Silencing of Suppressor of Cytokine Signaling-3 due to Methylation Results in Phosphorylation of STAT3 in Imatinib Resistant BCR-ABL Positive Chronic Myeloid Leukemia Cells

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulatorgene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinibmesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematologicalremission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patientstreated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profileof SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials andMethods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assayswere performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profileswere investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Geneexpression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1,2 and 3 were examined by Western blotting. Results: The IC50 for imatinib on K562 was 362nM compared to3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing theconcentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation ofthe SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed downregulationof both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but notK562. Conclusions: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells.Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNAmethylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying thedevelopment of imatinib resistant in CML patients.     

Contribution of BCR–ABL-independent activation of ERK1/2 to acquired imatinib resistance in K562 chronic myeloid leukemia cells

BCR–ABL tyrosine kinase, generated from the reciprocal chromosomal translocation t(9;22), causes chronic myeloid leukemia (CML). BCR–ABL is inhibited by imatinib; however, several mechanisms of imatinib resistance have been proposed that account for loss of imatinib efficacy in patients with CML. Previously, we showed that overexpression of the efflux drug transporter P-glycoprotein partially contributed to imatinib resistance in imatinib-resistant K562 CML cells having no BCR–ABL mutations. To explain an additional mechanism of drug resistance, we established a subclone (K562/R) of the cells and examined the BCR–ABL signaling pathway in these and wild-type K562 (K562/W) cells. We found the K562/R cells were 15 times more resistant to imatinib than their wild-type counterparts. In both cell lines, BCR–ABL and its downstream signaling molecules, such as ERK1/2, ERK5, STAT5, and AKT, were phosphorylated in the absence of imatinib. In both cell lines, imatinib effectively reduced the phosphorylation of all the above, except ERK1/2, whose phosphorylation was, interestingly, only inhibited in the wild-type cells. We then observed that phospho-ERK1/2 levels decreased in the presence of siRNA targeting BCR–ABL, again, only in the K562/W cells. However, using an ERK1/2 inhibitor, U0126, we found that we could reduce phospho-ERK1/2 levels in K562/R cells and restore their sensitivity to imatinib. Taken together, we conclude that the BCR–ABL-independent activation of ERK1/2 contributes to imatinib resistance in K562/R cells, and that ERK1/2 could be a target for the treatment of CML patients whose imatinib resistance is due to this mechanism. ( Cancer Sci 2009)     

重组变构人肿瘤坏死因子相关凋亡诱导配体逆转慢性粒细胞白血病细胞对伊马替尼耐药的机制研究

目的:探讨重组变构人肿瘤坏死因子相关凋亡诱导配体（CPT）逆转慢性粒细胞白血病（CML）细胞伊马替尼耐药的相关机制。方法:选择2016年至2020年内蒙古医科大学附属医院就诊的5例CML患者，分别采集初诊和伊马替尼耐药状态下的肝素化骨髓血标本，分离单个核细胞。初诊时采集的单个核细胞依次命名为A1~E1，伊马替尼耐药后采集的单个核细胞分别命名为A2~E2。使用人CML野生型K562细胞株（K562-W），采用低浓度伊马替尼小剂量逐步加量的方法，获得伊马替尼耐药的K562细胞（K562-R）。采用20 μg/L CPT培养K562-R细胞，设为CPT-K562-R细胞组。CCK-8法检测细胞对伊马替尼的半数抑制浓度（ IC50）。采用K562-W、K562-R细胞构建CML移植瘤裸鼠模型，将裸鼠分为K562-W、K562-R、CPT-K562-R移植瘤组，三组均经口灌注伊马替尼，CPT-K562-R组同时皮下注射CPT；比较三组裸鼠移植瘤在伊马替尼治疗前、治疗4周后的肿瘤体积，以及三组裸鼠的存活时间。蛋白质印迹法检测CML患者骨髓单个核细胞、K562细胞株及其移植瘤组织中酪氨酸蛋白激酶受体B4（EphB4）、髓细胞白血病蛋白1（Mcl-1）蛋白水平的变化。 结果:5例CML患者A2~E2细胞中EphB4蛋白表达水平均较A1~E1细胞增高（均 P<0.01）。K562-W、K562-R、CPT-K562-R细胞对伊马替尼的 IC50分别为（0.160±0.015）mg/L、（5.450±0.460）mg/L、（0.300±0.035）mg/L，差异有统计学意义（ F＝390.65， P<0.01）。K562-W组细胞中EphB4、Mcl-1蛋白均呈低水平表达（0.54±0.02和0.70±0.08）；K562-R组细胞中EphB4、Mcl-1蛋白表达水平升高（3.04±0.11和2.88±0.04）；CPT-K562-R组细胞中EphB4、Mcl-1蛋白表达水平下降（0.57±0.03和0.38±0.04）。伊马替尼治疗前，K562-W、K562-R和CPT-K562-R移植瘤组裸鼠移植瘤体积差异无统计学意义（ F＝0.39， P＝0.68），提示裸鼠移植瘤成瘤均衡；伊马替尼治疗结束后三组移植瘤体积差异有统计学意义（ F＝26.16， P<0.01）。K562-W、K562-R和CPT-K562-R移植瘤组裸鼠存活时间分别为（18.5±3.3）d、（10.0±2.4）d、（17.5±1.6）d，差异有统计学意义（ F＝20.45， P<0.01）。K562-W移植瘤组中EphB4、Mcl-1蛋白均呈低水平表达（0.55±0.06和0.67±0.06）；K562-R移植瘤组中EphB4、Mcl-1蛋白表达水平升高（1.95±0.08和6.21±0.53）；CPT-K562-R移植瘤组中EphB4、Mcl-1蛋白表达水平下降（0.59±0.04和0.37±0.04），且接近K562-W移植瘤组的水平。 结论:CPT可能通过抑制EphB4、Mcl-1表达，增强CML对伊马替尼的敏感性，这可能是一种伊马替尼治疗靶向通路。     

Imatinib and nilotinib induce apoptosis of chronic myeloid leukemia cells through a Bim-dependant pathway modulated by cytokines

It is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated by extracellular factors. Apoptosis induced by imatinib and nilotinib was determined in BCR-ABL expressing cell lines and primary CML CD34+ cells. Both molecules induced apoptosis of BCR-ABL expressing cells. This apoptosis was inhibited by protein synthesis inhibition in both K562 and CML CD34+ cells. In K562, 80% inhibition of the BCR-ABL auto-phosphorylation by either imatinib or nilotinib induced a two fold increase in Bim-EL expression and induction of apoptosis in 48 h. Bim accumulation preceded apoptosis induction which was completely abolished by depletion in Bim using shRNA. However, the anti-proliferative effect of imatinib was preserved in Bim-depleted cells. When K562 cells were cultured in a cytokine containing medium, the pro-apoptotic effect of nilotinib was decreased by 68% and this was related to a decrease in Bim-EL dephosphorylation and accumulation. Similarly, the presence of a combination of cytokines inhibited 88% of NIL- and 39% of IMA-induced apoptosis in primary CML CD34+ cells. In conclusion, both nilotinib and imatinib induce apoptosis through Bim accumulation independently of cell cycle arrest. However, the pro-apoptotic effect of both molecules can be attenuated by the presence of cytokines and growth factors, particularly concerning nilotinib. Thus BCR-ABL inhibition restores the cytokine dependence but is not sufficient to induce apoptosis when other signaling pathways are activated.     

Oridonin in combination with imatinib exerts synergetic anti-leukemia effect in Ph+ acute lymphoblastic leukemia cells in vitro by inhibiting activation of LYN/mTOR signaling pathway

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is triggered by constitutively activated BCR-ABL and SRC family tyrosine kinases.They account for the activations of multiple growth-signaling pathways, including Raf/MEK/ERK, Akt/mTOR and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for Ph+ leukemia and plays efficacious role in CML. However, imatinib has few inhibitory effects on SRC tyrosine kinase with response rate of Ph+ ALL lower and relapse more frequent and quicker compared with CML. Previous studies showed that oridonin inhibits proliferation and induces apoptosis in many tumor cells. However, the anticancer activity and mechanism of oridonin in Ph+ ALL is unknown. To investigate the anticancer activity of oridonin, we examined its role in constitutively activated Akt/mTOR, Raf/MEK/ERK, STAT5 and SRC pathway, mRNA level of bcr/abl gene, cell viability and apoptosis in Ph+ ALL SUP-B15 cells. Furthermore, we detected synergetic effect of oridonin plus imatinib. Our results showed that oridonin inhibiting activations of LYN (one of SRC family kinases) and ABL and their downstream Akt/mTOR, Raf/MEK/ERK and STAT5 pathways, downregulated Bcl-2 but upregulated Bax protein and then induced apoptosis in Ph+ ALL cells. Oridonin plus imatinib exerted synergetic effects by overcoming imatinib defect of upregulating Akt/mTOR and LYN signaling. Additionally, we examined the effect of oridonin on the signaling pathways in the primary specimens from Ph+ ALL patients. Our data showed that oridonin remarkably suppressed activations of Akt/mTOR, Raf/MEK and STAT5 pathway in these primary specimens and oridonin with imatinib exerted synergetic suppressive effects on mTOR, STAT5 and LYN signaling in one imatinib resistant patient specimen. Additional evaluation of oridonin as a potential therapeutic agent for Ph+ ALL seems warranted.     

CUEDC2 sensitizes chronic myeloid leukemic cells to imatinib treatment

CUEDC2, a newly reported protein, has been found to be ubiquitously expressed in human tissues and repress NF-κB activity. To study the role of CUEDC2 in chronic myeloid leukemia (CML), we explored the function of CUEDC2 in CML cells through using the CML cell line K562 and its imatinib resistant cells K562/G01. K562 cells expressed a relatively higher level of CUEDC2 compared to K562/G01 cells. Knockdown of CUEDC2 in K562 cells resulted in decreased cell apoptosis after imatinib treatment; when CUEDC2 was overexpressed in K562/G01 cells, imatinib induced more cell apoptosis. By analyzing the activity of NF-κB, the results indicated a negative association between the expression of CUEDC2 and NF-κB signaling pathway in these CML cells. Our data suggested that the expression level of CUEDC2 has an inverse correlation with imatinib resistance and activity of NF-κB signaling pathway in CML cells, CUEDC2 could regulate imatinib sensitivity in CML cells at least partially through NF-κB signaling pathway.     

Transcription suppression of SARI (suppressor of AP-1, regulated by IFN) by BCR-ABL in human leukemia cells

The BCR-ABL tyrosine kinase has been implicated in the dysregulation of oncogenes and tumor suppressor genes involved in chronic myelogenous leukemia (CML). Suppressor of activator protein-1, regulated by interferon (SARI), is a recently identified tumor suppressor gene whose expression has been reported to be suppressed in several malignant neoplasms. However, the expression of SARI in leukemia and the underlying regulatory mechanism remain elusive. In this study, we demonstrated that SARI mRNA expression was low in CML patients. In vitro, BCR-ABL kinase inhibitor imatinib mesylate or siRNA specific to BCR-ABL upregulated SARI mRNA expression in human leukemia cells. In addition, JAK/STAT signaling inhibitor AG490 and RAS/MAPK signaling inhibitor PD98059 upregulated SARI mRNA expression, but PI3K/AKT pathway inhibitor LY294002 had no such effect. Functionally, silencing of SARI in CML-derived cell line K562 partially decreased imatinib mesylate-induced apoptosis. Taken together, these data demonstrate that SARI mRNA expression is suppressed by BCR-ABL through the downstream signaling pathways, suggesting SARI as a potential therapeutic target in CML.     

Imatinib-resistant K562 cells are more sensitive to celecoxib, a selective COX-2 inhibitor: role of COX-2 and MDR-1

Selective inhibition of the BCR/ABL tyrosine kinase by imatinib (STI571, Glivec/Gleevec) is the therapeutic strategy in patients with chronic myelogenous leukemia (CML). Despite significant hematologic and cytogenetic responses with imatinib, mainly due to the mutations in the Abl kinase domain, resistance occurs in patients with advanced disease. In the present study on imatinib-resistant K562 cells (IR-K562), however, no such mutations in the Abl kinase domain were observed. Further studies revealed the over-expression of COX-2 and MDR-1 in IR-K562 cells suggesting the possible involvement of COX-2 in the development of resistance to imatinib. So, we sought to examine the effect of celecoxib, a selective COX-2 inhibitor, on IR-K562 cells. The results clearly indicate that celecoxib is more effective in IR-K562 cells with a lower IC50 value of 10 microM compared to an IC50 value of 40 microM in K562 cells. This increase in the sensitivity of IR-K562 cells towards celecoxib suggests that the development of resistance in IR-K562 cells is COX-2 dependent. Further studies revealed down-regulation of MDR-1 by celecoxib and a decline in p-Akt levels. Celecoxib-induced apoptosis of IR-K562 cells led to release of cytochrome c, PARP cleavage and decreased Bcl2/Bax ratio. Also, celecoxib at 1 microM concentration induced apoptosis in IR-K562 cells synergistically with imatinib by reducing the IC50 value of imatinib from 10 to 6 microM. In conclusion, the present study indicates over-expression of COX-2 and MDR-1 in IR-K562 cells and celecoxib, a COX-2 specific inhibitor, induces apoptosis by inhibiting COX-2 and down-regulating MDR-1 expression through Akt/p-Akt signaling pathway.     

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.     

Anti-Proliferative Effects of Dendrophthoe pentandra Methanol Extract on BCR/ABL-Positive and Imatinib-Resistant Leukemia Cell Lines

Background: Imatinib mesylate, a tyrosine kinase inhibitor specifically targeting the BCR/ABL fusion protein, induces hematological remission in patients with chronic myeloid leukemia (CML). However, the majority of CML patients treated with imatinib develop resistance with prolonged therapy. Dendrophthoe pentandra (L.) Miq. is a Malaysian mistletoe species that has been used as a traditional treatment for several ailments such as smallpox, ulcers, and cancers. Methods: We developed a resistant cell line (designated as K562R) by long-term co-culture of a BCR/ ABL positive CML cell line, K562, with imatinib mesylate. We then investigated the anti-proliferative effects of D. pentandra methanol extract on parental K562 and resistant K562R cells. Trypan blue exclusion assays were performed to determine the IC50 concentration; apoptosis and cell cycle analysis were conducted by flow cytometry. Results: D. pentandra extract had greater anti-proliferative effects towards K562R (IC50= 192 μg/mL) compared to K562 (500 μg/ mL) cells. Upon treatment with D. pentandra extract at the IC50. concentration: K562 but not K562R demonstrated increase in apoptosis and cell cycle arrest in the G2/M phase. Conclusion: D. pentandra methanol extract exerts potent anti-proliferative effect on BCR/ABL positive K562 cells.     

Adaphostin has significant and selective activity against chronic and acute myeloid leukemia cells

Adaphostin is a tyrphostin that was designed to inhibit Bcr/Abl tyrosine kinase by altering the binding site of peptide substrates rather than that of adenosine triphosphate, a known mechanism of imatinib mesylate (IM). However, it has been shown that adaphostin-mediated cytotoxicity is dependent on oxidant production and does not require Bcr/Abl. We have tested adaphostin against both Philadelphia chromosome (Ph)-positive (K562, KBM5, KBM5-R [IM resistant KBM5], KBM7, and KBM7-R [IM-resistant KBM7]) and Ph-negative (OCI/AML2 and OCI/AML3) cells, and against cells from patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Adaphostin significantly inhibited growth of all cell lines (50% inhibition of cell proliferation [IC50] 0.5-1 microM) except K562 (IC50 13 microM). Ph-positive IM-resistant cell lines showed significant cross resistance to adaphostin. Simultaneous or sequential treatment with adaphostin and IM did not exert a synergistic effect in any KBM line. Adaphostin induced superoxide and apoptosis in a dose-dependent and time-dependent fashion in both Ph-positive and Ph-negative cells. Adaphostin selectively inhibited colony growth of cells from CML (IM-sensitive and IM-resistant) and AML patients. Analysis of tyrosine phosphorylated proteins after treatment with adaphostin revealed alternate effects in different cells consistent with the modulation of multiple targets. In conclusion, adaphostin showed significant and selective activity against CML and AML cells and its development for clinical testing is warranted.     

(-)Gossypol and its combination with imatinib induce apoptosis in human chronic myeloid leukemic cells

Chronic myeloid leukemia (CML) is characterized by the presence of chimeric protein BCR-ABL associated with high tyrosine kinase (TK) activity, which leads to cell tumorogenicity, resistance to apoptosis, and differentiation. Gossypol is a natural polyphenolic compound isolated from cottonseed and has antiproliferative activity in a variety of cancer cell lines. (-)Gossypol is proved the potent component. Here we examined the growth inhibitory effect of (-)gossypol and its combination with imatinib in K562 cells. (-)Gossypol inhibited cell growth, promoted apoptosis, induced DeltaPsim loss, and cytochrome C release. Furthermore, (-)gossypol had a synergistic inhibitory effect on growth in K562 cells when combined with imatinib. Enhanced apoptosis, cytochrome C release, and caspase 3 cleavage as well as noticeable decrease of Mcl-1 and Bcl-XL were observed in K562 cells treated with both (-)gossypol and imatinib. These results suggest that (-)gossypol induced apoptosis in K562 cells through a mitochondria pathway and that the combination of imatinib and (-)gossypol might be an effective treatment for CML.     

Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways

A tyrosine kinase inhibitor, STI571, has been demonstrated to be effective for the treatment of chronic myelogenous leukemia (CML). STI571 inhibits tyrosine kinase activity of ABL and induces apoptosis of CML cells. However, drug resistance develops commonly in patients with blast phase CML, and has become a significant therapeutic problem. We examined the effects of aminopeptidase inhibitors on CML cell line (K562) and a STI571-resistant subline of K562. Ubenimex and the more potent aminopeptidase inhibitor, actinonin, inhibited proliferation of both K562 cells and STI571-resistant K562 cells and also induced their apoptosis in dose- and time-dependent manners. Ubenimex and actinonin induced the activation of caspase-3, and the induction of apoptosis was inhibited by pan-caspase inhibitor, indicating this apoptosis is caspase-dependent. We found that serine phosphorylation of both MAPK and glycogen synthase kinase-3β were suppressed by aminopeptidase inhibitors in parent K562 and STI571-resistant K562 cells. The expression level of cyclin D1 protein was also reduced by ubenimex and actinonin in both cell lines. These results indicated STI571-resistance does not confer the cross-resistance to aminopeptidase inhibitors in K562 cells and revealed the new findings of aminopeptidase inhibitor-induced intracellular signaling pathways.     

Selective inhibition of PDGFR by imatinib elicits the sustained activation of ERK and downstream receptor signaling in malignant glioma cells

Clinical studies using the tyrosine kinase inhibitor, imatinib mesylate (Gleevec?), in glioblastoma, have shown no major inhibition of tumor growth or extension of survival for patients, unlike those in chronic myeloid leukemia (CML) and gastrointestinal stromal tumors. The molecular mechanisms of action of imatinib in glioblastoma cells are still not well understood. In this study, we investigated the effects of imatinib on the platelet derived growth factor receptor (PDGFR) downstream signaling pathways as well as on other cellular functions in human glioblastoma cells. NIH3T3 fibroblast and K562 CML cells were used for comparison. Western blot analysis demonstrated that imatinib was more effective in inhibiting the activated rather than the quiescent forms of the target proteins. Furthermore, the imatinib treatment induced the sustained activation of extracellular signal-regulated kinase (ERK 1/2) signaling as well as components of other downstream signaling pathways, such as PI3K/Akt, STAT3 and p38MAPK. Prior stimulation of the malignant cells with exogenous PDGF-BB partially abrogated this activation. Further analysis indicated that the activation of ERK induced by the imatinib treatment was related to the S-phase re-entry of the cell cycle in one of the three glioma cells. Imatinib significantly inhibited cell migration but not cell growth. The combination treatment of imatinib with a MEK or PI3K inhibitor resulted in significant growth inhibition but did not inhibit cell migration beyond the inhibition achieved with the imatinib treatment alone. The treatment of glioma cells with small interfering RNA inhibiting PDGFRB, however, evoked enhanced Akt signaling. These results indicate that the imatinib treatment of malignant glioma does not result in significant inhibitory effects and should be used with caution.     

Inhibition of Abl tyrosine kinase enhances nerve growth factor-mediated signaling in Bcr-Abl transformed cells via the alteration of signaling complex and the receptor turnover

Receptor tyrosine kinase-mediated signaling is tightly regulated by a number of cytoplasmic signaling molecules. In this report, we show that Bcr-Abl transformed chronic myelogenous leukemia (CML) cell lines, K562 and Meg-01, express the receptor for nerve growth factor (NGF), TrkA, on the cell surface; however, the NGF-mediated signal is not particularly strong. Treatment with imatinib, a potent inhibitor of Bcr-Abl tyrosine kinase, downmodulates phosphorylation of downstream molecules. Upon stimulation with NGF, Erk and Akt are phosphorylated to a much greater degree in imatinib-treated cells than in untreated cells. Knockdown of expression of Bcr-Abl using small interfering RNA technique also enhanced NGF-mediated Akt phosphorylation, indicating that Bcr-Abl kinase modifies NGF signaling directly. Imatinib treatment also enhanced NGF signaling in rat adrenal pheochromocytoma cell line PC12 that expresses TrkA and c-Abl, suggesting that it is not only restoration of responsiveness to NGF after blocking oncoprotein activity, but also c-Abl tyrosine kinase per se may be a negative regulator of growth factor signaling. Furthermore, inhibition of Abl tyrosine kinase enhanced clearance of surface TrkA after NGF treatment and simultaneously enhanced NGF-mediated signaling, suggesting that as in neuronal cells 'signaling endosomes' are formed in hematopoietic cells. To examine the role of TrkA in CML cells, we studied cell growth or colony formation in the presence or absence of imatinib with or without NGF. We found that NGF treatment induces cell survival in imatinib-treated CML cell lines, as well as colony formation of primary CD34+ CML cells, strongly suggesting that NGF/TrkA signaling contributes to aberrant signaling in CML.     

Imatinib has the potential to exert its antileukemia effects by down-regulating hERG1 K+ channels in chronic myelogenous leukemia

Imatinib is a powerful protein tyrosine kinase (PTK) inhibitor that specifically targets BCR-ABL, KIT, and PDGFR kinases, has become the current first-line therapy for all newly diagnosed chronic myeloid leukemia (CML). Beside PTKs, PTK inhibitors alter the activity of a large number of voltage-dependent ion channels. hERG1 K(+) channels are highly expressed in leukemia cells and appear of exceptional importance in favoring leukemogenesis. The present study explored a possible regulatory effect of imatinib upon hERG1 K(+) channels as a means to uncover new molecular events involved in the antileukemic activity of this PTK inhibitor in CML. The results demonstrated that hERG1 was highly detected in K562 cells and primary CML cells, and down-regulated by imatinib at mRNA and protein levels. Furthermore, imatinib markedly reduced hERG currents in HEK293T-hERG cells, this effect was accompanied by inhibition of CML cell proliferation and apoptosis, as well as suppression of vascular endothelial growth factor (VEGF) secretion. Moreover, these antileukemia effects of imatinib were potentiated by E-4031, a specific hERG1 inhibitor. Together, these results provide evidence of a novel potential molecular mechanism of antileukemic activities by imatinib which, independent of targeting tyrosine kinase, highlight hERG1 K(+) channels as a therapeutic target for CML treatment.