Original Paper Received: August 6, 2014 Accepted after revision: October 30, 2014 Published online: February 17, 2015
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
Apoptosis-Related Gene Expression Profile in Chronic Myeloid Leukemia Patients after Imatinib Mesylate and Dasatinib Therapy Aline Fernanda Ferreira a Gislane L.V. de Oliveira a Raquel Tognon a Maria Dulce S. Collassanti e Maria Aparecida Zanichelli e Nelson Hamerschlak f Ana Maria de Souza a Dimas Tadeu Covas b, d Simone Kashima c, d Fabiola Attie de Castro a
a
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, e b Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, c Laboratório de Biologia Molecular, Centro Regional de Hemoterapia de Ribeirão Preto, e d INCTC, Instituto Nacional de Ciência e Tecnologia em Células Tronco e Terapia Celular, Ribeirão Preto, e e Instituto de Tratamento do Câncer Infantil-ITACI, e f Sociedade Beneficente Israelita e Brasileira Hospital Albert Einstein de São Paulo, São Paulo, Brazil
Abstract Background/Aims: We investigated the effects of tyrosine kinase inhibitors (TKIs) on the expression of apoptosis-related genes (BCL-2 and death receptor family members) in chronic myeloid leukemia (CML) patients. Methods: Peripheral blood mononuclear cells from 32 healthy subjects and 26 CML patients were evaluated before and after treatment with imatinib mesylate (IM) and dasatinib (DAS) by quantitative PCR. Results: Anti-apoptotic genes (c-FLIP and MCL-1) were overexpressed and the pro-apoptotic BIK was reduced in CML patients. Expression of BMF, A1, c-FLIP, MCL-1, CIAP-2 and CIAP-1 was modulated by DAS. In IM-resistant patients, expression of A1, c-FLIP, CIAP-1 and MCL-1 was upregulated, and BCL-2, CIAP-2, BAK, BAX, BIK and FASL expression was downregulated. Conclusion: Taken together, our results
© 2015 S. Karger AG, Basel 0001–5792/15/1334–0354$39.50/0 E-Mail [email protected] www.karger.com/aha
point out that, in CML, DAS interferes with the apoptotic machinery regulation. In addition, the data suggest that apoptosis-related gene expression profiles are associated with primary resistance to IM. © 2015 S. Karger AG, Basel
Introduction
Chronic myeloid leukemia (CML) is a myeloproliferative disease resulting from the clonal expansion of pluripotent hematopoietic stem cells [1–3]. The pathophysiology of CML is associated with a translocation between chromosomes 9 and 22, promoting the BCR-ABL fusion gene, which codifies the BCR-ABL oncoprotein [4, 5]. Tyrosine kinase activity of BCR-ABL is responsible for a phenotype involving malignant transformation of cells, which become apoptosis resistant [6]. Expression of BCRABL has been shown to block mitochondrial permeability and release of cytochrome c, thereby inhibiting casAline Fernanda Ferreira Departamento de Análises Clínicas, Toxicológicas e Bromatológicas Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo Av. do Café, s/n, Monte Alegre, Ribeirão Preto, SP 14040-903 (Brazil) E-Mail alineferreira591 @ gmail.com
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Key Words Apoptosis · BCR-ABL · Chronic myeloid leukemia · Gene expression · Tyrosine kinase inhibitors
Apoptotic Gene Expression in CML Cases after TKI
Table 1. Clinical and demographic characteristics of the CML pa-
tients Patient
Gender
Ethnic background
Age, years
Treatment
Response to TKI
Sokal index
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F F F F F M M F M M M F M M M M F F F F M M M F F M
C C NC C C C NC C C C C NC NC C C C C C C C C C C C C C
77 59 40 42 30 45 65 25 54 68 35 30 57 44 30 51 54 30 31 36 59 57 63 66 52 52
IM/DAS IM IM IM IM IM IM/DAS IM IM IM/DAS IM/DAS IM IM IM IM IM IM IM/DAS IM IM/DAS IM/DAS IM/DAS IM/DAS IM/DAS IM/DAS IM/DAS
R/CCR R CCR CCR CCR CCR CCR CCR CCR R/CCR CCR R CCR R R CCR CCR CCR CCR R R CCR R CCR CCR CCR
1.13 2.13 0.82 0.58 0.87 1.29 1.39 1.28 1.56 1.03 0.98 0.67 1.91 1.28 13.93 0.77 0.83 0.54 1.01 0.85 1.79 1.94 0.91 1.04 1.05 0.76
M = Male; F = female; C = Caucasian; NC = not Caucasian; R = IM resistant.
CML patients before and 12 months after treatment with IM and 6 months after treatment with DAS or a combination both.
Subjects, Materials and Methods Patients and Controls The 26 CML patients evaluated were under regular outpatient treatment at the Brigadeiro Hospital or Sociedade Beneficente Israelita e Brasileira Hospital Albert Einstein, both located in the city of São Paulo, Brazil. CML diagnosis was confirmed by Ph chromosome detection in bone marrow cytogenetic analyses and by identification of BCR-ABL rearrangement in the peripheral blood by real-time PCR. In the CML group, 13 patients were females and 13 were males. Four were not Caucasian (3 Afro-Brazilians and 1 mulatto) and 22 were Caucasian. Their median age was 48 years (range, 25–77 years). Demographic and clinical characteristics of the patients are described in table 1. All the 26 CML patients were
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pase activation and apoptosis [7]. In addition, BCR-ABL upregulates the expression of anti-apoptotic genes c-FLIP and BCL-XL, and nuclear factor (NF)-κB levels and Akt pathway activity are increased [8, 9]. BCL genes also have a role in primary and acquired drug resistance. Increased expression of BCL-2 and BCLXL has been observed in acute myeloid leukemia (AML) cell lines resistant to high levels of busulfan treatment, and elevated expression of BCL-W and MCL-1 was identified in cell lines which were treated with, and eventually resistant to, etoposide and cytosine arabinoside [10, 11]. CML can be treated with tyrosine kinase inhibitors (TKIs) imatinib (IM), dasatinib (DAS) and nilotinib (NIL), which changed the curative approach and improved treatment efficacy for CML, as well as through bone marrow transplantation [12–14]. Despite the promising results related to the use of TKIs, especially IM, mechanisms of leukemic cell resistance to IM have been described. Such resistance is caused mainly by the presence of mutations in the BCR-ABL catalytic region, Philadelphia (Ph) chromosome duplication, BCR-ABL overexpression or high levels of glycoprotein P [15]. The persistence of BCR-ABL-positive cells in CML patients after IM treatment indicates that the tyrosine kinase activity inhibition is not sufficient to eliminate all leukemic cells. Resistance was associated with increased levels of the Stat3 target genes BCL-XL, MCL-1 and survivin [16]. These data also motivated us to study the apoptotic gene profile in CML patients. Therefore, there must be secondary events or processes involved in disease pathogenesis, such as the apoptotic machinery deregulation in CML, which may contribute to TKI resistance. Thus, the description of such links is necessary for the development of more effective drugs to be used either as monotherapy or in combination with TKIs [17]. Although there are much data in the literature regarding the mechanism of action of TKIs, little is known of their effects on the apoptotic machinery. Elucidation of the potential of TKIs could contribute to less side effects and improved efficiency by guiding the development of new TKI adjuvant drugs for the treatment of CML patients [18]. In the present study, we evaluated mRNA expression of anti-apoptotic genes (A1, BCL-2, BCL-XL, BCL-W, cFLIP, CIAP-1, CIAP-2 and MCL-1) and pro-apoptotic genes (BAD, BAK, BAX, BID, BIK, BIMEL, BMF, BOK, FAS, FASL, PUMA and NOXA) in peripheral blood mononuclear cells (PBMCs) from healthy subjects and
PBMC Isolation PBMCs from patients and controls were isolated using Ficoll density gradient centrifugation (Histopaque-1077; Sigma-Aldrich, St. Louis, Mo., USA) [19]. The entire PBMC fraction from the CML patients was used since BCR-ABL is the only biological marker for CML cells, and since it is an intracellular marker, the separation of BCR-ABL-positive cells would cause cell destruction and loss of the sample. RNA Extraction, cDNA Synthesis and Quantitative Real-Time PCR PBMC RNA was extracted using 750 μl of TRIzol reagent (Invitrogen Life Technologies, Carlsbad, Calif., USA) following the manufacturer’s instructions. Two micrograms of total RNA were reverse transcribed using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, Calif., USA) according to the manufacturer’s instructions. After cDNA synthesis, real-time PCR was performed using SYBR Green PCR master mix (Applied Biosystems) in a 7500 real-time PCR system (Applied Biosystems) for quantification of the relative expression of apoptosis-related genes (A1, BAD, BAK, BAX, BCL-2, BCL-XL, BCL-XS, BCL-W, BID, BIK, BIMEL, BMF, BOK, c-FLIP, CIAP-1, CIAP-2, FAS, FASL, MCL-1, NOXA and PUMA). Primers for the detection of those genes were designed on the sequences obtained from the GenBank database (http://www.ncbi.nih.gov/genbank). β-ACTIN and GAPDH were used as housekeeping genes controls (online suppl. table 1; for all online suppl. material, see www.karger.com/ doi/10.1159/000369446). All amplification reactions were performed in a total volume of 15 μl containing 7.5 μl of SYBR Green reagent, 1 μl each of the forward and reverse primers (15 pmol) and 35 ng (1.5 μl) cDNA. PCR conditions included 50 ° C for 2 min, 95 ° C for 10 min, followed by 50–55 cycles at 95 ° C for 15 s at the gene-specific annealing temperature for 1 min and 72 ° C for 34 s. All tests were performed in duplicate. The threshold cycle values, defined by the default settings, were measured using 7500 Sequence Detection System Software, version 2.0.1 (Applied Biosystems). The results are presented as relative expression units (REU) [20]. The REU was calculated by the formule: REU = 10,000/2ΔCT.
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Western Blotting After RNA extraction, samples were submitted to serial extraction with TRIzol in order to obtain DNA and proteins. Subsequently, 20 μg of the obtained proteins were diluted in 5 μl of sample buffer [0.076 g of Tris, 5 ml of glycerol and 0.4 g of sodium dodecyl sulfate (SDS), 200 μl of β-mercaptoethanol and 1% of bromophenol blue at pH 6.8], heated at 100 ° C and submitted to separation via SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Hybond-P; Amersham Biosciences, Little Chalfont, UK). The membrane was blocked with Trisbuffered saline enriched with 5% non-fat milk and immunolabeled overnight with the polyclonal antibodies anti-A1 and antiBCL-W, as well as with the monoclonal antibodies anti-MCL-1 and anti-BAD (BD Biosciences, Franklin Lakes, N.J., USA). The immunocomplexes were labeled with horseradish peroxidase coupled to a secondary anti-IgG antibody at dilutions ranging from 1: 1,000 to 1: 2,000 for 45 min (GE Healthcare, Little Chalfont, UK) and detected using luminescent substrates (ECL Plus Western blotting detection system; GE Healthcare). The bands were detected by chemiluminescence and quantified using densitometry. The results are expressed as the integrated density value ratio between the target protein and the endogenous protein tubulin (Sigma-Aldrich, St. Louis, Mo., USA) or actin (Santa Cruz Biotechnology, Santa Cruz, Calif., USA; online suppl. fig. 1).
Statistical Analysis A mixed-model analysis was used in order to identify statistical differences between controls and CML patients (before and after treatment) in terms of apoptosis-related gene expression in PBMCs. The Mann-Whitney test was applied to compare the mRNA levels of target genes in IM-resistant CML patients with those observed in CML patients who achieved CCR. Spearman’s correlation coefficient was used in order to draw correlations between apoptotic gene expression and the patient’s Sokal index (SI). Values of p < 0.05 were regarded as significant. All analyses were performed using the Statgraphics Plus 5.0 software.
Results
Anti-Apoptotic Gene Expression in CML Patients before and after TKI Treatment Expression of the anti-apoptotic genes A1, BCL-2, BCL-XL, BCL-W and CIAP-1 in the control group was comparable to that seen in the CML group before and after IM treatment. The BCL-2 and BCL-XL expression in the control group was similar to that in CML patients treated with DAS (online suppl. tables 2 and 3). In the CML group treated with DAS, mean A1 expression was significantly higher before than after treatment (848.0 vs. 395.0, p = 0.005; fig. 1a,). Mean post-DAS BCLW expression was significantly lower than mean BCL-W expression in the control group (0.1 vs. 3.1, p = 0.03; fig. 1b). Ferreira et al.
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evaluated before and 12 months after treatment with 400 mg/day of IM (Gleevec; Novartis) to verify the effect of IM on apoptosisrelated gene expression in vivo. We analyzed 17 CML patients before and after IM therapy without excluding patients in remission. Over the period investigated, a complete cytogenetic remission (CCR) was achieved by 11 patients following treatment, and 15 were found to be resistant to IM. The patients resistant to IM were treated with 70 mg/day of DAS (Bristol-Meyers-Squib, Princeton, N.J., USA), and 12 of them were investigated before and after DAS therapy. The control group consisted of 32 healthy subjects: 16 females and 16 males. Twenty-six were Caucasian and 6 were not Caucasian (5 Afro-Brazilians and 1 mulatto). Their median age was 45 years (range, 23–77 years). The study design was approved by the Institutional Ethics Committee of the Sociedade Beneficente Israelita e Brasileira Hospital Albert Einstein of São Paulo (No. 06/385).
100
10,000
10 1 BCL-W (REU)
A1 (REU)
1,000 100 10
0.01 0.001 0.0001 0.00001
1 0.1
0.1
0.000001 Control
Pre-DAS
p = 0.26
a
1.0×10–07
Post-DAS
p = 0.005
p = 0.35
Control
Pre-DAS
p = 0.06
b
1,000
Post-DAS p = 0.61
p = 0.03 1,000 100 10
c-FLIP (REU)
c-FLIP (REU)
100
10
1
1 0.1 0.01 0.001 0.0001 0.00001 0.000001
0.1
1.0×10–07 Control
Pre-IM
p < 0.0001
100
100
10
Control
Pre-IM
Control
p = 0.002
Pre-DAS
p < 0.0001
p = 0.03
Post-DAS
10
1
Post-IM
p < 0.0001
Pre-DAS
p = 0.13 1,000
p < 0.0001
e
d
1,000
1
Control
p = 0.0002
p = 0.92
p < 0.0001
MCL-1 (REU)
MCL-1 (REU)
c
Post-IM
f
Post-DAS
p < 0.0001
p = 0.17
Apoptotic Gene Expression in CML Cases after TKI
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
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Fig. 1. Expression of A1, BCL-W, c-FLIP and MCL-1 anti-apoptotic genes in CML patients treated with TKIs. Expression of A1 (a), BCL-W (b), c-FLIP (c, d) and MCL-1 (e, f) in PBMCs from controls and CML patients before and after treatment with DAS (a, b, d, f) and IM (c, e). Horizontal bars represent mean REUs.
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1,000 100 CIAP-2 (REU)
10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-IM p = 0.05
a
Post-IM p = 0.13
p = 0.003 10,000 1,000 100
CIAP-2 (REU)
10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-DAS p = 0.48
Post-DAS p < 0.0001
b
p = 0.01 10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-DAS p = 0.79
c
Post-DAS p = 0.01
p = 0.04
Fig. 2. Modulation of CIAP-2 and CIAP-1 expression by IM and DAS. Expression of CIAP-2 (a, b) and CIAP-1 (c) in PBMCs from controls and CML patients before and after treatment with IM (a) and DAS (b, c). Horizontal bars represent mean REUs.
Ferreira et al.
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Pro-Apoptotic Gene Expression in CML Patients before and after TKI Treatment Mean BIK was lower in patients before (33.3, p = 0.007) and after IM treatment (2.0, p = 0.003) than in controls (360.0), and downregulated prior to treatment in CML patients receiving DAS (35.0, p = 0.04) in comparison with control subjects (mean = 360.0; fig. 3a). In addition, CML patients on DAS did not present alterations in BIK expression after treatment (fig. 3b). In IM-treated patients, mean BOK expression was lower after than before treatment (p = 0.06), as well as in the control group (5.3 vs. 1,073.0 and 39.2, p = 0.01; fig. 3c). In DAS-treated patients, mean BOK expression was significantly lower before and after treatment than in the controls (39.2 and 16.6 vs. 1,073.0, p = 0.03 and 0.02,
10,000
CIAP-1 (REU)
Among the CML patients treated with IM, mean relative c-FLIP expression before treatment was approximately 12 times higher than in the control group (88.3 vs. 6.9, p < 0.0001; fig. 1c). Mean c-FLIP expression was higher in post-DAS CML patients than in the control group (6.9 vs. 19.3, p = 0.13), with significant differences between the pre-DAS group (mean = 42.6) and the control group as well as the post-DAS group (p = 0.002; fig. 1d). As shown in figure 1e, mean MCL-1 expression was elevated in CML patients before (18.4, p < 0.0001) and after IM treatment (24.3, p < 0.0001) in comparison with the control subjects (4.7). In contrast, in CML patients treated with DAS, MCL-1 expression after treatment was considerably lower than prior to treatment (7.8 vs. 30.1, p < 0.0001). Expression of MCL-1 was comparable between CML patients after DAS treatment and controls (p = 0.17; fig. 1f). In CML patients treated with IM, mean CIAP-2 expression before (612.0, p = 0.05) and after treatment (799.0, p = 0.13) was increased compared to the controls (480.0, fig. 2a). However, in CML patients treated with DAS, mean CIAP-2 expression was lower after treatment than before DAS (47.6 vs. 661.0, p < 0.0001) and versus the control group (480.0, p = 0.01; fig. 2b). In CML patients treated with DAS, mean CIAP-1 expression was decreased after DAS treatment in comparison with before treatment (4.6 vs. 171.0, p = 0.01) as well as compared to the control group (120.6, p = 0.04; fig. 2c). In conclusion, c-FLIP and MCL-1 were upregulated in untreated CML patients compared to controls. We also observed that IM modulates MCL-1 expression whereas DAS modulates A1, c-FLIP, MCL-1, CIAP-2 and CIAP-1 expression.
BIK (REU)
BIK (REU)
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-IM p = 0.007
a
Post-IM
p = 0.04
100,000
10,000
10,000
BOK (REU)
BOK (REU)
p = 0.58
1,000
100 10 1
100 10 1
0.1
0.1
0.01 Control
Pre-IM
0.01
Post-IM
Control
p = 0.06
p = 0.12
c
100 10 BMF (REU)
1 0.1 0.01 0.001 0.0001 0.00001 0.000001 Pre-IM p < 0.0001 p = 0.0001
Post-IM
p = 0.02 10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-DAS p = 0.85
p = 0.61
Post-DAS p = 0.93
p = 0.03
p = 0.01
Control
Pre-DAS
d
1,000
FAS (REU)
Post-DAS
p = 0.09
100,000
1.0×10–07
Pre-DAS
b
p = 0.003
0.001
Control
p = 0.64
1,000
e
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
f
Post-DAS p = 0.004
p = 0.01
Apoptotic Gene Expression in CML Cases after TKI
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
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Fig. 3. TKI effects on pro-apoptotic genes in CML patients. Expression of BIK (a, b), BOK (c, d), FAS (e) and BMF (f) in PBMCs from controls and CML patients before and after treatment with IM (a, c, e) and DAS (b, d, f). Horizontal bars represent mean REUs.
Gene Expression Profiles Are Associated with IM Response Gene expression was analyzed in 11 patients who achieved CCR after IM therapy and in 15 who presented IM resistance. In contrast to CCR patients, IM-resistant patients presented significant overexpression of anti-apoptotic genes: A1 (median = 47.4, p = 0.04; fig. 4a), c-FLIP (median = 84.7, p = 0.04; fig. 4b), CIAP-1 (median = 31.5, p = 0.008; fig. 4c) and MCL-1 (median = 57.7, p = 0.03; fig. 4d). Expression of BCL-2 (fig. 4e) and CIAP-2 (fig. 4f) was lower in IM-resistant patients (median = 0.6 and 66.8) than in those achieving CCR (p = 0.04 and p = 0.0007, respectively). Expression of BCL-XL and BCL-W was similar between IM-resistant patients and those responsive to IM (online suppl. table 4). Gene expression analysis of IM-resistant patients and patients who achieved CCR revealed differences in the pro-apoptotic gene profiles (fig. 4h–j): BAK (median = 127.4 vs. 11.53, p = 0.02), BAX (median = 0.09 vs. 0.005, p = 0.008), BIK (median = 1.96 vs. 0.2, p = 0.02) and FASL 360
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
(median = 4.0 vs. 0.2, p = 0.03). Expression of BAD, BCLXS, BIMEL, BOK, BMF, FAS, NOXA and PUMA was not significantly different between the two groups (online suppl. table 4). In summary, we observed that IM-resistant patients presented high expression of A1, c-FLIP, MCL-1 and CIAP-1 and low levels of BCL-2, CIAP-2, BAK, BAX, BIK and FASL. Western Blot Results In this study, we used Western blots in order to evaluate the levels of a number of apoptotic proteins in CML patients before and after treatment with IM. The proteins selected for this analysis were the anti-apoptotic proteins A1, BCL-W and MCL-1, together with the proapoptotic protein BAD. The data confirm the data obtained from mRNA expression analysis of genes involved in apoptosis. The results are shown in online supplementary figure 1. Correlation between SI and Gene Expression We studied low-risk patients with an SI 1.2. In our CML patients, there was no significant correlation between the SI and gene expression of A1, BAD, BAK, BAX, BCL-2, BCL-XL, BCL-XS, BCL-W, BID, BIK, BIMEL, BMF, BOK, c-FLIP, CIAP-1, CIAP-2, FAS, FASL, MCL-1, NOXA or PUMA (online suppl. table 5). However, we observed a higher level of c-FLIP in patients with a high SI in comparison to those with a low SI (p = 0.03). IM-resistant patients with an intermediate risk present a higher level of CIAP-1 than patients with a low SI (p = 0.03). MCL-1 expression was higher in patients with low SI than in patients with intermediate and high SI (p = 0.03; data not shown). The IM-resistant group is composed of 15 patients, 3 of them present a low Sokal risk, 7 are classified as intermediate and 5 as high.
Discussion
The present study showed the effects of TKIs (IM/ DAS) on the expression of genes involved in apoptosis regulation in CML patients. We also demonstrated that apoptosis-related gene expression is deregulated in CML patients resistant to IM. The elevated expression of the anti-apoptotic molecules C-FLIP and MCL-1 in CML patients prior to treatFerreira et al.
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respectively), although there was no statistically significant difference between the values before and after treatment (fig. 3d). Mean FAS expression was higher in IM-treated patients before and after treatment than in controls (285.8 and 226.5 vs. 46.6, p < 0.0001 and p = 0.0001, respectively), although there was neither a statistically significant difference between FAS expression before and after treatment (fig. 3e), nor was there any difference in expression among the CML patients treated with DAS. The only significant difference observed in terms of mean BMF expression was that values were higher after than before DAS treatment and versus controls (1,403.0 vs. 429.0 and 93.2, p = 0.004 and p = 0.01, respectively; fig. 3f). In CML patients treated with IM, there was no difference in mean BMF expression before versus after treatment. Expression of the pro-apoptotic genes BAD, BAK, BAX, BCL-XS, BID, BIMEL, FASL, NOXA and PUMA did not differ among the groups studied (online suppl. tables 2 and 3). In summary, we observed that BIK expression is lower and FAS is higher in CML patients (before IM treatment) in comparison to the control group. IM did not interfere with pro-apoptotic gene expression in CML, and DAS modulates BMF mRNA levels in CML patients after treatment.
1,000 c-FLIP (REU)
100 10 1
CCR
a
100
10
R
b
p = 0.04
1,000 MCL-1 (REU)
CIAP-1 (REU)
100 10 1 CCR
CCR
BAK (REU)
CCR
R p = 0.02
1.0×10–05
CCR
R p = 0.0007
10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
CCR
h
FASL (REU)
BIK (REU)
R p = 0.02
i
R
1.0×100
f
BAX (REU) CCR
g
pression profile in CML patients according to IM response. Overexpression of A1 (a), c-FLIP (b), CIAP-1 (c) and MCL-1 (d) in IM-resistant CML patients after treatment. Low mRNA levels of BCL-2 (e), CIAP-2 (f), BAK (g), BAX (h), BIK (i) and FASL (j) in IM-resistant CML patients after treatment. Horizontal bars represent mean REUs. R = IM resistant.
CCR p = 0.03
1.0×10–10
R p = 0.04
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
1
1.0×1005
e
Fig. 4. Anti- and pro-apoptotic gene ex-
10
d
p = 0.008
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
100
R
CIAP-2 (REU)
BCL-2 (REU)
c 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
R p = 0.04
1,000
0.1
CCR
R p = 0.008
100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
j
CCR
R p = 0.03
ment might result from constitutive BCR-ABL-induced activation of STATs, which regulate gene transcription, by binding specifically to the regulatory elements of the DNA. The permanent activation of the STAT pathway in neoplasia, including CML, leads to inappropriate gene
regulation and therefore altered processes such as apoptosis and cell proliferation [21]. C-FLIP overexpression has also been associated with the pathogenesis of Hodgkin’s lymphoma and chronic lymphoid leukemia (CLL) [21–24]. Regarding MCL-1 levels, Aichberger et al. [25]
Apoptotic Gene Expression in CML Cases after TKI
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A1 (REU)
1,000
362
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
dent since this process was not observed in an BCR-ABLnegative leukemic cell line. Thus, these observations allow us to speculate that the reduction in c-FLIP, MCL-1, CIAP-1 and CIAP-2 found here may be associated with BCR-ABL inhibition by DAS. Munzert et al. [32] evaluated the expression of CIAP-1 and CIAP-2 genes in normal and CLL lymphocytes and noted overexpression of these genes in both groups, suggesting that neoplastic and healthy cells share a common pattern of expression of these genes [32]. An unexpected result seen in the present study was the low levels of BCL-2 observed in IM-resistant patients. Others studies have shown that BCL-2 may accelerate the onset of acute leukemia by promoting the survival of BCR-ABL-expressing cells, enabling them to accumulate additional genetic lesions [33, 34], and the blockade of apoptosis may be a key event in the progression of CML to blast crisis [33, 34]. In AML, high BCL-2 levels are associated with resistance to chemotherapy, decreased rates of complete remission and shortened survival [35–38]. Since our IM-resistant patients presented low levels of BCL-2, we suggest that at least in this group of patients the expression of BCL-2 may not be related or contribute to CML progression or lack of remission. The BAX gene is important in apoptosis induced by IM, and it is considered a good prognostic indicator in AML [39]. The presence of BCR-ABL rearrangement has been linked to downregulation of BAX. We may suggest that the resistance to IM observed in our patients may be linked to low levels of BAX, which can decrease its translocation and impair triggering of apoptotic signals. In a study using the TKI INNO-406, the authors verified an induction of apoptosis in Ph-positive cells after treatment, which was considered to be a consequence of the elevated activity of BIM, BAD, BMF and BIK [40]. Based on these data, we can speculate that the decrease in BIK in IM-resistant CML patients may be associated with a low effect of IM on CML cells. In terms of gene expression, the difference between the IM-resistant patients and those achieving CCR might be linked to the maintenance of tyrosine kinase activity of BCR-ABL and its amplification in leukemic cells. Cells that develop IM resistance, regardless of the mechanism involved, are selected for the ability to grow in the presence of this drug and become predominant in relation to the cells that are sensitive to IM, which undergo apoptosis [40]. Tipping et al. [41] suggested that there is an alternative oncogenic pathway of BCR-ABL activation in the leukemic cells of CML patients [41] Ferreira et al.
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attributed the high expression of MCL-1 to the increased survival and accumulation of BCR-ABL-positive leukemic cells in CML patients. An aberrant MCL-1 expression was not only shown in CML but also in individuals with AML, CLL and multiple myeloma [26, 27]. Our study reinforces the correlation between C-FLIP and MCL-1 overexpression and the pathogenesis of hematological neoplasia and BCR-ABL-positive cell resistance to apoptosis in CML. BIK expression was downregulated in CML patients in comparison to controls, as demonstrated in CLL and AML [11]. These authors associated the low levels of BIK to apoptosis resistance and leukemogenesis. Thus, we may suggest that its reduction in CML patients may also contribute to cell death resistance in leukemic cells. The anti-apoptotic gene MCL-1 presented, in our study, a higher expression in CML patients after IM treatment. Our hypothesis for these controversial data is that MCL-1 levels may be linked to the presence of primary IM resistance in the analyzed group who present high levels of MCL-1. Bueno-da-Silva et al. [28] showed that IM does not sensitize BCR-ABL-positive cell lines to apoptosis induced by chemotherapeutic agents. Based on these data, pharmacological inhibition of BCR-ABL by IM does not alter the resistance of BCR-ABL-positive cells to apoptosis [28]. These data allow us to suggest that the elevated levels of MCL-1 in our patients treated with IM are not associated to IM-induced deregulation of the apoptotic machinery. Regarding IM responses, the present investigation underscores the relation of anti-apoptotic gene expression with primary resistance to IM in CML patients. The higher c-FLIP expression observed in IM-resistant patients may be linked to the elevated TGF-β1 in BCRABL-positive cells [29, 30]. The complete eradication of CML is hindered by a small pocket of hematopoietic stem cells, which are resistant to IM, in part because they are not cycling [29]. Elevated c-FLIP expression was also associated with resistance to bortezomib in AML patients [30]. The results of the present study suggest that the resistance profiles of our patients may be related to the high anti-apoptotic gene expression observed. We also demonstrated in CML patients that DAS downregulated the expression of C-FLIP, MCL-1, CIAP-1 and CIAP-2 anti-apoptotic genes. The ability of DAS to induce leukemic cell apoptosis was observed by Snead et al. [31] after treating CML cells with this drug. They suggest that apoptosis induced by DAS is BCR-ABL depen-
which might confer a phenotype of resistance when the kinase plays an active role in the process of clone survival [42]. Based on these observations, we can suggest that apoptosis-related genes are potential therapeutic targets in the treatment of CML. In view of the existence of resistance to TKIs among CML patients, it is necessary to expand our knowledge on the activities of these drugs with the objective of eventually increasing their effectiveness in the treatment of this disease.
Acknowledgments We first wish to thank the CML patients who participated in this study. We are grateful to Zita Gregório for her technical assistance and help in collecting peripheral blood samples. This work was supported by FAPESP grant 2005/57746-8.
Disclosure Statement The authors declare that they have no competing interests.
1 Kantarjian HM, Smith TL, O’Brien S, Beran M, Pierce S, Talpaz M: Prolonged survival in chronic myelogenous leukemia after cytogenetic response to interferon-α therapy. Ann Intern Med 1995;122:254–261. 2 Frazer R, Irvine AE, McCullin MF: Chronic myeloid leukaemia in the 21st century. Ulster Med J 2006;76:8–17. 3 Melo JV, Barnes DJ: Chronic myeloid leukemia as a model of disease evolution in human cancer. Nature 2007;7:441–450. 4 Di Bacco A, Keeshan K, McKenna SL, Cotter TG: Molecular abnormalities in chronic myeloid leukemia: deregulation of cell growth and apoptosis. Oncologist 2000; 5: 405–415. 5 Weisberg E, Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin JD: Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nat Rev Cancer 2007;7:345–356. 6 Deininger MW, Vieira S, Mendiola R, Schultheis B, Goldman JM, Melo JV: BCR-ABL tyrosine kinase activity regulates the expression of multiple genes implicated in the pathogenesis of chronic myeloid leukemia. Cancer Res 2000;60:2049–2055. 7 Amarante-Mendes GP, Naekyung KC, Liu L, Huang Y, Perkins CL, Green DR, Bhalla K: Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3. Blood 1998;91: 1700– 1705. 8 Brumatti G, Weinlich R, Chehab CF, Yon M, Amarante-Mendes GP: Comparison of the anti-apoptotic effects of Bcr-Abl, Bcl-2 and Bcl-xL following diverse apoptogenic stimuli. FEBS Lett 2003;541:57–63. 9 Amarante-Mendes GP, McGahon AJ, Nishiola WK, Afar DE, Witte ON, Green DR: Bcl2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-xL. Oncogene 1998; 16: 1383–1390. 10 Yasui K, Mihara S, Zhao C, Okamoto H, Saito-Ohara F, Tomida A, Funato T, Yokomizo A, Naito S, Imoto I, Tsuruo T, Inaza-
Apoptotic Gene Expression in CML Cases after TKI
11
12
13
14 15
16
17
18
19 20
wa J: Alteration in copy numbers of genes as a mechanism for acquired drug resistance. Cancer Res 2004;64:1403–1410. Valdez BC, Murray D, Ramdas L, de Lima M, Jones R, Kornblau S, Betancourt D, Li Y, Champlin RE, Andersson BS: Altered gene expression in busulfan-resistant human myeloid leukemia. Leuk Res 2008; 32: 1684– 1697. Stamatović D, Balint B, Tukić L, Elez M, Tarabar O, Todorović M, Todorić-Zivanović B, Ostojić G, Tatomirović Z, Marjanović S, Malesević M: Allogeneic stem cell transplant for chronic myeloid leukemia as a still promising option in the era of the new target therapy. Vojnosanit Pregl 2012;69:37–42. Faderl S, Talpaz M, Estrov Z, Kantarjian H: Chronic myelogenous leukemia: biology and therapy. Ann Intern Med 1999;131:207– 291. Hehlmann R, Hochhaus A, Baccarani M: Chronic myeloid leukaemia. Lancet 2007;370: 342–350. Druker BJ, Lydon NB: Lessons learned from the development of abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105:3–7. Bewry NN, Nair RR, Emmons MF, Boulware D, Pinilla-Ibarz J, Hazlehurst LA: Stat3 contributes to resistance toward Bcr-Abl inhibitors in a bone marrow microenvironment model of drug resistance. Mol Cancer Ther 2008;7:3169–3175. Steinberg M: Dasatinib: a tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Clin Ther 2007;29:2289–2308. Doggrel SA: BMS-354825: a novel drug with potential for the treatment of imatinib-resistant chronic myeloid leukaemia. Expert Opin Biol Ther 2005;305:399–401. Boyum A: Separation of lymphocytes, lymphocyte subgroups and monocytes: a review. Lymphology 1977;10:71–76. Albesiano E, Messmer BT, Damle RN, Allen SL, Rai KR, Chiorazzi N: Activation-induced cytidine deaminase in chronic lymphocytic
21 22
23
24
25
26
27
28
leukemia B cells: expression as multiple forms in a dynamic, variably sized fraction of the clone. Blood 2003;102:3333–3339. Cools J, Maertens C, Marynen P: Resistance to tyrosine kinase inhibitors: calling on extra forces. Drug Resist Updat 2005;8:119–29. Nelson EA, Walker SR, Kepich A, Gashin LB, Hideshima T, Ikeda H, Chauhan D, Anderson KC, Frank DA: Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3. Blood 2008; 112: 5095– 5102. Mackus WJ, Kater AP, Grummels A, Evers LM, Hooijbrink B, Kramer MH, Castro JE, Kipps TJ, van Lier RA, van Oers MH, Eldering E: Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation. Leukemia 2005;19:427–434. Thomas RK, Kallenborn A, Wickenhauser C, Schultze JL, Draube A, Vockerodt M, Re D, Diehl V, Wolf J: Constitutive expression of cFLIP in Hodgkin and Reed-Sternberg cells. Am J Pathol 2002;160:1521–1528. Aichberger KJ, Mayerhofer M, Krauth MT, Skvara H, Florian S, Sonneck K, Akgul C, Derdak S, Pickl WF, Wacheck V, Selzer E, Monia BP, Moriggl R, Valent P, Sillaber C: Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides. Blood 2005;105:3303–3311. Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC: Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 1998; 91: 991–1000. Derenne S, Monia B, Dean NM, Taylor JK, Rapp MJ, Harousseau JL, Bataille R, Amiot M: Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-xL is an essential survival protein of human myeloma cells. Blood 2002; 100:194–199. Bueno-da-Silva AEB, Brumati G, Russo FO, Green DR, Amarante-Mendes GP: Bcr-Ablmediated resistance to apoptosis is independent of constant tyrosine-kinase activity. Cell Death Differ 2003;10:592–598.
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
363
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/2/2015 10:06:10 PM
References
364
34 Handa H, Hegde UP, Kotelnikov VM, Mundle SD, Dong LM, Burke P, Rose S, Gaskin F, Raza A, Preisler HD: Bcl-2 and c-myc expression, cell cycle kinetics and apoptosis during the progression of chronic myelogenous leukemia from diagnosis to blastic phase. Leuk Res 1997;21:479–489. 35 Bradbury DA, Russell NH: Comparative quantitative expression of bcl-2 by normal and leukaemic myeloid cells. Br J Haematol 1995;91:374–379. 36 Campos L, Rouault JP, Sabido O, Oriol P, Roubi N, Vasselon C, Archimbaud E, Magaud JP, Guyotat D: High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Blood 1993;81:3091–3096. 37 Maung ZT, MacLean FR, Reid MM, Pearson AD, Proctor SJ, Hamilton PJ, Hall AG: The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia. Br J Haematol 1994;88:105–109. 38 Porwit-MacDonald A, Ivory K, Wilkinson S, Wheatley K, Wong L, Janossy G: Bcl-2 protein expression in normal human bone marrow precursors and in acute myelogenous leukemia. Leukemia 1995;9:1191–1198.
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
39 Del Poeta G, Bruno A, Del Principe MI, Venditti A, Maurillo L, Buccisano F, Stasi R, Neri B, Luciano F, Siniscalchi A, de Fabritiis P, Amadori S: Deregulation of the mitochondrial apoptotic machinery and development of molecular targeted drugs in acute myeloid leukemia. Curr Cancer Drug Targets 2008; 8: 207–222. 40 Kuroda J, Kimura S, Strasser A, Andreeff M, O’Reilly LA, Ashihara E, Kamitsuji Y, Yokota A, Kawata E, Takeuchi M, Tanaka R, Tabe Y, Taniwaki M, Maekawa T: Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia. Cell Death Differ 2007;14:1667–1677. 41 Tipping AJ, Deininger MW, Goldman JM, Melo JV: Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate. Exp Hematol 2003;31:1073–1080. 42 Mahon FX, Deininger MW, Schutheis B, Chabrol J, Reiffers J, Goldman JM, Melo JV: Selection and characterization of BCR-ABL+ cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 2000;96:1070–1079.
Ferreira et al.
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/2/2015 10:06:10 PM
29 Moller GM, Frost V, Melo JV, Chantry A: Upregulation of the TGF beta signalling pathway by Bcr-Abl: implications for haemopoietic cell growth and chronic myeloid leukaemia. FEBS Lett 2007;581:1329–1334. 30 Riccioni R, Senese M, Diverio D, Riti V, Buffolino S, Mariani G, Boe A, Cedrone M, LoCoco F, Foà R, Peschle C, Testa U: M4 and M5 acute myeloid leukaemias display a high sensitivity to Bortezomib-mediated apoptosis. Br J Haematol 2007;139:194–205. 31 Snead JL, O’Hare T, Adrian LT, Eide CA, Lange T, Druker BJ, Deininger MW: Acute dasatinib exposure commits Bcr-Abl-dependent cells to apoptosis. Blood 2009;114:3459– 3463. 32 Munzert G, Kirchner D, Stobbe H, Bergmann L, Schmid RM, Döhner H, Heimpel H: Tumor necrosis factor receptor-associated factor 1 gene overexpression in B-cell chronic lymphocytic leukemia: analysis of NF-κB/Relregulated inhibitors of apoptosis. Blood 2002; 100:3749–3756. 33 Delia D, Aiello A, Soligo D, Fontanella E, Melani C, Pezzella F, Pierotti MA, Della Porta G: bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells. Blood 1992;79:1291–1298.
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Apoptosis-Related Gene Expression Profile in Chronic Myeloid Leukemia Patients after Imatinib Mesylate and Dasatinib Therapy Aline Fernanda Ferreira a Gislane L.V. de Oliveira a Raquel Tognon a Maria Dulce S. Collassanti e Maria Aparecida Zanichelli e Nelson Hamerschlak f Ana Maria de Souza a Dimas Tadeu Covas b, d Simone Kashima c, d Fabiola Attie de Castro a
a
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, e b Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, c Laboratório de Biologia Molecular, Centro Regional de Hemoterapia de Ribeirão Preto, e d INCTC, Instituto Nacional de Ciência e Tecnologia em Células Tronco e Terapia Celular, Ribeirão Preto, e e Instituto de Tratamento do Câncer Infantil-ITACI, e f Sociedade Beneficente Israelita e Brasileira Hospital Albert Einstein de São Paulo, São Paulo, Brazil
Abstract Background/Aims: We investigated the effects of tyrosine kinase inhibitors (TKIs) on the expression of apoptosis-related genes (BCL-2 and death receptor family members) in chronic myeloid leukemia (CML) patients. Methods: Peripheral blood mononuclear cells from 32 healthy subjects and 26 CML patients were evaluated before and after treatment with imatinib mesylate (IM) and dasatinib (DAS) by quantitative PCR. Results: Anti-apoptotic genes (c-FLIP and MCL-1) were overexpressed and the pro-apoptotic BIK was reduced in CML patients. Expression of BMF, A1, c-FLIP, MCL-1, CIAP-2 and CIAP-1 was modulated by DAS. In IM-resistant patients, expression of A1, c-FLIP, CIAP-1 and MCL-1 was upregulated, and BCL-2, CIAP-2, BAK, BAX, BIK and FASL expression was downregulated. Conclusion: Taken together, our results
© 2015 S. Karger AG, Basel 0001–5792/15/1334–0354$39.50/0 E-Mail [email protected] www.karger.com/aha
point out that, in CML, DAS interferes with the apoptotic machinery regulation. In addition, the data suggest that apoptosis-related gene expression profiles are associated with primary resistance to IM. © 2015 S. Karger AG, Basel
Introduction
Chronic myeloid leukemia (CML) is a myeloproliferative disease resulting from the clonal expansion of pluripotent hematopoietic stem cells [1–3]. The pathophysiology of CML is associated with a translocation between chromosomes 9 and 22, promoting the BCR-ABL fusion gene, which codifies the BCR-ABL oncoprotein [4, 5]. Tyrosine kinase activity of BCR-ABL is responsible for a phenotype involving malignant transformation of cells, which become apoptosis resistant [6]. Expression of BCRABL has been shown to block mitochondrial permeability and release of cytochrome c, thereby inhibiting casAline Fernanda Ferreira Departamento de Análises Clínicas, Toxicológicas e Bromatológicas Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo Av. do Café, s/n, Monte Alegre, Ribeirão Preto, SP 14040-903 (Brazil) E-Mail alineferreira591 @ gmail.com
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Key Words Apoptosis · BCR-ABL · Chronic myeloid leukemia · Gene expression · Tyrosine kinase inhibitors
Apoptotic Gene Expression in CML Cases after TKI
Table 1. Clinical and demographic characteristics of the CML pa-
tients Patient
Gender
Ethnic background
Age, years
Treatment
Response to TKI
Sokal index
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F F F F F M M F M M M F M M M M F F F F M M M F F M
C C NC C C C NC C C C C NC NC C C C C C C C C C C C C C
77 59 40 42 30 45 65 25 54 68 35 30 57 44 30 51 54 30 31 36 59 57 63 66 52 52
IM/DAS IM IM IM IM IM IM/DAS IM IM IM/DAS IM/DAS IM IM IM IM IM IM IM/DAS IM IM/DAS IM/DAS IM/DAS IM/DAS IM/DAS IM/DAS IM/DAS
R/CCR R CCR CCR CCR CCR CCR CCR CCR R/CCR CCR R CCR R R CCR CCR CCR CCR R R CCR R CCR CCR CCR
1.13 2.13 0.82 0.58 0.87 1.29 1.39 1.28 1.56 1.03 0.98 0.67 1.91 1.28 13.93 0.77 0.83 0.54 1.01 0.85 1.79 1.94 0.91 1.04 1.05 0.76
M = Male; F = female; C = Caucasian; NC = not Caucasian; R = IM resistant.
CML patients before and 12 months after treatment with IM and 6 months after treatment with DAS or a combination both.
Subjects, Materials and Methods Patients and Controls The 26 CML patients evaluated were under regular outpatient treatment at the Brigadeiro Hospital or Sociedade Beneficente Israelita e Brasileira Hospital Albert Einstein, both located in the city of São Paulo, Brazil. CML diagnosis was confirmed by Ph chromosome detection in bone marrow cytogenetic analyses and by identification of BCR-ABL rearrangement in the peripheral blood by real-time PCR. In the CML group, 13 patients were females and 13 were males. Four were not Caucasian (3 Afro-Brazilians and 1 mulatto) and 22 were Caucasian. Their median age was 48 years (range, 25–77 years). Demographic and clinical characteristics of the patients are described in table 1. All the 26 CML patients were
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
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pase activation and apoptosis [7]. In addition, BCR-ABL upregulates the expression of anti-apoptotic genes c-FLIP and BCL-XL, and nuclear factor (NF)-κB levels and Akt pathway activity are increased [8, 9]. BCL genes also have a role in primary and acquired drug resistance. Increased expression of BCL-2 and BCLXL has been observed in acute myeloid leukemia (AML) cell lines resistant to high levels of busulfan treatment, and elevated expression of BCL-W and MCL-1 was identified in cell lines which were treated with, and eventually resistant to, etoposide and cytosine arabinoside [10, 11]. CML can be treated with tyrosine kinase inhibitors (TKIs) imatinib (IM), dasatinib (DAS) and nilotinib (NIL), which changed the curative approach and improved treatment efficacy for CML, as well as through bone marrow transplantation [12–14]. Despite the promising results related to the use of TKIs, especially IM, mechanisms of leukemic cell resistance to IM have been described. Such resistance is caused mainly by the presence of mutations in the BCR-ABL catalytic region, Philadelphia (Ph) chromosome duplication, BCR-ABL overexpression or high levels of glycoprotein P [15]. The persistence of BCR-ABL-positive cells in CML patients after IM treatment indicates that the tyrosine kinase activity inhibition is not sufficient to eliminate all leukemic cells. Resistance was associated with increased levels of the Stat3 target genes BCL-XL, MCL-1 and survivin [16]. These data also motivated us to study the apoptotic gene profile in CML patients. Therefore, there must be secondary events or processes involved in disease pathogenesis, such as the apoptotic machinery deregulation in CML, which may contribute to TKI resistance. Thus, the description of such links is necessary for the development of more effective drugs to be used either as monotherapy or in combination with TKIs [17]. Although there are much data in the literature regarding the mechanism of action of TKIs, little is known of their effects on the apoptotic machinery. Elucidation of the potential of TKIs could contribute to less side effects and improved efficiency by guiding the development of new TKI adjuvant drugs for the treatment of CML patients [18]. In the present study, we evaluated mRNA expression of anti-apoptotic genes (A1, BCL-2, BCL-XL, BCL-W, cFLIP, CIAP-1, CIAP-2 and MCL-1) and pro-apoptotic genes (BAD, BAK, BAX, BID, BIK, BIMEL, BMF, BOK, FAS, FASL, PUMA and NOXA) in peripheral blood mononuclear cells (PBMCs) from healthy subjects and
PBMC Isolation PBMCs from patients and controls were isolated using Ficoll density gradient centrifugation (Histopaque-1077; Sigma-Aldrich, St. Louis, Mo., USA) [19]. The entire PBMC fraction from the CML patients was used since BCR-ABL is the only biological marker for CML cells, and since it is an intracellular marker, the separation of BCR-ABL-positive cells would cause cell destruction and loss of the sample. RNA Extraction, cDNA Synthesis and Quantitative Real-Time PCR PBMC RNA was extracted using 750 μl of TRIzol reagent (Invitrogen Life Technologies, Carlsbad, Calif., USA) following the manufacturer’s instructions. Two micrograms of total RNA were reverse transcribed using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, Calif., USA) according to the manufacturer’s instructions. After cDNA synthesis, real-time PCR was performed using SYBR Green PCR master mix (Applied Biosystems) in a 7500 real-time PCR system (Applied Biosystems) for quantification of the relative expression of apoptosis-related genes (A1, BAD, BAK, BAX, BCL-2, BCL-XL, BCL-XS, BCL-W, BID, BIK, BIMEL, BMF, BOK, c-FLIP, CIAP-1, CIAP-2, FAS, FASL, MCL-1, NOXA and PUMA). Primers for the detection of those genes were designed on the sequences obtained from the GenBank database (http://www.ncbi.nih.gov/genbank). β-ACTIN and GAPDH were used as housekeeping genes controls (online suppl. table 1; for all online suppl. material, see www.karger.com/ doi/10.1159/000369446). All amplification reactions were performed in a total volume of 15 μl containing 7.5 μl of SYBR Green reagent, 1 μl each of the forward and reverse primers (15 pmol) and 35 ng (1.5 μl) cDNA. PCR conditions included 50 ° C for 2 min, 95 ° C for 10 min, followed by 50–55 cycles at 95 ° C for 15 s at the gene-specific annealing temperature for 1 min and 72 ° C for 34 s. All tests were performed in duplicate. The threshold cycle values, defined by the default settings, were measured using 7500 Sequence Detection System Software, version 2.0.1 (Applied Biosystems). The results are presented as relative expression units (REU) [20]. The REU was calculated by the formule: REU = 10,000/2ΔCT.
356
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
Western Blotting After RNA extraction, samples were submitted to serial extraction with TRIzol in order to obtain DNA and proteins. Subsequently, 20 μg of the obtained proteins were diluted in 5 μl of sample buffer [0.076 g of Tris, 5 ml of glycerol and 0.4 g of sodium dodecyl sulfate (SDS), 200 μl of β-mercaptoethanol and 1% of bromophenol blue at pH 6.8], heated at 100 ° C and submitted to separation via SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Hybond-P; Amersham Biosciences, Little Chalfont, UK). The membrane was blocked with Trisbuffered saline enriched with 5% non-fat milk and immunolabeled overnight with the polyclonal antibodies anti-A1 and antiBCL-W, as well as with the monoclonal antibodies anti-MCL-1 and anti-BAD (BD Biosciences, Franklin Lakes, N.J., USA). The immunocomplexes were labeled with horseradish peroxidase coupled to a secondary anti-IgG antibody at dilutions ranging from 1: 1,000 to 1: 2,000 for 45 min (GE Healthcare, Little Chalfont, UK) and detected using luminescent substrates (ECL Plus Western blotting detection system; GE Healthcare). The bands were detected by chemiluminescence and quantified using densitometry. The results are expressed as the integrated density value ratio between the target protein and the endogenous protein tubulin (Sigma-Aldrich, St. Louis, Mo., USA) or actin (Santa Cruz Biotechnology, Santa Cruz, Calif., USA; online suppl. fig. 1).
Statistical Analysis A mixed-model analysis was used in order to identify statistical differences between controls and CML patients (before and after treatment) in terms of apoptosis-related gene expression in PBMCs. The Mann-Whitney test was applied to compare the mRNA levels of target genes in IM-resistant CML patients with those observed in CML patients who achieved CCR. Spearman’s correlation coefficient was used in order to draw correlations between apoptotic gene expression and the patient’s Sokal index (SI). Values of p < 0.05 were regarded as significant. All analyses were performed using the Statgraphics Plus 5.0 software.
Results
Anti-Apoptotic Gene Expression in CML Patients before and after TKI Treatment Expression of the anti-apoptotic genes A1, BCL-2, BCL-XL, BCL-W and CIAP-1 in the control group was comparable to that seen in the CML group before and after IM treatment. The BCL-2 and BCL-XL expression in the control group was similar to that in CML patients treated with DAS (online suppl. tables 2 and 3). In the CML group treated with DAS, mean A1 expression was significantly higher before than after treatment (848.0 vs. 395.0, p = 0.005; fig. 1a,). Mean post-DAS BCLW expression was significantly lower than mean BCL-W expression in the control group (0.1 vs. 3.1, p = 0.03; fig. 1b). Ferreira et al.
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evaluated before and 12 months after treatment with 400 mg/day of IM (Gleevec; Novartis) to verify the effect of IM on apoptosisrelated gene expression in vivo. We analyzed 17 CML patients before and after IM therapy without excluding patients in remission. Over the period investigated, a complete cytogenetic remission (CCR) was achieved by 11 patients following treatment, and 15 were found to be resistant to IM. The patients resistant to IM were treated with 70 mg/day of DAS (Bristol-Meyers-Squib, Princeton, N.J., USA), and 12 of them were investigated before and after DAS therapy. The control group consisted of 32 healthy subjects: 16 females and 16 males. Twenty-six were Caucasian and 6 were not Caucasian (5 Afro-Brazilians and 1 mulatto). Their median age was 45 years (range, 23–77 years). The study design was approved by the Institutional Ethics Committee of the Sociedade Beneficente Israelita e Brasileira Hospital Albert Einstein of São Paulo (No. 06/385).
100
10,000
10 1 BCL-W (REU)
A1 (REU)
1,000 100 10
0.01 0.001 0.0001 0.00001
1 0.1
0.1
0.000001 Control
Pre-DAS
p = 0.26
a
1.0×10–07
Post-DAS
p = 0.005
p = 0.35
Control
Pre-DAS
p = 0.06
b
1,000
Post-DAS p = 0.61
p = 0.03 1,000 100 10
c-FLIP (REU)
c-FLIP (REU)
100
10
1
1 0.1 0.01 0.001 0.0001 0.00001 0.000001
0.1
1.0×10–07 Control
Pre-IM
p < 0.0001
100
100
10
Control
Pre-IM
Control
p = 0.002
Pre-DAS
p < 0.0001
p = 0.03
Post-DAS
10
1
Post-IM
p < 0.0001
Pre-DAS
p = 0.13 1,000
p < 0.0001
e
d
1,000
1
Control
p = 0.0002
p = 0.92
p < 0.0001
MCL-1 (REU)
MCL-1 (REU)
c
Post-IM
f
Post-DAS
p < 0.0001
p = 0.17
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Fig. 1. Expression of A1, BCL-W, c-FLIP and MCL-1 anti-apoptotic genes in CML patients treated with TKIs. Expression of A1 (a), BCL-W (b), c-FLIP (c, d) and MCL-1 (e, f) in PBMCs from controls and CML patients before and after treatment with DAS (a, b, d, f) and IM (c, e). Horizontal bars represent mean REUs.
358
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1,000 100 CIAP-2 (REU)
10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-IM p = 0.05
a
Post-IM p = 0.13
p = 0.003 10,000 1,000 100
CIAP-2 (REU)
10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-DAS p = 0.48
Post-DAS p < 0.0001
b
p = 0.01 10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-DAS p = 0.79
c
Post-DAS p = 0.01
p = 0.04
Fig. 2. Modulation of CIAP-2 and CIAP-1 expression by IM and DAS. Expression of CIAP-2 (a, b) and CIAP-1 (c) in PBMCs from controls and CML patients before and after treatment with IM (a) and DAS (b, c). Horizontal bars represent mean REUs.
Ferreira et al.
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Pro-Apoptotic Gene Expression in CML Patients before and after TKI Treatment Mean BIK was lower in patients before (33.3, p = 0.007) and after IM treatment (2.0, p = 0.003) than in controls (360.0), and downregulated prior to treatment in CML patients receiving DAS (35.0, p = 0.04) in comparison with control subjects (mean = 360.0; fig. 3a). In addition, CML patients on DAS did not present alterations in BIK expression after treatment (fig. 3b). In IM-treated patients, mean BOK expression was lower after than before treatment (p = 0.06), as well as in the control group (5.3 vs. 1,073.0 and 39.2, p = 0.01; fig. 3c). In DAS-treated patients, mean BOK expression was significantly lower before and after treatment than in the controls (39.2 and 16.6 vs. 1,073.0, p = 0.03 and 0.02,
10,000
CIAP-1 (REU)
Among the CML patients treated with IM, mean relative c-FLIP expression before treatment was approximately 12 times higher than in the control group (88.3 vs. 6.9, p < 0.0001; fig. 1c). Mean c-FLIP expression was higher in post-DAS CML patients than in the control group (6.9 vs. 19.3, p = 0.13), with significant differences between the pre-DAS group (mean = 42.6) and the control group as well as the post-DAS group (p = 0.002; fig. 1d). As shown in figure 1e, mean MCL-1 expression was elevated in CML patients before (18.4, p < 0.0001) and after IM treatment (24.3, p < 0.0001) in comparison with the control subjects (4.7). In contrast, in CML patients treated with DAS, MCL-1 expression after treatment was considerably lower than prior to treatment (7.8 vs. 30.1, p < 0.0001). Expression of MCL-1 was comparable between CML patients after DAS treatment and controls (p = 0.17; fig. 1f). In CML patients treated with IM, mean CIAP-2 expression before (612.0, p = 0.05) and after treatment (799.0, p = 0.13) was increased compared to the controls (480.0, fig. 2a). However, in CML patients treated with DAS, mean CIAP-2 expression was lower after treatment than before DAS (47.6 vs. 661.0, p < 0.0001) and versus the control group (480.0, p = 0.01; fig. 2b). In CML patients treated with DAS, mean CIAP-1 expression was decreased after DAS treatment in comparison with before treatment (4.6 vs. 171.0, p = 0.01) as well as compared to the control group (120.6, p = 0.04; fig. 2c). In conclusion, c-FLIP and MCL-1 were upregulated in untreated CML patients compared to controls. We also observed that IM modulates MCL-1 expression whereas DAS modulates A1, c-FLIP, MCL-1, CIAP-2 and CIAP-1 expression.
BIK (REU)
BIK (REU)
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-IM p = 0.007
a
Post-IM
p = 0.04
100,000
10,000
10,000
BOK (REU)
BOK (REU)
p = 0.58
1,000
100 10 1
100 10 1
0.1
0.1
0.01 Control
Pre-IM
0.01
Post-IM
Control
p = 0.06
p = 0.12
c
100 10 BMF (REU)
1 0.1 0.01 0.001 0.0001 0.00001 0.000001 Pre-IM p < 0.0001 p = 0.0001
Post-IM
p = 0.02 10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
Control
Pre-DAS p = 0.85
p = 0.61
Post-DAS p = 0.93
p = 0.03
p = 0.01
Control
Pre-DAS
d
1,000
FAS (REU)
Post-DAS
p = 0.09
100,000
1.0×10–07
Pre-DAS
b
p = 0.003
0.001
Control
p = 0.64
1,000
e
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
f
Post-DAS p = 0.004
p = 0.01
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Fig. 3. TKI effects on pro-apoptotic genes in CML patients. Expression of BIK (a, b), BOK (c, d), FAS (e) and BMF (f) in PBMCs from controls and CML patients before and after treatment with IM (a, c, e) and DAS (b, d, f). Horizontal bars represent mean REUs.
Gene Expression Profiles Are Associated with IM Response Gene expression was analyzed in 11 patients who achieved CCR after IM therapy and in 15 who presented IM resistance. In contrast to CCR patients, IM-resistant patients presented significant overexpression of anti-apoptotic genes: A1 (median = 47.4, p = 0.04; fig. 4a), c-FLIP (median = 84.7, p = 0.04; fig. 4b), CIAP-1 (median = 31.5, p = 0.008; fig. 4c) and MCL-1 (median = 57.7, p = 0.03; fig. 4d). Expression of BCL-2 (fig. 4e) and CIAP-2 (fig. 4f) was lower in IM-resistant patients (median = 0.6 and 66.8) than in those achieving CCR (p = 0.04 and p = 0.0007, respectively). Expression of BCL-XL and BCL-W was similar between IM-resistant patients and those responsive to IM (online suppl. table 4). Gene expression analysis of IM-resistant patients and patients who achieved CCR revealed differences in the pro-apoptotic gene profiles (fig. 4h–j): BAK (median = 127.4 vs. 11.53, p = 0.02), BAX (median = 0.09 vs. 0.005, p = 0.008), BIK (median = 1.96 vs. 0.2, p = 0.02) and FASL 360
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(median = 4.0 vs. 0.2, p = 0.03). Expression of BAD, BCLXS, BIMEL, BOK, BMF, FAS, NOXA and PUMA was not significantly different between the two groups (online suppl. table 4). In summary, we observed that IM-resistant patients presented high expression of A1, c-FLIP, MCL-1 and CIAP-1 and low levels of BCL-2, CIAP-2, BAK, BAX, BIK and FASL. Western Blot Results In this study, we used Western blots in order to evaluate the levels of a number of apoptotic proteins in CML patients before and after treatment with IM. The proteins selected for this analysis were the anti-apoptotic proteins A1, BCL-W and MCL-1, together with the proapoptotic protein BAD. The data confirm the data obtained from mRNA expression analysis of genes involved in apoptosis. The results are shown in online supplementary figure 1. Correlation between SI and Gene Expression We studied low-risk patients with an SI 1.2. In our CML patients, there was no significant correlation between the SI and gene expression of A1, BAD, BAK, BAX, BCL-2, BCL-XL, BCL-XS, BCL-W, BID, BIK, BIMEL, BMF, BOK, c-FLIP, CIAP-1, CIAP-2, FAS, FASL, MCL-1, NOXA or PUMA (online suppl. table 5). However, we observed a higher level of c-FLIP in patients with a high SI in comparison to those with a low SI (p = 0.03). IM-resistant patients with an intermediate risk present a higher level of CIAP-1 than patients with a low SI (p = 0.03). MCL-1 expression was higher in patients with low SI than in patients with intermediate and high SI (p = 0.03; data not shown). The IM-resistant group is composed of 15 patients, 3 of them present a low Sokal risk, 7 are classified as intermediate and 5 as high.
Discussion
The present study showed the effects of TKIs (IM/ DAS) on the expression of genes involved in apoptosis regulation in CML patients. We also demonstrated that apoptosis-related gene expression is deregulated in CML patients resistant to IM. The elevated expression of the anti-apoptotic molecules C-FLIP and MCL-1 in CML patients prior to treatFerreira et al.
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respectively), although there was no statistically significant difference between the values before and after treatment (fig. 3d). Mean FAS expression was higher in IM-treated patients before and after treatment than in controls (285.8 and 226.5 vs. 46.6, p < 0.0001 and p = 0.0001, respectively), although there was neither a statistically significant difference between FAS expression before and after treatment (fig. 3e), nor was there any difference in expression among the CML patients treated with DAS. The only significant difference observed in terms of mean BMF expression was that values were higher after than before DAS treatment and versus controls (1,403.0 vs. 429.0 and 93.2, p = 0.004 and p = 0.01, respectively; fig. 3f). In CML patients treated with IM, there was no difference in mean BMF expression before versus after treatment. Expression of the pro-apoptotic genes BAD, BAK, BAX, BCL-XS, BID, BIMEL, FASL, NOXA and PUMA did not differ among the groups studied (online suppl. tables 2 and 3). In summary, we observed that BIK expression is lower and FAS is higher in CML patients (before IM treatment) in comparison to the control group. IM did not interfere with pro-apoptotic gene expression in CML, and DAS modulates BMF mRNA levels in CML patients after treatment.
1,000 c-FLIP (REU)
100 10 1
CCR
a
100
10
R
b
p = 0.04
1,000 MCL-1 (REU)
CIAP-1 (REU)
100 10 1 CCR
CCR
BAK (REU)
CCR
R p = 0.02
1.0×10–05
CCR
R p = 0.0007
10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
CCR
h
FASL (REU)
BIK (REU)
R p = 0.02
i
R
1.0×100
f
BAX (REU) CCR
g
pression profile in CML patients according to IM response. Overexpression of A1 (a), c-FLIP (b), CIAP-1 (c) and MCL-1 (d) in IM-resistant CML patients after treatment. Low mRNA levels of BCL-2 (e), CIAP-2 (f), BAK (g), BAX (h), BIK (i) and FASL (j) in IM-resistant CML patients after treatment. Horizontal bars represent mean REUs. R = IM resistant.
CCR p = 0.03
1.0×10–10
R p = 0.04
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
1
1.0×1005
e
Fig. 4. Anti- and pro-apoptotic gene ex-
10
d
p = 0.008
10,000 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
100
R
CIAP-2 (REU)
BCL-2 (REU)
c 1,000 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
R p = 0.04
1,000
0.1
CCR
R p = 0.008
100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 1.0×10–07
j
CCR
R p = 0.03
ment might result from constitutive BCR-ABL-induced activation of STATs, which regulate gene transcription, by binding specifically to the regulatory elements of the DNA. The permanent activation of the STAT pathway in neoplasia, including CML, leads to inappropriate gene
regulation and therefore altered processes such as apoptosis and cell proliferation [21]. C-FLIP overexpression has also been associated with the pathogenesis of Hodgkin’s lymphoma and chronic lymphoid leukemia (CLL) [21–24]. Regarding MCL-1 levels, Aichberger et al. [25]
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A1 (REU)
1,000
362
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dent since this process was not observed in an BCR-ABLnegative leukemic cell line. Thus, these observations allow us to speculate that the reduction in c-FLIP, MCL-1, CIAP-1 and CIAP-2 found here may be associated with BCR-ABL inhibition by DAS. Munzert et al. [32] evaluated the expression of CIAP-1 and CIAP-2 genes in normal and CLL lymphocytes and noted overexpression of these genes in both groups, suggesting that neoplastic and healthy cells share a common pattern of expression of these genes [32]. An unexpected result seen in the present study was the low levels of BCL-2 observed in IM-resistant patients. Others studies have shown that BCL-2 may accelerate the onset of acute leukemia by promoting the survival of BCR-ABL-expressing cells, enabling them to accumulate additional genetic lesions [33, 34], and the blockade of apoptosis may be a key event in the progression of CML to blast crisis [33, 34]. In AML, high BCL-2 levels are associated with resistance to chemotherapy, decreased rates of complete remission and shortened survival [35–38]. Since our IM-resistant patients presented low levels of BCL-2, we suggest that at least in this group of patients the expression of BCL-2 may not be related or contribute to CML progression or lack of remission. The BAX gene is important in apoptosis induced by IM, and it is considered a good prognostic indicator in AML [39]. The presence of BCR-ABL rearrangement has been linked to downregulation of BAX. We may suggest that the resistance to IM observed in our patients may be linked to low levels of BAX, which can decrease its translocation and impair triggering of apoptotic signals. In a study using the TKI INNO-406, the authors verified an induction of apoptosis in Ph-positive cells after treatment, which was considered to be a consequence of the elevated activity of BIM, BAD, BMF and BIK [40]. Based on these data, we can speculate that the decrease in BIK in IM-resistant CML patients may be associated with a low effect of IM on CML cells. In terms of gene expression, the difference between the IM-resistant patients and those achieving CCR might be linked to the maintenance of tyrosine kinase activity of BCR-ABL and its amplification in leukemic cells. Cells that develop IM resistance, regardless of the mechanism involved, are selected for the ability to grow in the presence of this drug and become predominant in relation to the cells that are sensitive to IM, which undergo apoptosis [40]. Tipping et al. [41] suggested that there is an alternative oncogenic pathway of BCR-ABL activation in the leukemic cells of CML patients [41] Ferreira et al.
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attributed the high expression of MCL-1 to the increased survival and accumulation of BCR-ABL-positive leukemic cells in CML patients. An aberrant MCL-1 expression was not only shown in CML but also in individuals with AML, CLL and multiple myeloma [26, 27]. Our study reinforces the correlation between C-FLIP and MCL-1 overexpression and the pathogenesis of hematological neoplasia and BCR-ABL-positive cell resistance to apoptosis in CML. BIK expression was downregulated in CML patients in comparison to controls, as demonstrated in CLL and AML [11]. These authors associated the low levels of BIK to apoptosis resistance and leukemogenesis. Thus, we may suggest that its reduction in CML patients may also contribute to cell death resistance in leukemic cells. The anti-apoptotic gene MCL-1 presented, in our study, a higher expression in CML patients after IM treatment. Our hypothesis for these controversial data is that MCL-1 levels may be linked to the presence of primary IM resistance in the analyzed group who present high levels of MCL-1. Bueno-da-Silva et al. [28] showed that IM does not sensitize BCR-ABL-positive cell lines to apoptosis induced by chemotherapeutic agents. Based on these data, pharmacological inhibition of BCR-ABL by IM does not alter the resistance of BCR-ABL-positive cells to apoptosis [28]. These data allow us to suggest that the elevated levels of MCL-1 in our patients treated with IM are not associated to IM-induced deregulation of the apoptotic machinery. Regarding IM responses, the present investigation underscores the relation of anti-apoptotic gene expression with primary resistance to IM in CML patients. The higher c-FLIP expression observed in IM-resistant patients may be linked to the elevated TGF-β1 in BCRABL-positive cells [29, 30]. The complete eradication of CML is hindered by a small pocket of hematopoietic stem cells, which are resistant to IM, in part because they are not cycling [29]. Elevated c-FLIP expression was also associated with resistance to bortezomib in AML patients [30]. The results of the present study suggest that the resistance profiles of our patients may be related to the high anti-apoptotic gene expression observed. We also demonstrated in CML patients that DAS downregulated the expression of C-FLIP, MCL-1, CIAP-1 and CIAP-2 anti-apoptotic genes. The ability of DAS to induce leukemic cell apoptosis was observed by Snead et al. [31] after treating CML cells with this drug. They suggest that apoptosis induced by DAS is BCR-ABL depen-
which might confer a phenotype of resistance when the kinase plays an active role in the process of clone survival [42]. Based on these observations, we can suggest that apoptosis-related genes are potential therapeutic targets in the treatment of CML. In view of the existence of resistance to TKIs among CML patients, it is necessary to expand our knowledge on the activities of these drugs with the objective of eventually increasing their effectiveness in the treatment of this disease.
Acknowledgments We first wish to thank the CML patients who participated in this study. We are grateful to Zita Gregório for her technical assistance and help in collecting peripheral blood samples. This work was supported by FAPESP grant 2005/57746-8.
Disclosure Statement The authors declare that they have no competing interests.
1 Kantarjian HM, Smith TL, O’Brien S, Beran M, Pierce S, Talpaz M: Prolonged survival in chronic myelogenous leukemia after cytogenetic response to interferon-α therapy. Ann Intern Med 1995;122:254–261. 2 Frazer R, Irvine AE, McCullin MF: Chronic myeloid leukaemia in the 21st century. Ulster Med J 2006;76:8–17. 3 Melo JV, Barnes DJ: Chronic myeloid leukemia as a model of disease evolution in human cancer. Nature 2007;7:441–450. 4 Di Bacco A, Keeshan K, McKenna SL, Cotter TG: Molecular abnormalities in chronic myeloid leukemia: deregulation of cell growth and apoptosis. Oncologist 2000; 5: 405–415. 5 Weisberg E, Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin JD: Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nat Rev Cancer 2007;7:345–356. 6 Deininger MW, Vieira S, Mendiola R, Schultheis B, Goldman JM, Melo JV: BCR-ABL tyrosine kinase activity regulates the expression of multiple genes implicated in the pathogenesis of chronic myeloid leukemia. Cancer Res 2000;60:2049–2055. 7 Amarante-Mendes GP, Naekyung KC, Liu L, Huang Y, Perkins CL, Green DR, Bhalla K: Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3. Blood 1998;91: 1700– 1705. 8 Brumatti G, Weinlich R, Chehab CF, Yon M, Amarante-Mendes GP: Comparison of the anti-apoptotic effects of Bcr-Abl, Bcl-2 and Bcl-xL following diverse apoptogenic stimuli. FEBS Lett 2003;541:57–63. 9 Amarante-Mendes GP, McGahon AJ, Nishiola WK, Afar DE, Witte ON, Green DR: Bcl2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-xL. Oncogene 1998; 16: 1383–1390. 10 Yasui K, Mihara S, Zhao C, Okamoto H, Saito-Ohara F, Tomida A, Funato T, Yokomizo A, Naito S, Imoto I, Tsuruo T, Inaza-
Apoptotic Gene Expression in CML Cases after TKI
11
12
13
14 15
16
17
18
19 20
wa J: Alteration in copy numbers of genes as a mechanism for acquired drug resistance. Cancer Res 2004;64:1403–1410. Valdez BC, Murray D, Ramdas L, de Lima M, Jones R, Kornblau S, Betancourt D, Li Y, Champlin RE, Andersson BS: Altered gene expression in busulfan-resistant human myeloid leukemia. Leuk Res 2008; 32: 1684– 1697. Stamatović D, Balint B, Tukić L, Elez M, Tarabar O, Todorović M, Todorić-Zivanović B, Ostojić G, Tatomirović Z, Marjanović S, Malesević M: Allogeneic stem cell transplant for chronic myeloid leukemia as a still promising option in the era of the new target therapy. Vojnosanit Pregl 2012;69:37–42. Faderl S, Talpaz M, Estrov Z, Kantarjian H: Chronic myelogenous leukemia: biology and therapy. Ann Intern Med 1999;131:207– 291. Hehlmann R, Hochhaus A, Baccarani M: Chronic myeloid leukaemia. Lancet 2007;370: 342–350. Druker BJ, Lydon NB: Lessons learned from the development of abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105:3–7. Bewry NN, Nair RR, Emmons MF, Boulware D, Pinilla-Ibarz J, Hazlehurst LA: Stat3 contributes to resistance toward Bcr-Abl inhibitors in a bone marrow microenvironment model of drug resistance. Mol Cancer Ther 2008;7:3169–3175. Steinberg M: Dasatinib: a tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Clin Ther 2007;29:2289–2308. Doggrel SA: BMS-354825: a novel drug with potential for the treatment of imatinib-resistant chronic myeloid leukaemia. Expert Opin Biol Ther 2005;305:399–401. Boyum A: Separation of lymphocytes, lymphocyte subgroups and monocytes: a review. Lymphology 1977;10:71–76. Albesiano E, Messmer BT, Damle RN, Allen SL, Rai KR, Chiorazzi N: Activation-induced cytidine deaminase in chronic lymphocytic
21 22
23
24
25
26
27
28
leukemia B cells: expression as multiple forms in a dynamic, variably sized fraction of the clone. Blood 2003;102:3333–3339. Cools J, Maertens C, Marynen P: Resistance to tyrosine kinase inhibitors: calling on extra forces. Drug Resist Updat 2005;8:119–29. Nelson EA, Walker SR, Kepich A, Gashin LB, Hideshima T, Ikeda H, Chauhan D, Anderson KC, Frank DA: Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3. Blood 2008; 112: 5095– 5102. Mackus WJ, Kater AP, Grummels A, Evers LM, Hooijbrink B, Kramer MH, Castro JE, Kipps TJ, van Lier RA, van Oers MH, Eldering E: Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation. Leukemia 2005;19:427–434. Thomas RK, Kallenborn A, Wickenhauser C, Schultze JL, Draube A, Vockerodt M, Re D, Diehl V, Wolf J: Constitutive expression of cFLIP in Hodgkin and Reed-Sternberg cells. Am J Pathol 2002;160:1521–1528. Aichberger KJ, Mayerhofer M, Krauth MT, Skvara H, Florian S, Sonneck K, Akgul C, Derdak S, Pickl WF, Wacheck V, Selzer E, Monia BP, Moriggl R, Valent P, Sillaber C: Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides. Blood 2005;105:3303–3311. Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC: Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 1998; 91: 991–1000. Derenne S, Monia B, Dean NM, Taylor JK, Rapp MJ, Harousseau JL, Bataille R, Amiot M: Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-xL is an essential survival protein of human myeloma cells. Blood 2002; 100:194–199. Bueno-da-Silva AEB, Brumati G, Russo FO, Green DR, Amarante-Mendes GP: Bcr-Ablmediated resistance to apoptosis is independent of constant tyrosine-kinase activity. Cell Death Differ 2003;10:592–598.
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
363
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/2/2015 10:06:10 PM
References
364
34 Handa H, Hegde UP, Kotelnikov VM, Mundle SD, Dong LM, Burke P, Rose S, Gaskin F, Raza A, Preisler HD: Bcl-2 and c-myc expression, cell cycle kinetics and apoptosis during the progression of chronic myelogenous leukemia from diagnosis to blastic phase. Leuk Res 1997;21:479–489. 35 Bradbury DA, Russell NH: Comparative quantitative expression of bcl-2 by normal and leukaemic myeloid cells. Br J Haematol 1995;91:374–379. 36 Campos L, Rouault JP, Sabido O, Oriol P, Roubi N, Vasselon C, Archimbaud E, Magaud JP, Guyotat D: High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Blood 1993;81:3091–3096. 37 Maung ZT, MacLean FR, Reid MM, Pearson AD, Proctor SJ, Hamilton PJ, Hall AG: The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia. Br J Haematol 1994;88:105–109. 38 Porwit-MacDonald A, Ivory K, Wilkinson S, Wheatley K, Wong L, Janossy G: Bcl-2 protein expression in normal human bone marrow precursors and in acute myelogenous leukemia. Leukemia 1995;9:1191–1198.
Acta Haematol 2015;133:354–364 DOI: 10.1159/000369446
39 Del Poeta G, Bruno A, Del Principe MI, Venditti A, Maurillo L, Buccisano F, Stasi R, Neri B, Luciano F, Siniscalchi A, de Fabritiis P, Amadori S: Deregulation of the mitochondrial apoptotic machinery and development of molecular targeted drugs in acute myeloid leukemia. Curr Cancer Drug Targets 2008; 8: 207–222. 40 Kuroda J, Kimura S, Strasser A, Andreeff M, O’Reilly LA, Ashihara E, Kamitsuji Y, Yokota A, Kawata E, Takeuchi M, Tanaka R, Tabe Y, Taniwaki M, Maekawa T: Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia. Cell Death Differ 2007;14:1667–1677. 41 Tipping AJ, Deininger MW, Goldman JM, Melo JV: Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate. Exp Hematol 2003;31:1073–1080. 42 Mahon FX, Deininger MW, Schutheis B, Chabrol J, Reiffers J, Goldman JM, Melo JV: Selection and characterization of BCR-ABL+ cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 2000;96:1070–1079.
Ferreira et al.
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29 Moller GM, Frost V, Melo JV, Chantry A: Upregulation of the TGF beta signalling pathway by Bcr-Abl: implications for haemopoietic cell growth and chronic myeloid leukaemia. FEBS Lett 2007;581:1329–1334. 30 Riccioni R, Senese M, Diverio D, Riti V, Buffolino S, Mariani G, Boe A, Cedrone M, LoCoco F, Foà R, Peschle C, Testa U: M4 and M5 acute myeloid leukaemias display a high sensitivity to Bortezomib-mediated apoptosis. Br J Haematol 2007;139:194–205. 31 Snead JL, O’Hare T, Adrian LT, Eide CA, Lange T, Druker BJ, Deininger MW: Acute dasatinib exposure commits Bcr-Abl-dependent cells to apoptosis. Blood 2009;114:3459– 3463. 32 Munzert G, Kirchner D, Stobbe H, Bergmann L, Schmid RM, Döhner H, Heimpel H: Tumor necrosis factor receptor-associated factor 1 gene overexpression in B-cell chronic lymphocytic leukemia: analysis of NF-κB/Relregulated inhibitors of apoptosis. Blood 2002; 100:3749–3756. 33 Delia D, Aiello A, Soligo D, Fontanella E, Melani C, Pezzella F, Pierotti MA, Della Porta G: bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells. Blood 1992;79:1291–1298.
