Review/Praca poglądowa
Crosstalk between BCR/ABL and RNAi
Interakcje pomiędzy BCR/ABL a RNAi
Sylwester Głowacki *, Ewelina Trela, Janusz Błasiak
DepartmentofMolecularGenetics,FacultyofBiologyandEnvironmentalProtection,UniversityofLodz,head:drhab.
KatarzynaWoźniak,Łódź,Poland
Introduction
Chronicmyeloidleukemia(CML)isprobablyoneofthemost extensively studied human malignancies. It is associated withaconsistentchromosomalabnormality– thePhiladel- phia(Ph)chromosome[1].ThePhchromosomeresultsfrom areciprocaltranslocationt(9;22)(q34;q11)whichinvolvesthe
ABL proto-oncogeneon chromosome9 and BCR(for break- pointclusterregion)onchromosome22[2].Thistransloca- tion creates the BCR/ABL fusion gene which encodes the BCR/ABL protein,atyrosine kinaseprotein thatis believed to be the principal cause of the CML pathogenesis [1, 3].
Molecular signaling in CML affected by BCR/ABL is highly complex and activates multiple signal transduction path- ways (see [1,2] for more details). The activity of BCR/ABL article info
Articlehistory:
Received:12.11.2012 Accepted:27.06.2013 Availableonline:11.07.2013
Keywords:
Leukemia
Epigenetics
BCR/ABL
RNAi
siRNA
miRNA
Słowakluczowe:
białaczka
epigenetyka
BCR/ABL
RNAi
siRNA
miRNA
abstract
Chronicmyeloidleukemia(CML)isamalignantdiseaseofprogenitormyeloidcellscau- sedbychromosomaltranslocationthatresultsintheformingofdiminutivePhiladephia chromosome that harbors BCR/ABL fusion oncogene. The product of this oncogene, a tyrosine kinase, altersseveral important regulatory pathways related to cell growth anddifferentiationthusleadingtocancertransformation.MajorformofCMLtherapyis basedontyrosinekinaseinhibitors,firstofallimatinib(IM).Somepatientsdevelopresis- tancetoIMinthecourseoftreatment.Intheprocessofleukemogenesistheactivityof miRNAs–oneofgroupsofRNAsinvolvedinRNAinterference(RNAi)–isaltered.Signa- turesof altered miRNAsactivitymay serveas a prognosticfactor inthe development andtherapyofseveraldiseases.Moreover,othergroupofRNAsinvolvedinRNAi–siRNA – might be valuable addition to array of specific therapeutics targeted the BCR/ABL kinase.
©2013PolskieTowarzystwoHematologówiTransfuzjologów,InstytutHematologiii Transfuzjologii.PublishedbyElsevierUrban&PartnerSp.zo.o.Allrightsreserved.
*Correspondingauthorat:KatedraGenetykiMolekularnej,WydziałBiologiiiOchronyŚrodowiska,ul.Pomorska141/143,90-263Łódź, Poland.
E-mailaddress:sglowa@biol.uni.lodz.pl(S.Głowacki).
ContentslistsavailableatSciVerseScienceDirect
Acta Haematologica Polonica
journalhomepage:www.elsevier.com/locate/achaem
0001-5814/$–seefrontmatter©2013PolskieTowarzystwoHematologówiTransfuzjologów,InstytutHematologiiiTransfuzjologii.PublishedbyElsevierUrban&PartnerSp.zo.o.Allrightsreserved.
http://dx.doi.org/10.1016/j.achaem.2013.07.001
leadstoderegulated cellularproliferation,decreasedadher- ence of leukemia cells to the bone marrow, and reduced apoptosis what results in the malignant expansion of pluripotentstemcellsinbonemarrow[3].CMLissubdivided into three phases: chronic phase (CP), which is the initial phase and disease, is usually diagnosed at this stage.
Subsequently,itadvancestoanacceleratedphase(AP).The last CML phase is called blast crisis (BC) [1]. Since CML is caused by BCR/ABL it was possible to design an effective targeted molecular therapy which selectively inhibits the aberrant BCR/ABL protein [4]. Imatinib mesylate (imatinib, IM,STI-571,Glivec®,NovartisPharma)isaBCR/ABLtyrosine kinase inhibitorthat is used for the treatment of CML. IM blockstheATP-bindingsiteandstabilizestheinactiveform of BCR/ABL thus blocking downstream signal transduction pathwaysaffectedbythisprotein[4].
RNA interference (RNAi) is a process, in which small non-coding RNAs(ncRNAs) incorporatedinto multi-protein RNA-inducedsilencingcomplex(RISC) repressgeneactivity inasequence-specificmannerat transcriptional,post-tran- scriptionalortranslationallevel [5,6].Theprocessof RNA- mediatedgenesilencinghasanessentialrole incell devel- opment, differentiation, proliferation, apoptosis, maintain- ingofchromatinstructure,virusresistanceandoncogenesis [7]. Thereare threetypes of smallncRNAsincluding micro RNA (miRNA), small interfering RNA (siRNA) and Piwi- interactingRNA(piRNA)[8].Inourreviewwewillfocuson siRNA and miRNAs as best understood groups of small ncRNAs,and theirroleinprocessesofleukemogenesisand interaction with signaling and regulatory pathways, in whichBCR/ABLkinaseisactive.
Biogenesis of miRNA and siRNA
MicroRNAsaresmall,approximately22 ntlong,RNAsthat are derived from endogenous hairpin-shaped transcripts through the action of the Drosha and Dicer proteins, and subsequentlyboundbyAgo-familyproteinsformingmature RISC complexes [9] (Fig. 1). Some of miRNA genes are located in introns of pre-mRNAs [10]. Other miRNA genes are clustered in the genome with an arrangement and expression pattern indicating polycistronic primary tran- scripts[11].TheprimarytranscriptsofmiRNA(pri-miRNAs) are usuallygenerated by RNA polymerase II[12]. They are processedinthenucleusbyMicroprocessorcomplex,which containsDroshaandDGCR8,alsoknownasPasha,proteins [9, 13]. The pre-miRNAsare transported tothe cytoplasm, wheretheyundergofurtherprocessingbytheDicerprotein, which cleaves dsRNA and releases a22-nucleotide mature double-stranded miRNAs.Onestrandof the miRNAduplex is subsequently loaded onto the Ago protein, and then incorporated into fully functional RISC complex, while the otherone,calledpassengerRNA,beingdegraded[9,14,15].
SmallinterferingRNAsareslightlyshorter thanmiRNAs andare around21ntlong.OriginallysiRNAswerebelieved to be exclusively derived from the exogenous RNA [6].
However, endogenous siRNA (endo-siRNA) were recently discovered in Drosophila melanogaster, mouse oocytes and somestem cells(reviewedin[9]).SimilarlytomiRNAsthey
formRISCcomplexeswithAgo-familyproteins[16],butthey differ in their biogenesis. Their origin stems from long double-strandedRNAsareprocessedwithDicerbutwithout Drosha activity (Fig. 2) [9]. Exogenous siRNAs may come from processedviral RNA[17], whileendo-siRNAoriginates from precursors created through transcription of sense- antisense pairs of transposons, or from long, self-paired, double-strandedfragmentofmRNAs[18].
RNA interference mediated by mi- and siRNAs
Thecoreactivityofmi-andsiRNAsissimilar:theyallowfor specificmodulationofgeneexpressionthroughguidingthe activity of RISC complexes. The complex contains Ago, which isbelievedtobeacoreelementof itwithits ability tobindtosingle-strandedRNA(ssRNA)anddsRNA [19,20].
After recognition of complementarity between targeted mRNAandmi-orsiRNAtemplate,RNaseactivityofRISCis induced,leadingtodegradationofmRNA[21].
The miRNAs can direct the RISC to downregulate gene expression by mRNA cleavage or translational repression.
Thepathway of silencingisdetermined bythe kindof the target: theRISCwillspecifycleavage ofthe targetmRNAif themRNAhassufficientcomplementaritytothemRNAorit will repress productive translation if the mRNA does not Fig.1–BiogenesisofmiRNA.miRNAsarederivedfrom endogenousprecursorsprocessedbythemicroprocessor complex,exportedtocytoplasm,processedbyDICERand loadedontoAGOproteinformingmatureRISCcomplexes
have sufficient complementarity to be cleaved. [19].
Recently, it was also shown that miRNAs could induce transcriptional silencing in human cells, directing Ago1, Polycombgroup(PcG)andhistonemethyltransferaseenhan- cer of zeste homolog1 (EZH2) recruitment to specific DNA sequences,alteringhistonemethylationstatus[22].Overthe several past years it was shown that miRNAs are able to repress the expression of important cancer-related genes andexpressionofmiRNAgenescontributetothepathogen- esisofhumanmalignances[23].
SmallinterferingRNAscanrepressgeneactivitythrough transcriptional gene silencing in human cells [5]. This mechanism needs an mRNA transcribed with 50 extended untranslated region overlapping with gene promoter, and probably involves some modification in methylation status [24, 25]. For example, after exposure to the siRNA, the targeted promoter exhibits higher levels of the silencing histonemethylmarksH3lysine-9di-methylation(H3K9me2) and histone H3 lysine-27 tri-methylation (H3K27me3) [26].
However, the role that DNA methylation plays in this
transcriptional silencing remains uncertain, as siRNA mediated transcriptionalgenesilencingwasobservedinthe absence of increased DNA methylation at the targeted promoter,buttheseobservationswerenotconfirmedinother studies[27,28].Inaddition,thequestionhowexactlysiRNAs recognize and modulate transcriptional gene silencing through histone methylationremains unanswered[24]. Pre- sumably,transcriptionalgenesilencinginvolvesDNAmethyl- transferase3a(DNMT3a),Ago1aswellasAgo2,HDAC-1and EZH2and/orG9a[24,26,29–31].DespitetheroleofsiRNAin transcriptionalgenesilencing,theywerealsoreportedtoact ingeneactivationinhumancells[29,32].
Interaction between miRNAs and BCR/ABL
There is growing evidence that miRNAs may play an important role in BCR/ABL-dependent malignancies (see Table I and Fig. 3). For example, miR-203 controls the expressionoftheABL1geneaswellasthefusiononcogene Fig.2–BiogenesisofsiRNA.siRNAscanbederivedbothfromendogenousprecursorsencodedinthegenomeaswellasfrom exogenousdouble-strandedRNAs.ProcessingofendogenousprecursorsoccurswithoutDroshaactivity.Afterbeing exportedintocytoplasmdoublestrandedmiRNAprecursorsareprocessedbyDICERcomplexandloadedontoAGOprotein
BCR/ABL.Itwasdemonstratedthatincreasedlevels ofmiR- 203 expression resulted in a decreased expression of BCR/
ABL[33].Moreover,aninhibitionofBCR/ABLexpressionwith miR-203sensitized BCR/ABL-positivecells toIMaction [34].
Anotherstudysuggeststhepresence ofareciprocalregula- toryloopbetweenBCR/ABLandmiR-451inducingtheinhibi- tionof miR-451bytheBCR/ABLactivity.Because miR-451is BCR/ABL negative regulator, this results in the increased expression of BCR/ABL.Moreover, this process may leadto synergy in the activity of miRNA and BCR/ABL tyrosine inhibitorIM[35].Otherinteractionsmayincludesynergyin oncogenic activity between some miRNAs and BCR/ABL kinase itself. One such case is miR-125b that was found over-expressed up to 90 times in acute myeloid leukemia cells with chromosomal translocation t(2;11)(p21;q23), and could accelerate leukemia development even when co- expressedwithBCR/ABL[36].
Recent findings show that specific miRNAs may play a fundamental role in the activation of oncogenic signal transductionpathwaysinCMLcells.Adecreasedexpression of miR-326 was reportedto lead tounrestricted activity of Smotransducer, akey player intheHedgehog pathway,in CMLcells[37].Similarly,miR-138actsinminicircuitrywith BCR/ABL and GATA1, an important factor in regulation of erythroidandmegakaryocyticdevelopment.Overexpression ofmiR-138decreasesBCR/ABLactivity.Conversely,IMtreat- ment increases miR-138 activity, which in turn activates GATA1[38].
As shown above miRNAs can directly modulate the expressionofBCR/ABL.However,thetyrosinekinaseactiv- ity of BCR/ABL was also shown to contribute to the expressionofdistinct miRNAs.Although changesinactiv- ityofmanymiRNAs areobservedinCMLcells,notallare BCR/ABL-dependent.It was shownthat hsa-miR-10a, hsa- miR-150, andhsa-miR-151 were down-regulated, whereas hsa-miR-96 was up-regulated in CML CD34+ cells. Among
these miRNAs,only activity ofhsa-miR-150 andhsa-miR- 151 was related to BCR/ABL kinase function, which was assessed by inactivation of BCR/ABL by IM [39]. It was reported that miR-17-92 cluster was expressed in a BCR/
ABL-dependent manner, and expression of pri-miR-17-92 wasalsomediatedbymyelocytomatosisoncogene(c-MYC).
Inaddition,miR-17-92miRNAswereoverexpressedinCML cellsinCP, but notinBC[40]. Anotherstudyshowed that miR-31, miR-155, miR-564 and, miR-328 despite possessing distinct targets, were downregulated and this effect was dependentonBCR/ABLtyrosinekinaseactivity[41,42].More- over, thedownregulationof miR-328wasalso dependenton BCR/ABL in dose-dependent manner [41]. BCR/ABL was reported to upregulate some miRNAs and in consequence inhibitnegativegrowthregulatorsandpromotingcellgrowth.
AsanexampleofthesemaybemiR-130aand-130b.Expres- sionofmiR-130aandmiR-130bwasobservedtobeBCR/ABL- dependent, but the exact mechanism by which BCR/ABL affects the expression of these miRNAs remains unknown.
However,apparentlyafterstimulationoftheactivityofthese miRNAs by BCR/ABL inhibition of CCN3 negative growth regulatoroccurspromotingcellproliferation.[43].
Micro RNAs and therapy of BCR/ABL dependent leukemias
TheuseofIMrevolutionizedtreatmentofCML[4].Despitethe clinicalsuccessobtainedwithIM,resistancetothisdrugisan emergingproblem[3]. SinceabnormalexpressionofmiRNAs wasdescribedinleukemias,miRNAsmaymodulateresponse to IM treatment in CML patients. It was reported that lentivirus-mediated over-expressionof polycistronicmiRNAs of miR-17-92 cluster enhanced response to IM [40]. It was also shown that miR-150 and miR-146a were upregulated, whilemiR-142-3pandmiR-199b-5pweredownregulatedafter TableI–MicroRNAs(miRNAs)expressedincellsharboringBCR/ABLkinase
miRNA Feature Reference
miR-7 DownregulatedinIM-respondingCMLpatients [44]
miR-10a DownregulationinCML,notBCR/ABL-dependent.TargetofthismiRNA isagrowthfactor
[36]
polycistronicmiR17-92cluster DownregulatedbyIMactionandsiRNAtargetedatBCR/ABL. [37]
miR-23a/26a/29a/29c DownregulatedinIM-respondingCMLpatients [44]
miR-30a InhibitedbyIM,actsasinhibitorofautophagy [45]
miR-30b/30c DownregulatedinIM-respondingCMLpatients [44]
miR-31 BCR/ABL-dependentdownregulationinCMLcells [39]
miR-96 BCR/ABL-dependentdownregulationinCMLcells [36]
miR-100/126# DownregulatedinIM-respondingCMLpatients [44]
miR-130a/b DownregulatedafterBCR/ABLknockdown,negativelyregulatesanti- growthfactorCCN3
[40]
miR-134/141 DownregulatedinIM-respondingCMLpatients [44]
miR-138 DecreasesactivityofBCR/ABL,sensitizestoIMaction [38]
miR-150 BCR/ABL-dependentdownregulationinCMLcells [36,41,42]
miR-151 BCR/ABL-dependentdownregulationinCMLcells [36]
miR-155 BCR/ABL-dependentdownregulationinCMLcells [39]
miR-183/191/196b/199a DownregulatedinIM-respondingCMLpatients [44]
miR-203 Lostthroughdeletioninsomehematopoeticmalignancies.ThismiRNA downregulatesABL1,includinginfusionwithBCR.
[33]
miR-224/326/422b/520a DownregulatedinIM-respondingCMLpatients [44]
miR-564 BCR/ABL-dependentdownregulationinCMLcells [39]
14 daysof IMtherapy [46]. Moreover,this study alsofound thataberrantexpressionlevelofthesemiRNAswasreturning towardnormallevels asseenin healthydonors,suggesting thatmiRNAs,byregulatingdistinctgenes,maybeinvolvedin BCR/ABL-driven leukemogenesis [46]. Another study, which was aimed at identifying miRNAs and their related targets associated with CML pathogenesis, revealed that miR-150, miR-20a,miR-17, miR-19a,miR-103, miR-144, miR-155, miR- 181a, miR-221 and miR-222 were deregulated in CML [47].
However,stating iftheyfunctionindependenceorindepen- denceofBCR/ABLneedsfurtherinvestigation[47].MicroRNAs mayalsoplayaroleinthedevelopmentofIM-resistance. It was shown that IM inhibits expression of miR-30a, which functions asinhibitorof autophagythrough downregulation ofBeclin1andATG5expression.Autophagy,inturn,isoneof waysof avoiding cell deathinduced byIM treatment.Thus this miRNA may serve as a basis for mechanism of IM- resistance. It was shown that decreased levels of miR-30a correlatedwithincreasedlevelsofABL1andBCR/ABLexpres- sion[48].
Since growing numbers of patients are suffering from resistance to IMit is necessarytofind markers which will enable identification of patients who may develop such resistance. One kind of such markers might be specific signatures of altered miRNA activity. It was revealed that a group of 19 miRNAs may function as predictors for IM resistance in newly diagnosed CML patients [44]. Among them18weredownregulatedandonlyonewasupregulated [44]. Another study showed that miR-451 was downregu- lated inIM-resistantpatientascompared toIM-responding patients suggesting that miR-451 expression levels are correlated with response to IM therapy [49]. Nevertheless, furtherstudiesarerequiredtoconfirmandvalidatetherole ofmiRNAsasnovelmarkersinpredictingIMresistance.
Inrecentyearsanewareaofpotentialdrugdevelopment emerged, that aims synthetic siRNAs targeted at specific oncogenesasameansofsuppressingtheiractivity.Several attempts wereperformed for evaluation of validity of that methodfortreatmentofBCR/ABL-inducedleukemias.Itwas demonstratedthatdeliveryofdouble-strandedRNAtargeted at BCR/ABLoncogeneresultedinRNAithatcausedBCR/ABL inhibitionanddeathofleukemiccells[50].ActivityofRNAi- based inhibitors appears to be even more specific than classical tyrosine inhibitors. snRNA designed for targeting specificvariantsof BCR/ABL– formedbyb3a2 breakpoint– inducedcelldeathonlyincellscarryingthisspecificvariant of that gene [51]. Successes in inducing cell death in leukemic cells with siRNA targeted at BCR/ABL led to evaluation of combined treatments with classical tyrosine inhibitorsandsiRNAs.DeliveryofIMtoK-562cellscarrying BCR/ABL together with siRNA targeted at this oncogene resulted in 3-fold decrease in IM EC50[52]. Synergism was alsoobservedbetweenactionofsiRNAstargetedatBCR/ABL and otheroncogenes [53]. Synthetic siRNAswerealsoused for overcomingIMresistanceinpatientwithCMLrecurring after IMtherapy.TheseresultssuggestthatsiRNAsmaybe feasible inCML therapy [54]. Similarresults wereobtained invitroforleukemiccellsderivedfrompatientwithrelapsed acutemyeloidleukemia,whoexhibitedPh+chromosomede novoduringclinicaltreatment.DeliveryofsiRNAresultedin cell-deathinductionincellsresistanttoIM[55].Moreover,it was demonstrated that siRNAs could overcome IM-resis- tance basedboth onBCR/ABLoverexpressionand its point mutations[56].
Future perspectives
Current findings indicate that many intertwined pathways involving BCR/ABL kinase activity and miRNA epigenetic modulationofgeneexpressionarepresentinleukemiccells.
Those mutual influences may be positive or negative in nature.Becauserecentadvancesinunderstandingtheroleof smallnon-codingRNAsprovideaplentyofnewlyidentified miRNAsandsiRNAsthisareaofresearchmay createanew opportunitiesforstudyingofmechanismsofcancertransfor- mation. Moreover, this may lead to development of new therapies, or to reinforcement of treatment potential of currentlyuseddrugs.However,therearesomeobstacles.The sole task of annotating snRNAs involved in modulating Fig.3–InteractionbetweenBCR/ABLandmiRNAs.miRNAs
mayinhibitactivityofBCR/ABLormaybeinhibitedbythis kinase.Thismayleadtosynergisticinteractionsbetween somemiRNAsandkinaseinhibitorssuchasimatinib(IM).
Moreover,asitisinthecaseofmiR-451negative interactionsbetweenBCR/ABLandmiRNAsmayform reciprocalinteractionsresultinginmutualdownregulation.
BCR/ABLmayalsoactwithsynergyonothermiRNAs,asin thecaseofpromotingcellgrowthwithmiR-130,anegative regulatorofCCN3,whichfunctionsasacellgrowth inhibitor
cellular metabolism is prone to error and many wrongly annotatedsnRNAswithoutanyreal functionalmeaningare present in databases [9]. This may make finding of new interactionsbetweenBCR/ABLandsnRNAscomplicated.
Nevertheless, there is no doubt that epigenetics in general,andsnRNAs specifically, willbeoneofmajorarea of interests in coming years. In case of many leukemias, especially CML, genetic components of cancertransforma- tion are relatively well understood. Yet still many differ- ences indisease development aswell as therapy response are observedbetweendifferentpatients.Itisapparentthat mechanisms of epigenetic nature are one of major factor responsible for that differences. Thus unraveling the com- plex web of mutual interactions between genetic and epigeneticfactorsinvolvedindeterminingthewayinwhich normalcellsundergocancertransformationisessentialfor futuredevelopmentofdiagnosingandtreatingofleukemias.
Authors' contributions/Wkład autorów
Accordingtoorder.
Conflict of interest/Konflikt interesu
Nonedeclared.
Financial support/Finansowanie
Nonedeclared.
Ethics/Etyka
Thework described inthis article has been carriedout in accordance with The Code of Ethics of the World Medical Association(Declaration of Helsinki)for experimentsinvol- ving humans; EU Directive 2010/63/EU for animal experi- ments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.
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