Altered intracellular signaling by imatinib increases the anti‐cancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia cells

Tyrosine kinase inhibitors (TKI), including imatinib (IM), improve the outcome of CML therapy. However, TKI treatment is long‐term and can induce resistance to TKI, which often leads to a poor clinical outcome in CML patients. Here, we examined the effect of continuous IM exposure on intracellular energy metabolism in K562 cells, a human Philadelphia chromosome‐positive CML cell line, and its subsequent sensitivity to anti‐cancer agents. Contrary to our expectations, we found that continuous IM exposure increased sensitivity to TKI. Cancer energy metabolism, characterized by abnormal glycolysis, is linked to cancer cell survival. Interestingly, glycolytic activity was suppressed by continuous exposure to IM, and autophagy increased to maintain cell viability by compensating for glycolytic suppression. Notably, increased sensitivity to TKI was not caused by glycolytic inhibition but by altered intracellular signaling, causing glycolytic suppression and increased autophagy, as evidenced by suppression of p70 S6 kinase 1 (S6K1) and activation of AMP‐activated protein kinase (AMPK). Using another human CML cell line (KCL22 cells) and BCR/ABL+ Ba/F3 cells (mimicking Philadelphia chromosome‐positive CML cells) confirmed that suppressing S6K1 and activating AMPK increased sensitivity to TKI. Furthermore, suppressing S6K1 and activating AMPK had a synergistic anti‐cancer effect by inhibiting autophagy in the presence of TKI. The present study provides new insight into the importance of signaling pathways that affect cellular energy metabolism, and suggests that co‐treatment with agents that disrupt energy metabolic signaling (using S6K1 suppressors and AMPK activators) plus blockade of autophagy may be strategies for TKI‐based CML therapy.     

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)     

Effect of imatinib mesylate on chronic myelogenous leukemia hematopoietic progenitor cells

Treatment of chronic myelogenous leukemia (CML) has been greatly enhanced by the development of Imatinib mesylate, a specific inhibitor of the BCR/ABL tyrosine kinase. While it is clear that imatinib effectively targets BCR/ABL positive hematopoietic cells, studies examining its effect on primitive hematopoietic progenitors are much more limited. As CML arises in a primitive hematopoietic progenitor cell, it is especially important to understand the effect of imatinib on these cells. Here we review studies investigating the effect of imatinib on the proliferation and viability of primitive and committed hematopoietic progenitors in CML. We describe evidence that BCR/ABL positive progenitors may persist in patients responding to imatinib and discuss problems of resistance to imatinib. Finally we discuss studies evaluating new approaches to overcome resistance of CML progenitor cells to imatinib.     

The tyrosine kinase inhibitors imatinib and AG957 reverse multidrug resistance in a chronic myelogenous leukemia cell line

The K562 cell line derived from a chronic myelogenous leukemia (CML) patient exhibits ATP-dependent exclusion of the multidrug resistance (MDR)-type drugs. The protein tyrosine kinases inhibitors, imatinib mesylate and AG957 allowed for increased doxorubicin and calcein-AM accumulation in these cells. Maximal modulation was achieved at 3 and 10 microM imatinib and AG957, respectively. This imatinib concentration is comparable to the plasma steady state levels observed in patients. Although the increase in cellular accumulation followed a time course similar to apoptotic manifestations induced by these drugs, the two phenomena seem independent. There was no correlation between the levels of MDR reversal and apoptosis in clones derived from the K562 cell line. Moreover, whereas protein kinase inhibitors induced apoptosis in only a fraction of the cells, the MDR reversal occurred in all of them. Inhibition of apoptosis by a non-specific inhibitor of caspases was not associated with MDR reversal. The consequence of these findings is that combination of tyrosine kinase inhibitors with antileukemic drugs is likely to have the added beneficial effect of allowing MDR-type drugs better access to cells.     

甲磺酸伊马替尼治疗慢性粒细胞白血病的临床观察

目的:总结甲磺酸伊马替尼(IM)治疗Ph阳性慢性粒细胞白血病(CML)患者的临床观察体会。方法:收集110例Ph阳性CML慢性期(CP)患者、6例加速期(AP)患者和4例急变期(BC)患者分别口服IM 400、600或800 mg/d。通过血液学、细胞遗传学和分子遗传学指标来判断疗效。结果:CP患者完全血液学反应(CHR)率、主要细胞遗传学反应(MCyR)率和完全细胞遗传学反应(CCyR)率分别为98.2%、90.9%和80.9%;AP分别为66.7%、33.3%和16.7%,P值分别为0.002、0.000和0.000。其中CP患者中肝肾功能不全的患者需减少IM剂量。27例经干扰素治疗失败的CP患者IM治疗仍有效。第1年高随访(1次/月)CP患者CCyR达81.9%,非高随访则为63.6%,P=0.005 2。服药前肾功能不全和肝功能不全较脏器功能正常的CP患者服药6个月内发生3~4级血液学毒副反应概率增高,服药6个月后有所减少。结论:IM对CP患者包括干扰素治疗失败的有较高疗效,对AP和BC患者有一定近期疗效。肝肾功能不全的患者易出现血液学毒副反应,需要药物剂量调整。     

Imatinib mesylate resistance through BCR-ABL independence in chronic myelogenous leukemia

Imatinib mesylate (IM) binds to the BCR-ABL protein, inhibiting its kinase activity and effectively controlling diseases driven by this kinase. IM resistance has been associated with kinase mutations or increased BCR-ABL expression. However, disease progression may be mediated by other mechanisms that render tumor cells independent of BCR-ABL. To demonstrate this potential, IM-resistant cells were found in chronic myelogenous leukemia patients with continuous BCR-ABL gene expression but undetectable BCR-ABL protein expression. These cells were unresponsive to IM and acquired BCR-ABL-independent signaling characteristics. IM resistance in some patients may be mediated through loss of kinase target dependence.     

P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate.

Imatinib (Glivec), STI571) is an intracellular acting drug that demonstrates high activity against BCR-ABL-positive chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). However, many patients, especially with advanced disease, develop drug resistance. Here, we show by a novel high-performance liquid chromatography-based method that intracellular levels of imatinib decrease in P-glycoprotein (Pgp)-positive leukemic cells. In a model of K562 cells with gradually increasing Pgp expression, a Pgp-dependent decline of intracellular imatinib levels was observed. Decreased imatinib levels were associated with a retained phosphorylation pattern of the Bcr-Abl target Crkl and loss of effect of imatinib on cellular proliferation and apoptosis. The modulation of Pgp by cyclosporin A (CSA) readily restored imatinib cytotoxicity in these cells. Finally, we provide first data showing a biological effect of Pgp modulation in the imatinib treatment of a patient with BCR-ABL-positive ALL. MDR1 overexpression must therefore be considered as an important clinical mechanism in the diversity of resistance development to imatinib treatment.     

Sphingosine kinase-1 mediates BCR/ABL-induced upregulation of Mcl-1 in chronic myeloid leukemia cells

The signaling mechanisms responsible for BCR/ABL-induced regulation of Mcl-1 expression in chronic myelogenous leukemia (CML) cells remain unclear. In this study, we show that BCR/ABL could upregulate sphingosine kinase-1 (SPK1) expression via multiple signal pathways, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and Janus kinase 2 (JAK2), leading to increase cellular SPK1 activity in CML cells. Retrovirus-mediated overexpression of bcr-abl gene in NIH-3T3, Ba/F3 and HL-60 cells results in upregulation and increased cellular activity of SPK1, whereas treatment of CML cells with specific inhibitors of the BCR/ABL, PI3K, MAPK and JAK2 pathways decreases BCR/ABL-induced SPK1 expression and cellular activity. BCR/ABL also induces upregulation of Mcl-1 expression in CML cells. Inhibition of SPK1 by adenovirus-mediated transfer of small interfering RNA or N,N-dimethylsphingosine reduced expression of Mcl-1 in CML cells. Our data indicated that BCR/ABL induces SPK1 expression and increases its cellular activity, leading to upregulation of Mcl-1 in CML cells. SPK1 silencing enhances the STI571-induced apoptosis of CML cell lines. It is suggested that SPK1 may be a potential therapeutic target in CML.     

慢性粒细胞白血病伊马替尼与尼洛替尼代谢组学差异性研究

目的 应用代谢组学技术研究服用伊马替尼与尼洛替尼的慢性粒细胞白血病(CML)患者血浆代谢差异,探讨其对CML患者血浆内源性代谢物干预作用的差异性.方法 选取2015年3月至2015年6月在苏州大学附属第一医院就诊的获得最佳疗效的CML慢性期患者34例,按服用药物分为伊马替尼组(19例)和尼洛替尼组(15例).采用回顾性病例对照研究方法,应用气相色谱-质谱(GC-MS)技术,对两组患者血浆样本中内源性小分子代谢物进行全面检测,采集代谢物指纹图谱,利用多变量数据分析及t检验筛选差异代谢物,并通过MetPA分析平台探讨差异代谢物相关的代谢通路.结果 应用GC-MS代谢组学方法可以区分伊马替尼与尼洛替尼.结合变量权重重要性(VIP)值及t检验筛选到差异代谢物,伊马替尼组中苯丙氨酸、吲哚丙酸、肉豆蔻酸、半乳糖、葡萄糖、软脂酸、色氨酸、硬脂酸、邻苯二甲酸及邻苯二甲酸酯含量较低,其VIP值分别为1.633(t=6.099,P<0.001)、1.338(t=4.367,P<0.001)、1.557(t=6.716,P<0.001)、1.154(t=3.056,P=0.005)、1.941(t=2.196,P=0.035)、1.207(t=3.785,P=0.001)、1.625(t=6.398,P<0.001)、1.555(t=6.553,P<0.001)、1.633(t=7.679,P<0.001)、1.633(t=8.374,P<0.001).主要涉及色氨酸代谢、苯丙氨酸代谢及半乳糖代谢通路.结论 应用代谢组学方法找到了CML患者伊马替尼与尼洛替尼的血浆内源性差异代谢物,这些差异可能与药物成分配比及药物体内代谢差异有关.     

CLP induces apoptosis in human leukemia K562 cells through Ca regulating extracellular-related protein kinase ERK activation

The cyclic lipopeptide (CLP) has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in CLP-induced apoptosis are still uncharacterized in human leukemic K562 cells. The current study investigated the molecular mechanism of action of CLP, purified from Bacillus natto T-2. CLP-induced a sustained increase in concentration of intracellular Ca²⁺. This increase in [Ca²⁺]i was associated with CLP-induced cell apoptosis and ERK phosphorylation. CLP-induced cell apoptosis was reversed by PD98059 (an inhibitor of ERK), but not by SB203580 (an inhibitor of p38) and SP200125 (an inhibitor of JNK), suggesting that the action of CLP on K562 cells was via ERK, but not via p38 and JNK. On the other hand, pretreatment with Bapta-AM, a well-known calcium chelator, partially blocked CLP-induced apoptosis, indicating that the elevation of [Ca²⁺]i may play an important role in the apoptosis. Collectively, in K562 cells, CLP-induced an increase in [Ca²⁺]i which evoked ERK phosphorylation. This ERK phosphorylation subsequently activated Bax, cytochrome c and caspase-3 leading to apoptosis.     

p21 Confers resistance to imatinib in human chronic myeloid leukemia cells

Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21^Cip1, initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21^Cip1 expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21^Cip1 in the response to imatinib in CML.     

Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a neoplasm of myeloid progenitor cells expressing Bcr-Abl fusion protein. However, some patients with CML are less likely to respond to imatinib, the inhibitor of Bcr-Abl kinase. Recent studies showed that mTOR pathway can increase responses to imatinib. The analysis of mTOR pathway in CML may provide new insights into possible targets of novel therapies. Therefore, we examined the expression of mTOR pathway molecules in bone marrow cells from CML patients and effect of rapamycin on K562 cells in?vitro. Western blot analysis showed the visibly higher phosphorylation of mTOR (70.6%), 4E-BP1 (76.5%) and p70S6K (73.5%) in bone marrow cells from CML patients. Moreover, treatment of CML cell line (K562) with rapamycin resulted in a decrease of phosphorylation of mTOR, 4E-BP1 and p70S6K. In?vitro, the cell viability in groups with rapamycin treatment displayed a significant decrease in a dose-dependent manner by MTT. The data presented an increase of G0/G1 phase cells and decrease of S phase cells after rapamycin treatment, and the decreased expression of cyclinD1, higher expression of p21 at mRNA level was also detected in K562 with rapamycin. Treatment with 20 nmol/l or more rapamycin could increase apoptotic cells, decrease expression of bcl-2 and activate caspase-3. In conclusion, the mTOR pathway might be involved in chronic myelogenous leukemia. Inhibition of mTOR pathway could interfer with cell proliferation and increase cell apoptosis in K562 cells. It suggested that mTOR might be an important therapeutic target for myelogenous leukemia.     

丝裂原活化蛋白激酶信号转导系统对Vp-16诱导分化作用的影响

目的:探讨丝裂原活化蛋白激酶(mitogen-acti-vated protein kinases,MAPKs)信号转导系统对依托泊苷(Vp-16)诱导K562细胞分化作用的影响。方法:采用四甲基偶氮唑盐(MTT)法测定细胞增殖活性;流式细胞仪解析细胞周期;硝基四氮唑蓝(NBT)还原实验检测细胞向单核/巨噬系统分化。结果:0·1~0·8μg/mL的Vp-16抑制K562细胞增殖,引起细胞G2/M期阻滞,诱导细胞向单核/巨噬系统分化;细胞外信号调节激酶(extracellular signal-regulated kinases,ERK)抑制剂PD98059降低Vp-16的诱导分化作用,P<0·05;p38丝裂原活化蛋白激酶(p38mitogen-activated protein kina-ses,p38MAPK)抑制剂SB203580增强Vp-16的作用,P<0·05;而C-JUN氨基末端激酶(c-jun N-terminal ki-nases,JNK)抑制剂SP600125对Vp-16的诱导分化作用无明显影响,P>0·05。结论:在Vp-16诱导K562细胞向单核/巨噬系统分化过程中,ERK正向,p38MAPK负向调节Vp-16的诱导分化作用。