BCR/ABL-specific CD8<Superscript>+</Superscript> T cells can be detected from CML patients,but are only expanded from healthy donors

The BCR/ABL p210 fusion protein has long been considered an ideal target antigen for the development of immunotherapeutic strategies in chronic myeloid leukaemia (CML) due to its central role in malignant transformation and to its unique novel amino acid sequence solely expressed in leukaemia cells. However, the feasibility to expand BCR-ABL-specific T cells remains still controversial. Using BCR/ABL peptide/MHC tetramers, significantly higher frequencies of tetramer positive cells were detected in the peripheral blood of HLA-A*0301 (mean 0.38%) and HLA-B*0801 (mean 0.28%) CML patients than in healthy donors (P = 0.0025 and 0.0026, respectively). However, following stimulation with autologous peptide-pulsed DCs, BCR/ABL-specific T cells were only expanded from some healthy donors, suggesting that CML patients may have a specific immune deficit with respect to the BCR/ABL antigen. Professor A. I. Dodi passed away recently. This paper is an original contribution from the meeting which took place 28–29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. A. I. “Tony” Dodi (29 January 2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

Quantitative detection of BCR–ABL fusion gene and its application in monitoring chronic myeloid leukemia treatment

The BCR–ABL fusion gene in chromosome translocation, t (9; 22), and its product, p210BCR/ABL oncogenic tyrosine kinase, is the underlying molecular mechanism that leads to the development of CML. Quantitative detection of BCR–ABL fusion gene has become a reliable approach to diagnose and monitor CML. The aim of this study was to evaluate a Roche t (9; 22) kit in CML diagnosis, monitoring treatment responses, and identification of relapse. Using BCR–ABL fusion gene-expressing K562 cells, a series of standard samples were prepared and used to establish a curve for the calculation of BCR–ABL fusion gene expression in patient samples. Our results indicate that PCR detection system with aforementioned kit has good reproducibility. In addition, the relative concentration of BCR–ABL measured by PCR was in agreement with the patient’s response to the Imatinib treatment and bone marrow morphology remission. Furthermore, we found that the relative concentration of BCR–ABL fusion gene increased 1–3 months before CML relapse was clinically and cytogenetically diagnosed, suggesting that the PCR-based BCR–ABL fusion gene detection with t (9; 22) kit is able to diagnose the recurrence of CML at least 1 month earlier than the classic cytogenetic analysis. In conclusion, detection of BCR–ABL fusion gene expression in CML using Roche t (9; 22) kit has great clinical value in the primary diagnosis, monitoring treatment responses, and identification of relapse in CML patients.     

Arrangement of chromosome 11 and 22 territories,EWSR1 and FLI1 genes,and other genetic elements of these chromosomes in human lymphocytes and Ewing sarcoma cells

Standard and repeated fluorescence in situ hybridization and high-resolution cytometry were used to study topographical parameters of chromosome 11 and 22 territories, EWSR1 and FLI1 genes, and other genetic elements of these chromosomes in human lymphocytes and Ewing sarcoma cells. HSA 11 and its elements (BCL1, FLI1, centromere) were found, on average, more peripherally in comparison with HSA 22 and investigated elements (BCR, EWSR1, centromere). After the elimination of fluctuations of chromosome territories in nuclear volume, it was found that genetic elements in most cases adhered to their territories. The investigated genetic elements of HSA 11 were found close to each other relative to the large molecular lengths among them. This finding indicates a higher degree of chromatin condensation of at least a part of HSA 11 compared with HSA 22. In general, there is no correlation between the physical and molecular distance of two loci of the same chromosome territory. The topographical parameters of the EWSR1 and FLI1 genes do not differ substantially for G(0)-lymphocytes, stimulated lymphocytes and Ewing sarcoma cells. The fusion genes pertaining to both derivative chromosomes 11 and 22 in Ewing sarcoma cell nuclei are shifted to the midway position between the native EWSR1 and FLI1 genes. Comparing results obtained for the EWSR1/FLI1 and ABL1/BCR genes in samples of patients suffering from Ewing sarcoma or chronic myelogenous leukaemia, it can be concluded that the mean positions of the fusion genes are determined by the final structure of the chimeric chromosomes and do not depend on the location of the translocation event.     

Oncogenic activation of c-ABL by mutation within its last exon.

The c-ABL proto-oncogene is a predominantly nuclear localized tyrosine kinase. A random mutagenesis scheme was used to isolate c-ABL mutants whose expression produced a transformed phenotype in rodent fibroblast cells. An in-frame deletion within the central region of the last exon was identified in one ABL mutant. The mechanism of c-ABL oncogenic activation by mutation within the last exon differs both functionally and structurally from those of v-ABL and BCR/ABL. This class of ABL mutants shows increased tyrosine phosphorylation of cellular proteins in vivo but low levels of autophosphorylation. Last-exon ABL mutants are distinguished from v-ABL or BCR/ABL by their inability to transform primary bone marrow cells or support the growth of transformed pre-B cells. These findings define a new mechanism of oncogenic activation for the ABL kinase through mutations in the last exon which do not require amino-terminal deletions or mutations within the src homology regions.     

In vitro inhibition of murine hematopoietic progenitors and stromal cells by vinorelbine

Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine bone marrow. The in vitro growth of colony-forming units–granulocyte/macrophage (CFU-GM), burst forming units–erythroid (BFU-E) and colony-forming units–mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 μg/ml) suppressed all of the different progenitor cells by 100%. A comparison of the dose–response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing doses of VRB. The appearence of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity was seen in cultures exposed to 0.05 and 0.075 μg/ml; and a marked loss at the dose of 0.1 μg/ml. Our results show that VRB has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected at a similar dose (0.025 μg/ml), suggesting that the stroma is more resistant to this drug. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.     

Deletion of ABL/BCR on der(9) associated with severe basophilia

Chronic basophilic leukemia is a rare form in chronic myeloid leukemia patients. Only limited number of reports are available. Herein, we describe a patient who presented with fatigue, weight loss, leucocytosis, prominent basophilia, and mild eosinophilia. On biopsy, bone marrow was hypercellular with marked basophils. The immunophenotype showed abnormal expression of CD7, which is suggestive of basophilic maturation. Chromosomal analysis from GTG-banded metaphases revealed Ph positivity, and fluorescence in situ hybridization (FISH) with BCR/ABL dual color, dual fusion probe showed single fusion on the der(22) chromosome and ABL/BCR fusion was deleted on the der(9) chromosome. The deletion (ABL/BCR) on der(9) may be associated with basophilia which may be also indicative of the transformation of CML to acute myeloid leukemia.     

TGF-beta1-induced PI3K/Akt/NF-kappaB/MMP9 signalling pathway is activated in Philadelphia chromosome-positive chronic myeloid leukaemia hemangioblasts

Overwhelming evidence from chronic myeloid leukaemia (CML) research indicates that patients harbour quiescent CML stem cells that are responsible for blast crisis. While the haematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated. We have previously isolated fetal liver kinase-1-positive (Flk1(+)) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph(+)) patients with hemangioblast property. Here, we show that these cells behave abnormally comparing with the hemangioblasts in healthy donors. These Ph(+) putative CML hemangioblast up-regulated TGF-β1 and result in activating matrix metalloproteinase-9 to enhance s-KitL and s-ICAM-1 secretion. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor-κB signalling pathway was involved in CML pathogenesis. These findings provide direct evidence for the first time that hemangioblasts beyond HSCs play a critical role in the progression of CML.     

Differences in oncogenic potency but not target cell specificity distinguish the two forms of the BCR/ABL oncogene.

Two forms of activated BCR/ABL proteins, P210 and P185, that differ in BCR-derived sequences, are associated with Philadelphia chromosome-positive leukemias. One of these diseases is chronic myelogenous leukemia, an indolent disease arising in hematopoietic stem cells that is almost always associated with the P210 form of BCR/ABL. Acute lymphocytic leukemia, a more aggressive malignancy, can be associated with both forms of BCR/ABL. While it is virtually certain that BCR/ABL plays a central role in both of these diseases, the features that determine the association of a particular form with a given disease have not been elucidated. We have used the bone marrow reconstitution leukemogenesis model to test the hypothesis that BCR sequences influence the ability of activated ABL to transform different types of hematopoietic cells. Our studies reveal that both P185 and P210 induce a similar spectrum of hematological diseases, including granulocytic, myelomonocytic, and lymphocytic leukemias. Despite the similarity of the disease patterns, animals given P185-infected marrow developed a more aggressive disease after a shorter latent period than those given P210-infected marrow. These data demonstrate that the structure of the BCR/ABL oncoprotein does not affect the type of disease induced by each form of the oncogene but does control the potency of the oncogenic signal.     

IRF-4 suppresses BCR/ABL transformation of myeloid cells in a DNA binding-independent manner

Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4-interacting protein found in the cytoplasm. However, IRF-4 tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain.     

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H₂O₂-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70 kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL^T315I). We also proved that Apg-2 inhibition aggravated H₂O₂ induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL^T315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.     

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.     

Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells

In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration–effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 μmol/L) than for human cord blood cells (IC50 = 4 μmol/L). Lead was 10–15 times more toxic to human hematopoietic cells (IC50 = 61 μmol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 μmol/L; B6C3F1, IC50 = 536 μmol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 μmol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 μmol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nucleotide sequence of both reciprocal translocation junction regions in a patient with Ph positive acute lymphoblastic leukaemia, with a breakpoint within the first intron of the BCR gene.

Breakpoints on chromosome 22 in the translocation t(9;22) found in Philadelphia positive acute lymphoblastic leukaemia patients fall within two categories. In the first the breakpoint is localized within the breakpoint cluster region of the BCR gene, analogous to the chromosome 22 breakpoint in chronic myeloid leukaemia. The second category has a breakpoint 5' of this area, but still within the BCR gene. We have previously shown that these breakpoints occur within the first intron of the BCR gene and cloned the 9q+ junction from such a patient. We have now determined the sequences around the breakpoints on both translocation partners from this patient as well as the germline regions. The chromosome 9 ABL sequence around the breakpoint shows homology to the consensus Alu sequence whereas the chromosome 22 BCR sequence does not. At the junction there is a 6 bp duplication of the chromosome 22 sequence which is present both in the 9q+ and in the 22q- translocation products. Possible mechanisms for the generation of the translocation are discussed.     

Influence of glutathione on zinc-mediated cellular toxicity

The effect of zinc on various pulmonary cell lines has been studied by measuring the depletion of total cellular glutathione after exposure to zinc(II) chloride at different concentrations. Total cellular glutathione (cGS) was measured at 31 ± 3 nmol/mg, 3.8 ± 0.6 nmol/mg, and 3.7 ±1.2 nmol/mg protein in A549, L2, and 11Lu cells, respectively. After treatment with buthionine sulfoximine (BSO), the cGS levels decreased by 20% in A549 cells and below 0.2 nmol/mg in L2 and 11Lu cells. Exposure of A549 cells to 25–200 μM ZnCl₂ for 4 h alone decreased the cGS content to 60–80%. There was little additional effect in BSO-pretreated cells. In L2 and 11Lu cells, the decrease of cGS was 70–85% following exposure to 15–150 μM ZnCl₂ for 2 h. If BSO was also used, the decrease in cGS was 85–95% in L2 cells and 75–85% in 11Lu cells. Exposure to 25–250 μM ZnCl₂ for 2 h diminished protein synthesis as determined by radiolabeled methionine incorporation, with half-maximum inhibition (EC₅₀) from 40–160 μM ZnCl₂. To attain similar EC₅₀ values in BSO-pretreated cells, only about half the zinc concentrations were required as compared to cells without pretreatment. The decrease of cGS was accompanied by an increased ratio of oxidized : reduced glutathione that was more pronounced in cells with low glutathione content.     

Nuclear structure and gene activity in human differentiated cells

The nuclear arrangement of the ABL, c-MYC, and RB1 genes was quantitatively investigated in human undifferentiated HL-60 cells and in a terminally differentiated population of human granulocytes. The ABL gene was expressed in both cell types, the c-MYC gene was active in HL-60 cells and down-regulated in granulocytes, and expression of the RB1 gene was undetectable in HL-60 cells but up-regulated in granulocytes. The distances of these genes to the nuclear center (membrane), to the center of the corresponding chromosome territory, and to the nearest centromere were determined. During granulopoesis, the majority of selected genetic structures were repositioned closer to the nuclear periphery. The nuclear reposition of the genes studied did not correlate with the changes of their expression. In both cell types, the c-MYC and RB1 genes were located at the periphery of the chromosome territories regardless of their activity. The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromosome territory than the studied genes. Close spatial proximity of the c-MYC and RB1 genes with centromeric heterochromatin, forming the chromocenters, correlated with gene activity, although the nearest chromocenter of the silenced RB1 gene did not involve centromeric heterochromatin of chromosome 13 where the given gene is localized. In addition, the role of heterochromatin in gene silencing was studied in retinoblastoma cells. In these differentiated tumor cells, one copy of the RB1 gene was positioned near the heterochromatic chromosome X, and reduced RB1 gene activity was observed. In the experiments presented here, we provide evidence that the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis may be realized through heterochromatin-mediated gene silencing.     

Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH)

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.