Imatinib (STI571) inhibits DNA repair in human leukemia oncogenic tyrosine kinase-expressing cells

BCR/ABL oncogene, as a result of chromosome aberration t(9;22), is the pathogenic principle of almost 95% of human chronic myeloid leukemia (CML). Imatinib (STI571) is a highly selective inhibitor of BCR/ABL oncogenic tyrosine kinase used in leukemia treatment. It has been suggested that BCR/ABL may contribute to the resistance of leukemic cells to drug and radiation through stimulation of DNA repair in these cells. To evaluate further the influence of STI571 on DNA repair we studied the efficacy of this process in BCR/ABL-positive and -negative cells using single cell electrophoresis (comet assay). In our experiments, K562 human chronic myeloid leukemia cells expressing BCR/ABL and CCRF-CEM human acute lymphoblastic leukemia cells without BCR/ABL expression were employed. The cells were exposed for 1 h at 37 degrees C to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 5 microM, mitomycin C (MMC) at 50 microM or to gamma-radiation at 15 Gy with or without a 24 h preincubation at 1 microM of STI571. The MTT cells survival after 4 days of culture showed that STI571 enhanced the cytotoxity of the examined compounds in the K562 line. Further it was found, that the inhibitor decreased the efficacy of DNA repair challenged by each agent, but only in the K562 expressing BCR/ABL. Due to the variety of DNA damage induced by the employed agents in this study we can speculate, that BCR/ABL may stimulate multiple pathways of DNA repair. These results extend our previous studies performed on BCR/ABL-transformed mouse cells onto human cells. It is shown that BCR/ABL stimulated DNA repair in human leukemia cells. In conclusion we report that STI571 was found to inhibit DNA repair and abrogate BCR/ABL-positive human leukemia cells therapeutic resistance.     

Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 microM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.     

Reciprocal t(9;22) ABL/BCR Fusion Proteins: Leukemogenic Potential and Effects on B Cell Commitment

Background

t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph⁺) determine formation of different fusion genes that are associated with either Ph⁺ acute lymphatic leukemia (Ph⁺ ALL) or chronic myeloid leukemia (CML). The “minor” breakpoint in Ph⁺ ALL encodes p185^BCR/ABL from der22 and p96^ABL/BCR from der9. The “major” breakpoint in CML encodes p210^BCR/ABL and p40^ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96^ABL/BCR and p40^ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL.

Methodology

All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR.

Principal Findings

Both p96^ABL/BCR and p40^ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96^ABL/BCR and to a minor extent p40^ABL/BCR forced the B-cell commitment of SL-cells and UCBC.

Conclusions/Significance

Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.     

Identification of a melanoma antigen, PRAME, as a BCR/ABL-inducible gene

In order to elucidate molecular events in BCR/ABL-induced transformation, we adopted a polymerase chain reaction (PCR)-based technique of differential display and compared mRNA expression in human factor-dependent cells, TF-1, with that in factor-independent cells, ID-1, which were established from TF-1 cells by transfection of BCR/ABL. Cloning and sequencing of a gene which was upregulated in ID-1 cells revealed that the gene was identical to a melanoma antigen, PRAME. Our present study demonstrated that PRAME was markedly expressed in primary leukemic cells with chronic myeloid leukemia (CML) in blastic crisis and Philadelphia (Ph)+-acute lymphoblastic leukemia (ALL), in which BCR/ABL played an important role as a pathogenic gene. Moreover, comparison of PRAME expression among CD34+ cells with CML in blastic, accelerated, and chronic phases revealed a higher expression in CML in advanced phases. Thus PRAME was considered to be a good candidate for a marker of Ph+-leukemic blast cells as well as a new target antigen of leukemic blast cells that cytotoxic T cells can recognize.     

BCR/ABL downregulates DNA-PK<Subscript>CS</Subscript>-dependent and upregulates backup non-homologous end joining in leukemic cells

Non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are the main mechanisms involved in the processing of DNA double strand breaks (DSBs) in humans. We showed previously that the oncogenic tyrosine kinase BCR/ABL stimulated DSBs repair by HRR. To evaluate the role of BCR/ABL in DSBs repair by NHEJ we examined the ability of leukemic BCR/ABL-expressing cell line BV173 to repair DNA damage induced by two DNA topoisomerase II inhibitors: etoposide and sobuzoxane. DNA lesions induced by sobuzoxane are repaired by a NHEJ pathway which is dependent on the catalytic subunit of protein kinase dependent on DNA (DNA-PK_CS; D-NHEJ), whereas damage evoked by etoposide are repaired by two distinct NHEJ pathways, dependent on or independent of DNA-PK_CS (backup NHEJ, B-NHEJ). Cells incubated with STI571, a highly specific inhibitor of BCR/ABL, displayed resistance to these agents associated with an accelerated kinetics of DSBs repair, as measured by the neutral comet assay and pulsed field gel electrophoresis. However, in a functional NHEJ assay, cells preincubated with STI571 repaired DSBs induced by a restriction enzyme with a lower efficacy than without the preincubation and addition of wortmannin, a specific inhibitor of DNA-PK_CS, did not change efficacy of the NHEJ reaction. We suggest that BCR/ABL switch on B-NHEJ which is more error-prone then D-NHEJ and in such manner contribute to the increase of the genomic instability of leukemic cells.     

Determination of BCR/ABL translocation in radiation-associated acute myeloid leukemia patients by fluorescence in situ hybridization

We report the results of BCR/ABL translocation analysis on interphase leukemic cells of 33 acute myeloid leukemia (AML) patients by fluorescence in situ hybridization. Of these, there were 13 persons exposed to ionizing radiation due to the Chernobyl accident with radiation-associated AML and 20 patients with spontaneous disease. BCR/ABL translocation which was detected in 4 and I case respectively may play an important role in radiation-induced leukemigenesis.     

Global effects of BCR/ABL and TEL/PDGFRbeta expression on the proteome and phosphoproteome: identification of the Rho pathway as a target of BCR/ABL

Many leukemic oncogenes form as a consequence of gene fusions or mutation that result in the activation or overexpression of a tyrosine kinase. To identify commonalities and differences in the action of two such kinases, breakpoint cluster region (BCR)/ABL and TEL/PDGFRbeta, two-dimensional gel electrophoresis was employed to characterize their effects on the proteome. While both oncogenes affected expression of specific proteins, few common effects were observed. A number of proteins whose expression is altered by BCR/ABL, including gelsolin and stathmin, are related to cytoskeletal function whereas no such changes were seen in TEL/PDGFRbeta-transfected cells. Treatment of cells with the kinase inhibitor STI571 for 4-h reversed changes in expression of some of these cytoskeletal proteins. Correspondingly, BCR/ABL-transfected cells were less responsive to chemotactic and chemokinetic stimuli than non-transfected cells and TEL/PDGFRbeta-transfected Ba/F3 cells. Decreased motile response was reversed by a 16-h treatment with STI571. A phosphoprotein-specific gel stain was used to identify TEL/PDGFRbeta and BCR/ABL-mediated changes in the phosphoproteome. These included changes on Crkl, Ras-GAP-binding protein 1, and for BCR/ABL, cytoskeletal proteins such as tubulin, and Nedd5. Decreased phosphorylation of Rho-GTPase dissociation inhibitor (Rho GDI) was also observed in BCR/ABL-transfected cells. This results in the activation of the Rho pathway, and treatment of cells with Y27632, an inhibitor of Rho kinase, inhibited DNA synthesis in BCR/ABL-transfected Ba/F3 cells but not TEL/PDGFRbeta-expressing cells. Expression of a dominant-negative RhoA inhibited both DNA synthesis and transwell migration, demonstrating the significance of this pathway in BCR/ABL-mediated transformation.     

Alternative forms of the BCR-ABL oncogene have quantitatively different potencies for stimulation of immature lymphoid cells.

The Philadelphia chromosome (t9:22;q34:q11) is found in more than 90% of patients with chronic myelogenous leukemia, in 10 to 20% of patients with acute lymphocytic leukemia, and in 1 to 2% of patients with acute myelogenous leukemia. Alternative chimeric oncogenes are formed by splicing different sets of BCR gene exons on chromosome 22 across the translocation breakpoint to a common set of ABL oncogene sequences on chromosome 9. This results in an 8.7-kilobase mRNA that encodes the P210 BCR-ABL gene product commonly found in patients with chronic myelogenous leukemia or a 7.0-kilobase mRNA that produces the P185 BCR-ABL gene product found in most Philadelphia chromosome-positive patients with acute lymphocytic leukemia. To compare the efficiency of growth stimulation by these two proteins, we derived cDNA clones for each with identical 5' and 3' untranslated regions and expressed them from retrovirus vectors. Matched stocks were compared for potency to transform immature B-lymphoid lineage precursors. The growth-stimulating effects of P185 for this cell type were found to be significantly greater than those of P210. Structural changes in BCR may regulate the effectiveness of the ABL tyrosine kinase function, as monitored by lymphocyte growth response. Changes in mitogenic potency may help to explain the more acute leukemic presentation usually associated with expression of the P185 BCR-ABL oncogene.     

BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP(-/-) mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.     

Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance

The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.     

Selective induction of apoptosis in Philadelphia chromosome-positive chronic myelogenous leukemia cells by an inhibitor of BCR - ABL tyrosine kinase, CGP 57148

The BCR - ABL tyrosine kinase has been implicated as the cause of Philadelphia chromosome (Ph1)-positive leukemias. We report herein that CGP 57148, a selective inhibitor of the ABL tyrosine kinase, caused apoptosis specifically in bcr - abl-positive chronic myelogenous leukemia (CML) cells, K562 and KYO-1. Upon treatment with CGP 57148, CRKL, a specific substrate for BCR - ABL that propagates signals via phosphatidylinositol-3' kinase (PI3K), was dephosphorylated, indicating inhibition of BCR - ABL tyrosine kinase at the cellular level. Likewise, MAPK/ERK, a downstream mediator of Ras, was also dephosphorylated. Caspase activation and cleavage of retinoblastoma protein (pRB) accompanied the development of CGP 57148-induced apoptosis. Inhibition of caspase suppressed apoptosis and the cleavage of pRB, and in turn arrested cells in the G1 phase. These results indicate that CGP 57148 shows apoptogenic and anti-proliferative effects on bcr - abl-positive cells by blocking BCR - ABL-initiated signaling pathways.     

Enhancement of imatinib-induced apoptosis of BCR/ABL-expressing cells by nutlin-3 through synergistic activation of the mitochondrial apoptotic pathway

The BCR/ABL tyrosine kinase inhibitor imatinib is highly effective for treatment of chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). However, relapses with emerging imatinib-resistance mutations in the BCR/ABL kinase domain pose a significant problem. Here, we demonstrate that nutlin-3, an inhibitor of Mdm2, inhibits proliferation and induces apoptosis more effectively in BCR/ABL-driven Ton.B210 cells than in those driven by IL-3. Moreover, nutlin-3 drastically enhanced imatinib-induced apoptosis in a p53-dependent manner in various BCR/ABL-expressing cells, which included primary leukemic cells from patients with CML blast crisis or Ph+ ALL and cells expressing the imatinib-resistant E255K BCR/ABL mutant. Nutlin-3 and imatinib synergistically induced Bax activation, mitochondrial membrane depolarization, and caspase-3 cleavage leading to caspase-dependent apoptosis, which was inhibited by overexpression of Bcl-XL. Imatinib did not significantly affect the nutlin-3-induced expression of p53 but abrogated that of p21. Furthermore, activation of Bax as well as caspase-3 induced by combined treatment with imatinib and nutlin-3 was observed preferentially in cells expressing p21 at reduced levels. The present study indicates that combined treatment with nutlin-3 and imatinib activates p53 without inducing p21 and synergistically activates Bax-mediated intrinsic mitochondrial pathway to induce apoptosis in BCR/ABL-expressing cells.     

The two major imatinib resistance mutations E255K and T315I enhance the activity of BCR/ABL fusion kinase

The resistance to the tyrosine kinase inhibitor imatinib in BCR/ABL-positive leukemias is mostly associated with mutations in the kinase domain of BCR/ABL, which include the most prevalent mutations E255K and T315I. Intriguingly, these mutations have also been identified in some patients before imatinib treatment. Here we examined the effects of these mutations on the kinase activity of a BCR/ABL kinase domain construct that also contained the SH3 and SH2 domains. When expressed in COS7 cells, the BCR/ABL construct with either E255K or T315I exhibited not only the resistance to imatinib but also the increase in activity to induce autophosphorylation as well as tyrosine phosphorylation of various cellular proteins, which included STAT5. The mutant kinases also showed increased activities in in vitro kinase assays. These results raise a possibility that the major imatinib resistance mutations E255K and T315I may confer the growth advantage on leukemic cells to expand in the absence of selective pressure from imatinib treatment.     

STI571 reduces NER activity in BCR/ABL-expressing cells

Nucleotide-excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) photoproducts. We investigated the influence of an anticancer drug, STI571, on the efficacy of NER in removing UV-induced DNA damage. STI571 is used mostly in the treatment of chronic myeloid leukemia and inhibits activity of the BCR/ABL oncogenic tyrosine kinase, which is a hallmark of this disease. NER activity was examined in the BCR/ABL-expressing cell lines K562 and BV173 of myeloid and lymphoid origin, respectively, as well as in CCRF-CEM cells, which do not express BCR/ABL. A murine myeloid parental 32D cell line and its counterpart transfected with the BCR/ABL gene were also tested. NER activity was assessed in the cell extracts by use of an UV-irradiated plasmid as a substrate and by a modified single-cell gel electrophoresis (comet) assay on UV-treated nucleoids. Additionally, quantitative PCR was performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. Results obtained from these experiments indicate that STI571 decreases the efficacy of NER in leukemic cells expressing BCR/ABL. Therefore, STI571 may overcome the drug resistance associated with increased DNA repair in BCR/ABL-positive leukemias.     

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.     

在BCR/ABL基因组上敲入mRNA不稳定元件可逆转K562细胞恶性转化

融合基因 BCR/ABL在慢性粒细胞白血病的恶性转化过程中起着主导作用 .针对融合基因的 3′端构建了一个定点基因打靶质粒 ,p F2 .neo.abl(1 - 4) ,将一段可引发核内 RNA降解的元件 ,URE,定点整合到融合基因 poly(A)位点的上游 .打靶质粒经脂质体转染 K562细胞后 ,在 96孔板上进行 40 0 μg/ml G41 8筛选 ,neor克隆进一步在 2 4孔板上扩增 .以特异性引物经基因组 PCR及Southern印迹分析对阳性克隆进行检测 .研究发现阳性克隆在 96孔板内 3周其增殖状况良好 ,但在 2 4孔板内扩增一周后迅速发生死亡现象 .观察单个阳性克隆在正常培养液增殖情况 ,发现 5d后其细胞周期被完全阻抑 .研究结果说明 ,在转录后期 m RNA水平控制 BCR/ABL融合基因的表达可以抑制慢性粒细胞白血病的恶性转化 .