移植性慢性髓系白血病裸鼠模型的初步建立

慢性髓系白血病（chronic myeloid leukemia,CML）是一种起源于造血干细胞的恶性克隆性疾病,CML干细胞被认为是导致疾病发生、发展并最终急变的根源,目前尚缺乏稳定的动物模型证明CML干细胞的存在。本研究旨在通过建立CML裸鼠模型,探讨人CML细胞在BABL/c裸小鼠体内的生物学行为,并使CML干细胞在裸鼠体内富集成为可能。对4至6周龄的BALB/c裸鼠进行切脾（splenectomy,S）,环磷酰胺腹腔注射（cytoxan intrap-eritoneal injection,C）及全身亚致死剂量照射（sublethal irradiation,I）等预处理（SCI）后,经尾静脉接种（5-8）×10^7个人CML慢性期患者单个核细胞。对4至6周龄的BALB/c裸鼠进行全身致死剂量照射（lethal irradiation）后,经尾静脉接种5×10^6同源裸鼠骨髓细胞和（5-8）×10^7个人CML慢性期患者单个核细胞。应用RT-PCR、塑胶包埋病理切片以及流式细胞术等检测裸鼠各脏器及骨髓中人CML细胞浸润情况,并比较两种建模方法的优劣。结果表明,CML细胞能浸润至经SCI预处理的裸鼠骨髓体内,但目前成功率还很低,仅为通过BABL/c裸鼠建立人CML动物模型的一个开端。而经致死剂量预处理裸鼠,CML细胞未能浸润至骨髓。结论：人CML慢性期白血病细胞能在经SCI预处理的裸鼠体内形成白血病,为建立CML动物模型寻找到新的方向。     

BCR/ABL转基因动物模型研究进展及应用

转基因动物模型为研究慢性粒系白血病的发病机理与治疗提供了一个良好平台。利用不同的启动子或四环素调控系统已建立了一系列的BCR/ABL转基因动物模型,其中相当一部分动物模型能很好的模拟人的CML特征,并在CML的发病机理与治疗的研究中广泛应用。本文就目前BCR/ABL转基因动物模型的研究进展、优缺点及应用作一综述。     

BCR/ABL转基因动物模型研究进展及应用

转基因动物模型为研究慢性粒系白血病的发病机理与治疗提供了一个良好平台.利用不同的启动子或四环素调控系统已建立了一系列的BCR/ ABL转基因动物模型,其中相当一部分动物模型能很好的模拟人的CML特征,并在CML的发病机理与治疗的研究中广泛应用.本文就目前BCR/ABL转基因动物模型的研究进展、优缺点及应用作一综述.     

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.     

Methotrexate exacerbates tumor progression in a murine model of chronic myeloid leukemia

Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). 32Dp210 tumor cells were found to be sensitive to methotrexate (MTX) in vitro, whereas cells expressing the tyrosine-22 DHFR gene were protected from MTX at up to micromolar concentrations. MTX administered at low dose (2 mg/kg/day) did not protect normal C3H-He/J mice from 32Dp210 tumor infused intravenously, with drug toxicity limiting the administration of higher doses. Animals engrafted with transgenic tyrosine-22 DHFR marrow were protected from greater MTX doses (up to 6 mg/kg/day). However, the increased doses of MTX afforded by drug-resistance gene expression surprisingly resulted in decreased survival of the transplanted tumor-bearing animals, with increased levels of tumor detected in peripheral blood. This apparent exacerbation of tumor progression by MTX was not observed in DHFR transgenic mice in which all cells and tissues contain the drug-resistance gene. This suggests that increased tumor progression in MTX-administered animals resulted from MTX sensitivity of a nonhematopoietic host component, thus allowing tumor expansion. We conclude that MTX exacerbates tumor progression in the 32Dp210 model of CML, and that based on this model alternate DHFR inhibitors combined with drug-resistant DHFR or other chemotherapeutic agent/drug-resistance gene combinations may be required for the application of drug-resistance gene expression to the treatment of CML.     

Bcr/Abl融合基因阳性、Flk1+CD34-慢性粒细胞性白血病干细胞的分离纯化及其生物学特性的研究

目的分离慢性粒细胞白血病(ChronicMyelogenousLeukemia,CML)患者骨髓中表达BCR/ABL肿瘤基因的细胞亚群,并研究其生物学特性。方法淋巴细胞分离液分离CML患者骨髓单个核细胞,免疫磁珠分选CD45-、GlyA-及CD34-细胞,极限稀释法获得单克隆细胞,流式细胞术鉴定其免疫表型,体外定向诱导分化检测其干细胞特性,并检测其Bcr/Abl融合基因的表达情况。结果CML患者骨髓源单克隆扩增细胞不表达造血细胞和内皮细胞的特征性表型,但Bcr/Abl融合基因阳性;诱导后的细胞能同时向造血细胞及血管内皮细胞分化。结论Bcr/Abl融合基因的产生至少发生在比造血干细胞更为早期的血液血管干细胞水平上,即CML至少起源于血液血管干细胞。     

慢性粒细胞白血病慢性期与急变期基因表达差异的研究

目的　研究慢性粒细胞白血病 (CML)不同临床阶段的基因表达差异 ,为阐明CML演变的可能机制提供依据。方法　应用DNA芯片技术 ,对CML慢性期与急变期骨髓单个核细胞的基因表达差异进行了检测。结果　在检测的 1176个基因中 ,有 6 8个基因显示急变期较慢性期明显上调 ,其中转录因子 16个 (2 3.5 % ) ,细胞表面抗原 15个 (2 2 .1% ) ,细胞调节蛋白 13个 (19.1% ) ,其它 2 4个(35 3% )。在差异表达的基因中 ,有 17个基因仅在急变期表达 ,而在慢性期不表达 ,11个 (16 .2 % )与G蛋白基因有关。结论　CML慢性期与急变期骨髓单个核细胞基因表达谱存在显著差异 ,基因高表达是CML急变期的特征之一。G蛋白相关基因在CML急变过程中可能起着重要作用。     

CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells

Chronic myelogenous leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, and this is associated with the cell migration defects in CML. We proposed that tyrosine kinase inhibitors, imatinib or INNO-406, may restore CXCR4 expression and cause the migration of CML cells to bone marrow microenvironment niches, which in turn results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib, INNO-406, or IFN-alpha increased CXCR4 expression and migration. This increase in CXCR4 levels on CML progenitor cells was likewise found in samples from CML patients treated with imatinib or IFN-alpha. Imatinib induced G0-G1 cell cycle block in CML cells, which was further enhanced in a mesenchymal stem cell (MSC) coculture system. MSC coculture protected KBM-5 cells from imatinib-induced cell death. These antiapoptotic effects were abrogated by the CXCR4 antagonist AMD3465 or by inhibitor of integrin-linked kinase QLT0267. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to bone marrow stroma, causing the G0-G1 cell cycle arrest and hence ensuring the survival of quiescent CML progenitor cells.     

Growth Arrest of BCR-ABL Positive Cells with a Sequence-Specific Polyamide-Chlorambucil Conjugate

Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model.     

吲哚美辛诱导慢性髓细胞白血病细胞凋亡的研究

目的　观察和确定吲哚美辛 (IN)对慢性髓细胞白血病 (CML)细胞凋亡的诱导作用 ,并部分揭示其分子机制 ,以期筛选一种新的抗白血病药物。方法　以CML细胞株K5 6 2及来自 6例初治Ph CML患者骨髓原代培养细胞为研究材料 ,在不同时间点 ,用不同浓度的IN进行干预 ,利用细胞形态学、流式细胞仪、DNA电泳、逆转录 聚合酶链反应 (RT PCR)等技术 ,确定IN对CML细胞凋亡和增殖的影响。结果　①IN能诱导K5 6 2细胞和原代培养的CML细胞凋亡 ,并有抑制白血病细胞增殖的作用 ;②IN对足叶乙甙诱导K5 6 2细胞凋亡具有协同作用 ;③IN能下调K5 6 2细胞中bcl 2mRNA水平表达 ,对baxmRNA水平无明显影响。结论　IN能诱导CML细胞凋亡和抑制白血病细胞增殖 ,IN对足叶乙甙的抗白血病效应具有增敏作用。bcl 2基因表达下调可能为IN诱导CML细胞凋亡的重要机制之一。     

Differentiation and maturation of thy-1 negative bone marrow cells. I. Effects of the thymus and radioresistant helper functions on the maturation of precursor cells specific for heterologous erythrocytes

The effects of adult thymectomy (ATx) and preimmunization on the differentiation of cells responsible for delayed footpad reaction (DFR), plaque forming cells (PFC) and cell mediated lympholysis (CML) were examined in lethally irradiated and thy-1 negative bone marrow cell reconstituted C57BL/6 recipients. ATx reduced the degrees of immune responses in irradiated and reconstituted mice. When the recipients had been preimmunized, all of DFR, PFC and CML became detectable even in irradiated and reconstituted ATx mice. Preimmunization also evoked early maturation of precursor cells for CML. Therefore, it was suggested that radioresistant helper effects, presumably in the presence of antigens, could promote the differentiation and maturation of T cell precursors in bone marrow in the absence of the thymus. We also demonstrated differences in restoration periods of such responses after lethal irradiation and reconstitution. One or two weeks following irradiation and reconstitution, DFR was first detectable. On the other hand, the generation of PFC was detected later than 2 weeks after bone marrow cell reconstitution, and it took over 4 weeks for thy-1 negative bone marrow cells to raise CML. T cells responsible for DFR may have lower dependency on the thymus than those for PFC and CML.     

Relationship between HLA-DR expression by normal myeloid progenitor cells and inhibition of colony growth by prostaglandin E. Implications for prostaglandin E resistance in chronic myeloid leukemia.

The expression of HLA-DR antigens by normal myeloid progenitor cells (CFU-GM) has been linked to inhibition of colony growth by prostaglandin E (PGE), while resistance to the inhibitory effects of PGE in chronic myeloid leukemia (CML) has been attributed to a lower fraction of HLA-DR+ CFU-GM in this disease. However, we have previously shown that virtually all CFU-GM in normal bone marrow (NBM) as well as CML peripheral blood express HLA-DR antigens, which raises the possibility that these surface molecules may not be the sole determinants of a progenitor cell's sensitivity to PGE. In order to evaluate the relationship between HLA-DR expression and prostaglandin inhibition, we partially purified NBM progenitor cells using fluorescence-activated cell sorting to prepare cell fractions with high and low HLA-DR antigen density. Normal progenitor cells with high DR density tended to form monocyte colonies in agar culture, whereas the low DR density fraction was enriched for granulocyte colony-forming cells. Inhibition by PGE was greatest in the high DR+ fraction and was largely restricted to monocyte progenitor cells. Inhibition of CFU-GM by PGE was less in CML than in NBM, but this decreased inhibition correlated with a significantly lower number of monocyte-CFU in CML. These data suggest that high HLA-DR antigen density may select for normal progenitor cells that are committed to monocyte differentiation and are, therefore, more likely to be inhibited by PGE. The relative deficit of monocyte progenitor cells in CML may partially explain the phenomenon of PGE resistance in this disease.     

Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells

Chronic myelogenous leukemia (CML) results from transformation of a long-term hematopoietic stem cell (LTHSC) by expression of the BCR-ABL fusion gene. However, BCR-ABL–expressing LTHSCs are heterogeneous in their capacity as leukemic stem cells (LSCs). Although discrepancies in proliferative, self-renewal, and differentiation properties of normal LTHSCs are being increasingly recognized, the mechanisms underlying heterogeneity of leukemic LTHSCs are poorly understood. Using a CML mouse model, we identified gene expression differences between leukemic and nonleukemic LTHSCs. Expression of the thrombopoietin (THPO) receptor MPL was elevated in leukemic LTHSC populations. Compared with LTHSCs with low MPL expression, LTHSCs with high MPL expression showed enhanced JAK/STAT signaling and proliferation in response to THPO in vitro and increased leukemogenic capacity in vivo. Although both G₀ and S phase subpopulations were increased in LTHSCs with high MPL expression, LSC capacity was restricted to quiescent cells. Inhibition of MPL expression in CML LTHSCs reduced THPO-induced JAK/STAT signaling and leukemogenic potential. These same phenotypes were also present in LTHSCs from patients with CML, and patient LTHSCs with high MPL expression had reduced sensitivity to BCR-ABL tyrosine kinase inhibitor treatment but increased sensitivity to JAK inhibitors. Together, our studies identify MPL expression levels as a key determinant of heterogeneous leukemia-initiating capacity and drug sensitivity of CML LTHSCs and suggest that high MPL–expressing CML stem cells are potential targets for therapy.     

BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is induced by the oncogenic BCR-ABL1 tyrosine kinase and can be effectively treated for many years with tyrosine kinase inhibitors (TKIs). However, unless CML patients receive life-long TKI treatment, leukemia will eventually recur; this is attributed to the failure of TKI treatment to eradicate leukemia-initiating cells (LICs). Recent work demonstrated that FoxO factors are critical for maintenance of CML-initiating cells; however, the mechanism of FoxO-dependent leukemia initiation remained elusive. Here, we identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. BCL6 represses Arf and p53 in CML cells and is required for colony formation and initiation of leukemia. Importantly, peptide inhibition of BCL6 in human CML cells compromises colony formation and leukemia initiation in transplant recipients and selectively eradicates CD34(+) CD38(-) LICs in patient-derived CML samples. These findings suggest that pharmacological inhibition of BCL6 may represent a novel strategy to eradicate LICs in CML. Clinical validation of this concept could limit the duration of TKI treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation.     

Tyrosine kinase inhibitors: a cure for chronic myeloid leukemia?

Chronic myeloid leukemia (CML), a malignant transformation of hematopoietic cells, accounts for one-fifth of all leukemias and will be diagnosed in 4,400 individuals in the United States this year. CML has three phases: chronic, accelerated, and blastic. Interferon, hydroxyurea, busulfan, and bone marrow and stem cell transplantation are used to treat CML, but individuals who are in the accelerated phases or blast crisis usually respond poorly to treatment. A new tyrosine kinase inhibitor, STI 571, is being evaluated in clinical trials. STI 571 is highly bioavailable in oral form and produced minimal toxicities in phase I studies. Further research is needed to determine the rate of response and survival data, but STI 571 holds great promise in the treatment of CML.     

慢性髓系白血病首发血小板显著增多

本研究探讨以血小板显著增多首发的慢性髓系白血病（CML）临床、细胞遗传学及分子生物学特征。应用骨髓细胞涂片、骨髓活检观察细胞形态学改变;RT-PCR检测bcr／abl融合基因;常规染色体核型分析及FISH检测细胞遗传学变化。结果发现：以血小板显著增多为首发表现的CML是一组具有独特临床和生物学特点的疾病,骨髓细胞涂片和骨髓活检表明,骨髓增生活跃,以巨核系异常增生为主,血小板大片戍堆,可见圆形核小巨核细胞,中等度白细胞增多,经细胞遗传学和分子生物学检测均证实存在有Ph染色体和（或）表达bcr／abl融合基因,对此类患者应该早期进行积极治疗,甚至进行分子生物学水平的干预;而原发性血小板增多症（ET）患者则不宜过多地使用化疗药物,否则反而诱致白血病的发生。结论：对血小板明显增多的患者应及时进行Ph染色体及bcr／abl融合基因表达水平的检测．这对于ET及CML的诊断和鉴别诊断极为重要,以避免误诊、误治。     

MIP-1α/CCL3-mediated maintenance of leukemia-initiating cells in the initiation process of chronic myeloid leukemia

In the initiation process of chronic myeloid leukemia (CML), a small number of transformed leukemia-initiating cells (LICs) coexist with a large number of normal hematopoietic cells, gradually increasing thereafter and eventually predominating in the hematopoietic space. However, the interaction between LICs and normal hematopoietic cells at the early phase has not been clearly delineated because of the lack of a suitable experimental model. In this study, we succeeded in causing a marked leukocytosis resembling CML from restricted foci of LICs in the normal hematopoietic system by direct transplantation of BCR-ABL gene–transduced LICs into the bone marrow (BM) cavity of nonirradiated mice. Herein, we observed that BCR-ABL⁺lineage⁻c-kit⁻ immature leukemia cells produced high levels of an inflammatory chemokine, MIP-1α/CCL3, which promoted the development of CML. Conversely, ablation of the CCL3 gene in LICs dramatically inhibited the development of CML and concomitantly reduced recurrence after the cessation of a short-term tyrosine kinase inhibitor treatment. Finally, normal hematopoietic stem/progenitor cells can directly impede the maintenance of LICs in BM in the absence of CCL3 signal.Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm (MPN) resulting from the neoplastic transformation of hematopoietic stem cells (HSCs). CML undergoes a triphasic process, a chronic phase, an accelerated phase, and a terminal blast crisis (Lahaye et al., 2005). More than 90% of CML cases are associated with the presence of the Philadelphia chromosome. This chromosome arises from a reciprocal translocation between chromosomes 9 and 22 and forms the breakpoint cluster region with a constitutively activated tyrosine kinase, BCR-ABL fusion protein (Ren, 2005; Melo and Barnes, 2007). This protein is a pathogenic protein in CML (Sawyers, 1999), and maintenance of BCR-ABL–expressing leukemia-initiating cells (LICs) in the BM is crucial for initiating the chronic phase of CML (Koschmieder et al., 2005).Zhang et al. (2012) observed several characteristic changes in the BM microenvironment of mice developing CML-like myeloproliferative disease, such as BM hypercellularity and myeloid cell infiltration into spleen (SP). Moreover, they detected an altered chemokine/cytokine expression pattern in the BM, including down-regulation of SDF-1/CXCL12 and up-regulation of MIP-1α/CCL3, MIP-1β/CCL4, IL-1α, IL-1β, and TNF. They further obtained similar observations on human CML patients. Based on these observations, they proposed that altered chemokine/cytokine expression in BM may contribute to the preferential proliferation of LICs in the BM microenvironment, to displace the normal hematopoietic cells, although they did not clarify the molecular and cellular mechanisms in more detail.Chemokines are produced by a wide variety of hematological and stromal cells and exhibit diverse activities on various types of BM-derived cells. Evidence is accumulating to indicate that a CC chemokine, MIP-1α/CCL3, has direct inhibitory activities on normal hematopoietic stem/progenitor cell (HSPC) growth (Graham et al., 1990; Dunlop et al., 1992; Maze et al., 1992; Broxmeyer et al., 1993). Induction of BCR-ABL expression in vivo can cause the aberrant expression of CCL3 in the BM (Zhang et al., 2012). Moreover, CCL3-mediated signal can regulate the in vitro proliferation of normal HSPCs and LICs in distinct ways (Eaves et al., 1993; Chasty et al., 1995), depending on the kinase activity of Abl protein (Wark et al., 1998). Furthermore, IFN-α–induced CCL3 production by BM-derived stromal cells enhanced β1 integrin–dependent adhesion of LICs to the stromal cells to restore normal hematopoiesis in CML (Bhatia et al., 1995). These observations suggest that CCL3 can contribute to the interaction between LICs and normal hematopoietic system in the initiation process of CML development (Zhang et al., 2012), but its precise roles remain unclear because of the lack of a suitable experimental model.Murine CML-like myeloproliferative disease can be induced by transferring human-derived BCR-ABL oncogene–transduced primitive BM cells to a lethally irradiated host (Pear et al., 1998; Li et al., 1999). This experimental model has been widely used to examine the in vivo leukemogenic role of the BCR-ABL oncogene in CML development. However, in this model, lethal irradiation completely breaks down the normal hematopoietic system to enable intravenously injected BCR-ABL⁺ leukemic cells to home to the BM to grow and develop CML. Thus, this model is not helpful in elucidating the role of the BM microenvironment in CML development. Furthermore, lethal irradiation induced a temporal leukopenia, a condition that can have a profound impact on CML pathology by compensatory overproduction of various growth factors (Singh et al., 2012). Hence, to observe the course of CML development under the steady-state, an inducible BCR-ABL transgenic mouse, which can express the BCR-ABL gene under the control of a Tet-regulated 3′ enhancer of the murine stem cell leukemia gene, was established (Koschmieder et al., 2005). This well-designed transgenic model enables the study of the function of LICs in the condition closely resembling that in CML patients. However, in this experimental model, it is not easy to selectively tag leukemia cells with mutated BCR-ABL gene for the examination of leukemia cell trafficking. Moreover, it is laborious to introduce a gene mutation into either leukemia cells or normal hematopoietic cells.To circumvent these problems, we initially attempted to establish an experimental CML model under nonirradiated conditions. We transduced c-kit⁺lineage⁻Sca-1⁺ (KLS⁺) HSPCs with BCR-ABL oncogene using retroviral vector and injected the resultant cells directly into the BM cavity in nonirradiated immune-deficient nude mice. In the early phase of this model, only <500 BCR-ABL⁺KLS⁺ LICs are presumed to coexist with a large number of normal residual hematopoietic cells. However, this procedure succeeded in the development of a CML-like disease with a marked leukocytosis and splenomegaly in nonirradiated and BM-preserved host. Moreover, LICs moved to the contralateral site of BM while expanding in the injected site of BM. Thus, this novel model is quite helpful to clarify the interaction between normal hematopoietic system and leukemic cells, particularly in the early phase of CML development, and the trafficking of LICs to other hematopoietic tissues. By using this model, we have obtained definitive evidence to indicate an indispensable role of leukemia cell–derived CCL3 in the maintenance of LICs in BM for subsequent CML development.     

Chronic myeloid leukemia: mechanisms of blastic transformation

The BCR-ABL1 oncoprotein transforms pluripotent HSCs and initiates chronic myeloid leukemia (CML). Patients with early phase (also known as chronic phase [CP]) disease usually respond to treatment with ABL tyrosine kinase inhibitors (TKIs), although some patients who respond initially later become resistant. In most patients, TKIs reduce the leukemia cell load substantially, but the cells from which the leukemia cells are derived during CP (so-called leukemia stem cells [LSCs]) are intrinsically insensitive to TKIs and survive long term. LSCs or their progeny can acquire additional genetic and/or epigenetic changes that cause the leukemia to transform from CP to a more advanced phase, which has been subclassified as either accelerated phase or blastic phase disease. The latter responds poorly to treatment and is usually fatal. Here, we discuss what is known about the molecular mechanisms leading to blastic transformation of CML and propose some novel therapeutic approaches.