provdng three new prognostc markers that would dentfy those wth worse outcome for ths dsease (Fgures b( )). DISCUSSION Our data allow us to propose a model whereby aberrant sgnalng through mtor leads to an upregulaton n eifb, whch drectly drves the translaton of a subset of mrnas that contan structured -UTRs, ncludng those nvolved n tumorgeness and chemoresstance, such as, BCL and ERCC (Fgure ). Importantly, we dentfed eifb and two of ts targets, and ERCC, as markers of poor prognoss n DLBCL, suggestng that the ncreased expresson and actvaton of ths proten drves tumorgeness (Fgures, and ). The data ndcate that the ncrease n eifb expresson occurs at the level of translaton (Supplementary Fgure and Fgure ) and, n agreement wth prevous data, that ths s downstream of mtor sgnalng (Fgures and ). It has been suggested that although sgnalng through mtorc s requred for the selectve upregulaton of termnal olgopyrmdne tract contanng mrnas, ths pathway does not drectly control the translaton of mrnas wth long and/or structured -UTRs. However, we show that n DLBCL, upregulaton of eifb levels by the mtor pathway ncreases the translaton of selected mrnas that contan structured -UTRs (Fgures, and ). Our data are supported by studes, whch show that slencng of eifb n HeLa cells reduces the translaton of selected mrnas that contan structured -UTRs. We demonstrate that hgh expresson correlated wth poor patent survval (Fgure ). The cellular role of s complex, wth opposng effects beng attrbuted for n tumor necross factor and FAS sgnalng to apoptoss. In addton, mutatons that nactvate functon are assocated wth prolonged survval of patents wth pancreatc cancer. It s well establshed that ncreased expresson of BCL s assocated wth DLBCL and t has been shown prevously that hgh expresson of BCL s assocated wth poor survval,, partcularly for germnal center B-cell-lke such as DLBCL. The data presented here suggest that ncreased translaton of BCL also contrbutes to the elevated levels of ths proten n DLBCL (Fgures, and ). Smlarly, we show that ERCC expresson s ncreased n DLBCL (Fgures, and ). ERCC s an essental component of the nucleotde excson repar pathway that s used to repar damaged DNA followng exposure to therapeutc agents such as csplatn. In addton, t has been shown recently that eifb s present as part of a gene expresson sgnature of acqured resstance to csplatn n patents wth gastrc cancer. Inhbton of these DNA repar pathways, as part of combnaton therapy wth mtor nhbtors, could therefore provde alternatve treatment optons for patents wth DLBCL that respond poorly to conventonal therapy. In ths regard, a recent tral has been establshed to evaluate mtor nhbtors for the treatment of patents wth relapsed or refractory DLBCL. In summary, our data show that ncreased eifb expresson makes a sgnfcant contrbuton to the development of tumorgeness n DLBCL. Targetng the protens that are translatonally Leukema () & Macmllan Publshers Lmted

10 upregulated and whch have not been prevously dentfed as assocated wth ths dsease, for example, or ERCC, n addton to drect methods to reduce eifb expresson or actvty, could provde new therapeutc optons, partcularly for patents who do not respond to conventonal treatment. CONFLICT OF INTEREST The authors declare no conflct of nterest. ACKNOWLEDGEMENTS Ths work was funded by grants from the LLR (formerly LRF; EH, KH, LCC and PG) and MRC program fundng (AEW, MB and MM). TS was funded by HSH Prnce Albert II of Monaco and the Government of the Prncpalty of Monaco. AEW holds a BBSRC Professoral Fellowshp and MB holds a MRC Senor Research Fellowshp. RVS was funded by the BBSRC. REFERENCES Stoneley M, Wlls AE. Aberrant regulaton of translaton ntaton n tumorgeness. Curr Mol Med ; :. Braunsten S, Karpsheva K, Pola C, Goldberg J, Hochman T, Yee H et al. A hypoxa-controlled cap-dependent to cap-ndependent translaton swtch n breast cancer. Mol Cell ; :. 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