Reduction of Raf Kinase Inhibitor Protein Expression by Bcr-Abl Contributes to Chronic Myelogenous Leukemia Proliferation

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells, and Ras activity enhances the oncogenic ability of Bcr-Abl. However, the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction, resulting in blocking the MAP kinase pathway. In the present study, we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl⁺ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status, resulting in inhibited proliferation of CML cells. Moreover, RKIP up-regulated cell cycle regulator FoxM1 expression, resulting in G₁ arrest via p27^Kip1 and p21^Cip1 accumulation. In colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte, colony-forming unit-granulocyte macrophage, and burst-forming unit erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions, and inhibited colony formation of Bcr-Abl⁺ progenitor cells, whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl⁺ progenitor cells. Thus, Bcr-Abl represses the expression of RKIP, continuously activates pERK1/2, and suppresses FoxM1 expression, resulting in proliferation of CML cells.     

A PDE Model for Imatinib-Treated Chronic Myelogenous Leukemia

We derive a model for describing the dynamics of imatinib-treated chronic myelogenous leukemia (CML). This model is a continuous extension of the agent-based CML model of Roeder et al. (Nat. Med. 12(10), 1181–1184, 2006) and of its recent formulation as a system of difference equations (Kim et al. in Bull. Math. Biol. 70(3), 728–744, 2008). The new model is formulated as a system of partial differential equations that describe various stages of differentiation and maturation of normal hematopoietic cells and of leukemic cells. An imatinib treatment is also incorporated into the model. The simulations of the new PDE model are shown to qualitatively agree with the results that were obtained with the discrete-time (difference equation and agent-based) models. At the same time, for a quantitative agreement, it is necessary to adjust the values of certain parameters, such as the rates of imatinib-induced inhibition and degradation.     

Hck和Lyn激酶与慢性髓细胞性白血病的关系

Src家族激酶（Sre—family kinase,SFK）是一组非受体型酪氨酸蛋白激酶,其家族成员Hck （hemopoietic cell kinase）和Lyn（v-yes-1 Yanaguchi sarcoma viral related oncogene homolog）激酶在慢性髓细胞性白血病（chronic myelogenous leukemia,CML）细胞中被BCR—ABL异常激活,并作用于多种细胞内信号转导分子,影响细胞的增殖、凋亡和迁移。深入研究SFK在CML发生和发展中的作用,对进一步认识CML发病机制及其靶向性治疗有着重要的实际意义。     

Strategic Treatment Interruptions During Imatinib Treatment of Chronic Myelogenous Leukemia

Although imatinib is an effective treatment for chronic myelogenous leukemia (CML), and nearly all patients treated with imatinib attain some form of remission, imatinib does not completely eliminate leukemia. Moreover, if the imatinib treatment is stopped, most patients eventually relapse (Cortes et al. in Clin. Cancer Res. 11:3425–3432, 2005). In Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008), the authors presented a mathematical model for the dynamics of CML under imatinib treatment that incorporates the anti-leukemia immune response. We use the mathematical model in Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008) to study and numerically simulate strategic treatment interruptions as a potential therapeutic strategy for CML patients. We present the results of numerous simulated treatment programs in which imatinib treatment is temporarily stopped to stimulate and leverage the anti-leukemia immune response to combat CML. The simulations presented in this paper imply that treatment programs that involve strategic treatment interruptions may prevent leukemia from relapsing and may prevent remission for significantly longer than continuous imatinib treatment. Moreover, in many cases, strategic treatment interruptions may completely eliminate leukemic cells from the body. Thus, strategic treatment interruptions may be a feasible clinical approach to enhancing the effects of imatinib treatment for CML. We study the effects of both the timing and the duration of the treatment interruption on the results of the treatment. We also present a sensitivity analysis of the results to the parameters in the mathematical model.     

Hematopoietic Stem Cells,Leukemic Stem Cells and Chronic Myelogenous Leukemia

Blood-related cancers, or leukemias, have been shown to arise from a rare subset of cells that escape normal regulation and drive the formation and growth of the tumor. The finding that these so-called cancer stem cells, or leukemic stem cells (LSC), can be purified away from the other cells in the tumor allows their precise analysis to identify candidate molecules and regulatory pathways that play a role in progression, maintenance, and spreading of leukemias. The analyses of the other, numerically dominant, cells in the tumor, while also interesting, do not directly interrogate these key properties of malignancies. Mouse models of human myeloproliferative disorder and acute myelogenous leukemia have highlighted the remarkable conservation of disease mechanisms between both species. They can now be used to identify the LSC for each type of human leukemia and understand how they escape normal regulation and become malignant. Given the clinical importance of LSC identification, the insights gained through these approaches will quickly translate into clinical applications and lead to improved treatments for human leukemias.     

The Mechanism of Action of Splenic Irradiation in Chronic Myelogenous Leukemia

The mechanism of action of splenic irradiation in the induction of a remission in chronic myelogenous leukemia was investigated in six patients using a leukocyte kinetic approach. The leukocytes were labelled in vitro with radioactive diisopropylfluorophosphate-32 and returned to the circulation. The effect of treatment on the rate of change of leukocyte specific activity was determined. The results suggest (1) that irradiation of the spleen damages granulopoietic cells as they cycle back and forth between the spleen, blood and other extravascular compartments; (2) that damage to exchangeable granulopoietic cells in transit through the irradiated spleen may explain the long remission often encountered after this form of therapy.     

Multiple Chromosome Abnormalities Following Bone Marrow Transplant for Chronic Myelogenous Leukemia

A 44-year-old female was diagnosed in the chronic phase of chronic myelogenous leukemia (CML) and was confirmed to be Philadelphia chromosome positive by a bone marrow cytogenetic study. No additional cytogenetic abnormalities were found. The patient's cell counts were initially well controlled with hydrox-yurea. She then received an unrelated 6 of 6 HLA matched allo-geneic bone marrow transplant (BMT) from a male donor. The patient underwent myeloablative therapy with thiotepa and five fractions of total body radiation prior to the transplant. About four weeks after transplantation, the patient developed biopsy-proven graft-versus-host disease of the skin and GI tract. A blood sample was drawn at that time for cytogenetic analysis. Among 34 analyzed cells, 22 were normal male donor cells. The remaining 12 cells did not have the t(9;22), but had numerous structural abnormalities. While many cells were missing an X chromosome, other abnormalities, including deletions, rearrangements, dicentrics, acentric fragments, rings and marker chromosomes were non-clonal. No clinical evidence of progression from CML chronic phase was found, suggesting that the non-clonal abnor-malities were therapy related.     

慢性髓细胞性白血病病人骨髓单个核细胞中差异表达基因的筛选

目的:筛选慢性髓细胞性白血病(CML)病人骨髓单个核细胞与正常人的差异表达基因,探讨CML的发病机制.方法:提取正常人和CML病人单个核细胞的RNA,逆转录成cDNA并用地高辛标记,应用全基因组表达谱基因芯片对差异表达基因进行研究,采用Jubilant病理/疾病分类法对CML相关差异表达基因进行分析.结果:共筛选出CM...     

Differences in structural elements of Bcr-Abl oncoprotein isoforms in Chronic Myelogenous Leukemia

in silico modeling, using Psipred and ExPASy servers was employed to determine the structural elements of Bcr-Abl oncoprotein(p210^BCR-ABL) isoforms, b2a2 and b3a2, expressed in Chronic Myelogenous Leukemia (CML). Both these proteins are tyrosinekinases having masses of 210-kDa and differing only by 25 amino acids coded by the b3 exonand an amino acidsubstitution(Glu903Asp). The secondary structure elements of the two proteins show differences in five α-helices and nine β-strands whichrelates to differences in the SH₃, SH₂, SH1 and DNA-binding domains. These differences can result in different roles played by thetwo isoforms in mediating signal transduction during the course of CML.     

Modeling Imatinib-Treated Chronic Myelogenous Leukemia: Reducing the Complexity of Agent-Based Models

We develop a model for describing the dynamics of imatinib-treated chronic myelogenous leukemia. Our model is based on replacing the recent agent-based model of Roeder et al. (Nat. Med. 12(10):1181–1184, 2006) by a system of deterministic difference equations. These difference equations describe the time-evolution of clusters of individual agents that are grouped by discretizing the state space. Hence, unlike standard agent-base models, the complexity of our model is independent of the number of agents, which allows to conduct simulation studies with a realistic number of cells. This approach also allows to directly evaluate the expected steady states of the system. The results of our numerical simulations show that our model replicates the averaged behavior of the original Roeder model with a significantly reduced computational cost. Our general approach can be used to simplify other similar agent-based models. In particular, due to the reduced computational complexity of our technique, one can use it to conduct sensitivity studies of the parameters in large agent-based systems.     

Isolation and Characterization of a Novel Nucleoside from the Urines of Chronic Myelogenous Leukemia Patients

Abstract

From 24 hour collections of urines of chronic myelogenous leukemia (CML) patients, a novel nucleoside was isolated. It was assigned the structure, 5′-deoxyinosine (I) on the basis of UV, NMR and mass spectrometry and by comparison of the spectral data and HPLC and TLC mobilities with those of the authentic sample. Another nucleoside, 5′-deoxy-5′-methylthioadenosine sulfoxide previously isolated from the urines of immunodeficient children was also found in the urine of a CML patient. Possible origin and significance of both of these nucleosides are discussed.     

Real-Time Analysis of Imatinib- and Dasatinib-Induced Effects on Chronic Myelogenous Leukemia Cell Interaction with Fibronectin

Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML), the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7) and of control BCR-ABL-negative leukemia cells (HEL, JURKAT) with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor) and dasatinib (dual ABL/SRC family kinase inhibitor), on cell binding to fibronectin is described. Both imatinib and low-dose (several nM) dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM) had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated) LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy.     

Studies on the Longevity,Sequestration and Release of the Leukocytes in Chronic Myelogenous Leukemia

The intravascular life-span of leukocytes labelled in vitro with radioactive di-isopropylfluorophosphate was studied in 12 patients with chronic myelogenous leukemia (CML). In relapse, leukocyte specific activity (LSA) disappeared slowly; in remission, LSA curves approached normal and only a small proportion of LSA disappeared slowly. The level of maturation of the leukocytes that persisted in the blood was investigated by a leukocyte fractionation technique which excluded immature myeloid cells from leukocyte samples. The influence of extracorpuscular factors upon the pattern of disappearance of LSA was investigated by means of cross-transfusion experiments, and LSA curves obtained with in vitro and in vivo labelling were compared. The results suggest that: (1) the intravascular life-span of the mature leukemic neutrophil is prolonged in relapse and in remission; (2) intrinsically abnormal leukocytes are sequestered in an extravascular pool(s) but recycling occurs; (3) extracorpuscular factors modify the LSA curves; (4) exchange of leukocytes between intravascular and extravascular pools may not occur in relapse; and (5) the intravascular and extravascular pools constitute a self-sustaining pool(s) not replenished from a non-miscible precursor pool.     

伊马替尼血药浓度监测指导慢性粒细胞白血病患者的研究

目的 观察是否可以通过对伊马替尼(Imatinib,IM)进行血药浓度监测提高疗效,减少药物不良反应。方法 选取2013~2018年就诊于我院的慢性粒细胞白血病(chronic myelogenous leukemia, CML)患者,分为试验组(药物监测组),对照组(常规经验治疗组)。对服药3个月、6个月、12个月、18个月,进行疗效及不良反应评估及比较。结果 共有51人入选此次临床试验。其中试验组35人,对照组16人。结果 服用伊马替尼400mg/d时,血药浓度568.00~3 989.66ng/ml,均数(标准差):1 716.46ng/ml(788.96);服用伊马替尼300mg/d时,血药浓度720.89~1 497.11ng/ml,均数(标准差):971.67ng/ml(204.02)。达到主要分子学反应(major molecular response, MMR)的伊马替尼血药浓度高于未达到稳态时的伊马替尼血药浓度。两组不良反应评级有统计学差异。试验组III级及以上不良反应发生率明显小于对照组。结论 伊马替尼的稳态血浆药物谷浓度存在较大个体差异,这种个体差异与疗效和不良反应存在相关性。通过治疗药物监测(therapeutic drug monitoring, TDM)可以在确保疗效的同时,减少伊马替尼在治疗慢性粒细胞白血病中的不良反应。结果尚需大样本临床试验进一步验证。伊马替尼药物代谢个体差异的原因需要大样本遗传药理学研究进一步探讨。     

Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, ³H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.     

BCR-ABL Gene Expression Is Required for Its Mutations in a Novel KCL-22 Cell Culture Model for Acquired Resistance of Chronic Myelogenous Leukemia

Acquired resistance through genetic mutations is a common phenomenon in several cancer therapies using molecularly targeted drugs, best exemplified by the BCR-ABL inhibitor imatinib in treating chronic myelogenous leukemia (CML). Overcoming acquired resistance is a daunting therapeutic challenge, and little is known about how these mutations evolve. To facilitate understanding the resistance mechanisms, we developed a novel culture model for CML acquired resistance in which the CML cell line KCL-22, following initial response to imatinib, develops resistant T315I BCR-ABL mutation. We demonstrate that the emergence of BCR-ABL mutations do not require pre-existing BCR-ABL mutations derived from the original patient as the subclones of KCL-22 cells can form various BCR-ABL mutations upon imatinib treatment. BCR-ABL mutation rates vary from cell clone to clone and passages, in contrast to the relatively stable mutation rate of the hypoxanthine-guanine phosphoribosyltransferase gene. Strikingly, development of BCR-ABL mutations depends on its gene expression because BCR-ABL knockdown completely blocks KCL-22 cell relapse on imatinib and acquisition of mutations. We further show that the endogenous BCR-ABL locus has significantly higher mutagenesis potential than the transduced randomly integrated BCR-ABL cDNA. Our study suggests important roles of BCR-ABL gene expression and its native chromosomal locus for acquisition of BCR-ABL mutations and provides a new tool for further studying resistance mechanisms.     

DNA甲基化通过锌指蛋白185调控慢性粒细胞白血病细胞的增殖

锌指蛋白185(ZNF185)属于LIM结构域蛋白,参与细胞的增殖和分化,在多种肿瘤细胞中具有抑癌基因的功能.ZNF185在正常人血液系统细胞中高表达,但目前对白血病细胞的作用未见研究.采用Western blot检测人外周血中性粒细胞、急性粒细胞白血病细胞系HL-60和慢性粒细胞白血病细胞系K562细胞中ZNF185的表达,发现ZNF185在HL-60和K562细胞中的表达水平显著低于外周血中性粒细胞.为了阐明ZNF185对慢性粒细胞白血病细胞增殖的影响,从人外周血中性粒细胞克隆ZNF185编码序列,转染K562细胞,MTT检测细胞增殖,发现过表达ZNF185显著抑制K562细胞的增殖.甲基化特异PCR分析表明:ZNF185启动子在HL-60和K562细胞中高甲基化,用5-氮杂-2′-脱氧胞苷处理K562细胞,促进ZNF185的表达,显著抑制细胞增殖.研究结果表明,ZNF185启动子高甲基化导致其在K562细胞中的表达降低和细胞增殖抑制作用减弱.可能是慢性粒细胞白血病发生或发展的原因之一.