|Year : 2019 | Volume
| Issue : 3 | Page : 1004-1008
Decreased expression of microRNA-451 is associated with imatinib mesylate resistance in patients with chronic myeloid leukemia
Khaled A Khalifa1, Omaima M Abbas2, Raafat M Abdlfattah3, Suzan A El-Hassanein4, Amira M. F. Shehata1, Sally S Mandour Esawy2
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, National Liver Institute, Menoufia, Egypt
2 Department of Clinical Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Medical Oncology, National Cancer Institute, Cairo University, Cairo, Egypt
4 Department of Clinical Oncology, Faculty of Medicine, Menoufia University, National Liver Institute, Menoufia, Egypt
|Date of Submission||23-Mar-2019|
|Date of Acceptance||27-Apr-2019|
|Date of Web Publication||17-Oct-2019|
Sally S Mandour Esawy
Department of Clinical Pathology, National Liver Institute, Menoufia University, Menoufia
Source of Support: None, Conflict of Interest: None
To measure the expression levels of microRNA-451 (miR-451) in patients with chronic myeloid leukemia (CML) and to correlate miR-451 expression levels with response to imatinib mesylate (IM)-based therapeutic protocol.
miRNAs are regulatory RNAs that have been classified as crucial players of cancer initiation and progression. Recent studies have illustrated an association between certain hematological malignancies and dysregulated miRNAs expressions; other studies have focused on the utilization of miRNAs as molecular biomarkers of cancer detection, outcome, and response to therapy.
Patients and methods
Real-time quantitative PCR technique was used to estimate the plasma miR-451 expression levels of 10 newly diagnosed patients and 30 patients with CML who received first-line therapy with imatinib. European Leukemia Net-recommended response criteria were used to classify patients according to their response to imatinib treatment.
Our results revealed up-regulation of miR-451 expression levels in imatinib treated as compared with newly diagnosed patients with CML (P = 0.003). MiR-451 expression was significantly upregulated in IM-responder when compared with the IM-resistant group (P < 0.001). Moreover, we demonstrated that miR-451 was significantly down-regulated in IM-resistant in comparison with the healthy control group (P < 0.001), whereas no significant difference was detected between IM-responder group and healthy controls (P = 0.620).
miR-451 can be used as a promising biomarker in patients with CML for prediction of imatinib treatment response and may be employed as a novel therapeutic tool.
Keywords: chronic myeloid leukemia, imatinib mesylate, microRNA-451, real-time PCR
|How to cite this article:|
Khalifa KA, Abbas OM, Abdlfattah RM, El-Hassanein SA, Shehata AM, Mandour Esawy SS. Decreased expression of microRNA-451 is associated with imatinib mesylate resistance in patients with chronic myeloid leukemia. Menoufia Med J 2019;32:1004-8
|How to cite this URL:|
Khalifa KA, Abbas OM, Abdlfattah RM, El-Hassanein SA, Shehata AM, Mandour Esawy SS. Decreased expression of microRNA-451 is associated with imatinib mesylate resistance in patients with chronic myeloid leukemia. Menoufia Med J [serial online] 2019 [cited 2020 May 31];32:1004-8. Available from: http://www.mmj.eg.net/text.asp?2019/32/3/1004/268805
| Introduction|| |
Chronic myeloid leukemia (CML) represents an entity of myeloproliferative neoplasm with characteristic reciprocal translocation which leads to the development of Philadelphia chromosome containing the breakpoint cluster region-Abelson murine leukemia (BCR-ABL1) fusion gene . This results in expression of an oncoprotein with increased tyrosine kinase activity and constitutive activation of proteins in several signal transduction pathways with subsequent enhanced leukemic cells growth and suppression of cellular death .
The knowledge of molecular biology and abnormal signaling of CML supported the design of small molecules that specifically targeted the tyrosine kinase activity of BCR-ABL1. In 1996, imatinib mesylate (IM) (formerly STI571) was described by Druker et al.  as the first tyrosine kinase inhibitor (TKI). After IM approval by the Food and Drug Administration in 2001, it was the first drug to be successfully prescribed to treat patients with CML .
IM competes with ATP for binding to the BCR-ABL1 kinase domain, thus blocking phosphorylation of tyrosine residues on its substrates. Thus, the oncogenic proliferative signal is effectively interrupted, and the disease is controlled particularly when therapy is started early in chronic phase (CP) .
Although IM therapy noticeably improved patient survival when used in early-stage disease, IM resistance could occur, especially for accelerated phase and blastic phase, leading to progression and relapse . The cause of IM resistance was first explained by BCR-ABL1-dependent mechanism owing to BCR-ABL1 point mutations that affected the binding site of IM .
BCR-ABL1 did not mutate in about half of patients with IM-resistant CML, and so BCR-ABL1-independent IM resistance mechanisms were suggested . Deregulated expressions of microRNAs (miRNAs) were one of the most important and widely investigated mechanisms in those patients ,,.
miRNAs are a set of small noncoding RNAs that appear to control the expression of coding genes at the posttranscriptional level. Recent evidences suggest that aberrant expression of miRNAs may be implicated in the acquisition of cancer cell resistance to therapeutic agents. The modification of these miRNAs can restore signaling pathways and optimize chemotherapies by inhibition of tumor cell growth and enhancing the drug sensitivity ,.
MicroRNA-451 (miR-451) was known to be implicated in many types of cancers by inhibiting proliferation, triggering apoptosis, and reversing of chemotherapeutic resistance . The potential role of miR-451 in CML was examined by Lopotová et al.  who suggested the presence of a reciprocal regulatory mechanism between BCR-ABL and miR-451 that maintained the leukemic state of CML cells. Thus, the aim of this study was to measure the expression levels of miR-451 in patients with CML and to correlate miR-451 expression levels with response to IM-based therapeutic protocol.
| Patients and Methods|| |
The present study was carried on 40 patients with CP CML who were selected from Clinical Oncology Department, Menoufia University, and National Cancer Institute, Cairo University, during the period from August 2017 to December 2018. We retrospectively analyzed 30 patients with CP CML who were classified according to the European Leukemia Net-recommended response criteria . Twelve patients were characterized as IM-responders, whereas 18 were IM-resistant patients. In addition, samples from 10 newly diagnosed patients with CP CML were collected before chemotherapy and analyzed. Follow-up duration of newly diagnosed patients was at least 12 months from time of diagnosis. All studied patients received imatinib as first-line therapy, and all IM-resistant patients received second-generation TKI drug. As a control group, 10 apparent healthy adult persons with matched age and sex were included.
Laboratory analyses were conducted at Clinical Pathology Department, Faculty of Medicine, and National Liver Institute, Menoufia University. The study was approved by Ethics Research Committee of Menoufia University, and informed consent from each patient and control was taken in accordance with Declaration of Helsinki.
For diagnosis and follow-up, complete blood count, peripheral blood smear, bone marrow aspiration, and molecular analysis of BCR-ABL1 by real-time quantitative PCR were done.
For assessment of miR-451 expression levels, peripheral blood samples were collected into K2 EDTA tubes and centrifuged at 4000 rpm for 10 min, and supernatants for RNA extraction were preserved at −80°C until further analysis.
Extraction of total RNA from plasma samples was done by miRNeasy Mini Kit (Qiagen, Hilden, Germany); in accordance with the manufacturer's recommendations. Spectrophotometric determination of RNA purity was performed by NanoDrop 2000 (Thermo Scientific, Wilmington, Delaware, USA).
Complementary DNA (cDNA) was manufactured by miScript II RT Kit (Qiagen). Reverse-transcription master mix per reaction was prepared and included 4 μl miScript HiSpec Buffer, 2 μl miScript Nucleics Mix, 2 μl miScript Reverse Transcriptase Mix. Overall, 12 μl of RNA of each sample was added to the previous mix, incubated for 60 min at 37°C and for 5 min at 95°C, and then the final cDNA was stored at –20°C until PCR analysis.
PCR quantification experiments were performed by Qiagen Rotor-Gene Q using the miScript SYBR Green PCR kit (Qiagen) and specific MiScript Primer Assays (for has-miR-451a MS00004242; for RNU6 MS00033740; Qiagen). Overall, 2.5 μl of diluted cDNA (10 μl of cDNA + 50 μl of RNase-free water) was added to the following reaction mix (12.5 μl of SYBR Green PCR Master Mix, 2.5 μl of miScript Primer Assay, 2.5 μl of miScript Universal Primer, and 5 μl of RNase-free water) to reach a total volume of 25 μl. The cycling circumstances of the reactions were as follows: 95°C for 15 min for initial activation, followed by 40 cycles at 94°C for 15 s for denaturation, 55°C for 30 s for annealing, and 70°C for 30 s for extension.
RNU6 was employed as an internal reference, and the relative expression level of miR-451 was calculated by the 2− ΔΔ Ct method [ΔΔCt=ΔCt (sample)−ΔCt (control)]; ΔCt = Ct (miR-451)–Ct (RNU6); Δ, Delta; CT, threshold cycle.
The relationships between different characteristics of patients with CML were assessed using χ2, analysis of variance, and Kruskal–Wallis tests. Mann–Whitney and Kruskal–Wallis tests were used to compare the expression levels of miR-451 in different CML groups and controls. Results were analyzed by SPSS program (version 20; IBM Corporation, Armonk, New York, USA). When P value was less than 0.05, the result was considered statistically significant.
| Results|| |
The study included 12 IM-responder patients with CP CML (six males and six females, with a median age of 38.5 years and range, 17–61 years), 18 IM-resistant patients with CP CML (12 males and six females, with a median age of 42.5 years and range, 23–67 years), and 10 newly diagnosed patients with CP CML (four males and six females, with a median age of 33 years and range, 16–74 years).
No statistically significant differences were detected between different CML groups regarding age, sex, spleen size, hemoglobin concentration, leukocytes count, platelets count, peripheral blood basophils %, bone marrow blasts %, and BCR-ABL1% at diagnosis (all P's > 0.05). Statistically significant differences between the studied groups were found with respect to serum lactate dehydrogenase levels, uric acid levels, BCR-ABL1% at 12 months, and miR-451 expression levels (P = 0.021, P = 0.006, P < 0.001, and P < 0.001, respectively) [Table 1].
|Table 1: Clinicopathological characteristics of patients with chronic myeloid leukemia at diagnosis|
Click here to view
We reclassified all patients with CML according to imatinib response and then compared miR-451 expression levels between IM-responder group (n = 17; 12 retrospectively analyzed IM-responder patients plus five newly diagnosed patients who were categorized as IM-responder), IM-resistant group (n = 23; 18 retrospectively analyzed IM-resistant patients plus five newly diagnosed patients who were classified as IM-resistant), and control group.
Our results showed that miR-451 expression was significantly up-regulated in IM-responder group when compared with the IM-resistant group (P < 0.001).
Furthermore, we demonstrated that miR-451 was significantly down-regulated in IM-resistant in comparison with the healthy control group (P < 0.001), whereas no significant difference was detected between IM-responder group and healthy controls (P = 0.620) [Table 2] and [Figure 1].
|Table 2: Comparison between imatinib mesylate-responder, imatinib mesylate-resistant, and controls groups according to microRNA-451 expression levels|
Click here to view
|Figure 1: MiR-451 expression levels in IM-responder, IM-resistant, and controls groups. IM, imatinib mesylate; miR-451, microRNA-451.|
Click here to view
We analyzed miR-451 expression levels of newly diagnosed patients with CP CML in comparison with imatinib-treated patients with CP CML who accomplished major molecular response (MMR). MiR-451 expression levels were found to be up-regulated in imatinib-treated CP CML as compared with newly diagnosed patients with CP CML (P = 0.003) [Table 3] and [Figure 2].
|Table 3: Comparison between newly diagnosed and imatinib-treated groups according to microRNA-451 expression levels|
Click here to view
|Figure 2: MiR-451 expression levels in newly diagnosed and imatinib-treated groups. miR-451, microRNA-451.|
Click here to view
| Discussion|| |
The current CML therapies include interferon, TKIs, and bone marrow transplantation. IM is one of the commonly used TKI in CML management. However, IM resistance represents an obstacle to achieve MMR. The development of IM resistance was explained by many theories; one of them was aberrant miRNAs expressions leading to BCR-ABL independent survival of CML cells .
Prediction and management of IM resistance is essential step for proper selection of treatment strategy and for better long-term patient outcomes. Numerous miRNAs were investigated and validated as early detectors of IM resistance .
Based on the essential role of miR-451 in CML, our study was planned to test the potential importance of miR-451 as a predictive biomarker of IM resistance.
Our results revealed up-regulation of miR-451 expression levels in imatinib-treated CP CML with MMR as compared with newly diagnosed CP CML. These findings were in accordance with Ferreira et al.  who designed quantitative PCR array for studying expressions of multiple miRNAs including miR-451. Our data can be explained by the proposed reciprocal regulatory loop between BCR-ABL1 and miR-451, which is responsible for blocking miR-451 expression and maintaining leukemic cell survival ,.
Iraci et al.  identified three human miRNAs, miR-451, miR-515-3p, and miR-760, that specifically targeted BCR-ABL and suppressed both its translation and its expression in CML cells.
In the context of the tumor suppressor role of miR-451, many studies suggested that BCR-ABL1 fusion gene and myc oncogene were important targets for miR-451. Hence, a regulatory loop was suggested which could explain the down-regulation of miR-451 in IM-resistant patients with CML and provide an alternative therapeutic approach for those patients.
Regarding the regulatory pathway between myc and miR-451, Liu et al.  studied miR-144/451 and myc expressions in imatinib-resistant CML cells. Interestingly, they reported that miR-144/451 expression was significantly down-regulated owing to the increased myc expression and that restoration of miR-144/451 could stop the proliferation of CML cells.
The present study showed that miR-451 expression was significantly up-regulated in IM-responder patients when compared with the IM-resistant group. These findings were consistent with results reported by Soltani et al.  who investigated the expression levels of miR-451 in peripheral white blood cells of patients with CML who received standard imatinib therapy.
Moreover, we demonstrated that miR-451 was significantly down-regulated in IM-resistant in comparison with the healthy control, whereas no significant difference was detected between IM-responder group and healthy controls. These findings were in agreement with Zhang et al.  who investigated the dynamics of miRNA expression levels during the progression of CML.
| Conclusion|| |
Our results emphasized the dysregulated expression of miR-451 in patients with CML and represented miR-451 as a predictive biomarker of imatinib resistance.
MiR-451 can be considered as a tumor suppressor gene in CML that may be implicated in the molecular biology and response to imatinib therapy. Future studies may clarify the role of miR-451 as a therapeutic target and provide promising opportunities for patients who do not respond to conventional therapies.
Financial support and sponsorship
National Liver Institute.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jabbour E, Kantarjian H. Chronic myeloid leukemia: 2018 update on diagnosis, therapy and monitoring. Am J Hematol 2018; 93
Soverini S, Mancini M, Bavaro L, Cavo M, Martinelli G. Chronic myeloid leukemia: the paradigm of targeting oncogenic tyrosine kinase signaling and counteracting resistance for successful cancer therapy. Mol Cancer 2018; 17
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, et al
. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996; 2
Schwetz BA. News from the Food and Drug Administration. JAMA 2001; 285
Sacha T. Imatinib in chronic myeloid leukemia: an overview. Mediterr J Hematol Infect Dis 2014; 6
Quintás-Cardama A, Kantarjian HM, Cortes JE. Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer Control 2009; 16
Ma L, Shan Y, Bai R, Xue L, Eide CA, Ou J, et al
. A therapeutically targetable mechanism of BCR-ABL-independent imatinib resistance in chronic myeloid leukemia. Sci Transl Med 2014; 6
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
Mosakhani N, Mustjoki S, Knuutila S. Down-regulation of miR-181c in imatinib-resistant chronic myeloid leukemia. Mol Cytogenet 2013; 6
Kaehler M, Ruemenapp J, Gonnermann D, Nagel I, Bruhn O, Haenisch S, et al
. MicroRNA-212/ABCG2-axis contributes to development of imatinib-resistance in leukemic cells. Oncotarget 2017; 8
Jin J, Yao J, Yue F, Jin Z, Li D, Wang S. Decreased expression of microRNA-214 contributes to imatinib mesylate resistance of chronic myeloid leukemia patients by upregulating ABCB1 gene expression. Exp Ther Med 2018; 16
Li H, Yang BB. Friend or foe: the role of microRNA in chemotherapy resistance. Acta Pharmacol Sin 2013; 34
Rolfo C, Fanale D, Hong DS, Tsimberidou AM, Piha-Paul SA, Pauwels P, et al
. Impact of microRNAs in resistance to chemotherapy and novel targeted agents in non-small cell lung cancer. Curr Pharm Biotechnol 2014; 15
Pan X, Wang R, Wang ZX. The potential role of miR-451 in cancer diagnosis, prognosis, and therapy. Mol Cancer Ther 2013; 12
Lopotová T, Zácková M, Klamová H, Moravcová J. MicroRNA-451 in chronic myeloid leukemia: miR-451–BCR-ABL regulatory loop? Leuk Res 2011; 35
Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF, et al
. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood 2013; 122
Di Stefano C, Mirone G, Perna S, Marfe G. The roles of microRNAs in the pathogenesis and drug resistance of chronic myelogenous leukemia (review). Oncol Rep 2016; 35
Jurkovicova D, Lukackova R, Magyerkova M, Kulcsar L, Krivjanska M, Krivjansky V, Chovanec M. microRNA expression profiling as supportive diagnostic and therapy prediction tool in chronic myeloid leukemia. Neoplasma 2015; 62
Ferreira LAM, Capannacci J, Hokama NK, Nogueira CR, Ceccarelli M, Cerulo L, et al
. Circulating microRNAs expression profile in newly diagnosed and imatinib treated chronic phase – chronic myeloid leukemia. Leuk Lymphoma 2018; 60
Scholl V, Hassan R, Zalcberg IR. miRNA-451: a putative predictor marker of imatinib therapy response in chronic myeloid leukemia. Leuk Res 2012; 36
Iraci N, Valli E, Gherardi S, Soverini S, Kalebic T, Baccarani M, et al
. Suppression of Bcr-Abl expression in CML by a panel of miRNAs (abstract). Blood 2009; 114
Liu L, Wang S, Chen R, Wu Y, Zhang B, Huang S, et al.
Myc induced miR-144/451 contributes to the acquired imatinib resistance in chronic myelogenous leukemia cell K562. Biochem Biophys Res Commun 2012; 425
Soltani I, Douzi K, Gharbi H, Benhassine I, Teber M, Amouri H, et al
. Downregulation of miR-451 in Tunisian chronic myeloid leukemia patients: potential implication in imatinib resistance. Hematology 2017; 22
Zhang J, Jiang Y, Han X, Roy M, Liu W, Zhao X, Liu J. Differential expression profiles and functional analysis of plasma miRNAs associated with chronic myeloid leukemia phases. Future Oncol 2018; 15
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]