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。     

Galangin increases the cytotoxic activity of imatinib mesylate in imatinib-sensitive and imatinib-resistant Bcr-Abl expressing leukemia cells

Resistance to imatinib mesylate is an emergent problem in the treatment of Bcr-Abl expressing myelogenous leukemias and additional therapeutic strategies are required. We observed that galangin, a non-toxic, naturally occurring flavonoid was effective as anti-proliferative, and apoptotic agent in Bcr-Abl expressing K562 and KCL22 cells and in imatinib mesylate resistant K562-R and KCL22-R cells. Galangin induced an arrest of cells in G0-G1phase of cell cycle and a decrease in pRb, cdk4, cdk1, cycline B levels; moreover, it was able to induce a monocytic differentiation of leukemic Bcr-Abl+ cells. Of note, galangin caused a decrease in Bcl-2 levels and markedly increased the apoptotic activity of imatinib both in sensitive or imatinib-resistant Bcr-Abl+ cell lines. In contrast, flavonoids unable to modify the Bcl-2 intracellular levels, such as fisetin and chrysin, did not increase the apoptotic effect of imatinib. These data suggest that galangin is an interesting candidate for a combination therapy in the treatment of imatinib-resistant leukemias.     

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)     

Distinct biological impact of dephosphorylation vs. downregulation of p210Bcr-Abl: implications for imatinib mesylate response and resistance

Imatinib mesylate suppresses phosphorylation of its kinase target, Bcr-Abl. We hypothesized that loss of p210Bcr-Abl (the kinase target) may lead to imatinib mesylate resistance. We studied K562 cells [chronic myelogenous leukemia (CML) blast crisis line] and MO7E/MBA-1 cells (with MBA-1 cells representing MO7E cells stably transfected with BCR-ABL). Imatinib mesylate resistance developed when p210Bcr-Abl expression was abolished. Furthermore, K562 cells were significantly more growth suppressed after imatinib mesylate exposure than after downregulation of Bcr-Abl expression. Signaling pathways which were functional in the absence of Bcr-Abl expression (NF-kappaB and mitogen-activated protein kinase activation or the growth factor pathway) were disrupted when p210Bcr-Abl was present but dephosphorylated, suggesting that an intact, but enzymatically inactive Bcr-Abl, may interfere with critical growth/signaling pathways. Downregulation of p210Bcr-Abl may be a mechanism by which imatinib mesylate resistance emerges. Samples from three of 15 patients with imatinib mesylate-resistant CML blast crisis had undetectable levels of p210Bcr-Abl. We conclude that retention of a dephosphorylated p210Bcr-Abl has a biologic impact distinct from that of downregulation/loss of p210Bcr-Abl and, in a subset of patients, loss of the target of the kinase inhibitor may lead to imatinib mesylate resistance.     

Valproate enhances imatinib-induced growth arrest and apoptosis in chronic myeloid leukemia cells

BACKGROUND: The objective of this study was to evaluate the ability of the clinically available histone deacetylase (HDAC) inhibitor valproate to enhance the cytotoxicity of the Bcr-Abl inhibitor imatinib in imatinib-resistant cell lines. METHODS: Interactions between imatinib, and valproate have been examined in imatinib-sensitive and -resistant chronic myeloid leukemia (CML)cell lines (K562, KCL-22, CML-T1) and in bone marrow mononuclear cells (MNCs) derived from imatinib-resistant CML patients. RESULTS: In imatinib-sensitive cell lines, cotreatment with imatinib 0.5 muM and valproate 5 microM for 48 hours potently enhanced imatinib-induced growth arrest and apoptosis. In resistant cell lines and in primary MNCs derived from imatinib-rsistant patients, valproate restored sensitivity to the cytotoxic effects of imatinib. Coexposure of cells to valproate and imatinib was associated with repression of several genes involved in Bcr-Abl transformation. In particular, the combination valproate-imatinib downregulated the expression of Bcr-Abl and the antiapoptotic protein Bcl-2, which is particularly overexpressed in imatinib-resistant clones. CONCLUSIONS: Data from this study suggested that administration of the clinically available HDAC inhibitor valproate may be a powerful strategy to enhance cytotoxic effects of imatinib in those patient resistant to imatinib or in which complete cytogenetic remission has been not reached.     

N-Benzyladriamycin-14-valerate (AD 198) cytotoxicty circumvents Bcr-Abl anti-apoptotic signaling in human leukemia cells and also potentiates imatinib cytotoxicity

Bcr-Abl activity in chronic myelogenous leukemia (CML) results in dysregulated cell proliferation and resistance against multiple cytotoxic agents due to the constitutive activation of proliferative signaling pathways. Currently, the most effective treatment of CML is the inhibition of Bcr-Abl activity by imatinib mesylate (Gleevec). Imatinib efficacy is limited by development of resistance through either expression of Bcr-Abl variants that bind imatinib less avidly, increased expression of Bcr-Abl, or expression of multidrug transport proteins. N-Benzyladriamycin-14-valerate (AD 198) is a novel antitumor PKC activating agent that triggers rapid apoptosis through PKC-delta activation and mitochondrial depolarization in a manner that is unaffected by Bcl-2 expression. We demonstrate that Bcr-Abl expression does not confer resistance to AD 198. Further, AD 198 rapidly induces Erk1/2 and STAT5 phosphorylation prior to cytochrome c release from mitochondria, indicating that proliferative pathways are active even as drug-treated cells undergo apoptosis. At sub-cytotoxic doses, AD 198 and its cellular metabolite, N-benzyladriamycin (AD 288) sensitize CML cells to imatinib through a supra-additive reduction in the level of Bcr-Abl protein expression. These results suggest that AD 198 is an effective treatment for CML both in combination with imatinib and alone against imatinib-resistant CML cells.     

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.     

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.     

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.     

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.     

Janus kinase 2: a critical target in chronic myelogenous leukemia

The Bcr-Abl tyrosine kinase is the causative factor in most chronic myelogenous leukemia (CML) patients. We have shown that Bcr-Abl is associated with a cluster of signaling proteins, including Janus kinase (Jak) 2, growth factor receptor binding protein 2-associated binder (Gab) 2, Akt, and glycogen synthase kinase (GSK)-3beta. Treatment of CML cell lines and mouse Bcr-Abl+ 32D cells with either Jak2 short interfering RNA or Jak2 kinase inhibitor AG490 inhibited pTyr Gab2 and pSer Akt formation, inhibited the activation of nuclear factor-kappaB, and caused the activation of GSK-3beta, leading to the reduction of c-Myc. Importantly, BaF3 cells expressing T315I and E255K imatinib-resistant mutants of Bcr-Abl underwent apoptosis on exposure to AG490 yet were resistant to imatinib. Similar to wild-type Bcr-Abl+ cells, inhibition of Jak2 by Ag490 treatment resulted in decrease of pSer Akt and c-Myc in imatinib-resistant cells. These results identify Jak2 as a potentially important therapeutic target for CML.     

Therapy options in imatinib failures

Chronic myelogenous leukemia (CML) is defined by the presence of the constitutively active tyrosine kinase breakpoint cluster region/Abelson (Bcr-Abl), which activates numerous signal transduction pathways leading to uncontrolled cell proliferation. The development of the Bcr-Abl-targeted imatinib represents a paradigm shift in the treatment of CML, because treatment with imatinib resulted in significantly better patient outcome, response rates, and overall survival compared with previous standards. Despite this advance, not all patients benefit from imatinib because of resistance and intolerance. Resistance to imatinib can develop from a number of mechanisms that can be defined as Bcr-Abl-dependent (e.g., most commonly resulting from point mutations in the Abl kinase domain) and Bcr-Abl-independent mechanisms (including the constitutive activation of downstream signaling molecules, e.g., Src family kinases), which could result in the activation of the pathway regardless of Bcr-Abl inhibition. Clearly, new treatment approaches are required for patients resistant to or intolerant of imatinib, which can be dose escalated in patients who demonstrate resistance. This does not result in long-term responses. Hematopoietic stem cell transplantation is limited by the availability of matched donors and the potential for morbidity. Dasatinib, a dual Bcr-Abl/Src kinase inhibitor, has shown efficacy against all imatinib-resistant Bcr-Abl mutations except for T315I. A large trial program showed that dasatinib is effective in patients previously exposed to imatinib and has a manageable safety profile in all phases of CML and Philadelphia chromosome-positive acute lymphoblastic leukemia, resulting in its approval. Nilotinib, an analogue of imatinib, also has demonstrated activity in a similar patient population. These agents and less clinically advanced strategies are discussed in this review.     

Re-Expression of Bone Marrow Proteoglycan-2 by 5-Azacytidine is associated with STAT3 Inactivation and Sensitivity Response to Imatinib in Resistant CML Cells

Background: Epigenetic silencing of tumor suppressor genes (TSG) is involved in development andprogression of cancers. Re-expression of TSG is inversely proportionate with STAT3 signaling pathways.Demethylation of DNA by 5-Azacytidine (5-Aza) results in re-expression of silenced TSG. Forced expression ofPRG2 by 5-Aza induced apoptosis in cancer cells. Imatinib is a tyrosine kinase inhibitor that potently inhibits BCR/ABL tyrosine kinase resulting in hematological remission in CML patients. However, majority of CML patients treatedwith imatinib would develop resistance under prolonged therapy. Methods: CML cells resistant to imatinib weretreated with 5-Aza and cytotoxicity of imatinib and apoptosis were determined by MTS and annexin-V, respectively.Gene expression analysis was detected by real time-PCR, STATs activity examined using Western blot and methylationstatus of PRG2 was determined by pyrosequencing analysis. Result: Expression of PRG2 was significantly higher inK562-R+5-Aza cells compared to K562 and K562-R (p=0.001). Methylation of PRG2 gene was significantly decreasedin K562-R+5-Aza cells compared to other cells (p=0.021). STAT3 was inactivated in K562-R+5-Aza cells which showedhigher sensitivity to imatinib. Conclusion: PRG2 gene is a TSG and its overexpression might induce sensitivity toimatinib. However, further studies are required to evaluate the negative regulations of PRG2 on STAT3 signaling.     

Cotreatment with vorinostat enhances activity of MK-0457 (VX-680) against acute and chronic myelogenous leukemia cells

PURPOSE: We determined the effects of vorinostat (suberoylanalide hydroxamic acid) and/or MK-0457 (VX-680), an Aurora kinase inhibitor on the cultured human (HL-60, OCI-AML3, and K562) and primary acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and mutant forms of Bcr-Abl. EXPERIMENTAL DESIGN: Following exposure to MK-0457 and/or vorinostat, apoptosis, loss of viability, as well as activity and levels of Aurora kinase and Bcr-Abl proteins were determined. RESULTS: Treatment with MK-0457 decreased the phosphorylation of Aurora kinase substrates including serine (S)10 on histone H3 and survivin, and led to aberrant mitosis, DNA endoreduplication as well as apoptosis of the cultured human acute leukemia HL-60, OCI-AML3, and K562 cells. Combined treatment with vorinostat and MK-0457 resulted in greater attenuation of Aurora and Bcr-Abl (in K562) kinase activity and levels as well as synergistically induced apoptosis of OCI-AML3, HL-60, and K562 cells. MK-0457 plus vorinostat also induced synergistic apoptosis of BaF3 cells with ectopic overexpression of wild-type or mutant Bcr-Abl. Finally, cotreatment with MK-0457 and vorinostat induced more loss of viability of primary AML and imatinib-refractory CML than treatment with either agent alone, but exhibited minimal toxicity to normal CD34+ progenitor cells. CONCLUSIONS: Combined in vitro treatment with MK-0457 and vorinostat is highly active against cultured and primary leukemia cells. These findings merit in vivo testing of the combination against human AML and CML cells, especially against imatinib mesylate-resistant Bcr-AblT315I-expressing CML Cells.     

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.