- Email: [email protected]
STAT Pathway on Dasatinib-Induced Apoptosis for CML Cell Model K562
SOHO Supplement 2015
Investigating the Role of JAK/STAT Pathway on Dasatinib-Induced Apoptosis for CML Cell Model K562 Ceyda Tunakan Dalgıç,1 Burçin Tezcanlı Kaymaz,2 Melda Cömert Özkan,3 Ays¸egül Dalmızrak,2 Fahri S¸ahin,3 Güray Saydam3 Abstract We aimed to evaluate the cytotoxic and apoptotic effects of dasatinib (BMS-354825) on K562 chronic myeloid leukemia (CML) cells and to examine the roles of STAT genes on dasatinib-induced apoptosis. The results showed that dasatinib decreased proliferation and induced apoptosis in K562 cells in a dose- and time-dependent manner. mRNA and protein levels of STAT5A and STAT5B genes were significantly reduced in dasatinib-treated K562 cells. These data indicated that STAT inhibition by dasatinib might be therapeutic in JAK/STAT pathwayeassociated malignancies after confirmation with clinical studies. Clinical Lymphoma, Myeloma & Leukemia, Vol. 15, No. S1, S161-6 ª 2015 Elsevier Inc. All rights reserved. Keywords: Apoptosis, Chronic myeloid leukemia, Dasatinib, JAK/STAT pathway, STAT5
Introduction Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem cells that arises from the reciprocal translocation between the breakpoint cluster region (BCR) gene on chromosome 22, and the Abelson (ABL) murine leukemia virus gene on chromosome 9, t(9;22)(q34;q11), resulting in the formation of Philadelphia (Ph) chromosome. The Ph chromosome encodes the BCR-ABL fusion protein, which has constitutive tyrosine kinase activity, leading to leukemogenesis.1 Imatinib mesylate, a selective inhibitor of the ABL tyrosine kinase, has demonstrated a remarkable efficacy in the treatment of CML by inducing cytogenetic remissions in over 75% of chronic-phase patients as first-line therapy.2 However, drug resistance and early relapses frequently occur in a considerable proportion of patients—a main limitation for prolonged survival.3 Resistance to imatinib is caused primarily by point mutations in the kinase domain of BCR-ABL that block drug binding.4
1
Department of Internal Medicine Department of Medical Biology 3 Department of Hematology _ Ege University Medical Faculty, Izmir, Turkey 2
Submitted: Dec 6, 2014; Accepted: Feb 3, 2015; Epub: Feb 17, 2015 Address for correspondence: Ceyda Tunakan Dalgıç, Department of Internal Medicine, _ 35100 Izmir, Turkey Fax: þ902323437876; e-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2015.02.012
In general, imatinib resistance can be subdivided into BCR-ABLdependent and -independent mechanisms. BCR-ABL-dependent mechanisms include over expression or amplification of the BCRABL gene and point mutations within the BCR-ABL kinase domain that interfere with imatinib binding. BCR-ABLindependent mechanisms include factors influencing the concentration of imatinib within the cell—for example, by alterations in drug influx and efflux and activation of BCR-ABL-independent pathways, such as members of the Src kinase family.5 To overcome this problem, more selective second-generation ABL tyrosine kinases have been developed. For these patients, other tyrosine kinase inhibitors (TKIs) such as nilotinib and dasatinib (BMS-354825) are used as second-generation therapy; these are approved for the treatment of CML after imatinib failure or intolerance. Currently marketed second-generation drugs include nilotinib, dasatinib, bosutinib, and ponatinib. Dasatinib is a thiazolylaminopyrimidine developed as the hydrochloride salt. It was discovered with a program directed toward immunosuppressive drugs and is 325-fold more potent against cells expressing wild-type BCR-ABL compared to imatinib.6,7 Dasatinib is a multitargeted inhibitor of BCR-ABL and the Src kinase family and also has inhibitory activity against additional downstream kinases.7,8 Dasatinib exclusively binds the active conformation of ABL kinase, in contrast to most TKIs.9 Dasatinib prevents the gained imatinib resistance, responsible for multidrug-resistant genes’ overexpression and BCR-ABL kinase region mutations by activating signaling pathways of the Src kinase family (LYN, HCK) and by inhibiting BCR-ABL, Src kinase
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S161
Role of JAK/STAT Pathway on Apoptosis family (SRC, LCK, YES, FYN), c-KIT, EPHA2, and PDGFR kinases. BCR-ABL activates multiple signalling pathways, including rat sarcoma (Ras), myelocytomatosis (myc), phosphoinositide 3-kinase/ Akt (PI3K/Akt), and Janus kinase/signal transducers and activator of transcription (JAK/STAT), which leads to uncontrolled proliferation and inhibition of apoptosis. Because the critical role of JAK/ STAT pathway has been demonstrated in myeloid differentiation, novel therapeutic strategies have been focused on targeting this pathway. STAT proteins are a family of latent cytoplasmic transcription factors that are involved in several cellular processes, such as proliferation, survival, apoptosis, and differentiation.10 Constitutive or aberrant activation of STATs was hypothesized to cause cellular transformation, in particular leukemogenesis.11,12 Seven mammalian STAT proteins have been discovered: STAT1 to STAT4, STAT5A, STAT5B, and STAT6.13 After dimerization and phosphorylation, STATs migrate into the nucleus, where they are activated, thus effecting cellular processes such as transcriptional regulations of several growth factors and oncoproteins.14 In the pathway, constitutive JAK activation leads to persistent activation of STATs, and in consequence, several cancer cells exhibit permanent STAT activity.15 Among STATs, STAT5A and STAT5B have also been shown to be necessary for the development of malignancy, and they exhibit regulatory roles in the development of leukemia.16 Until now, the efficacy of dasatinib in inducing apoptosis of leukemic cells has not been widely investigated in a point of JAK/ STAT pathway. The aim of this study was to investigate the apoptotic case of leukemic cells and to evaluate the transcription and translation levels of STAT5A and STAT5B, which are the potential targets of JAK/STAT pathway after dasatinib treatment on the model CML K562 cell line in order to clarify the underlying mechanism of dasatinib.
Materials and Methods Culturing Conditions of Cells Human chronic myelogenous leukemia cell line K562 were purchased from ECACC (European Collection of Cell Cultures). Cells were cultured in RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal calf serum, 100 U of penicillinestreptomycin per milliliter, and 1% L-glutamine at 37 C in humidified air containing 5% CO2. Cells with 95% survival rates and 80% confluence were used in experimental analyses.
Cell Proliferation Assay Cell viability and proliferation of untreated controls and dasatinib-treated cells were assessed by the Cell Proliferation Kit II (XTT; Roche Applied Science, Mannheim, Germany). K562 cells were seeded into 96-well plates at a density of 30 103 cells in 100 mL RPMI 1640 medium per well. Then cells were treated with increasing doses of dasatinib for a duration of 72 hours. After the proliferation assay, absorbance of each sample was measured spectrophotometrically with an enzyme-linked immunosorbent assay reader (ELISA; Thermo, Vantaa, Finland). All experiments were performed in triplicate. The obtained results were evaluated with the GraphPad Prism 5.01 (GraphPad Software, La Jolla, CA); cell proliferation curves were generated, and IC50 value was calculated for K562 cells.
Quantitative Real-Time Reverse TranscriptaseePolymerase Chain Reaction (qRT-PCR) Assay In expression analyses experiments, dasatinib was reverse transfected onto the cells, and at the end of 96 hours, each day’s cells were collected, including untreated control cells. In this manner, after 24 to 96 hours, total RNA extraction was performed from dasatinib-treated and -untreated control cells according to the manufacturer’s protocol (MagnaPure LC RNA Isolation Kit; Roche Applied Science). The amount and quality of RNAs were measured by a nanoDrop spectrophotometer, and 100 ng RNA was reverse transcribed into cDNA via Transcriptor High Fidelity cDNA Synthesis kit (Roche Applied Science) after the instructions. STAT5A and STAT5B mRNA expression levels were assessed by the LightCycler Fast Start DNA Master Hybridization Probes Kit in the qRT-PCR instrument LightCycler v2.0 in accordance with the G6PDH housekeeping gene set (Roche Applied Science). The primers and hybridization probes used for the amplification of STAT5A (NM_003152) and STAT5B (NM_012447) were as follows: STAT5A, F: 50 - GAAGCTGAACGTGCACATGAATC-30 , R: 50 -GTAGGGACAGAGTCTTCACCTGG-30 , FL: ACAGGA CTGTGAACTTCTCCTCTGTCACGG-FL, LC: CTCTGCA CCCCGCCGGTCAG-p. STAT5B, F: 50 -AGTTTGATTCTCA GGAAAGAATGT-30 , R: 50 -TCCATCAACAGCTTTAGCAGT-30 , FL: TTGGGAGACTTGAATTACCTTATCTACGT-FL, LC: TT CCTGATCGGCCAAAAGATGAA-p. The relative expression levels of target genes were determined by the proportion of the target value to reference value.
Western Blot Analyses Dasatinib Treatment Dasatinib (BMS-35482) was provided by Bristol-MyersSquibb (Princeton, NJ) and dissolved in dimethyl sulfoxide. Serial dilutions were prepared using serum-free RPMI 1640 medium. The experimental setup was generated with 3.3 nM of dasatinib treatment with IC50 (drug concentration causing 50% inhibition) value of K562 cells for 24 to 96 hours’ time course as well as with untreated control group cells. After 48 hours’ dasatinib treatment, cells were collected for apoptosis, gene expression, and Western blot analyses. All experiments were performed in triplicate, and the average of the results was taken.
S162
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
Cells were lysed in Complete Lysis-M Buffer containing Protease Inhibitor Cocktail Tablets (Roche Applied Science), and the supplied protein amounts were assessed by the Bradford method by use of bovine serum albumin concentration standards ranging between 0.25 and 2 mg/mL. Finally, 35 mg of each protein extract was resolved at 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to polyvinyl difluoride membranes using the iBlot dry transfer system (Invitrogen, Carlsbad, CA). The used primer antibody concentrations were 1:1000 diluted STAT5A (06-968; Upstate Biotechnology, Lake Placid, NY), STAT5B (06969; Upstate), and b-actin (#4967; Cell Signaling Technology, Danvers, MA). Primary antibody incubation, blotting, and
Ceyda Tunakan Dalgıç et al Figure 1 IC50 Dose Graphs for CML Cell Model K562
were calculated after taking the average values of triplicate absorbance measurements for each sample.
Statistical Analyses Dasatinib IC50 concentration and all cell proliferation experiment results were calculated with GraphPad Prism software v5.01. Relative mRNA expression levels gained at the end of qRT-PCR analyses and apoptosis assay results were evaluated by the Student t test in SPSS v15.0 software (IBM, Armonk, NY), with significance set at P < .05.
Results Dasatinib Reduced Proliferation of K562 Cells in a Time- and Dose-Dependent Manner
secondary antibody incubation were achieved by the iBlot Western Detection Kit (Invitrogen) for chromogenic detection of proteins in the iBlot system. The results were evaluated with the Chemi Smart 2000 gel imaging system (Vilber Lourmat, Marne-la-Vallée, France).
Apoptosis Analyses Apoptotic case of dasatinib -treated and -untreated cells were assessed by a photometric enzyme immunoassay Cell Death Detection ELISA Kit (Roche Applied Science) protocol that allows the detection of mono- and oligonucleosomes and thus measures apoptotic cell death. For this purpose, 5 104 cells were collected and resuspended in incubation buffer. At the end of the incubation process, for a duration of 30 minutes, the cell lysates were centrifuged, and 100 microliter of the supernatant was 1:10 prediluted with incubation buffer, reaching a final volume of 1 mL. Coating solution and incubation buffer were added into the wells of the MP modules and incubated for 90 minutes at room temperature. When conjugate and substrate solutions were added, the MP modules were put on a shaker and incubated at 300 rpm for 10 minutes. Finally, absorbance of each well was measured spectrophotometrically at 405 nm with an ELISA reader (Thermo, Vantaa, Finland). Apoptotic cell rates
The degree of cytotoxicity induced by dasatinib on human K562 cells was assessed by XTT cell proliferation assay, and the results demonstrated that dasatinib decreased cell proliferation significantly in a time- and dose-dependent manner. The IC50 value of dasatinib was calculated from the inhibition curve, and the value was determined to be 3.3 nM at hour 48 (Figure 1).
Dasatinib Triggers Transcriptional Down-Regulation in STAT5A and STAT5B After dasatinib treatment, leukemia cells were analyzed for differing expression patterns of STAT5A and STAT5B mRNA levels. After dasatinib treatment for 96 hours, a decrease at the STAT5A mRNA level was detected at 96 hours: STAT5A expression was significantly decreased by 1.5-fold (33.2% inhibition; P ¼ .02) compared to untreated control cells. However, STAT5B exhibited more dramatic expressional inhibition in response to dasatinib treatment. While STAT5B expression was down-regulated by 1.27fold (21.5% inhibition; P ¼ .07) at 72 hours, a significant 4.47-fold inhibition (77.6% inhibition; P < .001) was detected at 96 hours (Figure 2).
Effects of Dasatinib Induction on STAT Protein Levels According to Western blot results, translational changes were not detected with either STAT5A or STAT5B at the early stages of our experimental study as a result of dasatinib treatment compared to untreated control cells. However, at a time course of 96 hours,
Figure 2 STAT5A/STAT5B Gene Expressions in Dasatinib-Treated CML Cell Model K562. qRT-PCR Assay Was Performed in Triplicate, and Average mRNA Expression Levels Were Assessed by Rating to Reference Genes. After Dasatinib Treatment, (A) STAT5A and (B) STAT5B Expressional Down-Regulations Were Assessed. Statistical Analyses Were Performed by Student t Test
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S163
Role of JAK/STAT Pathway on Apoptosis Figure 3 Detection of STAT5A and STAT5B Protein Levels at 96 Hours. Detection of STAT5A and STAT5B Protein Levels at 96 Hours by Western Blot Analysis. b-Actin Was Used as Control
decreased, a significant increase was seen in the apoptotic cells in the time-course experiment. The absorbance levels obtained from cell death analysis showed significant increases in the dasatinib-treated cells compared to untreated control cells. Increased absorbance levels indicated that the number of apoptotic nucleosomes was increased in dasatinib-treated cells. When we compared the apoptosis rate of dasatinib-treated and -untreated cells, while 3.6-fold significant apoptosis induction was assessed at 72 hours (P ¼ .002), a 4.5-fold significant increase (P < .0001) was detected at 96 hours in the dasatinib-treated group. These results indicate that dasatinib is an effective TKI that triggers leukemic cell apoptosis partially on STAT5A and STAT5B (Figure 4).
Discussion expressional down-regulation was exhibited at both STAT5A and STAT5B proteins. In general, the obtained data from mRNA expression analysis and Western blot results were along the same lines: similar to transcriptional inhibition results, suppression at STAT5A and STAT5B protein levels were inhibited at the late stages of the experiment. While STAT5A protein expression level exhibited a moderate translational down-regulation at 96 hours, STAT5B protein expression was highly suppressed for the same time interval after dasatinib treatment compared to untreated control cells. In brief, both STAT5A and STAT5B protein expression levels were decreased at 96 hours’ treatment; however, STAT5B showed distinct translational inhibition in response to dasatinib treatment (Figure 3).
Dasatinib Triggers Apoptosis of Leukemic Cells Apoptosis analyses indicated that dasatinib induced leukemic cell apoptosis starting from the early stages of the experiment with an accelerated apoptosis rate compared to untreated control cells. After dasatinib treatment, while the percentage of living cells was
The main signal transduction pathways related in cell cycle, apoptosis, and leukemia development are JAK/STAT, Raf/MEK/ Erk, and PI3K/AKT. By the fusion gene BCR-ABL, tyrosine kinase activity occurs to activate Ras, Raf, PI3K, JNK/SAPK, Crkl, and STAT proteins, so cell proliferation is induced and apoptosis is inhibited. The STAT proteins are activated by many hematological cytokines and growth factors. They regulate cell cycle, apoptosis, and proliferation of different cells via accelerating gene transcription. Because STATs are constitutively active in certain hematooncologic diseases, they are suggested to play important roles in leukemogenesis.17 STAT5A and STAT5B genes, members of the JAK-STAT signaling pathway, are nuclear transcription factors that are responsible for activating the genes that exhibit increased expression in hematological malignancies and signal transduction. It is known that the abnormalities seen in the JAK/STAT pathway are not only involved in the development of solid cancers, such as those of lung, breast, head, and neck, but also affect leukemia and lymphoma. Because these transcription factors are overexpressed in tumor cells that proliferate uncontrollably,
Figure 4 Dasatinib-Treated and -Untreated CML K562 Cell Apoptosis Analysis. Dasatinib-Treated and -Untreated CML K562 Cell Apoptosis Analyses Was Performed for Detection of Histone ComplexeFormed DNA Fragments. Apoptosis Induction Was Detected in Dasatinib-Treated Leukemia Cell Cytoplasm by Measuring Quantitation of Mono- and Oligonucleosomes. Statistical Analyses Were Performed by Student t Test
S164
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
Ceyda Tunakan Dalgıç et al escape from the immune system, avoid apoptosis, and stimulate angiogenesis, they have become popular molecular therapeutic targets that seek to overcome leukemogenesis.18 Dasatinib is a novel potent oral multitargeted kinase inhibitor of BCR-ABL and Src family kinases and is a promising cancer therapeutic agent. However, the molecular mechanism of action of dasatinib is not yet fully understood.19 Scientist are trying to clarify the underlying molecular approaches of dasatinib-induced leukemic cell apoptosis. In a recent study, the authors evaluated the effect of the TKI dasatinib in leukemic cell lines bearing or not bearing c-kit mutations. It is known that activating mutations of the c-kit gene are frequently found in CBF (core binding factor) leukemias. Their data demonstrated that in the acute myeloid leukemia (AML) KASUMI-1 cell line bearing the N822K c-kit mutation, dasatinib was a potent suppressor of c-kit and Src kinase activity and inhibited the phosphorylation of their downstream target AKT, possibly through the Src-mediated VEGF/VEGFR receptor type 2 pathway. Dasatinib also effectively blocked proliferation and induced apoptosis through caspase-3 activation in KASUMI-1 cells. These data further encouraged the integration of dasatinib in the treatment of CBF AML with c-kit mutations in the context of clinical trials.20 In some other studies, the researchers pointed out the relationship between dasatinib treatment, STAT5 expressional regulation, and apoptosis. In 2004, Baskiewicz-Masiuk and Machali nski17 evaluated the effects on suppression of STAT5A and STAT5B expression on the clonogenicity and apoptosis of the chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells. Antisense oligodeoxynucleotides (ODNs) were applied to block STAT5A and STAT5B at the mRNA level, and qRT-PCR was used to determine STAT5 mRNA expression levels in the cells after ODN treatment. Perturbation of STAT5 expression decreased proliferative potential of the CML and the AML blasts as well as enhanced their apoptosis. Their studies showed that the STAT5 proteins might be critical in the regulation of growth and apoptosis of the CML and AML leukemic cells. The aim of our study was to investigate the apoptotic case of leukemic cells and to evaluate the transcriptional changes of STAT5A and STAT5B that are the members of JAK/STAT pathway after dasatinib treatment on CML model cell line K562. We examined the potential cytotoxic and apoptotic effects of dasatinib on K562 CML cells and investigated the possible mechanisms involved in cell death. The results of XTT cell proliferation assays revealed that dasatinib decreased proliferation of K562 cells in a dose- and time-dependent manner. When we compared the apoptosis rate of dasatinib-treated or -untreated cells, a 4.5-fold apoptosis induction was assessed at 96 hours in dasatinib-applied group (P < .0001). This difference was statistically significant compared to untreated controls and indicated that dasatinib induces apoptosis in K562 cells. Further, we analyzed STAT5 expression levels after dasatinib treatment in order to define the underlying molecular mechanism of dasatinib upon CML cells. Whereas STAT5A mRNA expression was decreased 1.5-fold (by 33.2% inhibition), STAT5B exhibited a 4.47-fold down-regulation (by 77.6% inhibition) at 96 hours. As for STAT5A and STAT5B protein expression level results, they were significantly suppressed at 96 hours, and these protein
expression results were in line with mRNA expression results. Similar to our results, a study of dasatinib and STAT5 signaling was reported in 2007. That study found that dasatinib inhibited tyrosine phosphorylation of Src family kinases, including Src, Hck, and Lyn, in K562 human CML cells. Significantly, downstream STAT5 signaling was also blocked by dasatinib, as shown by decreases in levels of phosphorylated STAT5 and STAT5 DNA-binding activities. In addition, dasatinib down-regulated expression of Stat5 target genes, including Bcl-x, Mcl-1, and cyclin D1. Consistent with those results, blockade of Stat5 signaling by dasatinib was accompanied by inhibition of cell proliferation and induction of apoptosis. Surprisingly, STAT5 DNA-binding activities were enhanced with increasing cell density, which was associated with resistance to apoptosis by dasatinib.19 In conclusion, one possible reason for dasatinib-induced leukemic cell apoptosis might be a significant decrease in STAT5A and STAT5B expression levels that are transcription factors and exhibit up-regulated expression in leukemia. Therefore, STAT5A and STAT5B are important molecular targets in the research of CML pathogenesis. In this study, we have shown for the first time that dasatinib triggered apoptosis through inhibiting the expression of STATs in any malignancy regarding JAK/STAT pathway. These findings are important because discovery or identification of novel agents targeting STAT5 might open new era for the treatment of different types of hematological malignancies associated with aberrant STAT expressions.
Clinical Practice Points In this study, we have shown for the first time that dasatinib
triggered apoptosis through inhibiting the expression of STATs in any malignancy regarding JAK/STAT pathway. These findings are important because discovery or identification of novel agents targeting STAT5 might open new era for the treatment of different types of hematological malignancies associated with aberrant STAT expressions.
Disclosure The authors have stated that they have no conflicts of interest.
References 1. Koca E, Haznedaroglu IC. Imatinib mesylate and the management of chronic myeloid leukemia (CML). Turk J Haematol 2005; 22:161-72. 2. O’Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348:994-1004. 3. Hughes T, Branford S. Molecular monitoring of chronic myeloid leukemia. Semin Hematol 2003; 40:62-8. 4. Shah N, Nicoll J, Nagar B, et al. Multiple BCRABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib in chronic phase and blast crisis chronic myelogenous leukemia. Leuk Res 2002; 2:117-25. 5. An X, Tiwari A, Sun Y, et al. BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review. Leuk Res 2010; 34:1255-68. 6. Manley P, Cowan-Jacob S, Mestan J. Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia. Biochim Biophys Acta 2005; 1754:3-13. 7. Olivieri A, Manzione L. Dasatinib: a new step in molecular target therapy. Ann Oncol 2007; 18:42-6. 8. Han L, Schuringa J, Mulder A, et al. Dasatinib impairs long-term expansion of leukemic progenitors in a subset of acute myeloid leukemia case. Ann Hematol 2010; 89:861-71.
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S165
Role of JAK/STAT Pathway on Apoptosis 9. Eck M, Manley P. The interplay of structural information and functional studies in kinase drug design: insights from BCR-Abl. Curr Opin Cell Biol 2009; 21:288-95. 10. Benekli M, Baer MR, Baumann H, et al. Signal transducer and activator of transcription proteins in leukemias. Blood 2003; 101:2940-54. 11. Garcia R, Jove R. Activation of STAT transcription factors in oncogenic tyrosine kinase signaling. J Biomed Sci 1998; 5:79-85. 12. Bowman T, Garcia R, Turkson J, et al. STATs in oncogenesis. Oncogene 2000; 19: 2474-88. 13. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994; 264:1415-21. 14. Bromberg JF. Activation of STAT proteins and growth control. Bioessays 2001; 23: 161-9. 15. Nasr R, Bazarbachi A. Chronic myeloid leukemia: “archetype” of the impact of targeted therapies. Pathol Biol 2012; 60:230-45.
S166
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
16. Levy DE, Gilliland DG. Divergent roles of Stat1 and Stat5 in malignancy as revealed by gene disruptions in mice. Oncogene 2000; 19:2505-10. 17. Baskiewicz-Masiuk M, Machali nski B. The role of the STAT5 proteins in the proliferation and apoptosis of the CML and AML cells. Eur J Haematol 2004; 72: 420-9. 18. Kaymaz BT, Selvi N, Gokbulut AA, et al. Suppression of STAT5A and STAT5B chronic myeloid leukemia cells via siRNA and antisenseoligonucleotide applications with the induction of apoptosis. Am J Blood Res 2013; 3:58-70. 19. Nam S, Williams A, Vultur A, et al. Dasatinib (BMS-354825) inhibits Stat5 signaling associated with apoptosis in chronic myelogenous leukemia cells. Mol Cancer Ther 2007; 6:1400-5. 20. Mpakou VE, Kontsioti F, Papageorgiou S, et al. Dasatinib inhibits proliferation and induces apoptosis in the KASUMI-1 cell line bearing the t(8; 21)(q22; q22) and the N822K c-kit mutation. Leuk Res 2013; 37:175-82.
Investigating the Role of JAK/STAT Pathway on Dasatinib-Induced Apoptosis for CML Cell Model K562 Ceyda Tunakan Dalgıç,1 Burçin Tezcanlı Kaymaz,2 Melda Cömert Özkan,3 Ays¸egül Dalmızrak,2 Fahri S¸ahin,3 Güray Saydam3 Abstract We aimed to evaluate the cytotoxic and apoptotic effects of dasatinib (BMS-354825) on K562 chronic myeloid leukemia (CML) cells and to examine the roles of STAT genes on dasatinib-induced apoptosis. The results showed that dasatinib decreased proliferation and induced apoptosis in K562 cells in a dose- and time-dependent manner. mRNA and protein levels of STAT5A and STAT5B genes were significantly reduced in dasatinib-treated K562 cells. These data indicated that STAT inhibition by dasatinib might be therapeutic in JAK/STAT pathwayeassociated malignancies after confirmation with clinical studies. Clinical Lymphoma, Myeloma & Leukemia, Vol. 15, No. S1, S161-6 ª 2015 Elsevier Inc. All rights reserved. Keywords: Apoptosis, Chronic myeloid leukemia, Dasatinib, JAK/STAT pathway, STAT5
Introduction Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem cells that arises from the reciprocal translocation between the breakpoint cluster region (BCR) gene on chromosome 22, and the Abelson (ABL) murine leukemia virus gene on chromosome 9, t(9;22)(q34;q11), resulting in the formation of Philadelphia (Ph) chromosome. The Ph chromosome encodes the BCR-ABL fusion protein, which has constitutive tyrosine kinase activity, leading to leukemogenesis.1 Imatinib mesylate, a selective inhibitor of the ABL tyrosine kinase, has demonstrated a remarkable efficacy in the treatment of CML by inducing cytogenetic remissions in over 75% of chronic-phase patients as first-line therapy.2 However, drug resistance and early relapses frequently occur in a considerable proportion of patients—a main limitation for prolonged survival.3 Resistance to imatinib is caused primarily by point mutations in the kinase domain of BCR-ABL that block drug binding.4
1
Department of Internal Medicine Department of Medical Biology 3 Department of Hematology _ Ege University Medical Faculty, Izmir, Turkey 2
Submitted: Dec 6, 2014; Accepted: Feb 3, 2015; Epub: Feb 17, 2015 Address for correspondence: Ceyda Tunakan Dalgıç, Department of Internal Medicine, _ 35100 Izmir, Turkey Fax: þ902323437876; e-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2015.02.012
In general, imatinib resistance can be subdivided into BCR-ABLdependent and -independent mechanisms. BCR-ABL-dependent mechanisms include over expression or amplification of the BCRABL gene and point mutations within the BCR-ABL kinase domain that interfere with imatinib binding. BCR-ABLindependent mechanisms include factors influencing the concentration of imatinib within the cell—for example, by alterations in drug influx and efflux and activation of BCR-ABL-independent pathways, such as members of the Src kinase family.5 To overcome this problem, more selective second-generation ABL tyrosine kinases have been developed. For these patients, other tyrosine kinase inhibitors (TKIs) such as nilotinib and dasatinib (BMS-354825) are used as second-generation therapy; these are approved for the treatment of CML after imatinib failure or intolerance. Currently marketed second-generation drugs include nilotinib, dasatinib, bosutinib, and ponatinib. Dasatinib is a thiazolylaminopyrimidine developed as the hydrochloride salt. It was discovered with a program directed toward immunosuppressive drugs and is 325-fold more potent against cells expressing wild-type BCR-ABL compared to imatinib.6,7 Dasatinib is a multitargeted inhibitor of BCR-ABL and the Src kinase family and also has inhibitory activity against additional downstream kinases.7,8 Dasatinib exclusively binds the active conformation of ABL kinase, in contrast to most TKIs.9 Dasatinib prevents the gained imatinib resistance, responsible for multidrug-resistant genes’ overexpression and BCR-ABL kinase region mutations by activating signaling pathways of the Src kinase family (LYN, HCK) and by inhibiting BCR-ABL, Src kinase
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S161
Role of JAK/STAT Pathway on Apoptosis family (SRC, LCK, YES, FYN), c-KIT, EPHA2, and PDGFR kinases. BCR-ABL activates multiple signalling pathways, including rat sarcoma (Ras), myelocytomatosis (myc), phosphoinositide 3-kinase/ Akt (PI3K/Akt), and Janus kinase/signal transducers and activator of transcription (JAK/STAT), which leads to uncontrolled proliferation and inhibition of apoptosis. Because the critical role of JAK/ STAT pathway has been demonstrated in myeloid differentiation, novel therapeutic strategies have been focused on targeting this pathway. STAT proteins are a family of latent cytoplasmic transcription factors that are involved in several cellular processes, such as proliferation, survival, apoptosis, and differentiation.10 Constitutive or aberrant activation of STATs was hypothesized to cause cellular transformation, in particular leukemogenesis.11,12 Seven mammalian STAT proteins have been discovered: STAT1 to STAT4, STAT5A, STAT5B, and STAT6.13 After dimerization and phosphorylation, STATs migrate into the nucleus, where they are activated, thus effecting cellular processes such as transcriptional regulations of several growth factors and oncoproteins.14 In the pathway, constitutive JAK activation leads to persistent activation of STATs, and in consequence, several cancer cells exhibit permanent STAT activity.15 Among STATs, STAT5A and STAT5B have also been shown to be necessary for the development of malignancy, and they exhibit regulatory roles in the development of leukemia.16 Until now, the efficacy of dasatinib in inducing apoptosis of leukemic cells has not been widely investigated in a point of JAK/ STAT pathway. The aim of this study was to investigate the apoptotic case of leukemic cells and to evaluate the transcription and translation levels of STAT5A and STAT5B, which are the potential targets of JAK/STAT pathway after dasatinib treatment on the model CML K562 cell line in order to clarify the underlying mechanism of dasatinib.
Materials and Methods Culturing Conditions of Cells Human chronic myelogenous leukemia cell line K562 were purchased from ECACC (European Collection of Cell Cultures). Cells were cultured in RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal calf serum, 100 U of penicillinestreptomycin per milliliter, and 1% L-glutamine at 37 C in humidified air containing 5% CO2. Cells with 95% survival rates and 80% confluence were used in experimental analyses.
Cell Proliferation Assay Cell viability and proliferation of untreated controls and dasatinib-treated cells were assessed by the Cell Proliferation Kit II (XTT; Roche Applied Science, Mannheim, Germany). K562 cells were seeded into 96-well plates at a density of 30 103 cells in 100 mL RPMI 1640 medium per well. Then cells were treated with increasing doses of dasatinib for a duration of 72 hours. After the proliferation assay, absorbance of each sample was measured spectrophotometrically with an enzyme-linked immunosorbent assay reader (ELISA; Thermo, Vantaa, Finland). All experiments were performed in triplicate. The obtained results were evaluated with the GraphPad Prism 5.01 (GraphPad Software, La Jolla, CA); cell proliferation curves were generated, and IC50 value was calculated for K562 cells.
Quantitative Real-Time Reverse TranscriptaseePolymerase Chain Reaction (qRT-PCR) Assay In expression analyses experiments, dasatinib was reverse transfected onto the cells, and at the end of 96 hours, each day’s cells were collected, including untreated control cells. In this manner, after 24 to 96 hours, total RNA extraction was performed from dasatinib-treated and -untreated control cells according to the manufacturer’s protocol (MagnaPure LC RNA Isolation Kit; Roche Applied Science). The amount and quality of RNAs were measured by a nanoDrop spectrophotometer, and 100 ng RNA was reverse transcribed into cDNA via Transcriptor High Fidelity cDNA Synthesis kit (Roche Applied Science) after the instructions. STAT5A and STAT5B mRNA expression levels were assessed by the LightCycler Fast Start DNA Master Hybridization Probes Kit in the qRT-PCR instrument LightCycler v2.0 in accordance with the G6PDH housekeeping gene set (Roche Applied Science). The primers and hybridization probes used for the amplification of STAT5A (NM_003152) and STAT5B (NM_012447) were as follows: STAT5A, F: 50 - GAAGCTGAACGTGCACATGAATC-30 , R: 50 -GTAGGGACAGAGTCTTCACCTGG-30 , FL: ACAGGA CTGTGAACTTCTCCTCTGTCACGG-FL, LC: CTCTGCA CCCCGCCGGTCAG-p. STAT5B, F: 50 -AGTTTGATTCTCA GGAAAGAATGT-30 , R: 50 -TCCATCAACAGCTTTAGCAGT-30 , FL: TTGGGAGACTTGAATTACCTTATCTACGT-FL, LC: TT CCTGATCGGCCAAAAGATGAA-p. The relative expression levels of target genes were determined by the proportion of the target value to reference value.
Western Blot Analyses Dasatinib Treatment Dasatinib (BMS-35482) was provided by Bristol-MyersSquibb (Princeton, NJ) and dissolved in dimethyl sulfoxide. Serial dilutions were prepared using serum-free RPMI 1640 medium. The experimental setup was generated with 3.3 nM of dasatinib treatment with IC50 (drug concentration causing 50% inhibition) value of K562 cells for 24 to 96 hours’ time course as well as with untreated control group cells. After 48 hours’ dasatinib treatment, cells were collected for apoptosis, gene expression, and Western blot analyses. All experiments were performed in triplicate, and the average of the results was taken.
S162
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
Cells were lysed in Complete Lysis-M Buffer containing Protease Inhibitor Cocktail Tablets (Roche Applied Science), and the supplied protein amounts were assessed by the Bradford method by use of bovine serum albumin concentration standards ranging between 0.25 and 2 mg/mL. Finally, 35 mg of each protein extract was resolved at 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to polyvinyl difluoride membranes using the iBlot dry transfer system (Invitrogen, Carlsbad, CA). The used primer antibody concentrations were 1:1000 diluted STAT5A (06-968; Upstate Biotechnology, Lake Placid, NY), STAT5B (06969; Upstate), and b-actin (#4967; Cell Signaling Technology, Danvers, MA). Primary antibody incubation, blotting, and
Ceyda Tunakan Dalgıç et al Figure 1 IC50 Dose Graphs for CML Cell Model K562
were calculated after taking the average values of triplicate absorbance measurements for each sample.
Statistical Analyses Dasatinib IC50 concentration and all cell proliferation experiment results were calculated with GraphPad Prism software v5.01. Relative mRNA expression levels gained at the end of qRT-PCR analyses and apoptosis assay results were evaluated by the Student t test in SPSS v15.0 software (IBM, Armonk, NY), with significance set at P < .05.
Results Dasatinib Reduced Proliferation of K562 Cells in a Time- and Dose-Dependent Manner
secondary antibody incubation were achieved by the iBlot Western Detection Kit (Invitrogen) for chromogenic detection of proteins in the iBlot system. The results were evaluated with the Chemi Smart 2000 gel imaging system (Vilber Lourmat, Marne-la-Vallée, France).
Apoptosis Analyses Apoptotic case of dasatinib -treated and -untreated cells were assessed by a photometric enzyme immunoassay Cell Death Detection ELISA Kit (Roche Applied Science) protocol that allows the detection of mono- and oligonucleosomes and thus measures apoptotic cell death. For this purpose, 5 104 cells were collected and resuspended in incubation buffer. At the end of the incubation process, for a duration of 30 minutes, the cell lysates were centrifuged, and 100 microliter of the supernatant was 1:10 prediluted with incubation buffer, reaching a final volume of 1 mL. Coating solution and incubation buffer were added into the wells of the MP modules and incubated for 90 minutes at room temperature. When conjugate and substrate solutions were added, the MP modules were put on a shaker and incubated at 300 rpm for 10 minutes. Finally, absorbance of each well was measured spectrophotometrically at 405 nm with an ELISA reader (Thermo, Vantaa, Finland). Apoptotic cell rates
The degree of cytotoxicity induced by dasatinib on human K562 cells was assessed by XTT cell proliferation assay, and the results demonstrated that dasatinib decreased cell proliferation significantly in a time- and dose-dependent manner. The IC50 value of dasatinib was calculated from the inhibition curve, and the value was determined to be 3.3 nM at hour 48 (Figure 1).
Dasatinib Triggers Transcriptional Down-Regulation in STAT5A and STAT5B After dasatinib treatment, leukemia cells were analyzed for differing expression patterns of STAT5A and STAT5B mRNA levels. After dasatinib treatment for 96 hours, a decrease at the STAT5A mRNA level was detected at 96 hours: STAT5A expression was significantly decreased by 1.5-fold (33.2% inhibition; P ¼ .02) compared to untreated control cells. However, STAT5B exhibited more dramatic expressional inhibition in response to dasatinib treatment. While STAT5B expression was down-regulated by 1.27fold (21.5% inhibition; P ¼ .07) at 72 hours, a significant 4.47-fold inhibition (77.6% inhibition; P < .001) was detected at 96 hours (Figure 2).
Effects of Dasatinib Induction on STAT Protein Levels According to Western blot results, translational changes were not detected with either STAT5A or STAT5B at the early stages of our experimental study as a result of dasatinib treatment compared to untreated control cells. However, at a time course of 96 hours,
Figure 2 STAT5A/STAT5B Gene Expressions in Dasatinib-Treated CML Cell Model K562. qRT-PCR Assay Was Performed in Triplicate, and Average mRNA Expression Levels Were Assessed by Rating to Reference Genes. After Dasatinib Treatment, (A) STAT5A and (B) STAT5B Expressional Down-Regulations Were Assessed. Statistical Analyses Were Performed by Student t Test
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S163
Role of JAK/STAT Pathway on Apoptosis Figure 3 Detection of STAT5A and STAT5B Protein Levels at 96 Hours. Detection of STAT5A and STAT5B Protein Levels at 96 Hours by Western Blot Analysis. b-Actin Was Used as Control
decreased, a significant increase was seen in the apoptotic cells in the time-course experiment. The absorbance levels obtained from cell death analysis showed significant increases in the dasatinib-treated cells compared to untreated control cells. Increased absorbance levels indicated that the number of apoptotic nucleosomes was increased in dasatinib-treated cells. When we compared the apoptosis rate of dasatinib-treated and -untreated cells, while 3.6-fold significant apoptosis induction was assessed at 72 hours (P ¼ .002), a 4.5-fold significant increase (P < .0001) was detected at 96 hours in the dasatinib-treated group. These results indicate that dasatinib is an effective TKI that triggers leukemic cell apoptosis partially on STAT5A and STAT5B (Figure 4).
Discussion expressional down-regulation was exhibited at both STAT5A and STAT5B proteins. In general, the obtained data from mRNA expression analysis and Western blot results were along the same lines: similar to transcriptional inhibition results, suppression at STAT5A and STAT5B protein levels were inhibited at the late stages of the experiment. While STAT5A protein expression level exhibited a moderate translational down-regulation at 96 hours, STAT5B protein expression was highly suppressed for the same time interval after dasatinib treatment compared to untreated control cells. In brief, both STAT5A and STAT5B protein expression levels were decreased at 96 hours’ treatment; however, STAT5B showed distinct translational inhibition in response to dasatinib treatment (Figure 3).
Dasatinib Triggers Apoptosis of Leukemic Cells Apoptosis analyses indicated that dasatinib induced leukemic cell apoptosis starting from the early stages of the experiment with an accelerated apoptosis rate compared to untreated control cells. After dasatinib treatment, while the percentage of living cells was
The main signal transduction pathways related in cell cycle, apoptosis, and leukemia development are JAK/STAT, Raf/MEK/ Erk, and PI3K/AKT. By the fusion gene BCR-ABL, tyrosine kinase activity occurs to activate Ras, Raf, PI3K, JNK/SAPK, Crkl, and STAT proteins, so cell proliferation is induced and apoptosis is inhibited. The STAT proteins are activated by many hematological cytokines and growth factors. They regulate cell cycle, apoptosis, and proliferation of different cells via accelerating gene transcription. Because STATs are constitutively active in certain hematooncologic diseases, they are suggested to play important roles in leukemogenesis.17 STAT5A and STAT5B genes, members of the JAK-STAT signaling pathway, are nuclear transcription factors that are responsible for activating the genes that exhibit increased expression in hematological malignancies and signal transduction. It is known that the abnormalities seen in the JAK/STAT pathway are not only involved in the development of solid cancers, such as those of lung, breast, head, and neck, but also affect leukemia and lymphoma. Because these transcription factors are overexpressed in tumor cells that proliferate uncontrollably,
Figure 4 Dasatinib-Treated and -Untreated CML K562 Cell Apoptosis Analysis. Dasatinib-Treated and -Untreated CML K562 Cell Apoptosis Analyses Was Performed for Detection of Histone ComplexeFormed DNA Fragments. Apoptosis Induction Was Detected in Dasatinib-Treated Leukemia Cell Cytoplasm by Measuring Quantitation of Mono- and Oligonucleosomes. Statistical Analyses Were Performed by Student t Test
S164
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
Ceyda Tunakan Dalgıç et al escape from the immune system, avoid apoptosis, and stimulate angiogenesis, they have become popular molecular therapeutic targets that seek to overcome leukemogenesis.18 Dasatinib is a novel potent oral multitargeted kinase inhibitor of BCR-ABL and Src family kinases and is a promising cancer therapeutic agent. However, the molecular mechanism of action of dasatinib is not yet fully understood.19 Scientist are trying to clarify the underlying molecular approaches of dasatinib-induced leukemic cell apoptosis. In a recent study, the authors evaluated the effect of the TKI dasatinib in leukemic cell lines bearing or not bearing c-kit mutations. It is known that activating mutations of the c-kit gene are frequently found in CBF (core binding factor) leukemias. Their data demonstrated that in the acute myeloid leukemia (AML) KASUMI-1 cell line bearing the N822K c-kit mutation, dasatinib was a potent suppressor of c-kit and Src kinase activity and inhibited the phosphorylation of their downstream target AKT, possibly through the Src-mediated VEGF/VEGFR receptor type 2 pathway. Dasatinib also effectively blocked proliferation and induced apoptosis through caspase-3 activation in KASUMI-1 cells. These data further encouraged the integration of dasatinib in the treatment of CBF AML with c-kit mutations in the context of clinical trials.20 In some other studies, the researchers pointed out the relationship between dasatinib treatment, STAT5 expressional regulation, and apoptosis. In 2004, Baskiewicz-Masiuk and Machali nski17 evaluated the effects on suppression of STAT5A and STAT5B expression on the clonogenicity and apoptosis of the chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells. Antisense oligodeoxynucleotides (ODNs) were applied to block STAT5A and STAT5B at the mRNA level, and qRT-PCR was used to determine STAT5 mRNA expression levels in the cells after ODN treatment. Perturbation of STAT5 expression decreased proliferative potential of the CML and the AML blasts as well as enhanced their apoptosis. Their studies showed that the STAT5 proteins might be critical in the regulation of growth and apoptosis of the CML and AML leukemic cells. The aim of our study was to investigate the apoptotic case of leukemic cells and to evaluate the transcriptional changes of STAT5A and STAT5B that are the members of JAK/STAT pathway after dasatinib treatment on CML model cell line K562. We examined the potential cytotoxic and apoptotic effects of dasatinib on K562 CML cells and investigated the possible mechanisms involved in cell death. The results of XTT cell proliferation assays revealed that dasatinib decreased proliferation of K562 cells in a dose- and time-dependent manner. When we compared the apoptosis rate of dasatinib-treated or -untreated cells, a 4.5-fold apoptosis induction was assessed at 96 hours in dasatinib-applied group (P < .0001). This difference was statistically significant compared to untreated controls and indicated that dasatinib induces apoptosis in K562 cells. Further, we analyzed STAT5 expression levels after dasatinib treatment in order to define the underlying molecular mechanism of dasatinib upon CML cells. Whereas STAT5A mRNA expression was decreased 1.5-fold (by 33.2% inhibition), STAT5B exhibited a 4.47-fold down-regulation (by 77.6% inhibition) at 96 hours. As for STAT5A and STAT5B protein expression level results, they were significantly suppressed at 96 hours, and these protein
expression results were in line with mRNA expression results. Similar to our results, a study of dasatinib and STAT5 signaling was reported in 2007. That study found that dasatinib inhibited tyrosine phosphorylation of Src family kinases, including Src, Hck, and Lyn, in K562 human CML cells. Significantly, downstream STAT5 signaling was also blocked by dasatinib, as shown by decreases in levels of phosphorylated STAT5 and STAT5 DNA-binding activities. In addition, dasatinib down-regulated expression of Stat5 target genes, including Bcl-x, Mcl-1, and cyclin D1. Consistent with those results, blockade of Stat5 signaling by dasatinib was accompanied by inhibition of cell proliferation and induction of apoptosis. Surprisingly, STAT5 DNA-binding activities were enhanced with increasing cell density, which was associated with resistance to apoptosis by dasatinib.19 In conclusion, one possible reason for dasatinib-induced leukemic cell apoptosis might be a significant decrease in STAT5A and STAT5B expression levels that are transcription factors and exhibit up-regulated expression in leukemia. Therefore, STAT5A and STAT5B are important molecular targets in the research of CML pathogenesis. In this study, we have shown for the first time that dasatinib triggered apoptosis through inhibiting the expression of STATs in any malignancy regarding JAK/STAT pathway. These findings are important because discovery or identification of novel agents targeting STAT5 might open new era for the treatment of different types of hematological malignancies associated with aberrant STAT expressions.
Clinical Practice Points In this study, we have shown for the first time that dasatinib
triggered apoptosis through inhibiting the expression of STATs in any malignancy regarding JAK/STAT pathway. These findings are important because discovery or identification of novel agents targeting STAT5 might open new era for the treatment of different types of hematological malignancies associated with aberrant STAT expressions.
Disclosure The authors have stated that they have no conflicts of interest.
References 1. Koca E, Haznedaroglu IC. Imatinib mesylate and the management of chronic myeloid leukemia (CML). Turk J Haematol 2005; 22:161-72. 2. O’Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348:994-1004. 3. Hughes T, Branford S. Molecular monitoring of chronic myeloid leukemia. Semin Hematol 2003; 40:62-8. 4. Shah N, Nicoll J, Nagar B, et al. Multiple BCRABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib in chronic phase and blast crisis chronic myelogenous leukemia. Leuk Res 2002; 2:117-25. 5. An X, Tiwari A, Sun Y, et al. BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review. Leuk Res 2010; 34:1255-68. 6. Manley P, Cowan-Jacob S, Mestan J. Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia. Biochim Biophys Acta 2005; 1754:3-13. 7. Olivieri A, Manzione L. Dasatinib: a new step in molecular target therapy. Ann Oncol 2007; 18:42-6. 8. Han L, Schuringa J, Mulder A, et al. Dasatinib impairs long-term expansion of leukemic progenitors in a subset of acute myeloid leukemia case. Ann Hematol 2010; 89:861-71.
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S165
Role of JAK/STAT Pathway on Apoptosis 9. Eck M, Manley P. The interplay of structural information and functional studies in kinase drug design: insights from BCR-Abl. Curr Opin Cell Biol 2009; 21:288-95. 10. Benekli M, Baer MR, Baumann H, et al. Signal transducer and activator of transcription proteins in leukemias. Blood 2003; 101:2940-54. 11. Garcia R, Jove R. Activation of STAT transcription factors in oncogenic tyrosine kinase signaling. J Biomed Sci 1998; 5:79-85. 12. Bowman T, Garcia R, Turkson J, et al. STATs in oncogenesis. Oncogene 2000; 19: 2474-88. 13. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994; 264:1415-21. 14. Bromberg JF. Activation of STAT proteins and growth control. Bioessays 2001; 23: 161-9. 15. Nasr R, Bazarbachi A. Chronic myeloid leukemia: “archetype” of the impact of targeted therapies. Pathol Biol 2012; 60:230-45.
S166
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
16. Levy DE, Gilliland DG. Divergent roles of Stat1 and Stat5 in malignancy as revealed by gene disruptions in mice. Oncogene 2000; 19:2505-10. 17. Baskiewicz-Masiuk M, Machali nski B. The role of the STAT5 proteins in the proliferation and apoptosis of the CML and AML cells. Eur J Haematol 2004; 72: 420-9. 18. Kaymaz BT, Selvi N, Gokbulut AA, et al. Suppression of STAT5A and STAT5B chronic myeloid leukemia cells via siRNA and antisenseoligonucleotide applications with the induction of apoptosis. Am J Blood Res 2013; 3:58-70. 19. Nam S, Williams A, Vultur A, et al. Dasatinib (BMS-354825) inhibits Stat5 signaling associated with apoptosis in chronic myelogenous leukemia cells. Mol Cancer Ther 2007; 6:1400-5. 20. Mpakou VE, Kontsioti F, Papageorgiou S, et al. Dasatinib inhibits proliferation and induces apoptosis in the KASUMI-1 cell line bearing the t(8; 21)(q22; q22) and the N822K c-kit mutation. Leuk Res 2013; 37:175-82.