Micro-RNA response to imatinib mesylate in patients with chronic myeloid leukemia

Micro-RNA response to imatinib mesylate in patients with chronicmyeloid leukemia

by Stephane Flamant, William Ritchie, Joelle Guilhot, Jeff Holst, Marie-Laure Bonnet,Jean-Claude Chomel, Francois Guilhot, Ali G. Turhan, and John E.J. Rasko

Haematologica 2010 [Epub ahead of print]

Citation: Flamant S, Ritchie W, Guilhot J, Holst J, Bonnet ML, Chomel JC, Guilhot F,Turhan AG, and Rasko JE. Micro-RNA response to imatinib mesylate in patients withchronic myeloid leukemia. Haematologica. 2010; 96:xxx doi:10.3324/haematol.2009.020636

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1

Micro-RNA response to imatinib mesylate in patients with chronic myeloid leukemia

Running Title: Deregulated miRNA in imatinib-treated CML patients

Stéphane Flamant1,8

, William Ritchie1, Joëlle Guilhot

2,3, Jeff Holst

1, Marie-Laure Bonnet

3, Jean-Claude

Chomel3,5

, François Guilhot

2-4, Ali G. Turhan

3,5 and John E. J. Rasko

1,6,7,§

1Gene & Stem Cell Therapy Program, Centenary Institute, Newtown, NSW 2042, Australia;

2Centre

d’Investigation Clinique INSERM 802, CHU de Poitiers, France; 3EA 3805/INSERM U935, Université

de Poitiers, France; 4Service d’Oncologie Hématologique et Thérapie Cellulaire, CHU de Poitiers,

France; 5Service d’Hématologie et Oncologie Biologique, CHU de Poitiers, France;

6Faculty of

Medicine, University of Sydney, Sydney, NSW, Australia; 7Cell and Molecular Therapies, Sydney

Cancer Centre, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia; 8present address:

Terry Fox Laboratory, Vancouver, BC V5Z1L3, Canada

Correspondence: John E. J. Rasko, Gene & Stem Cell Therapy Program, Centenary Institute,

Locked Bag No 6, Newtown, NSW 2042, Australia. E-mail: [email protected]

Phone: international +61.2.95656156. Fax: international +61.2.95656101

Acknowledgments: the authors would like to thank Wilfred Leung for providing technical assistance.

Funding: this work was supported by the Fondation de France contre la Leucémie, the Australian

National Health and Medical Research Council Project Grant (358300 to J.E.J.R.) and Training

fellowship (571156 to W.R.), the Rebecca L. Cooper Foundation, the Cell and Gene Trust, the Clive

and Vera Ramaciotti Foundation, and the Cancer Institute of NSW.

Key words: BCR-ABL, miRNA, imatinib, CML

DOI: 10.3324/haematol.2009.020636

2

Abstract

Background. Micro-RNAs (miRNAs) control gene expression by destabilizing targeted transcripts and

inhibiting their translation. Aberrant expression of miRNAs has been described in many human

cancers, including chronic myeloid leukemia (CML). Current first line therapy for newly diagnosed

CML is imatinib mesylate (IM), which typically produces a rapid hematological response. However the

effect of IM on miRNA expression in vivo has not been thoroughly examined.

Design and Methods. Using a TaqMan Low-Density Array system, we analyzed miRNA expression in

blood samples from newly diagnosed CML patients before and within the first 2 weeks of IM therapy.

Quantitative RT-PCR was used to validate IM-modulated miRNAs in sequential primary CML samples

(n=11, plus 12 additional validation patients). Bioinformatic target gene prediction analysis was

performed based on changes in miRNA expression.

Results. We observed increased expression of miR-150 and miR-146a, and reduced expression of

miR-142-3p and miR-199b-5p (3-fold median change) after 2 weeks of IM therapy. A significant

correlation (p<0.05) between the Sokal score and pre-treatment miR-142-3p levels was noted.

Expression changes in the same miRNAs were consistently found in an additional cohort of CML

patients, as compared to healthy subjects. Peripheral blood cells from chronic phase and blast crisis

patients displayed a 30-fold lower expression of miR-150 compared to normal samples, which is of

particular interest since c-Myb, a known target of miR-150, was recently shown to be necessary for

Bcr-Abl-mediated transformation.

Conclusion. We found that IM treatment of CML patients rapidly normalizes the characteristic miRNA

expression profile, suggesting that miRNAs may serve as a novel clinically useful biomarker in CML.

DOI: 10.3324/haematol.2009.020636

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Introduction

Micro-RNAs (miRNAs) are small, endogenous non-coding RNAs that post-transcriptionally regulate

gene expression through partial base-pairing with the 3’-untranslated region of target mRNAs,

resulting in mRNA destabilization and translational inhibition (1,2). These molecules regulate many

biological processes, such as cell differentiation, proliferation, apoptosis, and have been implicated in

cancers and leukemias (3,4). The overexpression of miRNAs can perturb normal hematopoiesis, as

first identified by Chen et al (5). The involvement of miRNAs in hematological malignancies was

initially suggested in chronic lymphocytic leukemia (CLL) (6). Subsequent expression profiling studies

identified miRNA signatures characterizing CLL outcome (7,8), acute lymphoblastic leukemia (9) and

acute myeloid leukemia (AML) associated with various cytogenetic abnormalities (10,11). For

example, increased expression of miR-155 has been detected in various leukemias and lymphomas

(12), and mice reconstituted with miR-155-expressing bone marrow cells were shown to develop a

myeloproliferative disorder (13). Similarly, over-expression of the miR-17~92 cluster promotes B-cell

lymphoma in a mouse model (14), and mice with increased expression of this cluster in lymphocytes

develop lymphoproliferative and autoimmune diseases (15).

The contribution of miRNAs to the development of, or response to therapy in chronic myeloid

leukemia (CML) has not been examined in depth. Venturini et al (16) recently described the increased

expression of the miR-17~92 cluster in chronic phase (CP) CML patients that was not found in blast

crisis (BC) samples. CML is characterized by the expression of the BCR-ABL fusion gene, resulting

from the t(9;22) (q34;q11) translocation. Through its constitutive tyrosine kinase (TK) activity, Bcr-Abl

activates a number of signaling pathways which lead to the leukemic phenotype (17). The TK inhibitor

imatinib mesylate (IM) is currently the first line therapy for newly diagnosed CML patients, leading to

the rapid clearance of leukemic cells in peripheral blood in >95% of cases (18). However, a subset of

patients do not respond to IM treatment, owing to intolerance or drug resistance.

To identify miRNAs implicated in CML we sought to determine the repertoire of miRNAs expressed in

leukemic cells from newly diagnosed patients with CML, prior to and within the first 2 weeks during IM

therapy. Using these early time points allowed us to monitor miRNA expression before the leukemic

cells became undetectable. We hypothesized that differentially expressed miRNAs would likely play a

role in the leukemic cells, and could provide useful novel biomarkers in CML. We used the TaqMan

DOI: 10.3324/haematol.2009.020636

4

Low Density Array (TLDA) microfluidic system to profile the expression of 365 human mature miRNAs

in sequential primary CML samples. This analysis led to the identification of several miRNAs

modulated in vivo by IM, which displayed increased (miR-150, miR-146a) or decreased expression

(miR-142-3p, miR-199b-5p) after the start of IM therapy as compared to pre-treatment levels. These

miRNAs were also differentially expressed in an additional cohort of CML CP patients, and we

showed for the first time that the expression of miR-150 was reduced in BC samples as well.

Furthermore, we found significant positive and negative correlations between miRNA expression

levels and clinical data before treatment. We discuss candidate target genes for these miRNAs, of

relevance in CML.

DOI: 10.3324/haematol.2009.020636

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Design and Methods

Patient samples

Blood samples were obtained from 10 CML patients in the Clinical Investigation Centre (CIC) –

INSERM 802 at Poitiers University Hospital (Table 1). Samples were collected at diagnosis or on the

day before the start of IM treatment (day 0), and when available, within 24 hours after the initiation of

IM therapy (day 1), and after one week (day 7) and two weeks (day 14) of IM therapy. One additional

patient (P06), for whom no pre-treatment sample was available, was included in this set, using miRNA

expression data obtained at day 1 to estimate the expression fold change at day 14. However this

patient was not used for the correlation analyses with pre-treatment clinical data. IM was administered

at a standard dose of 400 mg each day, at fixed hours. Additional blood or bone marrow samples

were obtained from CML patients at diagnosis prior to IM treatment or in blast crisis, as well as from

healthy volunteer donors. The study was approved by the scientific committee of the CIC-INSERM

802 (registration number CIC 101-2007) and each patient/donor gave written informed consent in

accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were

prepared by sedimentation over a Ficoll cushion. Cells were lysed in Trizol (Invitrogen, Carlsbad, CA)

and stored at -80ºC before RNA extraction.

TaqMan low-density array screening

Reverse transcription (RT) reaction was performed using human Megaplex™ RT primers (ABI Applied

Biosystems, Foster City, CA, USA), which contains a pool of 365 individual miRNA-specific

primers, according to manufacturer’s instructions. Real-time quantitative (q)RT-PCR was then carried

out on an ABI 7900HT real time PCR machine with the LDA thermal cycler block, using pre-defined

TLDA thermal cycling conditions. QRT-PCR data were extracted with SDS2.3 and RQ Manager Software

(ABI). In order to obtain comparative data across all time points, the 4 samples corresponding to each

time point were analyzed simultaneously, along with baseline (day 0) samples. Thresholds for the

determination of Ct values were manually set to 0.2, 0.1, or 0.05 for each miRNA across the 16

samples, depending on the quality of the amplification. Relative fold change was calculated using the

DOI: 10.3324/haematol.2009.020636

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Ct method, normalized to RNU48 expression, based on averaged Ct for each time point compared

to day 0.

MiRNA quantitative RT-PCR

Total RNA (100 ng) was treated with DNase I (Invitrogen), precipitated overnight and poly-adenylated

using E. coli poly(A) polymerase (Ambion, Austin, TX). After phenol/chloroform extraction and

overnight precipitation, poly(A)-tailed RNAs were reversed-transcribed using SuperScript III

(Invitrogen) and the oligo-d(T)/adapter primer : 5'-

GCGAGCACAGAATTAATACGACTCACTATAGGACGGCTTTTTTTTTTTTTTTVN-3'. MiRNA qRT-

PCR was then performed using 1 µl of 1/10-diluted cDNAs in a 10-µl reaction containing 0.3 µM of

the adapter-specific reverse primer 5'-GCGAGCACAGAATTAATACGACTCAC-3' and miRNA-specific

forward primer (Table S2, online supplementary data), 0.1X SybrGreen (Invitrogen) and 1X HotStart

Taq MasterMix (Qiagen, Valencia, CA). Endogenous control RNU48 was amplified using 5’-

TGATGATGACCCCAGGTAACTC-3’ (forward) and 5’-GAGCGCTGCGGTGATG-3’ (reverse). Real

time PCR was performed in triplicate reactions on a Rotorgene RG-3000 (Corbett, Sydney, Australia),

with the following settings : 50°C/2min, 95°C/13min, then 40 cycles of [95°C/15sec, 58°C/15sec

touchdown -1°C for 7 cycles, 72°C/15sec], followed by melt cycle. Expression of miR-155 was

measured using TaqMan detection assays #4373124 (miR-155) and #4373383 (RNU48), according

to manufacturer’s instructions (ABI).

Statistical analysis

The significance of fold change in miRNA expression was analyzed using the Wilcoxon signed rank

test applied to the Ct values. MiRNA differential expression analysis between unrelated samples

was conducted using the 2-tailed Mann-Whitney U test, and correlation analyses were computed

using the Spearman rank correlation test. Analyses were performed using GraphPad Prism v.5 (La

Jolla, CA, USA) and SAS v.9 (Cary, NJ, USA) Software. Results with a p-value < 0.05 were

considered significant.

DOI: 10.3324/haematol.2009.020636

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Target gene prediction analysis

TargetScan 5.0 (19) was searched amongst conserved predictions for genes targeted by highly

expressed miR-142-3p, miR-199b-5p, or both, but neither by lowly expressed miR-146 nor miR-150,

and vice-versa.

DOI: 10.3324/haematol.2009.020636

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Results

Identification of differentially expressed miRNAs in primary cells from CML patients treated with

imatinib

Screening of miRNA expression using the TLDA system was initially performed on 4 patients who had

samples taken at day 0, day 1, day 7 and day 14 of IM treatment (Table 1). In order to validate the

TLDA data from these patients, previously characterized miRNAs from the miR-17~92 cluster on

chromosome (Chr) 13 as well as its paralogous clusters, miR-106a~363 on Chr X and miR-106b~25

on Chr 7, were examined (16). Of the 13 miRNAs analyzed, 11 were consistently detected in all

samples, most of which displayed a slightly reduced expression after the first day of IM treatment

(Figure 1A). A transient increase in expression for some of these miRNAs was noted at day 7,

resulting essentially from atypical expression data in a single sample, that was not observed in the

other 3 samples. By day 14, ten miRNAs showed a variable reduction in expression level compared to

day 0. We selected 3 of these miRNAs and confirmed their reduced expression at day 1 and day 14

by quantitative (q)RT-PCR (Figure 1B). Altogether, these data suggest that expression of the miR-

17~92 cluster in vivo is decreased upon IM therapy, in line with the previous in vitro study, thus

providing an internal validation for our TLDA analysis.

We demonstrated the consistent expression of 141 mature miRNAs across all samples out of a

possible 365 represented on the TLDA analysis. Fifty-two miRNAs (37%) showed log2 variations of

>0.6 (i.e. >1.5-fold change) in expression from day 0 to day 14, of which 44 were decreased and 8

increased by day 14 (online supplementary Table S1). We selected a set of 18 miRNAs for further

analysis (Figure 1C), based upon their averaged fold change in expression (log2 variations >0.6 at

day 14; miR-18, -32, -98, -132, -148a, -199b-5p, -301a, -374a, -422b; see Table S1) or their known

involvement in various aspects of blood cell physiology (miR-23, -27, -142-3p, -143, -145, -146a, -155,

-222, -223). Interestingly, some of these miRNAs showed a progressive decrease (miR-142-3p, miR-

148a, miR-199b-5p, miR-222) or increase (miR-146a) in expression during IM therapy. miR-150 and

miR-181a, which are involved in hematopoiesis (5, 20), were also included in this set, as neither were

represented on the TLDA analysis. Subsequent analysis focused on miRNA expression changes

between day 0 and day 14. Of the 20 miRNAs selected, 13 were reliably and reproducibly amplified

DOI: 10.3324/haematol.2009.020636

9

by qRT-PCR in the 11 CML samples (Figure 2A). Six miRNAs showed a median fold change >3

between day 0 and day 14, with reduced expression of miR-142-3p, miR-199b-5p, as well as the

genomically clustered miR-143/miR-145, and increased expression of miR-146a and miR-150 after IM

treatment. Except for miR-143, expression variations reached statistical significance (Wilcoxon signed

rank test, Figure 2A and Table S3).

To further assess how these changes in miRNA expression differed from normal levels, we used the

Ct values to determine which miRNAs were significantly differentially expressed in CML samples

compared to PBMC samples obtained from 6 healthy donors (controls). The expression levels of miR-

150 and miR-146a, which are strongly correlated in leukemic cells (Table S4), showed a progressive

increase from day 0 to day 14, with highest levels in control samples (Figure 2B). Indeed, at day 14,

miR-146a expression was no longer signficantly different from controls. Conversely, while miR-142-3p

and miR-199b-5p expression levels were strongly correlated (Table S4), they displayed a progressive

decrease in expression from day 0 to day 14 samples, with the lowest levels found in controls (Figure

2C). In both cases, there was a significant difference in their expression levels between day-0 CML

and control samples. Furthermore, expression of miR-145, but not miR-143, was significantly different

between day 0 samples and controls, although these two miRNAs displayed a similar decrease in

their expression pattern from day 0 to day 14 (Figure 2D). Finally, the small difference in miR-181a

expression between day 0 samples and controls was statistically significant, whereas variations

displayed by miR-18 and miR-148a were of borderline significance (not shown).

Overall these results demonstrate a significantly increased expression of miR-150 and miR-146a, and

a decrease in miR-142-3p, miR-199b-5p, and miR-145 expression, in peripheral blood cells in newly

diagnosed CML patients after treatment with IM. Our data also demonstrate that their aberrant

expression in leukemic cells tends towards, or reaches normal control values after 14 days IM

treatment.

Correlation between miRNA expression levels and clinical data

White blood cell counts (WBC) before IM treatment strongly correlated with miR-143, miR-145 and

with miR-199b-5p expression levels (P<0.001, P<0.01 and P<0.05 respectively, Table 2,) . An inverse

correlation was observed between the WBC and miR-150 expression at day 0. These results suggest

DOI: 10.3324/haematol.2009.020636

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that low expression levels of miR-150, and high expression levels of miR-143/miR-145/miR-199b-5p

reflect the high leukocyte counts found in newly diagnosed CML patients. Platelet levels at day 0 were

strongly associated with miR-181a and miR-18 levels (P<0.01), as well as miR-155 (P<0.05). The

hemoglobin level at day 0 correlated with miR-27 and miR-148a expression (P<0.01, Table 2).

Importantly, there was no correlation between miRNA expression changes and the variations in

lymphocyte vs neutrophil composition at these 2 time points, suggesting that the observed changes in

miRNA expression do not reflect changes in peripheral blood composition (Table S6).

Although most of the day 0 samples were collected after initial diagnosis (Table 1), we sought to

examine whether miRNA expression levels correlated with the Sokal risk score (21). Analysis of the

clinical features at day 0 and diagnosis revealed that 9 out of 10 patients exhibited similar blood cell

counts at these two time points, whereas the last patient displayed large variations in WBC (patient

P11, Table 1). Given that, as shown above, the expression of some miRNAs correlated with the WBC,

we performed the correlation analysis on the remaining 9 patients only, and found that miR-142-3p

correlated to the Sokal score (Table 2). This result suggests that miR-142-3p expression levels at

diagnosis could be used for prognostic purposes in CML.

Furthermore, miR-18 fold change between day 0 and day 14 inversely correlated to the time before

the occurrence of a first complete hematological response (r=-0.7198, P=0.0125). Curiously, this

miRNA is decreased in patients treated with IM. This may indicate that the rapid inhibition of miR-18 is

detrimental to an early hematological remission. Finally, although only four patients in this study

presented with blasts in peripheral blood at diagnosis (Table 1), this group of patients showed higher

levels of miR-142-3p and miR-199b-5p expression, compared to the group without blasts (not shown).

Analysis of differentially expressed miRNAs in a separate CML test set

We showed that a number of miRNAs were significantly differentially expressed between pre-

treatment CML patients and healthy donors. This raised the question whether these miRNAs were

consistently differentially expressed in CML as compared to normal cells, which would be indicative of

a more direct role for these miRNAs in leukemic cells. We therefore measured the expression level of

selected miRNAs by qRT-PCR in samples from an additional 12 CML patients, including 6 patients at

diagnosis before IM treatment (chronic phase, CP) and 6 in blast crisis (BC), including 2 patients who

DOI: 10.3324/haematol.2009.020636

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relapsed after IM. miR-150 expression was significantly reduced by ~30-fold in both CP and BC

patients as compared to normal cells (Figure 3A), and miR-146a expression was significantly reduced

in CP samples (Figure 3B). On the other hand, miR-142-3p and miR-199b-5p displayed a significantly

higher expression in CP, but not BC samples, compared to controls (Figure 3C-D). miR-145 and miR-

181a expression was not significantly different between CP, BC, and control groups (not shown).

These results confirmed the consistent reduction in expression of miR-150 and miR-146a, and

increased expression of miR-142-3p and miR-199b-5p in CML samples at diagnosis, and showed that

low expression of miR-150 was maintained in BC phase.

Target gene prediction analysis

In order to provide insights regarding genes of relevance for CML that could be targeted by the

miRNAs identified in our study, we searched for predictions using TargetScan 5.0 (19), considering

genes simultaneously targeted by the highly expressed miR-142-3p and miR-199b-5p, but not by

miR-146a and miR-150, and vice-versa (Table 3). This approach relies on our recent observation that

miRNA targets are often targeted at multiple sites (22), which substantially enhances the specificity of

predictions. Putative target genes of miR-142-3p and miR-199b-5p included ARGHEF12, MAP3K11,

and MYH9, while the complementary analysis identified 2 known genes putatively co-targeted by miR-

146a and miR-150, including the metalloproteinase gene MMP16. Searching for genes targeted by

any individual miRNA but not the other 2 (e.g. genes targeted by miR-146a or miR-150, but not by

miR-142-3p nor miR-199b-5p) resulted in larger numbers of candidate genes (Table S5).

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Discussion

In this study, we analyzed the expression profile of miRNAs in patients newly diagnosed with CML

undergoing upfront therapy with IM. Of 141 mature miRNAs consistently detected, 52 were

differentially expressed by more than 1.5-fold, most of which exhibited reduced expression by day 14

of IM therapy compared to pre-treatment samples. We demonstrated the significantly increased

expression of miR-150 and miR-146a, and decreased expression of miR-142-3p and miR-199b-5p in

PBMCs of CML patients after 14 days of IM treatment. Expression levels of miRNAs tended to

normalize to levels seen in non-leukemic PBMCs by day 14 of IM treatment. The changes in miRNA

expression are likely attributable to the leukemic cells, since after 2 weeks of IM therapy, only one

patient had achieved complete hematological remission (Table 1). Therefore we identified miRNAs

that are modulated in vivo by IM in CML patients.

We found that miR-150, miR-146a, miR-142-3p and miR-199b-5p consistently exhibited aberrant

expression in an additional group of patients with CML at diagnosis compared to healthy subjects.

This raises the possibility that these miRNAs could be involved in Bcr-Abl-mediated leukemogenesis,

although further studies are required to show whether they display differential expression in more

primitive hematopoietic cells of CML patients during IM treatment. Interestingly, Venturini et al (16)

identified miR-142-3p as being decreased in K562 cells after IM treatment, although its differential

expression was not conclusively demonstrated in primary CML samples. Moreover, in CD34+ cells of

AML patients, a high expression of miR-199a, which belongs to the same miRNA family as miR-199b,

was recently shown to be associated with poor outcome (10). The CD34+ cells from AML samples

also exhibited reduced expression of miR-146 with respect to CD34+ cells from healthy subjects (10).

Recently, Agirre et al (23) reported decreased expression of miR-10a, miR-150 and miR-151, and

increased expression of miR-96, in both CD34+ and bone marrow mononuclear cells of CML patients

at diagnosis compared to healthy subjects. This is consistent with our data demonstrating decreased

expression of miR-150 in CML samples, and increased expression of miR-151 and miR-10a by day

14 following IM treatment (Table S1). These data suggest that the aberrant expression of miR-150,

miR-146a, miR-142-3p and miR-199b-5p identified in our study likely plays a role in leukemic cells,

and potentially in the more primitive hematopoietic compartment in chronic phase CML patients.

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miR-146a, miR-155 and miR-132 have been shown to be increased in monocytes in response to LPS

stimulation, suggesting their involvement in the regulation of the immune system (24). Since we found

an increased expression in both miR-132 and miR-146a by day 14 of IM treatment (Table S1), their

reduced expression in CML cells could play a role in impairing an immune response against CML

cells. Also of interest was the increased expression of miR-215 and miR-192 after IM treatment.

These miRNAs were recently shown to be increased by p53 in diverse settings, and to contribute to

subsequent cell cycle arrest (25). Given that IM treatment induced p53 expression in BCR-ABL-

expressing cells (26), it is therefore possible that miR-215 and miR-192 could play a role in IM-

induced, p53-mediated cell cycle regulation of CML cells.

miRNA expression levels before treatment correlated with various clinical parameters, including WBC,

platelet counts and hemoglobin levels (Table 2). In addition, we found a correlation between miR-142-

3p expression level in CML patients and the Sokal risk score at diagnosis. Expression of both miR-

150 and miR-146a was consistent for all but one patient (Figure 2A). In contrast to the other 10

patients, this patient exhibited decreased expression of these miRNAs by day 14 of IM treatment

compared to day 0. Interestingly, this patient was the only one who ceased IM treatment after 4

months, having achieved complete hematological remission, because of the occurrence of acute

hepatitis. Although it is only one case, it will be of interest to determine whether measuring miR-150

and miR-146a expression as early as within the first 2 weeks of IM therapy could be indicative of IM

intolerance later on. Of note, this patient was included in the TLDA analysis, which may explain, at

least in part, the high heterogeneity observed in the miRNA expression results.

We identified several genes predicted to be targets of both miR-142-3p and miR-199b-5p, and neither

miR-146a nor miR-150 (and vice-versa), that are known to be involved in hematopoiesis or leukemia,

including ARHGEF12, MAP3K11, and MYH9. The Rho guanine nucleotide exchange factor gene

ARHGEF12 (also known as LARG) was first identified as a fusion partner for MLL in acute myeloid

leukemia (27). MAP3K11 encodes a serine/threonine kinase involved in the activation of both the p38

MAPK and JNK signaling pathways (28). This gene was shown to interact functionally with BCR-ABL

in fibroblast and hematopoietic cell transformation (29). The gene MYH9 encodes a non-muscle

myosin heavy chain which is up-regulated in myeloid cells upon differentiation (30) and is mutated in

several hematopoietic disorders (31). Genes putatively co-targeted by miR-146a and miR-150

included MMP16/MT3-MMP, which encodes a metalloproteinase that confers basement membrane

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transmigration ability to cancer cells (32). Of note, it was recently suggested that miR-146b, another

member of the miR-146 family, targeted MMP16 in the human U373 glioma cells (33).

In addition to these predictions, a number of genes have already been validated as being targeted by

these miRNAs. Specifically, miR-150 targets the oncogene MYB (34, 35), which encodes a

transcription factor that is often over-expressed in hematological malignancies, including CML (36).

Furthermore, in a mouse model of CML blast crisis, it was recently found that c-Myb was required for

Bcr-Abl-dependent leukemogenesis (37). Interestingly, over-expression of miR-150 in K562 CML cells

strongly inhibits c-MYB protein expression (35). It is thus possible that c-Myb upregulation induced by

Bcr-Abl may be mediated, at least in part, by the reduced expression of miR-150. miR-146 was

recently shown to negatively regulate the NF- B pathway by inhibiting the expression of IRAK1 and

TRAF6 (24, 38). NF- B transcriptional activity is constitutively activated by Bcr-Abl and mediates Bcr-

Abl-induced transformation in cell lines and primary cells (39, 40). Furthermore, inhibition of NF- B

activity induces apoptosis of BCR-ABL-expressing cells (41). This suggests that reduced expression

of miR-146a could be involved in the constitutive activation of NF- B signaling induced by Bcr-Abl,

thereby contributing to its transforming and anti-apoptotic activity. In support of these ideas, both MYB

and TRAF6 were identified in our target gene prediction analysis as not being targeted by either miR-

142-3p or miR-199b-5p (Table S5). miR-199b-5p was recently reported to negatively regulate the

Notch pathway in meduloblastoma cells by inhibiting the Notch target gene HES1, leading to reduced

cell proliferation (42). Interestingly, constitutive activation of Notch in K562 cells was shown to inhibit

cell proliferation and colony-forming activity, along with increased HES1 mRNA levels (43). It is

therefore possible that the increased expression of miR-199b-5p could play a role in the inhibition of

Notch signaling in CML cells, contributing to their increased proliferative activity.

In summary, IM treatment results in a relatively rapid increase in the expression of miR-150 and miR-

146a, and decreased expression of miR-142-3p and miR-199b-5p in PBMCs of patients newly

diagnosed with CML. We showed that the aberrant expression level of these miRNAs was tending

towards normal levels after only 2 weeks of IM therapy. These miRNAs, by regulating certain

predicted and known target genes, may be involved in Bcr-Abl-driven leukemogenesis.

DOI: 10.3324/haematol.2009.020636

15

Authorship and Disclosures

SF, AGT, and JEJR designed the research; SF performed the experiments and analyzed data;

WR analyzed the TLDA results and performed the target gene prediction analysis; JG analyzed

data and performed statistical analyses; JH contributed design of the TLDA experiments and

provided conceptual input; M-LB and J-CC processed patient samples; FG and AGT provided

patient samples and clinical data; SF and JEJR wrote the manuscript.

The authors declare the following competing financial interests: AGT: Bristol Myers, Novartis

(Honoraria, travel expenses). JEJR: Sydney IVF (Approved pathology practitioner and member of

Ethics Committee), Gilead Biosciences (Honorarium for lectures, travel expenses).

DOI: 10.3324/haematol.2009.020636

16

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Table 1. Patients' clinical parameters at diagnosis and day 0.

Patient Id

Age Sex

Clinical features at diagnosis Clinical features at day 0 time before CHR (days after start

of IM)

WBC (10

6/mL)

Platelets (10

6/mL)

Blast (%)

Hemoglobin (g/dL)

Sokal risk score

WBC (10

6/mL)

Platelets (10

6/mL)

Hemoglobin (g/dL)

P06 50 F 446 223 0 9.7 0.78013 29 506 12 41

P07 76 M 86 288 2 15.2 1.03815 86 288 15.2 43

P11 62 F 172 326 0 10.8 0.93677 14 270 10.5 21

P12 58 M 31 200 0 13.2 0.69798 45 166 14.1 21

P14 48 M 162 354 2 13.2 0.91578 162 354 13.2 28

P17 49 M 565 405 4 9.4 1.47316 479 314 8.7 27

P20 58 M 119 731 4 9 1.67627 128 850 9.8 42

P24 50 M 37 220 0 16.1 0.64043 56 223 15.9 34

P26 74 M 18 148 0 14.9 0.83502 18 148 14.9 15

P30 45 F 15 859 0 12.7 0.78511 16 951 12.7 82

P43 70 M 63 195 0 14.3 0.79905 63 193 15.2 169

WBC indicates white blood cell counts, CHR, complete hematological response. For patients P07, P14, and P26, day 0 samples used in this study were those at diagnosis. The 4 patients whose samples were used in the TLDA screening are shown with grey background.

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Table 2. Correlation analysis between miRNA expression level and clinical data before IM therapy.

miRNA name WBC Platelet counts Hemoglobin SOKAL index1

miR-18 ns 0.8182** ns ns miR-27 ns ns 0.8061** ns miR-142-3p ns 0.6606* ns 0.7667* miR-143 0.8754*** ns ns ns miR-145 0.8182** ns ns ns miR-146 ns ns ns ns miR-148a ns -0.7455* 0.8182** ns miR-150 -0.6848* ns ns ns miR-155 ns 0.8571* ns ns miR-181a ns 0.8424** ns ns

miR-199-5p 0.6606* ns ns ns WBC indicates white blood cell counts, ns : no significant correlation. Data represent correlation coefficient r from Spearman correlation analysis using day 0 Ct values. 1This anaylsis was computed using 9 patients only (see text).

*P<0.05 ; **P<0.01 ; ***P<0.001.

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Table 3. Target gene predictions for differentially expressed miRNAs in CML.

Gene Symbol

Name Comment Reference

Targeted by miR-142-3p AND miR-199b-5p, but NOT miR-150 OR miR-146a

ACVR2A Activin A type II receptor Targeted by miR-15/16 in X. laevis embryos

44

ANK3 Ankyrin 3 -

ARHGEF12 Rho guanine nucleotide exchange factor (GEF) 12 (also called LARG for leukemia-associated rho GEF)

MLL fusion partner in acute myeloid leukemia

27

C18orf25 - -

CCNJ Cyclin J -

MAP3K11 Mitogen-activated protein kinase kinase kinase 11 (also called MLK3, PTK1, SPRK)

Interacts functionally with BCR-ABL for fibroblast and hematopoietic cell transformation

29

MYH9 Non-muscle myosin IIA Upregulated in myeloid cell lines upon induction of differentiation

30

MYO5A Myosin V heavy-chain -

RBM47 RNA binding motif protein 47 -

RHEB Ras homolog enriched in brain Association with mTOR is inhibited by PML

45

RNF38 Ring finger protein 38 -

ZNF618 Zinc finger protein 618 -

Targeted by miR-150 AND miR-146a, but NOT miR-142-3p OR miR-199b-5p

C12orf36 - -

MMP16 Matrix metallopeptidase 16 (membrane-inserted). Targeted by miR-146b in U373 glioblastoma cells

33

NFASC Neurofascin homolog (chicken) -

Upper part shows genes predicted by TargetScan 5.0 to be co-targeted by miR-142-3p AND miR-199b-5p, with no target sites for miR-146a nor miR-150. Lower part shows predictions using the opposite miRNA combination.

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Figure legends

Figure 1. Identification of miRNAs differentially expressed in CML patients during imatinib mesylate

(IM) therapy. (A) Average fold change compared to pretreatment levels of the miR-17~92 cluster

members using TaqMan low density array (TLDA) analysis (n=4). (B) QRT-PCR validation of the

TLDA results showing the fold change in miR-17-3p, miR-18, and miR-20 after 1 and 14 days of IM

treatment. (C) Fold change of a selection of 18 miRNAs from the TLDA analysis at day 1, 7, and 14,

relative to day 0 (set to 1). Data represent mean + SD of the 4 samples tested.

Figure 2. Validation of deregulated miRNAs in CML. (A) Fold change compared to pretreatment levels

of 13 miRNAs at day 14 following IM therapy analyzed by qRT-PCR (n = 11 patients). Fold change

significance was tested using a non-parametric t-test on paired samples (2-tailed Wilcoxon test); (*)

P<0.05; (**) P<0.01. (B-D) Statistical analysis of miRNA expression in the sequential CML samples

and healthy controls. The Ct values (Ct of RNU48 minus Ct of miRNA), which reflect logarithmic

scale changes, are shown for all samples. *P<0.05; **P<0.01; ns: no significant difference (2-tailed

Mann-Whitney test). Bar: median; cont: normal control PBMCs.

Figure 3. Analysis of miRNA expression in a subsequent test set of CML patients. Expression level

( Ct) of miR-150 (A), miR-146a (B), miR-142-3p (C), and miR-199b-5p (D) in 6 chronic phase (CP)

and 6 blast crisis (BC) CML patients. *P<0.05; **P<0.01; ns: no significant difference (2-tailed Mann-

Whitney test). Bar: median; cont: normal control PBMCs.

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1

Micro-RNA response to imatinib mesylate in patients with chronic myeloid leukemia

Stéphane Flamant1,8

, William Ritchie1, Joëlle Guilhot

2,3, Jeff Holst

1, Marie-Laure Bonnet

3,

Jean-Claude Chomel3,5

, François Guilhot

2-4, Ali G. Turhan

3,5, and John E. J. Rasko

1,6,7,§

Supplementary Data

The authors wish to include 6 tables as data supplements, and provide below a short description of

each of these items.

Online Supplementary Table S1. Fold change of miRNAs from TLDA analysis at day 1, 7, and 14

compared to day 0. Complete list of the 141 miRNAs reproducibly detected in CML samples by TLDA

analysis. Upper and lower parts represent miRNAs with log2 variations of > 0.6, i.e. mean fold change

> 1.5 by day 14 (52 miRNAs, 44 down, 8 up). Intermediate parts represent miRNAs with mean fold

change of 1.3-1.5 (36 miRNAs, 25 down,

Online Supplementary Table S2. Primer sequences used in this study for qRT-PCR validation

experiments.

Online Supplementary Table S3. miRNA median fold change from qRT-PCR analysis. Expression

fold change in IM-treated patients between day 14 and day 0 (n=11). P-values were calculated using

2-tailed Wilcoxon test on paired DCt values.

Online Supplementary Table S4. miRNA expression correlation analysis in CML samples. (A-B)

Spearman correlation analysis of miRNA expression levels at (A) day 0 and (B ) day 14. (C)

Spearman correlation analysis of miRNA expression fold change between day 0 and day 14 IM

treatment.

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2

Online Supplementary Table S5. Target gene predictions using various miRNA combinations. This

table contains the full list of conserved target genes from TargetScan v5.0 predicted to be targeted by

the following miRNA combinations : (A) miR-142-3p AND miR-199b-5p but NOT miR-146 NOT miR-

150 ; (B) miR-142-3p OR miR-199b-5p but NOT miR-146 NOT miR-150 ; (C) miR-146 AND miR-150

but NOT miR-142-3p NOT miR-199b-5p ; (D) miR-146 OR miR-150 but NOT miR-142-3p NOT miR-

199b-5p. For comparison, the predictions from combinations (A) and (C), presented in Table 3 in the

main text, were reproduced in this table.

Online Supplementary Table S6. Absence of correlation between miRNA expression fold changes

and blood cell composition changes. (A) Patients' blood cell composition, showing the percentages of

lymphocytes and neutrophils at day 0 and day 14 IM treatment. (B) Data represent Spearman

correlation coefficient r between miRNA fold changes and the cell composition ratio shown in (A)

between day 0 and day 14.

DOI: 10.3324/haematol.2009.020636

Table S1. Fold change of miRNAs from TLDA analysis at day 1, 7, and 14 compared to day 0.

miRNA assay Day 1 Day 7 Day 14Variation atday 14 vs

day 0 (ABI 'detector' code) Fold change SD Fold change SD Fold change SD

hsa-miR-199b-4373100 -0.539 0.871 -0.669 0.597 -3.800 0.745

hsa-miR-422a-4373200 -0.680 0.829 -2.529 1.560 -2.380 1.522

Reduced bymore than1.5-fold

hsa-miR-17-3p-4373120 -0.974 0.955 0.582 0.724 -2.050 1.298

hsa-miR-517a-4373243 -2.643 1.544 -1.390 1.328 -1.955 1.133

hsa-miR-143-4373134 -1.287 0.911 -0.477 0.610 -1.789 1.368

hsa-miR-32-4373056 -0.360 0.832 -1.436 1.781 -1.674 1.456

hsa-miR-378-4373024 -0.508 0.438 -0.898 0.440 -1.535 0.489

hsa-miR-379-4373023 -0.940 0.522 -1.277 0.269 -1.510 0.616

hsa-miR-145-4373133 0.137 0.520 -0.023 0.515 -1.483 1.028

hsa-miR-302a-4373275 -0.890 0.703 -2.165 0.784 -1.395 0.748

hsa-miR-518e-4373265 0.401 0.812 -0.768 1.442 -1.330 1.474

hsa-miR-301-4373064 -0.430 0.556 -0.931 1.194 -1.120 0.596

hsa-miR-19a-4373099 -0.342 0.787 -0.457 0.936 -1.046 0.798

hsa-miR-98-4373009 -0.691 1.748 -1.054 1.517 -1.042 1.108

hsa-miR-148a-4373130 -0.332 0.416 -0.551 0.447 -1.020 0.408

hsa-miR-550-4380954 0.133 0.462 0.083 0.402 -1.002 0.544

hsa-miR-422b-4373016 -0.994 0.418 -1.323 0.839 -0.984 0.465

hsa-miR-514-4373240 -0.548 1.141 -0.669 1.129 -0.899 0.894

hsa-miR-130b-4373144 -0.381 0.974 0.266 0.853 -0.887 0.907

hsa-miR-142-3p-4373136 -0.060 0.408 -0.469 0.560 -0.882 0.805

hsa-miR-223-4373075 -0.240 0.485 -0.187 0.513 -0.878 0.803

hsa-miR-20a-4373286 -0.094 0.668 -0.119 0.780 -0.863 0.622

hsa-miR-518c-4373247 0.337 1.243 -1.523 1.468 -0.859 1.211

hsa-miR-518b-4373246 -0.988 0.995 -0.325 0.678 -0.847 0.960

hsa-miR-302c-4373277 -0.220 0.733 -1.618 1.475 -0.846 0.665

hsa-miR-106b-4373155 -0.197 0.366 0.071 0.540 -0.841 0.445

hsa-miR-19b-4373098 -0.369 0.700 -0.154 0.824 -0.810 0.638

hsa-miR-27b-4373068 -0.675 1.039 -0.883 1.375 -0.792 1.015

hsa-miR-449-4373207 0.317 0.818 -0.097 1.089 -0.752 1.115

hsa-miR-367-4373034 -1.408 0.899 -1.760 1.344 -0.743 1.035

hsa-miR-221-4373077 -0.612 1.399 -1.774 1.929 -0.743 1.291

hsa-miR-374-4373028 -0.583 0.959 -0.462 0.955 -0.739 0.784

hsa-miR-18a-4373118 -0.180 0.558 0.277 0.698 -0.726 0.658

hsa-miR-25-4373071 -0.073 0.406 0.309 0.577 -0.721 0.541

hsa-miR-449b-4381011 -0.344 0.888 -0.138 0.449 -0.717 0.427

hsa-miR-659-4380924 0.733 0.312 0.338 0.510 -0.705 0.861

hsa-miR-23a-4373074 -0.563 0.351 -0.361 0.441 -0.700 0.600

hsa-miR-372-4373029 -0.064 0.960 -0.135 1.055 -0.699 0.903

hsa-miR-515-3p-4373241 0.827 0.672 1.119 0.563 -0.664 0.888

hsa-miR-660-4380925 -0.110 0.929 -0.156 0.974 -0.657 0.867

hsa-miR-99a-4373008 0.125 1.031 -0.010 0.822 -0.635 0.933

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hsa-miR-629-4380969 -1.005 0.610 -0.526 0.917 -0.633 0.753

hsa-miR-335-4373045 -0.232 1.591 -0.209 1.440 -0.623 1.188

hsa-miR-142-5p-4373135 -0.101 0.496 -0.095 0.618 -0.601 0.690

hsa-let-7c-4373167 -0.627 1.533 -0.141 1.231 -0.567 0.864

hsa-miR-365-4373194 0.255 0.426 -0.464 0.331 -0.528 0.398

Reduced bymore than1.3-fold

hsa-miR-20b-4373263 -0.252 1.212 -0.563 1.529 -0.525 0.827

hsa-miR-324-3p-4373053 -0.265 0.663 0.416 0.618 -0.525 0.590

hsa-miR-518d-4373248 0.314 0.358 -1.063 1.101 -0.503 0.562

hsa-miR-425-5p-4380926 -0.154 0.518 0.140 0.580 -0.498 0.535

hsa-miR-30b-4373290 -0.224 0.657 0.020 0.736 -0.486 0.591

hsa-miR-425-4373202 -0.134 0.313 -0.171 0.782 -0.485 0.528

hsa-miR-17-5p-4373119 -0.247 0.752 0.288 0.853 -0.476 0.663

hsa-miR-650-4381006 -1.213 0.452 -0.541 0.964 -0.475 0.571

hsa-miR-140-4373138 -0.217 0.506 -0.036 0.628 -0.461 0.623

hsa-miR-15b-4373122 -0.103 0.197 0.522 0.505 -0.453 0.304

hsa-miR-585-4381027 -0.146 0.280 -0.190 0.215 -0.449 0.192

hsa-miR-222-4373076 0.134 0.641 -0.275 0.802 -0.444 0.597

hsa-miR-509-4373234 -1.165 0.765 -0.132 0.795 -0.438 0.891

hsa-miR-320-4373055 -0.836 0.785 0.028 0.537 -0.436 0.539

hsa-miR-376a-4373026 -2.178 2.038 -0.139 1.536 -0.413 1.148

hsa-miR-93-4373012 -0.144 0.223 0.313 0.415 -0.407 0.317

hsa-miR-125b-4373148 -0.581 0.882 0.135 0.610 -0.404 0.658

hsa-miR-340-4373041 0.066 0.641 -0.452 0.795 -0.394 0.590

hsa-miR-484-4381032 -0.146 0.525 0.417 0.579 -0.388 0.518

hsa-miR-15a-4373123 -0.156 0.526 0.131 0.675 -0.386 0.624

hsa-let-7b-4373168 -0.095 0.297 0.445 0.592 -0.384 0.303

hsa-miR-328-4373049 -0.438 0.653 -0.072 0.722 -0.382 0.592

hsa-miR-191-4373109 -0.212 0.491 0.226 0.501 -0.382 0.561

hsa-miR-490-4373215 0.192 0.299 -0.164 0.324 -0.372 0.338Modified byless than1.3-fold hsa-miR-345-4373039 -0.047 0.236 0.271 0.213 -0.364 0.249

hsa-miR-510-4373235 0.478 0.448 -0.303 0.558 -0.364 1.006

hsa-miR-155-4373124 -0.293 0.810 -0.610 0.804 -0.351 0.550

hsa-miR-195-4373105 -0.162 0.511 -0.016 0.870 -0.344 0.473

hsa-miR-27a-4373287 -0.126 0.485 -0.016 0.645 -0.327 0.494

hsa-miR-103-4373158 -0.363 0.540 -0.020 0.613 -0.306 0.413

hsa-miR-199a-4378068 -0.474 1.150 -0.772 1.252 -0.305 0.904

hsa-miR-16-4373121 -0.250 0.570 0.346 0.823 -0.288 0.558

hsa-miR-21-4373090 -0.584 0.913 -0.931 0.892 -0.267 0.800

hsa-miR-194-4373106 -0.501 0.520 -0.848 1.146 -0.234 0.433

hsa-miR-197-4373102 -0.221 0.465 0.140 0.456 -0.232 0.577

hsa-miR-196a-4373104 0.244 0.836 -0.488 1.136 -0.230 0.824

hsa-miR-30a-5p-4373061 -0.246 0.526 0.255 0.575 -0.229 0.439

hsa-miR-451-4373209 -0.746 1.683 0.902 1.832 -0.226 1.348

hsa-miR-331-4373046 -0.133 0.568 0.473 0.581 -0.225 0.468

DOI: 10.3324/haematol.2009.020636

hsa-miR-126-4373269 -1.340 2.244 -0.080 1.752 -0.225 1.512

hsa-miR-30d-4373059 -0.158 0.591 0.388 0.640 -0.222 0.449

hsa-miR-520e-4373255 -0.049 0.606 0.184 0.594 -0.205 0.478

hsa-miR-302a-4378070 -0.714 0.787 0.026 0.799 -0.193 0.837

hsa-miR-324-5p-4373052 -0.097 0.634 0.175 0.604 -0.188 0.602

hsa-miR-186-4373112 -0.031 0.844 0.193 0.969 -0.186 0.785

hsa-miR-196b-4373103 -0.026 0.998 0.293 0.957 -0.166 0.942

hsa-miR-517c-4373264 1.309 1.587 0.023 1.317 -0.161 1.438

hsa-miR-30c-4373060 -0.304 0.502 0.297 0.584 -0.151 0.486

hsa-miR-362-4378092 -0.718 0.686 -0.465 0.882 -0.131 0.619

hsa-miR-24-4373072 -0.256 0.636 -0.103 0.724 -0.114 0.608

hsa-let-7a-4373169 -0.173 0.432 0.115 0.489 -0.093 0.394

hsa-miR-26b-4373069 -0.232 0.744 0.044 0.769 -0.066 0.671

hsa-miR-383-4373018 0.656 1.216 0.984 1.284 -0.060 1.283

hsa-miR-423-4373015 0.102 0.448 0.382 0.422 -0.053 0.452

hsa-miR-92-4373013 0.002 0.472 0.853 0.698 -0.048 0.369

hsa-miR-330-4373047 0.814 0.897 0.537 0.876 -0.042 1.015

hsa-miR-565-4380942 0.185 0.515 0.761 0.514 0.008 0.784

hsa-miR-181d-4373180 0.622 0.684 -0.635 1.011 0.035 0.702

hsa-miR-338-4373043 -0.349 0.708 -2.215 0.898 0.062 0.646

hsa-miR-100-4373160 0.829 1.143 0.578 1.137 0.065 1.092

hsa-miR-210-4373089 -0.198 1.082 0.315 1.335 0.091 1.087

hsa-let-7f-4373164 -0.818 1.139 -0.826 0.870 0.117 0.581

hsa-miR-501-4373226 -0.736 0.815 -0.192 1.115 0.117 0.807

hsa-miR-192-4373108 0.330 1.035 0.508 1.090 0.120 1.059

hsa-let-7g-4373163 -0.005 0.661 0.109 0.673 0.125 0.557

hsa-miR-130a-4373145 -0.617 1.312 0.307 0.984 0.133 0.892

hsa-miR-126-4378064 -0.586 1.851 0.061 1.673 0.138 1.442

hsa-miR-409-5p-4373197 -0.333 1.646 0.584 1.452 0.167 1.435

hsa-miR-28-4373067 -0.375 1.040 -0.062 1.015 0.223 0.794

hsa-miR-152-4373126 -0.839 1.496 0.490 1.369 0.233 1.183

hsa-miR-181b-4373116 0.227 0.311 0.851 0.567 0.243 0.296

hsa-miR-29c-4373289 0.063 0.898 -0.180 0.886 0.255 0.664

hsa-miR-26a-4373070 -0.178 0.774 0.185 0.740 0.256 0.652

hsa-miR-361-4373035 -0.473 0.835 -0.583 0.758 0.260 0.638

hsa-miR-630-4380970 -0.011 0.692 -0.041 0.994 0.277 0.624

hsa-miR-148b-4373129 -0.139 0.979 0.004 0.973 0.289 0.824

hsa-let-7d-4373166 0.431 0.579 0.891 0.821 0.394 0.575

hsa-miR-532-4380928 0.461 0.356 0.688 0.701 0.404 0.337

Increased bymore than1.3-fold

hsa-miR-596-4380959 0.678 0.273 0.774 0.531 0.406 0.284

hsa-miR-146b-4373178 0.027 0.990 -0.230 0.980 0.421 0.741

hsa-miR-296-4373066 -0.165 1.147 0.981 1.041 0.439 0.967

hsa-miR-99b-4373007 0.104 0.779 0.016 0.972 0.464 0.878

hsa-miR-200c-4373096 0.240 0.846 0.574 0.917 0.493 0.729

DOI: 10.3324/haematol.2009.020636

hsa-miR-642-4380995 -0.821 0.963 -2.074 1.502 0.494 0.686

hsa-miR-29a-4373065 0.022 0.737 0.023 0.682 0.560 0.642

hsa-miR-515-5p-4373242 -0.232 1.023 0.666 0.999 0.572 1.115

hsa-miR-125a-4373149 -0.001 0.606 0.480 0.528 0.575 0.561

hsa-miR-146a-4373132 -0.393 1.259 -0.249 1.337 0.657 0.987

hsa-miR-339-4373042 -0.060 0.780 0.163 0.850 0.685 0.603

Increased by

more than1.5-fold

hsa-miR-342-4373040 0.037 0.862 0.138 0.874 0.685 0.744

hsa-miR-151-4373179 -0.835 1.796 -0.079 1.888 0.768 1.476

hsa-miR-10a-4373153 0.825 1.329 0.988 1.346 0.791 1.374

hsa-miR-485-3p-4378095 1.877 1.210 0.032 1.673 0.885 1.227

hsa-miR-132-4373143 0.220 0.914 1.458 0.909 1.048 0.923

hsa-miR-215-4373084 2.013 1.715 1.277 1.502 2.568 1.165

SD indicates standard deviation. Fold change (log2) was calculated from the averaged !Ct of the 4 samples for each time

point, compared to day 0.

DOI: 10.3324/haematol.2009.020636

Table S2. Primer sequences used in this study for qRT-PCR validation experiments

miRNA namemiRNA mature sequence (5'-3')

q-PCR primer sequence (5'-3')

hsa-miR-181

UAAGGUGCAUCUAGUGCAGAUAG GGTTGGTTAAGGTGCATCTAGTG

hsa-miR-231

AUCACAUUGCCAGGGAUUUCC TAATTATCACATTGCCAGGGAT

hsa-miR-271

UUCACAGUGGCUAAGUUCCGC GGATTACGTTCACAGTGGCTAAG

hsa-miR-32 UAUUGCACAUUACUAAGUUGCA GGTTCAGCTTATTGCACATTACTAAGT

hsa-miR-98 UGAGGUAGUAAGUUGUAUUGUU GGGTGGTGAGGTAGTAAGTTGTA

hsa-miR-132 UAACAGUCUACAGCCAUGGUCG GGGTGTTAACAGTCTACAGCCAT

hsa-miR-142-3p UGUAGUGUUUCCUACUUUAUGGA GGGTGGTTGTAGTGTTTCCTACT

hsa-miR-143 UGAGAUGAAGCACUGUAGCUC CCGGAATTCTGAGATGAAGCACTG

hsa-miR-145 GUCCAGUUUUCCCAGGAAUCCCU AAGGTTGTCCAGTTTTCCCAGGAA

hsa-miR-146a UGAGAACUGAAUUCCAUGGGUU AACTGATGAGAACTGAATTCCATG

hsa-miR-148a UCAGUGCACUACAGAACUUUGU GGTTGTCAGTGCACTACAGAACTT

hsa-miR-150 UCUCCCAACCCUUGUACCAGUG AACCTGATCTCCCAACCCTTGTA

hsa-miR-1552

UUAAUGCUAAUCGUGAUAGGGGU -

hsa-miR-181a AACAUUCAACGCUGUCGGUGAGU GGTTGGTAACATTCAACGCTGTC

hsa-miR-199b-5p CCCAGUGUUUAGACUAUCUGUUC GTCAGGCCCAGTGTTTAGACTAT

hsa-miR-222 AGCUACAUCUGGCUACUGGGU GGAACAAGCTACATCTGGCTACT

hsa-miR-223 UGUCAGUUUGUCAAAUACCCCA CCACGTCTGTCAGTTTGTCAAA

hsa-miR-301a CAGUGCAAUAGUAUUGUCAAAGC GTCAGGCAGTGCAATAGTATTGTCA

hsa-miR-374a UUAUAAUACAACCUGAUAAGUG CCCTAGTTATAATACAACCTGATAAGTG

hsa-miR-422b ACUGGACUUGGAGUCAGAAGG ACTGCTCTGGACTTGGAGTCA

The overlap between the primer sequence and the mature miRNA is underlined. MiRNAs reproduciblyamplified are displayed on a grey background.1Primers for these assays detect isoforms a and b of the mature miRNA.

2This miRNA was tested using the corresponding ABI TaqMan assay.

DOI: 10.3324/haematol.2009.020636

Table S3. miRNA median fold change from qRT-PCR analysis.

miRNA Fold change Range P

miR-18 0.616 [0.281 - 3.580] 0.0558

miR-27 0.841 [0.665 - 2.789] 0.2291

miR-142-3p 0.321 [0.134 - 1.444] 0.0137

miR-143 0.344 [0.048 - 3.918] 0.0674

miR-145 0.297 [0.070 - 1.705] 0.0186

miR-146 3.249 [0.150 - 22.94] 0.0322

miR-148a 0.521 [0.295 - 1.395] 0.0537

miR-150 3.458 [0.247 - 79.34] 0.0137

miR-1551

0.920 [0.611 - 2.621] 0.8125

miR-181a 1.395 [0.595 - 4.857] 0.0674

miR-199b-5p 0.275 [0.071 - 1.283] 0.0068

miR-222 0.901 [0.387 - 2.694] 0.7646

miR-223 0.702 [0.253 - 2.549] 0.1748

Expression fold change between day 0 and day 14.P-values were calculated using 2-tailed Wilcoxon test on paired _Ct values.1This miRNA was tested using the corresponding ABI TaqMan assay on 7 samples only.

DOI: 10.3324/haematol.2009.020636

Table S4. miRNA expression correlation analysis in CML samples.

A - Before IM therapy (day 0)

miR-199b-5p miR-181a miR-150 miR-148a miR-146 miR-145 miR-143 miR-142-3p miR-18

miR-18 0.7939** -0.8545**

miR-142-3p 0.7818** 0.6485* -0.6485*

miR-143 -0.8024** -0.8085**

miR-145 0.6606*

miR-146 0.8909***

miR-148a -0.6727*

miR-150 -0.6606*

miR-181a

miR-199b-5p

B - After 14 days IM therapy

miR-199b-5p miR-181a miR-150 miR-148a miR-146 miR-145 miR-143 miR-142-3p miR-18

miR-18 0.6848* 0.8909***

miR-142-3p 0.8667** -0.6727* 0.6970*

miR-143 0.9030***

miR-145

miR-146 0.7842**

miR-148a 0.8303**

miR-150 -0.7576*

miR-181a

miR-199b-5p

C - Correlation analysis of miRNA expression fold change in CML samples

miR-199b-5p miR-181a miR-150 miR-148a miR-146 miR-145 miR-143 miR-142-3p miR-18

miR-18 0.7212* 0.7333*

miR-142-3p 0.8303** 0.7576*

miR-143 0.7818** 0.9273***

miR-145 0.7212* 0.7333*

miR-146 0.7576*

miR-148a

miR-150

miR-181a

miR-199b-5p

Correlation coefficients r from Spearman correlation analysis using (A) day 0 _Ct values, (B) day 14 _Ct values, and (C) miRNAexpression fold changes from day 0 to day 14. *P<0.05; **P<0.01; ***P<0.001; empty: not significant.

DOI: 10.3324/haematol.2009.020636

Table S5. Target gene predictions using various miRNA combinations, as follows

A. miR-142-3pAND miR-199b-

5pbut NOT miR-146 NOT miR-

150

B. miR-142-3p OR miR-199b-5pbut NOT miR-146 NOT miR-150

C. miR-146AND miR-150

butNOT miR-142-3p NOT miR-

199b-5p

D. miR-146 OR miR-150 butNOT miR-142-3p NOT miR-

199b-5p

ACVR2A AADACL1 CYLC2 KDELR2 PMAIP1 STAM C12orf36 ACBD3 MMP14

ANK3 ABCA1 D4S234E KIAA0355 PMP22 STAU1 MMP16 ADAM19 MMP16

ARHGEF12 ABCC1 DCUN1D4 KIAA0753 PNPLA6 STK4 NFASC ADIPOR2 MTCH2

C18orf25 ABCC5 DDR1 KIAA0831 PODXL STRN3 AKAP9 MYADM

CCNJ ACBD5 DDX3X KIAA1553 POGK STX12 ALAD MYB

MAP3K11 ACVR2A DDX3Y KIAA2018 POLS SULF1 ANKRD52 MYBL1

MYH9 ACVR2B DDX6 KIT POMGNT1 SYPL1 AP3M2 MYO6

MYO5A ADAMTS3 DEPDC1B KL PPARGC1A TAGAP APPL1 MYT1

RBM47 ADAMTSL3 DIRC2 KLF12 PPFIA1 TAOK1 ARL8A NEGR1

RHEB ADCY9 DKFZp667G2110 KLF13 PPFIBP1 TARDBP ATG12 NF2

RNF38 ADD3 DLC1 KLHL6 PPP1R10 TBC1D2B ATP8A2 NFASC

ZNF618 ADRBK2 DMTF1 KPNA4 PPP1R12A TBC1D8 BAG1 NFIC

AFF1 DRAM KPNB1 PPP1R2 TBL1X BASP1 NOVA1

AFF2 DYNC1LI2 LAMC1 PPP1R9A TBL1XR1 BCL11A NPAS4

AFTPH DYNC2LI1 LARGE PPP3CA TESK2 BCORL1 NRAS

AKAP1 E2F3 LARP4 PPP3R1 TET2 BIVM NSUN4

AKT1S1 ECE1 LCOR PRDM16 TEX2 BNC1 NUMB

ALS2 EDEM3 LEPREL1 PRLR TFG BSN PAPD5

ALS2CR2 EHD4 LIFR PROM1 TGFBR1 BTG2 PAPPA

AMOTL1 EHF LIMD2 PRPF40A TIMM10 BTRC PBX2

ANK3 EIF2C1 LIN7C PRRG1 TIPARP C12orf36 PCBP4

ANKRD11 EIF5B LLGL2 PTPN23 TIRAP C14orf43 PDCD4

ANKRD13C EML4 LMAN2 PTPRE TM9SF3 C17orf39 PDIA6

ANKRD42 EPB41L1 LMO3 PUM1 TMEM110 C1orf25 PFN2

ANKS1A EPHA7 LRP1B PURB TMEM115 C1orf71 PHOX2B

AP1G1 EPN1 LRP4 PVRL1 TMEM123 C5orf46 PIK3AP1

DOI: 10.3324/haematol.2009.020636

APPBP2 ERC1 LRRC1 PXN TMEM135 C6orf134 PIK3R1

ARHGAP12 ERG LRRC32 R3HDM2 TMEM200B CALU PKHD1

ARHGAP19 ERLIN1 LRRC59 RAB1A TMEM32 CARD10 PLP2

ARHGAP21 ETS1 LYSMD3 RAB21 TMEM55B CASK PM20D2

ARHGAP29 ETS2 M6PR RAB2A TMEM59 CC2D1B POLD3

ARHGEF12 ETV6 MAB21L1 RAB3A TMEM63B CCBP2 POM121

ARHGEF2 EXOC8 MAP2K4 RAB40C TMEM66 CCDC4 POM121C

ARID2 EXTL3 MAP3K11 RAC1 TNKS CD80 PPM1M

ARID5B FADS6 MAP3K7IP2 RAD23B TNRC18 CDAN1 PPP1R11

ARL1 FAM100B MAP3K7IP3 RANBP10 TOX3 CDC42BPA PRICKLE2

ARL15 FAM107B MAP4 RANBP2 TP53INP2 CDON PRKAR1A

ARL6IP6 FAM108C1 MAP4K3 RANBP3 TRPM4 CHD2 PRPS1

ARNTL FAM114A1 MARCH1 RARG TSEN34 CMTM6 PRRT2

ARRB2 FAM116A MARCH7 RASSF2 TSPAN5 CNTFR PSCD3

ASH1L FAM130A1 MARCKS RBBP4 TSPAN6 COL4A4 PSMD3

ASRGL1 FAM178A MARK3 RBM23 TST CPD PTGFRN

ATF7IP FAM44B MARK4 RBM24 TTBK1 CSF1R PTP4A1

ATG16L1 FBXO21 MCFD2 RBM27 TTC9 DGKG PTPRB

ATG4D FBXO33 MEF2D RBM47 TWF1 DLGAP1 PVRL2

ATP13A2 FBXO45 MFHAS1 RBPMS TXNL1 DLGAP2 RABGAP1

ATXN7 FBXO9 MGAT3 REEP2 UBAP1 DNAJB7 RASGEF1A

ATXN7L1 FKBP1A MGAT4A RERE UBE2G1 DNAL1 RC3H1

AUTS2 FLJ20160 MGAT4B RFWD3 UBE2Q1 DNPEP RCSD1

B3GNT1 FLJ20309 MICAL3 RGL2 UBL3 DSEL REPS2

BAAT FLRT3 MIER3 RGMA UNC84A DYRK1A RFTN2

BACH1 FMNL2 MINK1 RGMB UNKL EBF3 RIMBP2

BACH2 FNDC3A MKL2 RGS10 USP31 EDA RIMS2

BAT1 FNTB MN1 RHEB USP37 EDNRB RNASEL

BAZ1A FOXO1 MOBKL3 RHOBTB3 USP46 EIF4B RNF165

BCLAF1 FOXO4 MORF4L2 RICTOR USP6NL EIF4E ROBO1

BLCAP FSD1 MPP5 RIMS1 UTRN EIF4G2 RUNX1T1

DOI: 10.3324/haematol.2009.020636

BNC2 FSTL4 MRPL22 RLF UTX EIF5A2 SCN3B

BRWD3 FUT9 MRPS25 RND1 UTY ELK1 SEC23IP

BTBD3 FXR1 MTCH1 RNF11 VAMP3 ENTPD1 SEMA3G

BTBD7 FZD6 MTMR3 RNF38 VEGFA EP300 SGMS1

BTBD9 GAB1 MTMR9 ROCK1 VPS24 EPHB2 SH3BP5L

C10orf18 GANAB MYH10 ROCK2 VPS26A FAM134C SH3TC2

C10orf46 GARNL1 MYH9 ROD1 WAPAL FAM26E SHISA4

C10orf97 GARNL4 MYLK RRP15 WASL FAM65B SIAH2

C11orf9 GCNT2 MYO5A RUNX3 WDR44 FBXL10 SLC10A3

C13orf1 GFI1 MYST2 S1PR3 WDTC1 FBXW11 SLC2A3

C16orf70 GHR NAALADL2 SACS WIPF2 FBXW2 SLFN13

C17orf63 GIT1 NAB2 SAMD12 WIPI2 FLOT2 SLITRK3

C18orf25 GJA5 NARG1 SAR1A WIZ FOXD3 SMAD4

C19orf63 GLIPR1 NAT11 SAT1 WNK3 FOXP1 SNIP

C1GALT1 GNAQ NCOA2 SDC4 WNT2 FREQ SNX21

C1orf9 GNB2 NCSTN SEC24C XPO1 FRYL SORT1

C20orf194 GOLGA1 NLK SEMA3F YAF2 FTO SP8

C20orf3 GPATCH8 NOTUM SEMA6A YPEL1 GABRA4 SRrp35

C21orf66 GPD2 NPAS2 SERPINE1 ZBTB10 GABRG2 ST7L

C9orf5 GPR124 NR1D2 SFRS1 ZBTB41 GALNT10 ST8SIA4

C9orf72 GPR180 NR2F6 SGCD ZBTB46 GDI1 STRA13

CACNB2 GPR85 NR3C1 SH2B1 ZBTB5 GIGYF2 STRBP

CAPRIN1 GPR89A NTNG1 SH3GLB1 ZBTB8 GLE1 STX3

CAV1 GPRC5A NUDT11 SH3PXD2A ZCCHC14 GLIS3 STX5

CCDC120 GRB10 NUFIP2 SIRT1 ZCCHC24 GRID1 SYN2

CCDC43 GRIK3 NUP210 SLAMF8 ZCCHC4 GRSF1 SYT1

CCDC88A GSK3B NUPL1 SLC17A5 ZEB2 GTF3C2 TADA1L

CCDC88C GTF2A1 ODZ4 SLC1A3 ZFP2 HIG2 TAPT1

CCNJ HDLBP ONECUT2 SLC24A3 ZFYVE20 HNRNPD TBC1D20

CCNL1 HECTD1 OSGIN2 SLC25A22 ZFYVE27 HNRNPH3 TCF21

CCNT2 HEXIM1 OSR1 SLC25A44 ZMYND8 HNRNPU TDRKH

DOI: 10.3324/haematol.2009.020636

CD84 HGS OTX1 SLC30A7 ZNF215 IER5L TEX261

CDCA7L HIF1A P15RS SLC35E1 ZNF217 IGF2BP1 TMCC1

CDK7 HIPK2 PAN3 SLC35E4 ZNF238 IGSF1 TOM1

CDKN1C HLF PAQR9 SLC35F5 ZNF329 IPO9 TP53

CELSR1 HMCN1 PARD6B SLC37A3 ZNF395 IRF2BP2 TRAF6

CEP192 HMGB1 PARP12 SLC39A10 ZNF439 ITGB3 TRPS1

CFL2 HOXB6 PATZ1 SLC4A8 ZNF468 ITSN1 TSPYL5

CHRNE HSPA12A PAX3 SLC9A8 ZNF516 JAZF1 TTPAL

CIITA HSPA5 PBRM1 SLCO4C1 ZNF547 JMJD3 TXNDC4

CLCN5 IER3 PCGF3 SMARCA4 ZNF563 KBTBD3 UBE2R2

CLIC4 IKBKB PCYOX1 SMARCAD1 ZNF579 KCMF1 UBXD8

CLIP1 INPP5F PDE4B SMG1 ZNF594 KCNIP3 UHRF1

CLTA IPO8 PDE4D SNAI1 ZNF614 KCTD15 UPF1

CLTC ITCH PDE8A SNF1LK ZNF618 KIAA1310 USP3

CNN1 ITFG3 PDIK1L SNF1LK2 ZNF629 KIF24 USP47

CNOT6L ITGA3 PDPN SNN ZNF652 KLF7 UST

COG4 ITGA4 PGM1 SNX18 ZNF654 KPNA6 WASF2

COL24A1 ITGAV PGRMC2 SNX6 ZNF700 LFNG WDR40A

COL5A3 ITGB8 PHACTR4 SOCS6 ZNF701 LIN28 WWC2

COPG ITPKB PI4KA SOS2 ZNF706 LMO4 ZBTB2

COPS7A ITPR3 PICALM SOX11 ZNF710 LRP11 ZBTB4

CPEB2 JAG1 PIK3CD SOX4 ZNF740 LRP2 ZCCHC17

CRK JMJD1C PIK3R6 SPNS1 ZNF763 LRRC15 ZFYVE1

CRTAM JPH3 PKN2 SRL ZNF776 LRRTM2 ZMAT2

CSDC2 JUNB PLEKHH1 SRRM1 ZNF827 MAPK13 ZNF189

CSGALNACT1 KAT2B PLXNA2 SS18 ZNF831 MBTD1 ZNF229

CTNND1 KCNU1 PLXND1 ST6GAL1 ZSWIM4 MED1 ZNF532

CTTN KCTD16 MIB1 ZNF826

MLL2 ZNRF3

DOI: 10.3324/haematol.2009.020636

Table S6. Absence of correlation between miRNA expression fold changes and blood cell composition changes

A - Patients' blood cell composition at day 0 and day 14.

blood cell composition at day 0 blood cell composition at day 14Patient

Id neutrophils%

lymphocytes%

neutro/lympho

1neutrophils

%lymphocytes

%neutro/lympho

cellcomposition

ratio2

P07 69,0 7,0 9,86 80,0 12,0 6,67 0,68

P11 69,0 17,0 4,06 50,7 33,0 1,54 0,38

P12 70,0 6,0 11,67 78,0 19,0 4,11 0,35

P14 41,0 4,0 10,25 82,0 9,2 8,91 0,87

P20 63,0 3,0 21,00 63,0 1,0 63,00 3,00

P24 56,0 17,0 3,29 71,0 16,8 4,23 1,28

P26 68,0 6,0 11,33 73,5 16,3 4,51 0,40

P30 71,0 16,0 4,44 74,0 14,1 5,25 1,18

P43 68,0 9,0 7,56 77,6 13,3 5,83 0,77

Data for patient P17 at day 0 were not available.1Neutro/lympho represents the ratio of the percentages of neutrophils and lymphocytes, indicative of the relative proportion of each cell

population. 2Ratio of neutro/lympho value at day 14 vs day 0.

B - Spearman correlation analysis

miR-18 miR-142-3p miR-143 miR-145 miR-146 miR-148a miR-150 miR-181a miR-199b-5p

r -0,317 -0,300 0,167 0,200 0,033 0,050 0,300 -0,150 -0,200

p-value 0,410 0,437 0,678 0,613 0,948 0,912 0,437 0,708 0,613

Data represent Spearman correlation coefficient r between miRNA fold changes and the cell composition ratio shown in A between day 0 and day 14