PROGNOSE VAN ACUTE MYELOIDE LEUKEMIE - lib.ugent.be · AML-onderzoek op dit gebied binnen de...
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Academiejaar 2012-2013 DE ROL VAN MICRORNA IN DE ONTWIKKELING EN PROGNOSE VAN ACUTE MYELOIDE LEUKEMIE Freddy VAN DAMME Promotor: Prof. Dr. Jan Philippé Scriptie voorgedragen in de 2 de Master in het kader van de opleiding tot MASTER OF MEDICINE IN DE GENEESKUNDE
Transcript of PROGNOSE VAN ACUTE MYELOIDE LEUKEMIE - lib.ugent.be · AML-onderzoek op dit gebied binnen de...
Academiejaar 2012-2013
DE ROL VAN MICRORNA IN DE ONTWIKKELING EN
PROGNOSE VAN ACUTE MYELOIDE LEUKEMIE
Freddy VAN DAMME
Promotor: Prof. Dr. Jan Philippé
Scriptie voorgedragen in de 2de
Master in het kader van de opleiding tot
MASTER OF MEDICINE IN DE GENEESKUNDE
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Academiejaar 2012-2013
DE ROL VAN MICRORNA IN DE ONTWIKKELING EN
PROGNOSE VAN ACUTE MYELOIDE LEUKEMIE
Freddy VAN DAMME
Promotor: Prof. Dr. Jan Philippé
Scriptie voorgedragen in de 2de
Master in het kader van de opleiding tot
MASTER OF MEDICINE IN DE GENEESKUNDE
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Aan Kristien
aan Karel, Jan en Mieke
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Voorwoord
Mijn eerste en oprechte dank gaat uit naar Prof. Dr. Jan Philippé. Zijn beschikbaarheid,
motiverende begeleiding en vele suggesties zijn het fundament van dit werk. Tevens wil ik
Apr. Barbara Denys en Dr. Karl Vandepoele danken voor hun hulp en advies.
If humanity is to have a hopeful future, there is no
escape from the preeminent involvement and
responsibility of the single human soul, in all its
loneliness and frailty.
George F. Kennan (1904-2005)
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“De auteur en de promotor geven de toelating deze scriptie voor consultatie beschikbaar te
stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de
beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting
uitdrukkelijk de bron te vermelden bij het aanhalen van resultaten uit deze scriptie.“
Datum: 10 april 2013
Handtekening student : Handtekening promotor:
Freddy Van Damme Jan Philippé
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Inhoud
Abstract ................................................................................................................................................... 8
1 Inleiding ............................................................................................................................................... 9
1.1 Doel van de studie ......................................................................................................................... 9
1.2 Acute Myeloïde Leukemie ............................................................................................................ 9
1.3 MicroRNA .................................................................................................................................. 15
2 Methodiek .......................................................................................................................................... 18
3 Review Paper: “The Role of MicroRNAs in the Development of Chemoresistance Mechanisms in
Acute Myeloid Leukemia: any Hope for the Patient?” ......................................................................... 20
Abstract ............................................................................................................................................. 20
1 Introduction .................................................................................................................................... 20
2 A review of the role of some key microRNAs in AML ................................................................. 22
3 The Role of MicroRNAs in Chemoresistance Mechanisms .......................................................... 33
4 Review papers dealing with microRNA in AML .......................................................................... 38
5 Conclusions .................................................................................................................................... 40
4 Discussie ............................................................................................................................................ 42
5 Referenties ......................................................................................................................................... 45
6 Bijlagen (op CD) ................................................................................................................................ 53
Bijlage 1: Samenvattingen van de bestudeerde literatuur ................................................................. 53
Bijlage 2: Tabel MicroRNA‟s in AML ............................................................................................. 53
Bijlage 3: Humane hematopoiese en normale bloedcelwaarden ....................................................... 53
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Lijst van frequent gebruikte afkortingen
AK abnormaal karyotype
ALL acute lymfatische leukemie
AML acute myeloïde leukemie
APL acute promyelocytenleukemie
AraC cytarabine of cytosine arabinoside
ATRA all-trans retinoic acid
BI betrouwbaarheidsinterval
CA cytogenetisch abnormaal
CBFL core-binding factor leukemia
CDK cyclin-dependent kinase
CEBPA CCAAT/enhancer-binding protein-α
CLL chronische lymfatische leukemie
CR complete remissie
1,25-D3 1,25-dihydroxyvitamine-D3
FAB French-American-British
FLT3-ITD Fms-like tyrosine kinase – internal tandem duplication
HSC hematopoietische stamcel
LSC leukemische stamcel
MDR multiple drug resistance
miR microRNA
mRNA messenger RNA
MLL mixed lineage leukemia
NPM1 nucleophosmine1 gen
NPMc+ cytoplasmatisch nucleophosmine eiwit
RISC RNA-induced silencing complex
RNA ribonucleïnezuur
SCT stamceltransplantatie
siRNA small interfering RNA
3‟UTR 3’ untranslated region
wt wild type
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Abstract
Inleiding. De rol van microRNA‟s in de ontwikkeling van acute myeloïde leukemie (AML)
heeft gedurende de voorbij 5 tot 10 jaar toenemend aandacht gekregen. Dit is een relevant
onderzoeksdomein daar de prognose van patiënten met AML ongunstig is. De detectie van
specifieke microRNA‟s bij AML kan potentieel de diagnose, prognose en behandeling
verbeteren.
Methodologie. Dit werk is gebaseerd op een brede literatuurstudie over de rol van
microRNA‟s in AML en in het bijzonder bij chemoresistentie.
Resultaten. Specifieke patronen van geïnduceerde of onderdrukte microRNA‟s lijken
gecorreleerd te zijn met individuele AML-subklassen. Voor een beperkt aantal microRNA‟s
is een belangrijke rol in AML aangetoond. Deze zijn: miR-10, miR-29, miR-126, miR-155,
miR-181 en miR-223. Verhoogde expressie van miR-29b in AML is in verschillende studies
als gunstig beschreven. Dit microRNA onderdrukt de expressie van anti-apoptotische genen
en stimuleert de expressie van pro-apoptotische genen. Ook de miR-181-familie wordt
consistent als tumorsuppressor beschreven. Een hogere expressie van dit microRNA is
gecorreleerd met een significant betere prognose, maar het mechanisme hierbij is nog niet
opgehelderd. miR-223 heeft een sleutelfunctie in het blokkeren van de celcyclus-progressie
en in het induceren van granulocytaire differentiatie.
Eén van de belangrijke redenen van therapiefalen bij AML is de aanwezigheid chemo-
resistentie of multiple drug resistance (MDR). De microRNA‟s gecorreleerd met MDR lijken
sterk verschillend in de verschillende onderzoeken. Toch heeft de kennis van microRNA-
expressie in AML reeds geleid tot concrete klinische studies. Zo is zeer recent lenalidomide,
een immunomodulerende drug, in een muizenmodel aangewend om de expressie van de
tumorsuppressor miR-181a te versterken. Ook is de aanwezigheid van hogere gehaltes van de
tumorsuppressor miR-29b bij oudere uitbehandelde of bij zwakke patiënten belangrijk. De
aanwezigheid van hogere miR-29b expressie onder behandeling met decitabine leidde tot een
versterkt hypomethylerend en dus silencing effect op actieve oncogenen betrokken in AML.
Conclusie. In het toekomstige AML-onderzoek zal het belangrijk zijn de moleculaire
effecten van microRNA‟s in kritische pathways zoals apoptose, differentiatie, proliferatie,
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immuniteit en epigenetische gencontrole te doorgronden. Een beter moleculair inzicht kan
zeer gerichte microRNA-gerelateerde therapie mogelijk maken, die de huidige chemotherapie
effectiever maakt of ten dele kan vervangen.
1 Inleiding
1.1 Doel van de studie
Het doel van dit werk is het in kaart brengen en het samenvatten van de kernpunten van de
huidige kennis betreffende de rol van microRNA‟s bij de ontwikkeling van acute myeloïde
leukemie (AML). Als methodologie is gekozen voor een uitgebreide literatuurstudie van dit
onderzoeksgebied waarin een opmerkelijke dynamiek heerst. De aandacht gaat vooral naar de
rol van microRNA in de ontwikkeling, de diagnose, de prognose en de behandeling van
AML. Bijzondere aandacht gaat naar de potentiële rol van microRNA bij het ontwikkelen van
chemoresistentie bij leukemische blasten. De opgedane kennis dient in eerste instantie om het
AML-onderzoek op dit gebied binnen de vakgroep Klinische Biologie, Immunologie en
Microbiologie een sterker fundament te geven. Uit de literatuur blijkt immers welke
microRNA‟s het belangrijkste zijn bij de grootste subgroepen van AML. De literatuurstudie
leert ons in welke centra ter wereld er expertise is op het domein van AML-microRNA en
kan mee aanwijzingen geven aan de vakgroep: op welke subdomeinen is verder onderzoek
zinvol? Het overzicht maakt ook duidelijk met welke andere onderzoeksgroepen,
bijvoorbeeld binnen de Benelux, een samenwerking zinvol zou kunnen zijn. De
samenvattingen van de bestudeerde artikels (een 50-tal) zijn opgenomen in Bijlage 1. De
bestudeerde literatuur werd geïntegreerd in hoofdstuk 3. In overleg met de promotor is de
opgedane kennis samengevat in de vorm van een engelstalige Review Paper. Deze tekst is
een eerste voorstel van „final draft‟. De bedoeling is deze review paper in te sturen ter
publicatie in het tijdschrift “Leukemia Research”.
1.2 Acute Myeloïde Leukemie
Etiogenese
AML is de verzamelnaam van een heterogene groep van klonale maligniteiten van
hematopoietische progenitorcellen die ontstaan zijn uit de maligne transformatie van één
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stamcel of één voorlopercel (1). Hiervoor zijn minstens twee transformaties nodig. Een eerste
interfereert met de normale uitrijping van myeloblasten of verdere myeloïde voorlopers
waardoor deze geblokkeerd worden in een immature toestand, de tweede leidt tot een
ongecontroleerde proliferatie van deze onrijpe voorlopers (2). Dit leidt tot de accumulatie van
deze blasten in het beenmerg en later ook in het perifeer bloed. Een blastenpercentage in het
beenmerg hoger dan 20% is nodig voor de diagnose van „acute leukemie‟ tenzij er een
recurrente translocatie wordt gevonden (type t(15;17) of t(8;21) of inv(16)) (3). Meestal gaat
dit gepaard met een perifeer bloedbeeld dat ernstige anemie en trombopenie vertoont,
aangezien de snel prolifererende blasten-populatie het normale hematopoietische weefsel
verdringt (2). Deze beenmergpathologie verklaart dan ook de typische symptomen van acute
leukemie: bleekheid, snelle vermoeidheid en dyspnoe d’effort (anemie), recidiverende
infecties (granulocytopenie) en bloedingsneiging (trombopenie) (4). Afhankelijk van het type
AML kunnen ook andere symptomen optreden. Zo komt extramedullaire infiltratie vaker
voor in subtypes met monocytaire morfologie (AML-M5). Dit kan aanleiding geven tot
hypertrofisch tandvlees, lymfadenopathie, huidinfiltraties (chloromen) en hepato-
splenomegalie (2,5). In acute promyelocytenleukemie (APL of AML-M3) daarentegen is er
een hoge incidentie van DIC (disseminated intravascular coagulation) en een hoog risico op
spontane bloedingen in vitale organen. APL wordt dan ook als een medische urgentie
beschouwd en behandeld (2,5).
Incidentie - Prevalentie
AML komt vooral bij volwassenen voor (85%) en de incidentie ervan per 100.000 inwoners
stijgt met de leeftijd: van minder dan 1 tot meer dan 10 per 100.000 personen per jaar voor
jongeren en ouderen (> 65 jaar) respectievelijk (2). De mediane leeftijd bij diagnose is 66
jaar (6). De prevalentie is gemiddeld 3,8 gevallen per 100.000 inwoners en dit stijgt tot 17,9
gevallen per 100.000 personen ouder dan 65 jaar (1).
Klassieke FAB morfologische indeling (7)
Acute myeloïde leukemieën werden klassiek volgens een morfologische classificatie
ingedeeld op basis van de locatie van blokkade in de differentiatie binnen de myeloïde reeks.
Deze zogenaamde FAB (French-American-British) classificatie is weergegeven in Tabel 1.
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Tabel 1
FAB-classificatie van AML op basis van morfologie en differentiatie in de myeloïde lijn
(7).
M0 acute myeloblastenleukemie zonder uitrijping, < 3% aankleuring met Sudan Black
M1 acute myeloblastenleukemie zonder uitrijping, ≥ 3% aankleuring met Sudan Black
M2 acute myeloblastenleukemie met uitrijping
M3 acute promyelocytenleukemie (APL)
M4 acute myelomonocytenleukemie
M5 acute monocytenleukemie
M6 erythroleukemie
M7 megakaryoblastenleukemie
Cytogenetische en moleculaire indeling (WHO 2008)
De oorspronkelijke FAB classificatie van 1976 (7) op basis van morfologie werd in 2001 een
eerste maal en in 2008 verder geoptimaliseerd en vervangen door de huidige WHO AML-
indeling gebaseerd op acht subgroepen (8), zie Tabel 2. Deze indeling is gebaseerd op de
verworven kennis van de cytogenetische en moleculaire afwijkingen van de leukemische
cellen, opgedaan gedurende de voorbije 20 jaar. Dit is belangrijk daar deze indeling een
biologisch relevanter idee geeft van de leukemie en een duidelijkere voorspelling van de
prognose mogelijk maakt dan de oorspronkelijke morfologische FAB-classificatie. De
correlatie van cytogenetische en moleculaire eigenschappen van AML met klinische
prognose is in Tabel 3 weergegeven. Deze is voorgesteld als standaard door Döhner et al. in
naam van de Europese LeukemiaNet groep rond AML in 2010 (3).
Het overzicht in Tabel 2 toont aan dat AML een verzamelnaam is die een bijzonder
heterogene groep van subtypes omvat. Bij ongeveer 55% van de volwassen patiënten worden
specifieke cytogenetische afwijkingen gedetecteerd (9). Deze worden in de literatuur vaak als
CA-AML (cytogenetisch abnormaal) of als AK-AML (abnormaal karyotype) patiënten
aangeduid. Zoals blijkt uit Tabel 3 hebben deze chromosomale afwijkingen vaak een
prognostische significantie. Zo zijn b.v. t(15;17), t(8;21) of inv(16) meestal gecorreleerd met
een gunstige prognose.
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Tabel 2
WHO 2008-classificatie van AML en gerelateerde precursor neoplasmata, en acute
leukemieën van ambigue lineage (3,8).
1. AML met terugkomende genetische abnormaliteiten
a. AML met t(8;21)(q22;q22); RUNX1-RUNX1T1
b. AML met inv(16)(p13;1q22) of t(16;16)(p13.1;q22); CBFB-MYH11
c. APL met t(15;17)(q22;q22); PML-RARA *AML-M3 of APL
d. AML met t(9;11)(p22;q23); MLLT3-MLL
e. AML met t(6;9)(p23;q34); DEK-NUP214
f. AML met inv(3);(q21q26.2) of t(3;3)(q21;q26.2); RPN1-EVI1
g. AML (megakaryoblastisch) met t(1;22)(p13;q13); RBM15-MKL1
Provisoire entiteit: AML met gemuteerd NPM1
Provisoire entiteit: AML met gemuteerd CEBPA
2. AML met myelodysplasia-gerelateerde veranderingen
3. Therapie-gerelateerde myeloïde neoplasmata
4. AML, niet anders gespecificeerd (AML-NOS : Not Otherwise Specified)
a. AML met minimale differentiatie AML-M0
b. AML zonder maturatie AML-M1
c. AML met maturatie AML-M2
d. Acute myelomonocytaire leukemie AML-M4
e. Acute monoblastische/monocytaire leukemie AML-M5
f. Acute erythroïde leukemie AML-M6
i. Pure erythroïde leukemie
ii. Erythroleukemie, erythroïd/myeloïd
g. Acute megakaryoblastaire leukemie AML-M7
h. Acute basofylaire leukemie
i. Acute panmyelose met myelofibrose (synoniem: acute myelofibrose; acute
myelosclerose)
5. Myeloïd sarcoma (synoniem: extramedullaire myeloïde tumor; granulocytair sarcoma;
chloroma)
6. Myeloïde proliferaties gerelateerd aan Downsyndroom
a. Transiënte abnormale myelopoiese (synoniem: transiënte myeloproliferatieve
aandoening )
b. Myeloïde leukemie geassocieerd met Downsyndroom
7. Blastisch plasmacytoïd dendrietisch cel neoplasma
8. Acute leukemieën van ambigue lineage
a. Acute ongedifferentieerde leukemie
b. Gemengd fenotype acute leukemie van t(9;22)(q34;q11.2); BCR-ABL1
c. Gemengd fenotype acute leukemie van t(v;11q23); MLL herschikking
d. Gemengd fenotype acute leukemie, B/myeloïd, NOS
e. Gemengd fenotype acute leukemie, T/myeloïd, NOS
Provisoire entiteit: NK-cel lymfoblastaire leukemie/lymphoma
* De oorspronkelijke FAB subtypes zijn in deze tabel toegevoegd. De types AML-M0-M7
komen in de AML-NOS entiteit in dit systeem, met uitzondering van AML-M3. Zie ook
verdere commentaren in (3) over deze tabel.
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Tabel 3
Gestandaardiseerde rapportering voor de correlatie van cytogenetische en moleculair
genetische data in AML met klinische data (3).
Genetische groep Subsets
Gunstig t(8;21)(q22;q22); RUNX1-RUNX1T1
Inv(16)(p13.1; q22) of t(16;16)(p13.1;q22); CBFB-MYH11
Gemuteerd NPM1 zonder FLT3-ITD (normaal karyotype)
Biallelische mutatie in CEBPA (normaal karyotype)
Intermediair-I Gemuteerd NPM1 en FLT3-ITD (normaal karyotype)
NPM1-wt en FLT3-ITD (normaal karyotype)
NPM1-wt zonder FLT3-ITD (normaal karyotype)
Intermediair-II t(9;11)(p22;q23); MLLT3-MLL
Cytogenetische afwijkingen niet geclassificeerd als
gunstig of ongunstig
Ongunstig inv(3)(q21;q26.2) of t(3;3)(q21;q26.2); RPN1-EVI1
t(6;9)(p23;q34); DEK-NUP214
t(v;11)(v;q23); MLL herschikking
-5 of del(5q); -7;abnl(17p); complex karyotype
Bij ongeveer 45% van de AML-patiënten zijn er echter geen detecteerbare chromosomale
veranderingen; deze worden in de literatuur als CN-AML of NK-AML patiënten aangeduid,
zijnde cytogenetisch normaal of normaal karyotype patiënten (10). Dit is een bijzonder
heterogene groep. De leukemische blasten van deze patiënten tonen specifieke mutaties en/of
een deregulering in de expressie van specifieke genen (3). Zeer frequent voorkomende
mutaties zijn deze van de NPM1, CEBPA en FLT3-genen. Mutaties in deze genen zijn ook
gecorreleerd met de ziekteprognose, zie Tabel 3. Zo hebben patiënten met een FLT3-ITD
(interne tandem duplicatie in het FLT3-gen) een slechtere prognose dan patiënten met het
FLT3-wt-gen, terwijl de aanwezigheid van NPM1 of CEBPA mutatie meestal geassociëerd is
met een gunstige prognose (3). Naast de prognose voorspeld op basis van de gedetecteerde
chromosomale aberraties (cytogenetica) en de moleculair genetische afwijkingen (genmutatie
en expressie), kan de prognose ook voorspeld worden op basis van enkele klinische factoren.
Dit zijn: 1) de leeftijd bij het stellen van de AML-diagnose (hoe ouder, hoe slechter), 2) de
aanwezigheid van voorafbestaande afwijkingen passend bij een myelodysplasie voorfase
(slechtere prognose), en 3) vooral het resultaat van de eerste chemotherapiekuur: een vroege
remissie vertaalt zich in een significant beter eindresultaat (2).
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Behandeling
De huidige diagnose- en behandelingsmogelijkheden van AML bij volwassenen zijn recent
samengevat in een bijzonder gedetailleerde “Landmark Paper” van Döhner et al. in opdracht
van de Europese LeukemiaNet groep (3). De hoeksteen van de behandeling is nog steeds een
intensieve combinatie-chemotherapie ingedeeld in minstens twee fasen: 1) inductie-therapie,
en 2) consolidatie-therapie. De eerste fase beoogt remissie-inductie, d.i. het normaliseren van
het beenmerg zodat de morfologie ervan niet meer onderscheiden kan worden van normaal
beenmerg: het is normocellulair en er zijn minder dan 5% blasten. Bij deze patiënten is dan
een zogenaamde complete remissie (CR) bereikt. In de inductietherapie wordt meestal de
“3+7-standaard” toegepast: drie dagen met een anthracycline-behandeling (b.v.
daunorubicine) gevolgd door 7 dagen met cytosine-arabinoside (AraC) (3). Na deze therapie
zijn echter niet alle leukemiecellen gedood: er blijft een minimale ziekterest (minimal
residual disease - MRD) die onder de morfologische (lichtmicroscopische) detectiegrens ligt,
maar mogelijks niet onder de cytogenetische en moleculaire drempel van 109-10
10 residuele
tumorcellen (2). Om onder deze drempel te dalen wordt verder behandeld. Dit wordt de
consolidatie-therapie of postremissie-therapie genoemd. Afhankelijk van het type AML, de
leeftijd en patiëntspecifieke condities (comorbiditeit, herval), kan dit intensieve
conventionele chemotherapie zijn, langere-duur onderhoudsbehandeling, chemo-intensificatie
met b.v. AraC, eventueel gevolgd door een autologe- of allogene stamceltransplantatie als
laatste stap (3). De genezingskans is het hoogst na allogene stamceltransplantie (alloSCT).
Vanwege het hoge risico op mortaliteit verbonden met alloSCT, is deze therapie niet voor
alle patiënten geïndiceerd. Stamceltransplantatie zal voornamelijk toegepast worden bij
jongere personen met een hoogrisico-AML of bij herval, na het vinden van een geschikte
donor (2,3,5).
Ziekteprognose
In Nederland steeg de 5-jaarsoverleving bij AML met 8% in de periode van 1989-93 tot
2004-8: van 8% (95% - BI: 6-11%) tot 16% (95% - BI: 13-20%). De toegenomen overleving
na AML is echter sterk afhankelijk van de leeftijd van de patiënten: er werd een stijging van
20% naar 35% gemeten in de AML-populatie jonger dan 65 jaar in deze periode, terwijl de
overlevingswinst voor AML-patiënten ouder dan 65 jaar slechts met 1% was gestegen en nog
steeds lager was dan 10% in de periode 2004-8 (11). Dergelijke prognose-cijfers vindt men
ook wereldwijd. Döhner meldt een genezing van 20-75%, die zoals bovenvermeld zeer sterk
afhankelijk is van het cytogenetische en moleculair subtype, en uiteraard van de
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patiëntspecifieke factoren (leeftijd, algemene conditie, comorbiditeit). Voor patiënten ouder
dan 60 jaar werd een genezing door chemotherapie lager dan 10% vermeld (1).
1.3 MicroRNA
MicroRNA‟s zijn enkelstrengige, niet-coderende RNA-ketens met een lengte van 18-25
nucleotiden die de expressie van celproces-regulerende genen beïnvloeden (12). Zij regelen
de expressie van ten minste 5000 genen (13). Hun rol in fundamentele celprocessen zoals
ontwikkeling, differentiatie, proliferatie, overleving en apoptose is temporeel en zeer
weefselspecifiek (14). De rol van endogene microRNA‟s (lin-4, let-7) werd voor het eerst
beschreven door Lee, Feinbaum en Ambros in 1993 in hun genetische studie van C. elegans
mutanten met ontwikkelingsstoornissen (15). Functionele microRNA‟s worden gevormd in
een meerstapsproces in de nucleus en het cytoplasma (12,16). De eerste stap bestaat uit de
transcriptie van het microRNA gen door RNA Polymerase II wat een primair transcript of een
zogenaamd pri-microRNA geeft. Dit is typisch 1-3 kilobasen lang. Deze pri-microRNA‟s
worden in de kern omgevormd tot pre-microRNA‟s (hairpin intermediates) door de
ribonucleasen Drosha en DGCR8. De pre-microRNA‟s zijn 70-100 nucleotiden lang. Deze
worden vanuit de nucleus naar het cytoplasma getransfereerd door het proteïne Exportin-5. In
het cytoplasma worden de pre-microRNA‟s door het Dicer ribonuclease omgevormd in
mature dubbelstrengige korte microRNA‟s (18-25 bp, meestel 21-22 bp). Splitsing van deze
korte RNA-dubbelstrengen in enkelstrengen levert de finale microRNA -5p en -3p strengen.
Een van beide strengen wordt in het RNA-induced silencing complex (RISC) ingebouwd wat
toelaat dat deze hybridiseert met het doelwit (target) mRNA. Dit gebeurt op basis van de
complementariteit tussen de zogenaamde seed sequentie van het microRNA (positie 2 tot 8
van het 5‟ eind) met het 3’untranslated region (3‟UTR) van het doelwit mRNA. De
baseparing tussen het microRNA en het mRNA in deze stap zal resulteren in de afbraak
(slicing) van mRNA, of tot het onderdrukken van de mRNA translatie. Beide processen
resulteren in een post-transcriptionele genrepressie en leiden daarbij tot een gedaalde
proteïneconcentratie. Deze cytoplasmatische post-transcriptionele genrepressie door
microRNA‟s wordt momenteel als hun belangrijkste taak beschouwd (16). Recent zijn
microRNA‟s ook beschreven als activatoren van de eiwit translatie (17,18) en als regulatoren
van de transcriptie in de nucleus (19). Verder is ook aangetoond dat microRNA‟s de mRNA
translatie kunnen moduleren (decoy activity), onafhankelijk van het RISC-complex (20). De
vorming en werking van microRNA‟s is schematisch weergegeven in Figuur 1.
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Figuur 1: MicroRNA biogenese en effector pathways – uit (16).
a: transcriptie door RNA polymerase II leidt tot pri-microRNA, Drosha vormt hieruit pre-microRNA; b: pre-
microRNA transport van nucleus naar cytoplasma door Exportin 5; c: microRNA-duplex vorming door Dicer; d:
mature microRNA streng wordt geïncorporeerd in het RISC; e: microRNA-mRNA hybridisatie in het RISC
leidt tot mRNA afbraak, of tot inhibitie of activatie van de mRNA translatie, f: decoy activity: binding van
microRNA aan RNA-bindende proteïnen; g: regulatie van gen-transcriptie door microRNA.
De deregulering van microRNA in kanker werd voor het eerst beschreven door Calin et al. in
2002 in een genetische studie van de grote subgroep van patiënten met chronische
lymfatische leukemie (CLL) met 13q-deletie. Zij toonden aan dat miR-15 en miR-16, gelegen
op 13q14, onderdrukt waren in 68% van de CLL-patiënten (21). Kort na deze publicatie werd
aangetoond dat specifieke microRNA-patronen konden geassocieerd worden met andere
17
maligne kankers (22,23). Deze artikels initieerden een nieuwe focus binnen het oncologisch
onderzoek en sindsdien gaat er wereldwijd veel aandacht naar de rol van microRNA‟s in het
ontstaan, de diagnose, de prognose en behandeling van kanker (16). Op het gebied van CLL
en de rol van microRNA‟s in de CLL-etiogenese was de publicatie van de Croce-groep in
2011 een mijlpaal (24). Zij leverden hierbij een sluitende moleculaire verklaring voor de
gunstige prognose van de indolente CLL-vormen met 13q-deletie en bewezen dat de miR-
15a/miR-16-1 cluster onderdrukking op locus 13q14 het “primum movens” was in een
microRNA/TP53 circuit dat geassocieerd is met CLL-pathogenese en -outcome.
De rol van microRNA‟s met betrekking tot aspecten van AML krijgt sinds 2005 ook steeds
meer aandacht. De leidende researchgroep binnen dit onderzoeksdomein is zonder twijfel
deze van Marcucci en Croce van het Comprehensive Cancer Center van de Ohio State
University in de USA. Deze groep heeft baanbrekend werk verricht omtrent het belang van
microRNA in de etiogenese (9,25,26), de diagnose en prognose (8, 26-32) en in de
behandeling van AML (16,33,34,35). In een 2010 Nature Reviews artikel hebben Garzon et
al. (16) gepostuleerd dat microRNA‟s zowel als oncogen of als tumorsuppressorgen kunnen
fungeren. Zo kan een microRNA dat in normale fysiologische condities de expressie van een
oncogen onderdrukt, als een tumorsuppressor (gen) gedefinieerd worden. In pathologische
omstandigheden leidend tot mutatie van dit microRNA-gen, door deletie van dit gen, door
methylatie van de microRNA promotor, of door andere processen die de microRNA-
expressie onderdrukken, leidt dit tot gedaalde microRNA-expressie en dus tot een abnormaal
gestegen expressie van het target oncogen en kan dit bijdragen tot carcinogenese. Analoog
kan de gestegen expressie of amplificatie van een microRNA dat een tumorsuppressor
mRNA als doelwit heeft, leiden tot onderdrukking van deze tumorsuppressor en eveneens
leiden tot belangrijke stappen in de ontwikkeling en evolutie van kanker zoals apoptose-
inhibitie, celproliferatie, invasie en angiogenese. Zie Figuur 2.
18
Figuur 2: MicroRNA’s als oncogenen en tumorsuppressor-genen – uit (16).
a: een microRNA dat normaal de expressie van een oncogen onderdrukt, kan gedefinieerd worden als een
tumorsuppressor en is vaak afwezig in tumorcellen. Het verlies van dit microRNA door b.v. mutatie, deletie, of
biogenese-afwijkingen zal resulteren in een abnormaal verhoogde expressie van het target oncogen. Dit kan
bijdragen tot oncogene processen zoals gestegen proliferatie, angiogenese, invasie of gedaalde apoptose. b: de
amplificatie of overexpressie van een microRNA dat een tumorsuppressor onderdrukt kan bijdragen tot
tumorigenese door het stimuleren van b.v. proliferatie, angiogenese, invasie of door het onderdrukken van
apoptose of door het verhogen van genetische instabiliteit.
2 Methodiek
Deze thesis is gebaseerd op een grondig literatuuronderzoek. Daar microRNA-AML
onderzoek nieuw is binnen de vakgroep, was de eerste bedoeling de recente kennis over de
rol van microRNA‟s in de ontwikkeling, diagnose en prognose van AML breed in kaart te
brengen. Daarnaast werd er specifiek gezocht naar de potentiële rol van microRNA‟s in het
mechanisme van chemoresistentie of multiple drug resistance (MDR) in AML. Hiertoe
werden verschillende searches in PubMed gedaan. Typische zoekpatronen waren gebaseerd
op “AML and microRNA”, “AML and chemoresistance”, “AML and MDR”, “AML and
multiple drug resistance and microRNA”, “microRNA and resistance and leukemia”. Dit
leverde publicaties op waarbij door kruisreferenties verdere artikels geïdentificeerd werden.
Er werd ook specifiek op auteurs gezocht, b.v. “Legrand and leukemia”. Dit samen leverde
een honderdtal relevante artikels waarvan er ongeveer zestig recent waren gepubliceerd in
peer-reviewed journals. Deze werden in detail bestudeerd en samengevat (zie Bijlage 1).
19
Deze vormden de basis voor het schrijven van een eerste aanzet tot een Review Paper voor
het tijdschrift Leukemia Research.
20
3 Review Paper: “The Role of MicroRNAs in the Development of
Chemoresistance Mechanisms in Acute Myeloid Leukemia: any Hope for
the Patient?”
Freddy Van Damme, Karl Vandepoele, Barbara Denys, Jan Philippé*
Department of Clinical Biology, Immunology and Microbiology
Faculty of Medicine, Ghent University and Ghent University Hospital
De Pintelaan 185, 9000 Gent, Belgium.
Abstract
Acute myeloid leukemia (AML) is a heterogeneous group of different clonal disorders of
hematopoietic progenitor cells in the bone marrow. Today‟s AML treatment is strongly based
on cytotoxic multi-chemotherapy and may be followed by stem cell transplantation. In
general, attempts to increase response rates by the use of modulators of multiple drug
resistance (MDR) have failed. The role of microRNAs in the leukemogenesis process has
gained increased attention in the past 5-10 years. MicroRNA expression profiling is
suggested to be valuable in fine-tuning diagnosis and prognosis, in classifying AML
subtypes, and in treatment stratification. A number of studies focused on unraveling the
molecular mechanisms of microRNAs in the development of MDR with the goal to find
potential molecular switches able to modulate chemosensitivity. The current paper briefly
reviews a number of microRNAs which have been consistently associated with AML
development and focuses mainly on reviewing the current knowledge about the role of
microRNA in the development of chemoresistance in AML.
Keywords: AML, microRNA, chemoresistance, MDR
1 Introduction
Acute myeloid leukemia (AML) is a very heterogeneous group of different clonal disorders
of hematopoietic progenitor cells in the bone marrow, so called blasts (1). These can show
many different cytogenetic abnormalities and molecular aberrations (3,36). This
21
heterogeneity is acknowledged in the 2008 WHO classification for AML (8). The clonal
population of blasts has lost its ability to differentiate to final functional blood cells and is
proliferating malignantly (37). This leads to an accumulation of blasts in the bone marrow
which eventually will replace the normal hematopoietic tissue (1). AML incidence increases
from less than 1/100,000 persons/year for younger patients to more than 10/100,000
persons/year for patients above 60 years. An average age-adjusted incidence of 3.6 cases per
100,000 persons per year in the USA has been reported (6). In general, AML treatment is
based on cytotoxic chemotherapy and, depending on the specific cytogenetic and molecular
subtype and further clinical prognostic factors, leads to cure rates of 20-75% (1). Especially
the prognosis for older patients is dismal, with only about 10% of long-term (5-yr) survival
with current standard intensive chemotherapy (1). As a postremission strategy, autologous or
allogeneic stem cell transplantation (allo-SCT) in specific subgroups offers significant overall
survival for patients with intermediate- and high risk AML (3).
MicroRNAs are a class of small (18-25 nt) non-coding RNAs and their role in oncogenesis
has gained much attention in the past decade (9,12,26). The endogenous role of microRNAs
was first described by Lee, Feinbaum and Ambros in 1993 in their study of mutations in the
lin-4 and let-7 genes in the nematode C. elegans (15). They showed that the transcript of lin-4
is complementary to the 3‟UTR of the lin-14 and lin-28 genes, important regulators in the
larval development. The base-paring of the short lin-4 microRNA with the lin-14 mRNA,
leading to a double-stranded RNA, led to a decomposition of the lin-14 mRNA. As such, the
mechanism of post-transcriptional gene repression was first described. Since then, much
knowledge has been gathered on the mechanisms of formation and action of microRNAs (13,
14). Next to their predominant role in mRNA downregulation (12,38), recent papers have
also described miRNAs as activators of gene expression (17,18), of being able to bind to
ribonucleoproteins independent of RISC (with decoy activity) as modulators of regulation of
mRNA translation (20, 39), or can regulate gene expression at the transcriptional level by
binding directly to DNA (19). The first paper linking microRNA expression changes with
cancer was published by Calin et al. in 2002 (21). They described the downregulation of
miR-15 and miR-16 in the B-lymphocytes of a majority of chronic lymphatic leukemia
(CLL) patients, more specifically those with 13q deletions. This work was continued and a
small decade later, in 2011 (24), the Croce group published their landmark paper which
unraveled the detailed molecular pathway which associated the miR-15a/miR-16-1 cluster
downregulation with TP53, miR-34b/miR-34c and ZAP70 expression. As such, they
22
associated a microRNA/TP53 feedback circuit with pathogenesis and outcome of B-CLL and
presented a novel pathogenic model for association of 13q deletions with the indolent form of
CLL. Since the first paper of this group (21), the detection of distinctive microRNA
expression changes associated with cancer pathogenesis and prognosis has gained increasing
attention (16). This is particularly relevant as it is predicted that the human microRNAs
(about 1000 or more) control 20-30% of the human genome (13). Furthermore, it has been
proposed that microRNAs which normally downregulate the expression of an oncogene can
be defined as tumor suppressors and are often downregulated in cancer (16). Similarly, the
overexpression of microRNAs which downregulate tumor suppressors might contribute to
tumorigenesis. The current review paper aims at summarizing some of the important findings
on microRNA-AML association of the last decade and focuses on reviewing the involvement
of microRNAs with drug resistance mechanisms in AML. This might indicate how AML
diagnosis, prognosis and treatment might be improved. Hopefully rather sooner than later,
this might give hope to people hearing the adverse diagnosis of AML.
2 A review of the role of some key microRNAs in AML
miR-10a
Garzon et al. (31) identified distinctive microRNA signatures of AML with cytoplasmic
nucleophosmin (NPMc+), the result of an NPM1 mutation. Mutations of the NPM1 gene are
the most common genetic alteration in CN-AML (50-60%), and in the absence of FLT3-ITD
mutation, have been associated with higher complete remission rates and event-free survival
(3). In comparing NPMc+ AML bone marrow samples (n=55) versus NPM1-wt (n=30)
samples, a microRNA signature of upregulated miR-10a, miR-10b, and multiple let-7 and
miR-29 family members was identified. High expression of HOX genes is one of the most
distinguishing features of NPM1 AML (40). Whether the HOX imbedded microRNAs (e.g.
miR-10a and -10b) have a critical role in the expression of the HOX genes, remained to be
elucidated. Ovcharenko et al. (41) studied microRNA expression and their association with
NPM1 mutation in intermediate risk AML patients. It was shown that NPMc+ mutant AML
samples had a significant higher miR-10a expression in comparison to NPM1-wt samples. In
vitro studies using several human leukemia cell lines revealed a downregulation of MDM4
(murine double minute 4) gene expression after pre-miR-10a transfection. MDM4 is known
as a mediator of cellular stress that, amongst others, interferes with p53 activation. The
23
expression of MDM4 was also studied in intermediate risk AML patient samples with and
without NPM1 mutation. This revealed a trend (P = 0.07) for lower MDM4 expression in
AML patients with a mutated NPM1 versus patients with wild-type NPM1. Luciferase
reporter assays demonstrated that miR-10a has MDM4 as a direct target. The hypothesis was
that overexpression of miR-10a leads to MDM4 downregulation, which in turn leads to a
decrease of the MDM4-mediated repression of the p53-mediated transcriptional activation.
This p53 repression, as described in previous papers (42), occurs through a direct interaction
of MDM4 with p53 at its promoter binding sites. As such, lower levels of MDM4 lead to the
activation of p53, and so to the favorable prognosis of NPM1-mutant patients. The fact that
only a trend was observed for lower MDM4 expression in NPM1-mutant patient suggests that
additional mechanisms might contribute to oncogenic pathways in NPM1c+ AML patients.
miR-29a
Han et al. (43) used mouse bone marrow samples, normal human and AML bone marrow
cells as well as 293T cells (human embryonic kidney cells) in their study of the miR-29a-
induced self-renewal capacity of myeloid progenitors. It was shown that miR-29a expression
was high in hematopoietic stem cells (HSC) and was downregulated in progenitor cells. The
hypothesis was that miR-29a plays a unique role in the earliest stages of hematopoietic
development. An abnormal expression of this single microRNA might be sufficient to induce
proliferation and self-renewal in progenitors and might be a first step in malignant
transformation. Followed by further accumulating (epi)genetic „hits‟ during prolonged cell
life, this might lead to the final leukemic clone. To validate this hypothesis, mouse
hematopoietic stem / progenitor cells were modified to express miR-29a. This resulted in the
acquisition of a self-renewal capacity of myeloid progenitors, a granulocyte/macrophage
lineage bias amongst the myeloid progenitors, and the development of a myeloproliferative
disorder that progressed to AML. This demonstrated that miR-29a could be seen as a bona
fide oncogene, in contradiction to miR-29b which had been described as tumor suppressor
(44). Several tumor suppressor genes and regulators of the cell cycle were identified as
targets of miR-29a. One of these was HBP1, a known negative regulator of cell cycle
progression (45). However, knockdown of the HBP1 gene in mouse bone marrow cells using
short hairpin RNA did not yield the same effect as the ectopic overexpression of miR-29a.
This indicated that the function of miR-29a in leukemogenesis involves other targets that may
act alone or in concert with HBP1 downregulation. In a small population of human AML
LSC (Lin-CD34
+CD38
-) and non-LSC (Lin
-CD34
+CD38
+) blasts (n=12), the authors found a
24
significantly increased expression of miR-29a in all AML blasts samples, at levels similar to
those in multipotent progenitors. This was accompanied with a reduced HBP1 mRNA level in
89% (8/9) of the studied samples. Using control 293T cells versus 293T cells overexpressing
miR-29a, it was shown that the latter entered the S/G2 phase more rapidly. Based on this
result, the authors suggested that miR-29a might serve as a potential target for AML therapy.
Teichler et al. (46) studied the microRNA signature in AML patients with monosomy 7 (-7)
or deletion of 7p (del7q), known to have a poor prognosis (3). The group previously reported
that the nucleoprotein Ski was upregulated in AML, especially in -7/del7q, which presumably
represses the retinoic acid-induced myeloid differentiation (47). The hypothesis was that loss
of 7q explains the Ski upregulation and this is caused by reduced expression of microRNAs
located on 7q. Using AML samples (n=17) with isolated -7/-7q or complex karyotypes
including this deletion, versus normal bone marrow samples (n=8), it was shown that the only
microRNA that was downregulated and is localized on 7q, was miR-29a. By transfection
experiments in HL-60 cells with microRNA precursors known to have their locus on 7q, only
the miR-29a precursor effectively lowered the Ski protein level. Furthermore, also the
expression of the monocytic differentiation marker CD11b had increased after miR-29a
transfection. This effect could be enhanced by ATRA (all-trans retinoic acid) induction.
Luciferase reporter assays validated a functional binding site for miR-29a in the 3‟UTR of
SKI. The in vivo inverse relation between Ski and miR-29a expression was further analyzed
in AML samples (n=21). Samples with very high miR-29a expression revealed low Ski
expression (P=0.02). This suggests that miR-29a regulates Ski expression in AML and
suggests a tumor suppressor role for miR-29a in this pathway. Wang et al. (48) detected the
upregulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cells
and in CD34+ hematopoietic/progenitor cells. By gain-of-function and loss-of-function
experiments, it was demonstrated that both microRNAs promote the PMA (phorbol 12-
myristate 13-acetate)-induced monocytic and ATRA-induced granulocytic differentiation of
HL-60, THP-1 and NB4 cells. The data showed that both microRNAs directly inhibit the
cyclin T2 gene (CCTN2), preventing the release of hypophosphorylated retinoblastoma (Rb)
protein and resulting in the induction of monocytic differentiation. In addition, it was shown
that the CDK6 and TAB2 genes, targets of miR-29a and miR-142-3p respectively, are
involved in the regulation of both monocytic and granulocytic differentiation. A significant
decrease of miR-29a and miR-142-3p levels associated with a parallel increase in their target
protein levels in blasts from AML patients. Using lentivirus-mediated transfer, it was
demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic
25
stem/progenitor cells from healthy controls and AML patients downregulated the expression
of their targets and promoted myeloid differentiation. These findings confirmed that miR-29a
and miR-142-3p are key regulators of normal myeloid differentiation. As it was universally
observed that the two studied microRNAs were downregulated in AML M1 to M5 subtypes,
the authors stated that endogenous expression or ectopic implantation of miR-29a and miR-
142-3p may be a potential strategy in AML therapy.
miR-29b
Garzon et al. (33) studied the functional role of miR-29b in leukemogenesis. They
demonstrated that restoration of miR-29b expression in cell lines and primary AML samples
induced apoptosis and dramatically reduced tumorigenicity in a xenograft mouse model.
Transcriptome analysis after ectopic transfection of synthetic miR-29b into leukemia cells
indicated that miR-29b targets apoptosis, cell cycle, and proliferation pathways. In the
studied population of 45 primary AML samples, a significant enrichment for apoptosis genes,
including MCL-1, was found to be inversely correlated with miR-29b expression. It has been
described (49) that the Bcl-2 family is composed of both pro-apoptotic (e.g. Bcl-2, Mcl-1 and
Bcl-Xl) and anti-apoptotic proteins (BH3 proteins such as Bim, tBid, and Puma), and the
authors state that the balance of these two determines life or death for the cell. It was shown
that miR-29a and miR-29b not only directly target anti-apoptotic genes but also upregulate
pro-apoptotic genes, such as BIM (BCL2L11) and the tumor suppressor PDCD4 (50,51).
Based on the current work and the integration of that with previous knowledge, the final
hypothesis was that through the miR-29‟s targeting of MCL-1 and upregulation of the BIM
and PDCD4 transcripts, the impact of miR-29 on the survival of AML cells could be even
more robust. As such, the collected data supported a tumor suppressor role of miR-29b, and
the use of synthetic miR-29b might be considered as novel strategy to improve treatment
response in AML. Eyholzer et al. (52) studied the CEBPA (CCAAT/enhancer-binding
protein-α) mediated regulation of the tumor suppressor miR-29b. About 10% of the AML
patients show dominant-negative mutations in the CEBPA coding region (53). CEBPA has
been described as a master regulator active in the normal hematopoiesis process and crucial
for myeloid differentiation towards mature granulocytes (54,55). Previous literature showed
CEBPA to be a regulator of miR-223 (56) while CEBPA expression was suppressed by the
leukemic fusion proteins AML1-ETO, AML1-MDS1-EVI1 and CBFB-MYH11 in AML
patients with t(8;21), t(3;21) and inv(16) chromosomal rearrangements respectively
(57,58,59). Using Kasumi-1 cells conditionally expressing CEBPA, an 18-membered miR
26
signature was identified which associated with CEBPA upregulation. Focusing on the miR-29
cluster showed miR-29b to be suppressed in AML patients with impaired CEBPA function or
loss of chromosome 7q. It was demonstrated that CEBPA selectively regulates miR-29b
expression on its miR-29a/b1 locus on 7q32.3, while the 1q32.2 locus containing the miR-
29b2/c remains unaffected. As such, the authors provided a rationale for miR-29b
suppression in AML patients with disrupted CEBPA or aberrations on chromosome 7. Xiong
et al. (60) studied the expression of miR-29b in a cohort of 56 AML patients in relation to
MLLT11 expression. High expression of MLLT11, an MLL fusion partner, is a poor
prognostic biomarker in pediatric AML, adult CN-AML and adult MDS (3). They showed
that low miR-29b (and also miR-29a and -29c) expression was inversely related to MLLT11
expression (P < 0.05) in the AML patient population and the cell lines used (the leukemic cell
line REH and two lung cancer cell lines H157 and SKMES1). Their study focused on miR-
29b as only the expression of miR-29b had a significant predictive power for overall survival
in the studied cohort. Through transfection experiments the authors demonstrated that
MLLT11 expression was directly regulated by miR-29b. Furthermore they speculated that
deletion of the tumour suppressor miR-29b might be mechanistically responsible for the
pathogenesis of MDS/AML with -7q/monosomy 7 and that miR-29b may be a potential
prognostic biomarker for AML patients.
miR-125b, miR-126
Li et al. (61) studied the microRNA signatures associated with the four most common
rearrangements in AML, i.e. t(8;21)/AML1(RUNX1)-ETO(RUNX1T1), inv(16)/CBFB-
MYH11, t(15;17)/PML-RARA and the MLL/11q23 group. These count for about 30% of all
AML cases and are, except for the MLL group, associated with good outcome (31). It was
demonstrated that a minimum of two (miR-126-5p/126-3p), three (miR-224, miR-368, miR-
382) and seven microRNAs (miR-17-5p, miR-20a and the five abovementioned ones) could
accurately discriminate core-binding factor leukemias (CBFL: t(8;21) and inv(16)), t(15;17)
and MLL-rearrangement AMLs respectively, from each other. The precursor of the miR-126
gene is embedded in a 287-bp CpG island and it was shown that elevated expression of miR-
126 in CBF AML was associated with demethylation of the promoter and not with
amplification or mutation of the genomic locus. Gain-of-function and loss-of-function
experiments further showed that the microRNAs inhibited apoptosis and increased the
viability of AML cells. Enhanced colony-forming ability of mouse normal bone marrow
progenitor cells alone and particularly in cooperation with the fusion protein AML1-ETO was
27
obtained. Luciferase reported assays indicated the tumor suppressor PLK2 (Polo-like kinase
2) to be a bona fide target of miR-126. As such, these microRNAs would act as oncogene in
CBF AML development. As miR-126 was almost uniquely overexpressed in CBF AMLs, the
authors speculate that its overexpression may contribute to CBF leukemia as a second hit,
cooperating with the primary hit caused by the fusion proteins AML1-ETO or CBFB-
MYH11. This is supported by the fact that miR-126 inhibits apoptosis and enhances cell
proliferation alone, and particularly, in cooperation with the t(8,21) fusion gene. Further
studies would be needed to validate this hypothesis. Zhang et al. (62) studied microRNA
expression in pediatric AML and ALL (n=111, younger than 14 yrs) based on a test cohort of
36 patients and 7 normal controls and an independent validation cohort of 63 patients and 5
normal donors. Among other upregulated microRNAs (miR-100, -335, -146a, -99a), miR-
125b was highly expressed in the pediatric AML bone marrow samples versus controls,
especially in the M3 subtypes. The AML-M3 or acute promyelocytic leukemia (APL)
subgroup, with t(15;17)(q22;q12) and formation of the PML-RARA fusion gene, is
characterized by differentiation arrest at the promyelocytic stage of development. APL
represents about 10% of the pediatric AML cases (63). It was further demonstrated that miR-
125b expression in M3 patients was much higher in PML/RARA positive patients than in
PML/RARA negative patients, though miR-125b was high in both groups. This suggested
that miR-125b expression is associated with PML/RARA status and acts as a favorable
prognosticator in APL. For the M1 and M2 groups, the highest expressed microRNAs were
miR-335 and miR-126 respectively. Using the validation cohort, the expression of miR-126
was slightly higher in patients with t(8;21)(q22;q22) carrying the fusion gene AML-ETO than
AML-ETO negative ones. This might also imply that miR-126 expression associates with
prognosis of M2 patients. In a 2011 paper, the same authors (63) further studied miR-125b
upregulation in pediatric APL. In this study, miR-125b expression was analyzed in 169
pediatric bone marrow AML samples including 76 APL samples before therapy and 38 APL
samples after therapy. The data showed that miR-125b was highly expressed in the APL
samples, although also in the other subtypes a higher miR-125b was detected. The microRNA
expression was also analyzed in matched diagnosis-CR versus diagnosis-relapse pairs. This
showed that miR-125b expression had normalized in all complete remission patients while
the expression level in relapse patients was higher than in normal controls. The effects of
enforced expression of miR-125b were evaluated in leukemic cells and drug-resistant cell
lines (NB4, HL-60, K562). This showed that miR-125b could promote leukemic cell
proliferation and inhibit cell apoptosis by regulating the expression of Bak1 (BCL-2
28
antagonist/killer), a pro-death protein able to promote programmed cell death (64). These
findings indicate that miR-125b in APL may function as an oncogene by inhibiting cell
apoptosis and promoting cell proliferation.
miR-155
Garzon et al. (31) identified distinctive microRNA signatures of AML with NPMc+.
Comparing microRNA expression profiles of samples with NPMc+ and FLT3-ITD versus
samples with NPMc+ and FLT3-wt showed that only miR-155 was upregulated at a
significant level in FLT3-ITD mutated samples. Further experiments showed that the up-
regulation of miR-155 was independent from FLT3 signaling. This was demonstrated through
an unchanged miR-155 expression after either blocking of the FLT3 phosphorylation activity
or overexpressing FLT3-ITD in mouse myeloid progenitor cells. In another paper, Garzon et
al. (32) studied microRNA signatures associated with cytogenetics and prognosis in AML in
a cohort of 122 untreated adult AML patients. An independent set of 60 untreated AML
patients was used to validate the outcome signatures. This data showed that miR-155 was
significantly upregulated in FLT3-ITD versus FLT3-wt patients. The microRNA expression
was also compared between a cohort of relapsed and primary refractory patients and the
initial cohort of 122 patients. Similar microRNA signatures were obtained to those of the
untreated patients supporting the author‟s hypothesis that microRNA expression is largely
driven by cytogenetics. Whitman et al. (29) studied the association of FLT3-ITD with
outcome and gene- and microRNA expression signatures in a cohort of older (n=243, ≥ 60
yrs) CN-AML patients. FLT3-ITD is a known adverse prognostic marker in younger (< 60
yrs) CN-AML patients (3). Their data showed that presence of FLT3-ITD remained
associated with shorter disease free survival and overall survival in the group with age 60-69
years, but not in the subgroup of patients of 70 years or older. The most overexpressed
microRNA was miR-155 (2.8 times) which already had been associated with NPM1 mutation
and/or FLT3-ITD in previous papers (32, 65). Although some target genes of miR-155 have
been identified, e.g. the SHIP1 gene (66) as negative regulator of cell signaling in the
immune system and CEBPB (67) with a critical function in granulopoiesis, the molecular
pathways affected by miR-155 and their roles in oncogenesis remain to be elucidated.
miR-181
Debernardi et al. (68) correlated the expression of miR-181a with morphological sub-class
in AML in bone marrow and peripheral blood samples of 30 adult CN-AML patients. In this
29
study, the miR-181a expression levels were elevated in the M1 and M2 samples compared to
samples with M4 or M5 morphology. The authors referred to the fact that in normal human
bone marrow, in contrast, miR-181a has been reported to be preferentially expressed in B-
and T-cells, and in monocytes and granulocytes (69) which are more closely allied to the M4
and M5 leukemic subtypes. Marcucci et al. (27) studied CN-AML patients younger than 60
years (n=64) with high-risk molecular features like a FLT3-ITD, a wild-type NPM1, or both.
In this extensive correlation study, 12 microRNAs, of which 5 from the miR-181 family,
were associated with event-free survival in the training group. Based on the weighted levels
of the microRNAs as found in the training group, a calculated microRNA summary value (a
microRNA compound covariate predictor) was inversely associated with event-free survival
in the independent validation group (n=55). Using the results of gene-expression microarray
analysis, it was demonstrated that the expression levels of the miR-181 family were inversely
correlated with expression levels of predicted target genes involved in mechanisms of the
innate immunity. These included genes encoding toll-like receptors (e.g. TLR2, TLR4,
TLR8) and those encoding interleukin-1β and further effectors controlling the activation of
this cytokine (e.g. CARD family members). The hypothesis was that the downregulation of
the miR-181 family might contribute to an aggressive leukemia type through pathways
regulating the innate immunity. Garzon et al. (32) studied microRNA signatures associated
with cytogenetics and prognosis in AML in a cohort of 122 untreated adult AML patients. An
independent set of 60 untreated AML patients was used to validate the outcome signatures
using RT-qPCR. In a subtheme of this study, the microRNA expression profile of a subset of
patients (n=29) with multilineage dysplasia (MLD) was compared to a group of untreated de
novo AML patients (n=79). MLD-AML is mostly affecting older patients and is associated
with an unfavourable response to therapy (3, 70). This comparison revealed only miR-181a to
be downregulated in the MLD group. Wang et al. (71) studied the role of miR-181
interaction with p27Kip1
in human myeloid leukemia cell lines induced to differentiate by
1,25-dihydroxyvitamine-D3 (1,25-D3). Work described in previous papers (72,73) showed
that 1,25-D3 could induce (HL-60 cells) or enhance (U937 cells) the monocytic phenotype
and arrest their proliferation, predominantly in the G1 phase of the cell cycle. The mechanism
for this action so far was not understood. The authors provide evidence that exposure of HL-
60 and U937 cell lines to low concentrations of 1,25D3 decreases the expression of miR-181a
and miR-181b in a concentration and time dependent manner. p27Kip1
, an inhibitor of the cell
cycle controlling cyclin-dependent kinases (CDK4, CDK6), was identified as one of the
predicted targets of the miR-181s. Expression of pre-miR-181a in these cells led to the
30
abrogation of 1,25D-induced increase of p27Kip1
at both the mRNA and protein levels.
Transfection of the pre-miR-181a also blunted the effect of 1,25D3 on the expression of
monocytic differentiation markers, and reduced the G1 block in 1,25D3-treated cells. In
contrast, transfection of anti-miR-181a increased the 1,25D3-induced differentiation. These
data demonstrate that in cultured human myeloid cells the expression of p27Kip1
can be
regulated by at least two members of the miR-181 family. 1,25D3-induced repression of miR-
181s therefore may, amongst other mechanisms, contribute to the accumulation of p27Kip1
and the arrest of the cell cycle progression in the G1 phase. Schwind et al. (28) studied the
expression of miR-181a in a group of 187 CN-AML patients younger than 60 years. Another
group of 122 CN-AML patients (≥60 years) constituted an independent validation set for
outcome analysis. The presence of molecular prognosticators like FLT3-ITD, NPM1-wt and
others were also assessed. The results showed that higher miR-181a expression is associated
with a higher complete remission rate and a longer overall survival. The impact of miR-181a
expression was particularly striking in poor molecular risk patients with FLT3-ITD and
NPM1-wt where it yielded a higher complete remission rate and longer disease free survival.
Also in multivariate analysis, higher miR-181a expression was significantly associated with
better outcome, both in the whole patient cohort as in patients with FLT3-ITD and NPM1-wt.
These results were validated with the independent set of older CN-AML patients. In
agreement with previous data (27), a miR-181a-associated gene-expression profile showed an
enrichment of genes involved in innate immunity (e.g. TLR4 and IL-1B). Li et al. (30)
studied the role of miR-181 in the upregulation of the potential targets of this microRNA:
HOXA7, HOXA9, HOXA11 and PBX3, members of the so-called homeobox genes. The
aberrant overexpression of homeobox genes has been reported before in various AML
subtypes of intermediate or poor prognosis, including those bearing MLL rearrangements or
trisomy 8 (74,75). The upregulation of a gene signature composed of the four studied
homeobox genes associated with miR-181 downregulation was an independent predictor of
adverse overall survival on multivariate testing of 183 cytogenetic abnormal AML patients.
This was confirmed with an independent validation set of 271 CA-AML patients. In vitro and
in vivo studies showed that ectopic expression of miR-181b significantly promoted apoptosis
and inhibited viability and proliferation of leukemic cells and delayed leukemogenesis. Of the
four genes studied in depth, PBX3 was the only gene in four different calibration and
validation sets that consistently associated with poor survival and it was stronger repressed by
miR181a/b than the other genes. It was further proven that PBX3 is a direct target of miR-
181a/b and the PBX3 downregulation effect by miR-181a/b was shown on mRNA and
31
protein level in MLL bearing leukemic cells. The effects of miR-181a/b on cell viability,
growth and apoptosis could be reversed by forced expression of PBX3 in vivo and in vitro,
suggesting that PBX3 may play an important role in development and maintenance of MLL-
rearranged AML. The authors claim that restoring the expression of miR-181b or targeting
HOXA-PBX pathways directly may help improve the outcome of patients with adverse CA-
AML.
miR-221/222/223
Briochi et al. (76) elucidated a microRNA pathway which links the core-binding factor
fusion proteins with KIT upregulation in CBF AML. A common mutation associated with
CBF AML involves the KIT receptor (77). As such, high expression of KIT is a sign of CBF
leukemia. KIT or CD117, a proto-oncogene, is a receptor tyrosine kinase which upon binding
to stem cell factor activates its tyrosine kinase activity leading to activation of a pathway with
cell survival, proliferation and differentiation. Based on previous literature, the authors
hypothesized that miR-222/221 (with its locus on the X chromosome) represents the link
between CBF leukemia and KIT expression. The experimental work showed miR-222/221
upregulation after myeloid differentiation of normal bone marrow AC133+ stem progenitor
cells. CBFL blasts with high expression of KIT displayed a significant lower level of miR-
222/221 than non-CBFL blasts. Additionally, they found that the t(8;21) AML1-ETO fusion
protein binds on the miR-222/221 promoter and induces transcriptional repression of a
luciferase reporter containing the miR-221/221 promoter. Furthermore, a concomitant miR-
221/222 downregulation and KIT up-regulation was demonstrated in the 32D/WT1 mouse
cell model carrying the AML1-MTG16 (RUNX1-CBFA2T3) fusion protein. As such, the
authors provided a first hint that CBFL-associated fusion proteins may lead to KIT
upregulation by the down-regulation of the miR-222/221. In the same study, it was also
observed that miR-223 was down-regulated in CBFL samples. miR-223 is known as a
regulator of the myeloid differentiation also with its locus on the X chromosome (77,78). The
observation of down-regulation of miR-223 in t(8;21) AML was consistent with previous
literature describing the oncoprotein AML1-ETO as a direct transcriptional suppressor of
miR-223 expression utilizing an epigenetic silencing mechanism.. This work shows that the
CBF fusion proteins, in addition to directly targeting and downregulating the expression of
hematopoietic protein-coding sequences containing an AML1 consensus sequence (80,81),
can target microRNA genes such as miR-223 with a critical role in myelopoiesis.
32
Pullikan et al. (56) unravelled an autoregulatory negative feedback loop between the master
cell-cycle regulator E2F1 and miR-223 in AML. Previous papers had described that the
inhibition of E2F1 by CEBPα is pivotal for granulopoiesis (82). Recent studies described
miR-223, a transcriptional target of C/EBPα, as a critical player during granulopoiesis
(78,79). The authors built further on this knowledge and demonstrated that miR-223 blocks
the cell-cycle progression and is downregulated in different subtypes of AML. Experimental
work provided evidence that E2F1 binds to the miR-223 promotor in AML blasts and inhibits
miR-223 transcription. The data suggest that the binding of C/EBPα leads to the
transactivation of miR-233, which in turn blocks the translation of E2F1 leading to inhibition
of the cell-cycle progression and to myeloid differentiation. These findings suggest that
deregulation of C/EBPα by various mechanisms in AML results in weak binding of C/EBPα
to the miR-223 promotor, resulting in fewer miR-223 molecules in the cell cycle to inhibit
E2F1 translation (see Figure 3). The resulting overexpressed E2F1 could bind to the miR-
223 promotor and in turn lead to a further decrease in miR-223 expression through a negative
feedback loop. This leads to myeloid cell-cycle progression at the expense of differentiation.
Based on the fact that the myeloid master regulator C/EBPα has been shown to be
deregulated by different oncoproteins (e.g. AML1/ETO, BCR/ABL, PML/RARα, FLT3) in
AML (83), and that the CEBPA gene is mutated in about 10% of AML cases (3), the authors
speculate that C/EBPα deregulation may be one of the major steps in AML development. As
such, this work revealed a molecular network involving miR-223, CEBPA and E2F1 as major
components of the granulocytic differentiation program, which is deregulated in AML.
Therefore, manipulation of miR-223 could be a therapeutic target relevant in AML subtypes
with deregulated E2F1 inhibition.
33
Figure 3: A model for miR-223, C/EBPα and E2F1 in normal granulopoiesis and in
AML (56).
“During granulopoiesis (top panel), C/EBPα binds and transactivates miR-233 promotor, which in turn leads to
E2F1 repression and inhibition of cell-cycle progression resulting in myeloid differentiation. When C/EBPα is
deregulated by various mechanisms in AML (bottom panel), transactivation of miR-223 is inhibited, which
results in accumulation of E2F1. Overexpressed E2F1 binds to miR-223 promotor and inhibits miR-223
transcription through a negative feedback loop resulting in myeloid cell-cycle progression and block of
differentiation”. Copied from (56).
3 The Role of MicroRNAs in Chemoresistance Mechanisms
Multiple drug resistance (MDR) is considered a major contributor to the failing of
chemotherapy in leukemia and cancer in general (84-86). Drug resistance can result from
random drug-induced mutational events, epigenetic changes or karyotypic changes (87-90).
This can lead to a decreased uptake of drugs, to pathway changes affecting the capacity of
cytotoxic drugs to induce cell death (e.g. reduced apoptosis, increased DNA repair,
upregulation of glutathione-mediated drug detoxification), or can give rise to increased drug
efflux out of the cancer cell (91-93). Especially the expression of the family of ATP Binding
34
Cassette (ABC) transporters in relation to drug resistance in AML has been studied (94-97).
To date, the microRNA-based modulation of MDR in AML subtypes has not been studied
very intensively, and the mechanistic understanding of drug resistance remains largely
elusive. A limited number of papers dealing with microRNA in relation to drug resistance in
AML have been published in recent years and are summarized in the next paragraphs.
Mishra et al. (98) showed that a naturally occurring single nucleotide polymorphism (SNP)
in a microRNA binding site can lead to drug resistance. In particular they studied the
naturally occuring SNP C829T in human dihydrofolate reductase (DHFR) and showed this to
be a loss-of-function mutation resulting in methotrexate resistance. This SNP is located near a
miR-24 binding site in the 3‟UTR of DHFR. As a direct effect of this mutation, the inability
of miR-24 to downregulate the DHFR mRNA resulted in an increase of dihydrofolate
reductase yielding an increased methotrexate resistance. Zhao et al. (99) described the role of
miR-138 in MDR in the leukemia cell line HL-60. Parental HL-60 cells were compared to
vincristine-resistant HL-60 cells (HL-60-VCR) and to miR-138-transfected HL-60-VCR
cells. The HL-60-VCR cells showed a lower expression of miR-138 than the parental cells.
Three potential MDR mechanisms were studied: first a P-glycoprotein (P-gp) expression
increase; secondly an increased GST-mediated intracellular drug-detoxification, and thirdly a
decrease in apoptosis through regulation of genes in the Bcl-2-family: Bcl-2, Bcl-xL, Bax,
Bak. It was shown that VCR-resistance could be abrogated through the upregulation of miR-
138 in the HL-60-VCR cells. This loss of drug resistance was present for drugs known to be
transported by P-gp (e.g. vincristine and adriamycin) as well as for drugs known to be
unaffected by P-gp activity (e.g. 5-fluorouracil or cisplatin). Experimental data showed miR-
138 to target the MDR1 (ABCB1) mRNA, reducing the expression van P-gp significantly,
and re-establishing vincristine sensitivity. The expression of the multidrug transporter MRP
(ABCC1) had not altered after miR-138 upregulation. It was further shown that the MDR
mechanism for 5-fluorouracil and cisplatin could not be explained by an upregulation of the
GST-mediated drug detoxification mechanism. The third mode of the potential MDR-
mechamism (blocking of apoptosis) was also studied. This was done through adriamycin
(ADR)-induced apoptosis in miR-138-transfected HL-60/VCR cells. The upregulation of
miR-138 in these cells showed an accumulation of ADR in the cells, a reduced efflux of the
drug, and promoted the ADR-induced apoptosis. The upregulation of miR-138 was
associated with a decrease of Bcl-2 expression and increase of Bax, which could be
correlated with increased apoptosis. The authors concluded that miR-138 likely plays a role
35
in MDR in leukemia through regulation of MDR1/P-gp expression and through regulation of
an apoptosis pathway (BCL2). Li et al. (100) showed that anti-miR-21 enhanced the AraC
chemosensitivity of leukemic HL-60 cell by inducing an apoptosis mechanism. The anti-miR
alone was able to inhibit HL-60 cell viability while in combination with AraC, it sensitized
the cells to AraC-induced apoptosis. It was shown that anti-miR-21 effectively reduced the
miR-21 level which was accompanied with an upregulation of the studied target gene, the
PDCD4 tumor suppressor at both mRNA and protein level. The author‟s conclusion is that
anti-miR-21 enhances the chemosensitivity to AraC by inducing apoptosis in HL-60 cells
which might be partially due to upregulation of PDCD4. Feng et al. (101) studied the miR
signatures in leukemia cell lines with increasing resistance to doxorubicin (DOX) in
comparison to the parental cell line K562. MicroRNA-microarrays and qRT-PCR were used
to detect and confirm the differential microRNA expression. This showed that the expression
of miR-331-5p and miR-27a could be inversely correlated with the expression of MDR1/P-
gp in cells with increasing resistance to DOX. Transfection of DOX-resistant K562 and HL-
60 leukemia cell lines with miR-331-5p and miR-27a (individually or combined) led to an
increased sensitivity of these cells to DOX. This finding suggests that correction of a changed
microRNA modulation might be a therapeutic strategy to restore drug sensitivity. The authors
further demonstrated that both miRs were expressed at a lower concentration in a panel of
relapsed ALL and AML patients, in comparison to the population of patients diagnosed with
primary leukemia. This might indicate that relapse could be correlated, or is a consequence of
deregulation of miR-331-5p en miR-27a. Zhang et al. (63) studied miR-125b upregulation
in association with increased drug resistance in a pediatric AML population (n=176) of which
76 APL patient samples before therapy and 38 APL samples after therapy. This showed a
significant miR-125b upregulation in most of the AML subtypes in comparison to normal
bone marrow samples and a very high expression of miR-125b in the APL subgroup.
Transfection experiments indicated that miR-125b targets the tumor suppressor pro-death
protein Bak1 in leukemic cell lines (NB4, HL60, K562) which was confirmed by luciferase
reporter assays. An inverse association between miR-125b and Bak1 expression was
measured in about 70% of the APL samples. Dichotomisation of these samples in a high and
low miR-125b expressing group correlated with low and high Bak1 expression, respectively.
Bak1 expression was also increased in 60% of the complete remission patients after
chemotherapy. miR-125b was also found upregulated in leukemic drug-resistant cell lines,
and transfection of miR-125b-5p into HL-60 and NB4-R1 cells promoted cell proliferation
and significantly suppressed drug-induced apoptosis. An interesting observation was that
36
transfection with miR-125b-3p led to the opposite effect: an increase of drug-induced
apoptosis. Downregulation of the Bak1 protein by siRNA likewise led to similar effect of
decreased apoptosis after miR-125b upregulation. The results showed that miR-125b may
have a critical role in pediatric APL samples through targeting the tumor suppressor Bak1
and its dysregulation may be linked to increased drug resistance. This suggests that miR-125
may be a biomarker for malignancy and for the prediction of chemotherapy effectiveness in
APL. Gocek et al. (102) studied the miR-32 upregulation by 1,25-dihydroxyvitamine-D3
(1,25-D3) in human myeloid leukemia cells. Previous literature described that 1,25-D3
induced differentiation of AML cells was accompanied with an increased cell survival
capacity (103). The hypothesis was that miR-32 is a modulator of the pro-apoptotic protein
Bim and this is associated with changes in the ability of AraC to induce apoptosis in AML. It
was experimentally verified that miR-32 targets the 3‟UTR of the BCL1L11 gene encoding
for Bim. The reduction of the cellular level of miR-32, consequent increase in Bim
expression and potential increased AraC drug sensitivity was tested by preincubating AML
cells with anti-miR-32 followed by treatment of the cells with AraC. Necrosis and apoptosis
assays confirmed this anti-miR-32 effect and led to increased cell death induced by AraC,
DOX or daunomycin and abrogated the previously reported anti-apoptotic effect of 1,25D. As
such, the combination of differentiation (by 1,25-D3) with cytotoxic therapy (AraC) in AML
might be further enhanced by agents lowering the cellular miR-32 levels. Bai et al. (104)
studied the involvement of miR-21 in resistance to daunorubicine (DNR) in a K562 cell line.
Previous literature had described PTEN, a negative regulator of the PI3K/Akt pathway as a
direct target of miR-21 (105). Akt kinase phosphorylates several downstream proteins such as
e.g. Bcl2 in K562 cell (106) and as such is active in cell growth regulation. The hypothesis
was that overexpression of miR-21 may activate the PI3K/Akt pathway by reducing the
PTEN protein level. The authors showed that miR-21 expression was upregulated in a
daunorubicine-resistant cell line, while suppression of miR-21 led to enhanced sensitivity to
DNR. K562 cells with transduced miR-21 showed increased Akt phosphorylation, while
miR-21 knockdown in the K562/DNR resistant cell line resulted in reduced phosphorylation.
The role of PTEN in the induced DNR resistance was studied through transfection of PTEN
siRNA, followed by the treatment with various doses of DNR. This led to reduced PTEN,
increased pAkt levels and a cell survival pattern similar as obtained after miR-21
overexpression. As such, this work showed that overexpression of miR-21 activates the
PI3K/Akt pathway by decreasing the PTEN protein leading to increased daunorubicine
resistance. The authors state that this miR-21/PTEN/PI3K/Akt pathway may therefore be a
37
therapeutic target for drug sensitization in leukemia. Blum et al. (34) studied the clinical
response and miR-29b predictive significance in AML patients of 60 years or older, treated
with a 10-day schedule of decitabine, an azanucleoside DNA methyl transferase (DNMT)
inhibitor. Decitabine is applied in the treatment of MDS (107) and AML (108), based on its
capability to inhibit DNMT-induced aberrant hypermethylation and gene activation. miR-29b
was previously shown to target the DNA methyltransferases and miR-29b had been shown to
be downregulated in AML (109). A phase II clinical trial with single-agent decitabine was
conducted in a cohort of 53 patients with previously untreated AML. The data indicated that
higher levels of miR-29b were associated with improved clinical response. Following the
decitabine treatment schedule, complete remission was achieved in 52% of subjects with
normal karyotype and in 50% of those with complex karyotype with a median overall
survival and disease free survival of 55 and 46 weeks, respectively. Patients who responded
to decitabine had a higher pretreatment level of miR-29b than non-responders (P = 0.02).
This data suggests that higher baseline miR-29b expression in myeloid blasts leads to lower
DNMT levels and acts in synergy to induce increased sensitivity to the hypomethylating
effect of decitabine. Consistent with this finding, a trend to lower DNMT3a expression was
observed in decitabine responders compared to non-responders. Hickey at al. (110) recently
reported the upregulation of the anti-leukemic miR-181a by lenalidomide, a drug recently
applied in treatment of AML patients with CEBPA mutation (111, 112). This was a next step
after previous work showed that increased miR-181a expression is associated with improved
outcome in CN-AML (27). Within the studied adult population (<60 years) of primary CN-
AML with high-risk features, it was shown that miR-181a expression was increased in over
90% of the CEBPA mutated samples. CEBPA mutation was linked to inhibition of the
expression of the full-length C/EPBα-p42 isoform, while the expression of the truncated
C/EPBα-p30 isoform remained undisturbed or was even enhanced. The authors showed that
the presence of N-terminal CEBPA mutation and miR-181a expression are linked. The
truncated C/EPBα-p30 isoform was shown to bind to the miR-181a-1 promoter and
upregulate the miR-181a expression. In vitro experiments using THP-1 cells treated with
lenalidomide showed sensitization of these cells to AraC. This lenalidomide activity was
inhibited in the presence of the miR-181a antagomir, supporting that lenalidomide-induced
sensitivity to AraC is dependent on its ability to increase miR-181a expression. It was further
shown that the ectopic expression of miR-181a inhibited tumor growth in a xenograft mouse
model. Lenalidomide exhibited anti-tumorigenic activity paralleled by an increased miR-181a
expression. This collected data may provide a rationale for the improved clinical outcome
38
observed in CEBPA-mutated patients, and suggests that lenalidomide treatment enhancing
the C/EPBα-p30 protein levels, and in turn miR-181a, may sensitize AML blasts to
chemotherapy. To prove this principle, a Phase I trial (NCT01132586) of lenalidomide
followed by the AraC induction therapy was initiated by the authors.
In a couple of papers dealing with MDR in chronic myeloid leukemia (CML) or acute
lymphatic leukemia (ALL), identical microRNAs as described in the AML papers above,
were associated with chemoresistance. Therefore these papers are covered here as well.
Zimmerman et al. (113) profiled the microRNA expression in a model of Lyn kinase
mediated imatinib-resistant CML cells (Myl-R) and identified about 30 microRNAs whose
expression differed more than two-fold compared with drug-sensitive cells. Especially the
expression of the miR-181a-d family had significantly reduced (11-25 fold) in Myl-R cells.
Incubation of Myl-R cells with a Lyn inhibitor or nucleofection with Lyn-targeting siRNA
increased miR-181b and miR-181d expression. Sequence analysis of potential targets for
miR-181 predicted the anti-apoptotic Mcl-1 protein (myeloid cell leukemia-1) whose
expression was increased in Myl-R cells. Inhibition of Lyn or rescue of miR-181b expression
reduced the Mcl-1 expression in the Myl-R cells. Luciferase reported assays further
demonstrated that Mcl-1 is a direct target of miR-181b. Further data demonstrated that the
stimulation of Lyn kinase expression by 1,25-dihydroxyvitamine-D3 treatment in HL-60 cells
decreased miR-181b expression and increased Mcl-1 expression. This work suggests that
down-regulation of miR-181 upon Lyn activation could be a novel mechanism of drug
resistance in leukemia by increasing the Mcl-1 protein. This experimental observation
correlates well with the observation that low miR-181 expression is associated with poor
prognosis in AML, whereas high miR-181 expression is prognostic for event-free survival in
AML (27, 28). Han et al. (114) showed that downregulation of the same miR-27a might
promote relapse in ALL by regulating genes with roles in drug resistance pathways. The
polycomb gene BMI1 was identified as a bona fida target of miR27a in this work.
4 Review papers dealing with microRNA in AML
Havelange, Garzon and Croce (9) summarized the knowledge about microRNA
dysregulation in AML anno 2009 and focused on reviewing four major large-scale global
microRNA profiling studies. In these different studies, unfortunately, only a few miRs within
the full microRNA signature associated with an AML cytogenetic or molecular subclass are
equally up- or downregulated. In other words, the reproducibility of identifying up- or
39
downregulated microRNAs in identical subclasses of AML between different labs and
employing different patient cohorts, seems rather poor. The authors suggest that such
differences in reported microRNA signatures, might be explained mainly because of the
differences in overall cytogenetic and molecular AML subgroups in the studied patient
population. A second explanation may be the type of platforms used to interrogate microRNA
expressions. Such bias would be less evident, the authors state, when future research would
focus on well characterized cytogenetic and molecular subgroups of AML, as such
minimizing the biological heterogeneity. The authors further believe that microRNA studies
will have relevant therapeutic implications in the coming years. Synthetic microRNAs or
anti-miRs alone, or in association with chemotherapy, could become future AML therapies.
Synthetic microRNAs could be used to restore lost tumour suppressor microRNA expression,
or overexpressed oncogenic microRNAs could be silenced with antagomirs. Seca et al. (115)
reviewed microRNA patterns associated with AML subtypes as published in previous
literature. The knowledge at that time about microRNA patterns associated with t(8;21),
t(15;17), inv(16), MML-rearrangements, and NPM1, BAALC, CEBPA, MN1 en FTL3-
ITD/wt mutations are discussed. Similar to the previous review, the authors critically reflect
on the rather poor reproducibility of microRNA patterns associated with AML subtypes,
between different papers. Possible explanations are suggested which included the difference
in the studied AML populations with different cytogenetic and molecular features,
differences in sample preparation used for RNA extraction and purification, differences in
microRNA profiling methods and platforms used, and the absence of standardized
normalization procedures e.g.versus snRNA or tRNA. Garzon, Marcucci and Croce (16)
describe the role of microRNAs in tumorigenesis (“a paradigm shift“) and discuss the
rationale, the strategies and the challenges for the therapeutic targeting of microRNA in
cancer. The authors describe a number of microRNAs and the knowledge about their in vitro
and in vivo roles in cancer deeper. Examples are the miR-17-92 cluster, miR-155 and miR-
181a. The main portion of their paper deals with discussing future potential strategies of
microRNA-based therapeutics. Marcucci et al. (26) reviewed the prognostic and functional
role of microRNAs in AML in 2011. An overview of the most significant microRNAs
correlating with cytogenetics and molecular features in AML is given. Furthermore an
overview of the current knowledge about the functional roles of miR-155, miR-196b, miR-
223 and miR-29 is given in more detail. Havelange et al. (25) studied the functional
implications of microRNAs in AML by integrating microRNA and mRNA expression
profiling of bone marrow or peripheral blood samples of newly diagnosed AML patients.
40
Although not intended as a review article, this original article offers suggestions and opens
doors to future AML-microRNA research. Through network analysis linking microRNA and
mRNA interactions in e.g. apoptosis and inflammation/immunity networks, the data indicates
that mRNA transcripts are associated with several microRNAs. This suggests a combinatorial
effect in gene (dys)regulation by co-expressed endogenous microRNAs in AML. In other
words, a small group of microRNAs coordinately regulates an mRNA pathway transcriptome
by modulating the same group of genes within the pathway. Based on obtained in vitro
experimental results and potential discrepancies with AML patient samples in studying
molecular effects of miR-145, miR-23a, - 26a, and -128a, the authors reflect on potential
limitations of using cell lines for the validation of events as measured in primary AML
samples. An explanation for such discrepancies between true AML samples and cell lines
may be that many microRNA-mRNA interactions will occur only in the context of well-
defined cytogenetics and molecular subtypes of AML. Such very specific temporal and
molecular conditions may be very hard to reproduce by artificially manipulating in vitro
microRNA expression in specific cell lines.
5 Conclusions
Within the past 5-10 years, the role of microRNA dysregulation and its contribution to AML
pathogenesis is step-by-step being unraveled. In several studies, a limited number of
microRNAs has been consistently associated with the most abundant specific cytogenetic and
molecular aberrations in AML (9,31,26,61). The first mechanistic proposals explaining the
oncomiR and tumor suppressor miR action in pathways have been published (33,25,52,
56,76). MicroRNA up- and downregulation has been proposed as a contributor to improving
prognosis (26-30, 32,33,52,46,60) or has been described to affect resistance to drug therapy
(24,27,28,34,63,99,100-104,110,113) and the first clinical trials evaluating microRNA
expression modulation as a therapeutic option in AML have been reported (16,34,110).
Insight in the complex molecular mechanistic circuitry of specific microRNAs is slowly
being gathered. Several microRNAs act in a temporal and combinatorial way in the
regulation of a pathway transcriptome (e.g. immunity or apoptosis), and a same microRNA
can target different genes active in very different cell processes (25). This knowledge
challenges the idea to modulate single or a few microRNAs as an adjuvant to improve
outcome in cancer therapy. However, microRNAs such as miR-181a, miR-29b and miR-223
41
seem involved in crucial crossroads cell processes and as such their endo- or exogenous
modulation offers hope to restore malignant cell processes. Promising examples are miR-29b
expression levels in stratification of older AML patients for which decitabine treatment might
be a last option (34) or lenalidomide treatment leading to increased expression of the tumor
suppressor miR-181a potentially increasing the chemosensitivity of blasts to AraC
chemotherapy (110). The development of further fundamental insights in the action of
microRNAs in critical pathways affecting cell proliferation and differentiation may,
eventually, enable novel microRNA-targeting therapeutics and/or inhibit classical multidrug
resistance.
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42
4 Discussie
Gedurende de voorbije 5 tot 10 jaar is er toenemende aandacht voor het ontrafelen van de
kennis over de rol en mechanismen van microRNA‟s in het leukemogenese proces. Dit heeft
geleid tot het definiëren van tumorsuppressor microRNA‟s en oncogene microRNA‟s.
Eenzelfde microRNA kan in een bepaalde pathway de rol van tumorsuppressor vervullen
(b.v. suppressie van een oncogen), terwijl het in een andere pathway een oncogene rol kan
uitoefenen (b.v. onderdrukking van een tumorsuppressor-gen) (16). De rol van microRNA‟s
in de ontwikkeling van chemoresistentie of MDR krijgt ook een steeds belangrijker plaats in
het leukemie-onderzoek. Hierbij wordt de microRNA-expressie gecorreleerd met MDR en
wordt gezocht naar genen die door de veranderde microRNA expressie geïnduceerd of
onderdrukt worden in b.v. apoptose pathways of transporter systemen (63,99,101). De
microRNA expressie van AML patiënten kan ook gebruikt worden in het stratificeren van de
AML patiënten voor behandeling. Hierbij kan een verhoogde expressie van een specifiek
microRNA het behandelingsresultaat verbeteren. Deze kennis heeft dan ook geleid tot de
eerste klinische studies (16, 34, 110, 116) gebaseerd op microRNA-modulatie of microRNA-
expressieniveau.
Wat zijn de uitdagingen verbonden met microRNA-modulatie met het oog op de verbetering
van de prognose van AML patiënten?
Uit het literatuuroverzicht is het ten eerste duidelijk geworden dat één specifiek microRNA
betrokken kan zijn in de regulatie van verschillende celprocessen, b.v. immuniteit, apoptose,
epigenetische genregulatie, celproliferatie. Ten tweede is het ook aangetoond dat binnen deze
pathways (b.v. apoptose) verschillende microRNA‟s samenwerken om de verschillende
genen binnen dit specifiek celproces optimaal te moduleren (33). Deze complexe
samenwerking van microRNA‟s vormt een uitdaging voor het gebruik van microRNA-
targeting therapie. Hierdoor lijkt het aanneembaar dat modulatie van één specifiek
microRNA slechts een beperkt effect zal hebben op het beïnvloeden van een bepaalde
pathway. Daarom kan het zinvol zijn dat een dergelijke therapie zich eerst richt op specifieke
microRNA‟s die, volgens de recentste studies, in meerdere AML-subtypes een sleutelrol
lijken te vervullen. Zo‟n voorbeelden zijn de tumorsuppressoren miR-29b (33,52,60) en miR-
181 (27,28,30,32,68,71), miR-223 met een sleutelrol in het granulocytair differentiatieproces
(52,78,79) en het oncomir miR-126 dat sterk geïnduceerd wordt in core-binding factor
leukemieën, en geassocieerd is met het ontstaan hiervan (61). Een voorbeeld van een
43
microRNA-targeting therapie is onlangs (2013) beschreven (35). Hierbij werd, in een
muizenmodel, lenalidomide, een immunomodulerende drug, aangewend om de expressie van
de tumor suppressor miR-181a te verhogen, wat op zijn beurt leidde tot een verhoogde
gevoeligheid voor AraC therapie. Op basis van deze resultaten is een Fase I klinische studie
recent geïnitieerd (35). Een ander voorbeeld is de aanwezigheid van hogere miR-29b
expressie bij oudere uitbehandelde of zwakke patiënten. Dit microRNA is vaak onderdrukt in
AML (109). De aanwezigheid van een hoge miR-29b-expressie bij patiënten behandeld met
decitabine, een inhibitor van de DNA-methyltransferasen, leidde tot een versterkt
hypomethylerend en dus onderdrukkend effect op actieve oncogenen betrokken in AML. Dit
werd in een Fase II klinisch onderzoek in 2010 bevestigd (34). Andere voorbeelden zullen in
de nabije toekomst zeker volgen, mede door de grote interesse van de farmaceutische sector
voor microRNA-therapie (16,116). Het herstellen van miR-223-expressie kan ook een
therapeutische optie zijn. De verstoring van de transcriptiefactor C/EBPα is het primum
movens in een mechanisme dat leidt tot miR-223 onderdrukking, gevolgd door verhoogde
E2F1 expressie. Dit leidt tot een blokkering in de differentiatie en inductie van celproliferatie.
Dit mechanisme suggereert dat C/EBPα verstoring een belangrijke stap kan zijn in de
ontwikkeling tot AML (56). Het lijkt hierdoor zinvol de miR-223 expressie te herstellen, b.v.
door het gebruik van miR-223 mimics.
Welke zijn de verdere uitdagingen in het AML-microRNA-onderzoek ?
Vooreerst is er de grote heterogeniteit van AML-stalen die in diverse laboratoria op
microRNA-expressie worden onderzocht. Zoals aangegeven door Havelange (9) en Seca
(115), kan dit een belangrijke reden zijn om de waargenomen verschillen in AML subtype-
geassocieerde microRNA-patronen tussen de verschillende onderzoeksgroepen te verklaren.
Daarom kan het nuttig zijn meer homogene patiëntenpopulaties te bestuderen met een meer
beperkte cytogenetische en moleculaire variabiliteit. Het behandelingsresultaat zou dan
binnen dergelijke populatie kunnen gecorreleerd worden met microRNA- en mRNA-
signatuur, proteïne expressie en activiteit. Een tweede uitdaging is het gebruik van
leukemische cellijnen om in vivo associaties of gepostuleerde moleculaire mechanismen te
valideren. Vaak worden in leukemische cellijnen significante correlaties gevonden tussen
microRNA of mRNA en drugtransporter expressie (b.v. P-glycoproteïne) en geeft transporter
suppressie een significante daling van MDR (98,101). Deze in vitro resultaten zijn vaak niet,
of slechts matig, op AML-patiëntstalen overdraagbaar (3,63). Deze discrepanties kunnen
verklaard worden door het feit dat vele microRNA-mRNA interacties slechts plaatsvinden in
44
de context van zeer specifieke cytogenetische en moleculaire subtypes in AML (9). Een
grotere focus op het in vitro gebruik van AML stalen i.p.v. leukemie-cellijnen, kan hierin
misschien verbetering brengen.
De literatuur toont in elk geval een sterk groeiende aandacht voor microRNA in AML-
onderzoek. De eerste klinische onderzoeken zijn achter de rug. Of microRNA-modulerende
therapie, in de vervanging of als adjuvans tot de klassieke agressieve chemotherapie een
plaats krijgt in de standaard AML therapie, zal in de komende jaren duidelijk worden. In elk
geval zou dergelijke therapie met endogene biomoleculen een alternatief kunnen zijn.
Voornamelijk voor de groeiende populatie van oudere en zwakkere patiënten kan microRNA-
therapie de overleving en levenskwaliteit verbeteren.
45
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6 Bijlagen (op CD)
Bijlage 1: Samenvattingen van de bestudeerde literatuur
Bijlage 2: Tabel MicroRNA’s in AML
Bijlage 3: Humane hematopoiese en normale bloedcelwaarden
