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MYELOID NEOPLASIA
Brief report
Deregulation of TWIST-1 in the CD34⫹ compartment represents a novel
prognostic factor in chronic myeloid leukemia
*Erika Cosset,1,2 *Ghassan Hamdan,1,2 Sandrine Jeanpierre,1,3 Thibault Voeltzel,1,2 Karen Sagorny,1,2 Sandrine Hayette,4
François-Xavier Mahon,5 Charles Dumontet,1,2,6 Alain Puisieux,1-3 Franck E. Nicolini,6 and Véronique Maguer-Satta1,2
1Inserm Unité U590, Centre Léon Bérard, Lyon, France; 2Université de Lyon, Lyon, France; 3Centre Léon Bérard, Lyon, France; 4Hospices Civils de Lyon,
Centre Hospitalier Lyon Sud, Laboratory for Molecular Biology and Unité Mixte de Recherche 5239 Centre National de la Recherche Scientifique, Pierre-Bénite,
France; 5Inserm U876, Université Victor Segalen-2, Bordeaux, France; and 6Hospices Civils de Lyon, Hematology Department, E. Herriot Hospital, Lyon, France
The mechanisms of resistance to tyrosine kinase inhibitors (TKIs) in chronic
myeloid leukemia (CML) often remain obscure. Analysis of patient samples during
disease progression revealed the upregulation of the oncogene TWIST-1, also
measured in primary samples from TKIresistant patients. Moreover, we found
that TWIST-1 was overexpressed in CML
diagnostic samples of patients who later
developed cytogenetic resistance to imatinib, even those without any detectable
resistance mechanism. We confirmed the
up-regulation of TWIST-1 at both RNA and
protein levels in imatinib-resistant cell
lines, irrespective of any other resistance
mechanism. Analysis with specific small
interfering RNA suggested TWIST-1 involvement in the resistance phenotype.
Finally, the kinetics of TWIST-1 expres-
sion during the individual medical histories of CML patients indicated that
TWIST-1 expression is down-regulated by
TKIs and up-regulated with TKI resistance. We hypothesize that the overexpression of the TWIST-1 oncogene represents a novel key prognostic factor
potentially useful for optimizing CML
management in the TKI era. (Blood. 2011;
117(5):1673-1676)
Introduction
Chronic myeloid leukemia (CML) is a model for cancer stem cell
and tyrosine kinase inhibitor (TKI) resistance. If chronic phase
(CP) patients are discriminated by Sokal score1 at diagnosis, they
remain heterogeneous and lack a reliable molecular prognosis
marker. Imatinib has become the standard of CP-CML care,2 but
some rare Philadelphia-positive (Ph⫹) stem cells may be insensitive to TKIs,3-5 maintain detectable disease over years, and
represent a reservoir in which secondary molecular events occur
and initiate disease progression. The BMI-1 gene, involved in stem
cell self-renewal and maintenance, has been proposed to predict
CML disease progression.6 We hypothesized that TWIST-1, a
transcription factor involved in embryogenesis, undifferentiated
cell proliferation, and cell survival,7-9 might be involved in CML.
We analyzed its expression in CML cells from patients and in cell
lines and identified it as a unique predictive marker for TKI
resistance, especially in patients with no other detectable resistance
mechanism.
reaction (PCR) was performed using sequence-specific primers (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials
link at the top of the online article; LightCycler480II system, Roche
Diagnostics and QuantiFAST SYBR Green I-kit, QIAGEN) using a fast
protocol (activation, 5 minutes at 95°C; amplification,10 seconds at 95°C,
30 seconds at 60°C; melting, 10 seconds at 95°C, 10 seconds at 50°C).
Specific TWIST-small interfering RNA (siRNA; sequence coding regions
55-75) or a Scramble-negative control were transfected as duplexes
(RNAiMax, Invitrogen).7-9 Cells were counted (trypan blue staining),
controlled for transfer efficiency (flow cytometry BDFACSCalibur), or
analyzed for TWIST-1 silencing (RNA and protein). Western blot was
performed as described11 using monoclonal antibodies to ABL (BD
Biosciences PharMingen), TWIST-1 (Abnova), and Ku80 (Roche Diagnostics). Statistically, the Wilcoxon Mann-Whitney test, with R Version 2.9.2
software, assessed differences between CML phases, responding and
nonresponding patients or CD34⫹ and CD34⫺ fractions, and the Student
paired t test for differences between cell lines groups. In addition, we
performed an analysis of variance applying a bivariate analysis. Significant
P values (P ⬍ .05) are given in the text, supplemental Table 2, and figures.
Methods
Results and discussion
After informed consent in accordance with the Declaration of Helsinki and
local ethics committee bylaws (from the Délégation à la recherche clinique
des Hospices Civils de Lyon, Lyon, France), samples were obtained from
CML patients at diagnosis or during follow-up and from healthy allogeneic
donors. CML cell lines were cultured as described.10 CD34-purified cells
(StemCell Technologies) were placed in Tri-Reagent (Sigma-Aldrich).
Total RNAs were reverse-transcribed, and quantitative polymerase chain
Approximately 30% of CP-CML patients develop resistance to
TKIs, the mechanism(s) of which remain(s) obscure. Genome-wide
microarray analysis of CML cell lines has shown that TWIST-1,
elevated in other cancers, immature cells, and patients with drug
resistance,12 is deregulated in a CML-resistant cell line.13 We
analyzed TWIST-1 expression by quantitative PCR in purified
Submitted November 16, 2009; accepted November 16, 2010. Prepublished
online as Blood First Edition paper, December 1, 2010; DOI 10.1182/
blood-2009-11-254680.
The online version of this article contains a data supplement.
*E.C. and G.H. contributed equally to this study.
© 2011 by The American Society of Hematology
BLOOD, 3 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 5
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
1673
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1674
COSSET et al
BLOOD, 3 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 5
Figure 1. TWIST-1 expression in primary CD34ⴙ and CD34ⴚ selected CML cells. Gene expression in CD34 immuno-selected hematopoietic cells from CML patients.
Results are expressed as a ratio to normal (healthy donors) CD34⫺ cell values. (A) Comparative expression of TWIST-1, BMI-1, and BCR-ABL in CD34⫹ ( ) and CD34⫺ (f)
compartments from CML samples (n ⫽ 40). (B) Comparative expression of TWIST-1 in CD34⫹ (left panel) and CD34⫺ (right panel) cells in controls (healthy BM donors;
n ⫽ 10), in CP (n ⫽ 20), and in advanced-phase (n ⫽ 16) CML cells. Horizontal bars represent the medians. (C) Comparative expression of TWIST-1, in CD34⫹ (left panel) and
CD34⫺ (right panel) cells in CML patients responsive (Resp; n ⫽ 18) or nonresponsive (Non Resp; n ⫽ 25) to imatinib. Horizontal bars represent the medians. (D) Mean
TWIST-1 expression in CD34⫹ ( ) or CD34⫺ (f) CML cells from a total of 32 patients at diagnosis before any treatment. P1 to P11 indicate individual data from patient 1 (P1) to
patient 11 (P11). Their mutational status (NM indicates nonmutated; and M, mutated) or presence of chromosome Ph duplication (Dup Phi) status are indicated below the
graph. ND indicates not done; and UD, undetected.
immature (CD34⫹) and mature (CD34⫺) cells from patients at
different disease phases and after emergence of TKI resistance.
Compared with normal samples, the overall TWIST-1, BMI-1, and
BCR-ABL expressions increased (Figure 1A) in CD34⫹ and CD34⫺
cells of, respectively 66-fold (P ⬍ .001) and 74-fold (P ⬍ .001) for
TWIST-1 and 2.71-fold (P ⬍ .001) and 2.25-fold (P ⬍ .001) for
BMI-1 as reported.6 Along disease progression, TWIST-1 expression significantly increased in CD34⫹ cells (11-fold and up to
133-fold in chronic and advanced phases, respectively; Figure 1B)
and CD34⫺ cells (65-fold and 97-fold). Comparing TKI responder
and nonresponder samples, we observed an approximately 100fold increase in TWIST-1 expression, regardless of CD34 expression (P ⫽ .88; Figure 1C), strongly supporting its expression as a
predictive factor for CML response to TKIs, unlike BMI-1 (supplemental Figures 1A, 2B). In accordance with previous data, we
observed an increased expression of BCR-ABL between CD34⫹
and CD34⫺ cells (supplemental Figure 1A-B). We analyzed
TWIST-1 expression in CML samples at diagnosis in a series of
samples from 32 patients and observed an increased mean value at
diagnosis for nonresponders (24.65-fold and 107.44-fold) compared with responders (1.28-fold and 1.32-fold), respectively, for
CD34⫹ and CD34⫺ cells. Indeed, sensitive patients, before treatment (Figure 1D mean responder) or during treatment (Figure 1C
responder), always presented a level of TWIST-1 similar to that of
normal donors. The individual values of all patients (P1-P11) who
developed resistance (European LeukemiaNet criteria14) later in
their disease course had higher TWIST-1 expression than the mean
value of responding patients (Figure 1D; supplemental Table 2),
whereas only 2 had increased BMI-1 expression (supplemental
Figure 1C). Subsequently, we think that TWIST-1 represents a
unique and reliable predictive marker of TKI resistance in CML
regardless of the resistance origin (Figure 1D; supplemental Table
2). Compared with the responder mean values, all nonresponder
patient samples displayed a significant increased expression of
TWIST-1 in CD34⫹ cells (100% of nonresponder patients), whereas
only 8 of 11 tested patients (72% of nonresponder patients) had an
increased Twist-1 expression in their CD34⫺ compartment (supplemental Table 2). Nevertheless, the difference between responder
and nonresponder values of Twist-1 in CD34⫺ remained statistically significant (supplemental Table 2). This was confirmed by the
detection of TWIST-1 expression within mononuclear cells from
those patients plus one in accelerated phase and one in myeloid
blast crisis (supplemental Table 3). In addition, sorted CD19⫹ cells
from the patient 10 sample at diagnosis were negative for TWIST-1
expression despite a BCR-ABL positivity (not shown). In addition,
from the 21 TKI-responsive tested patients at diagnosis, only 3 and
4 slightly expressed TWIST-1 more than 2-fold, in their CD34⫹ and
CD34⫺ compartments, respectively (supplemental Table 2). Therefore, TWIST-1 measurement in CD34⫹ cells appears to be the most
reliable parameter to discriminate since diagnosis of imatinib
responders or nonresponders.
Our data obtained in primary CML samples confirmed TWIST-1
deregulation in the KCL22 CML cell line13 (resistant through
unknown mechanisms). Quantitative PCR showed a 23- and
40-fold TWIST-1 expression increase (Figure 2A), with resistance
in Lama-84 and KCL22, respectively, and confirmed at protein
level (Figure 2B). Interestingly, TWIST-1 overexpression seems to
be independent from BCR-ABL protein levels as observed in HEL,
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BLOOD, 3 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 5
DEREGULATION OF TWIST-1 IN THE CD34⫹ COMPARTMENT
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Figure 2. Involvement of TWIST-1 in CML response to
TKIs, KCL22, or Lama-84 cell lines was analyzed for
TWIST-1 expression. (A) Comparative expression by
quantitative PCR of TWIST-1 in Lama-84 and KCL-22
imatinib-sensitive (䡺) and imatinib-resistant (f) Ph1⫹
cell lines. Results are expressed as a ratio to normal
(healthy donors) CD34⫺ cell values. (B) TWIST-1, BCRABL, and KU80 (used as loading control) protein analysis
by Western blot in HEL expressing BCR-ABL (BA) or not
(WT), KCL-22 and Lama 84 imatinib-sensitive (S) and
-resistant (R) cell extracts. (C) Viability and viable cell
numbers in untreated and imatinib-treated (1.5␮M)
Lama84-resistant Ph1⫹ cell line, submitted to TWIST-1
siRNA or negative scramble as control. Cells were
incubated in 96-well plates in serum-free medium with
stem cell factor (100 ng/mL), in the presence of 50nM of
fluorescent siRNA. Results are presented as a percentage of viable cells or as total viable cell numbers ⫾ SEM.
After 2 days of culture, cell proliferation and viability were
determined by trypan blue counting. Fold proliferation
was determined by reference to the input number of
viable cells. Results represent the mean ⫾ SEM of
7 independent experiments. *Significant value (P ⬍ .05).
(D) TWIST-1 expression in KCL22-resistant cells
(KCL22R) transduced with a pSR vector empty or containing sh sequence against TWIST-1 or in KCl22-sensitive
cells (KCL22S) transduced with a pMIG vector empty or
containing TWIST-1 expressing sequence. After 3 days in
the presence of puromycin selection for KCL22RshTWIST-1 or after 48 hours for KCL22S-TWIST-1, cells
were incubated in the presence of imatinib at the indicated dose. After 3 days of culture, cell proliferation and
viability were determined by trypan blue counting. The
percentage of proliferation was determined by reference
to the nontreated number of viable cells ⫾ SEM from
5 experiments. (E) Follow-up analysis for TWIST-1 expression by quantitative PCR in the CD34⫺ fraction. Results
are expressed as a ratio to normal (healthy donors)
CD34⫺ cell values. The values obtained for the BCR-ABL/
ABL ratio are indicated as a percentage (IS) at each data
point for both patients. BC indicates blast crisis; and
CCyR, complete cytogenetic remission. †Deceased.
KCL22R, and Lama-84R cells10 (Figure 2B). TWIST-1 silencing by
RNAi in Lama-84R and KCL22R (supplemental Figure 2) cells
had no effect by itself but decreased cell numbers and viability in
response to imatinib (Figure 2C-D). Conversely, overexpression of
TWIST-1 in KCL22-sensitive cells (supplemental Figure 2B)
significantly improved their resistance to increasing doses of
imatinib (Figure 2D). Altogether, these data indicate that TWIST-1
expression is involved in imatinib resistance. Finally, we analyzed
TWIST-1 expression in CD34⫺ cells during disease progression;
and although it was highly expressed at diagnosis, its expression
significantly decreased after complete cytogenetic remission (Figure 2E)
and increased again at relapse, unlike BMI-1 (supplemental Figure 3).
In conclusion, this study demonstrates that TWIST-1 is deregulated in CML patients at diagnosis and during progression and in
cells innately imatinib-resistant through a yet unknown mechanism. Our results indicate that, with BCR-ABL monitoring, TWIST-1
could represent a powerful biomarker for early detection of TKI
resistance at diagnosis and during residual disease follow-up.
TWIST-1 might act as a survival factor providing a selective growth
advantage contributing to drug resistance, as reported in solid
tumors.12,15-17 TKI-resistant TWIST-1–overexpressing cells might
emerge from leukemic Ph⫹ stem cells, but further work is
necessary to evaluate the link with BCR-ABL.
Acknowledgments
The authors thank S. Joly (U590), K. Chabane (Centre Hospitalier
Lyon Sud), and E. Gadolet (Hematology department, Hôpital
Edouard Herriot) for technical assistance and S. Morisset for
statistical analysis (Association ARCHE, Hôpital Edouard Herriot).
This work was supported in part by Inserm (V.M.-S., A.P.) and
Novartis Pharma France (K.S.).
Authorship
Contribution: E.C., G.H., S.J., and K.S. performed experiments and
analyzed data; S.H. provided routine laboratory clinical data following
international guidelines; T.V. contributed to research design and analyzed data; F.-X.M. provided cell lines and proofread the manuscript;
C.D. contributed to discussions and proofread the manuscript; A.P.
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1676
BLOOD, 3 FEBRUARY 2011 䡠 VOLUME 117, NUMBER 5
COSSET et al
provided TWIST-1-vectors and proofread the manuscript; F.E.N. followed up the patients, provided primary cells, analyzed the data, and
wrote the paper; and V.M.-S. performed research design, analyzed data,
and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Véronique Maguer-Satta, Inserm Unité U590,
Centre Léon Bérard, 28 Rue Laennec, 69373 Lyon Cedex 08,
France; e-mail: [email protected]; and Franck Nicolini,
Hospices Civils de Lyon, Hematology department, E. Herriot
Hospital, 5 place d’Arsonval, 69437, Lyon, France; e-mail:
[email protected].
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2011 117: 1673-1676
doi:10.1182/blood-2009-11-254680 originally published
online December 1, 2010
Deregulation of TWIST-1 in the CD34+ compartment represents a novel
prognostic factor in chronic myeloid leukemia
Erika Cosset, Ghassan Hamdan, Sandrine Jeanpierre, Thibault Voeltzel, Karen Sagorny, Sandrine
Hayette, François-Xavier Mahon, Charles Dumontet, Alain Puisieux, Franck E. Nicolini and Véronique
Maguer-Satta
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