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PubMed2001

Cooperation of BCR-ABL and AML1/MDS1/EVI1 in blocking myeloid differentiation and rapid induction of an acute myelogenous leukemia.

The development of acute myelogenous leukemia (AML), which is characterized by a block of myeloid differentiation, is a multi-step process that involves several genetic abnormalities, but the molecular mechanisms by which these genetic alterations cooperate in leukemogenesis are poorly understood. The human chronic myelogenous leukemia (CML) is a model for multi-step leukemogenesis. BCR-ABL, a constitutively active tyrosine kinase, is a fusion protein generated by the t(9;22)(q34;q11) translocation found in the vast majority of CML patients. BCR-ABL efficiently induces a myeloproliferative disorder (MPD) in mice, but progression to CML blast phase requires additional mutations. The AML1/MDS1/EVI1 (AME) transcription factor fusion protein, is a product of the human t(3;21)(q26;q22) translocation found as a secondary mutation in some cases of CML during the blast phase. We have previously shown that AME can induce an AML in mice but with a greatly extended latency, suggesting a requirement for additional mutations. Here we demonstrate that AME alone does not block myeloid differentiation in vivo during the 4-month pre-leukemia stage, yet co-expression of BCR-ABL and AME in mice can block myeloid differentiation and rapidly induce an AML. Our results suggest that block of myeloid differentiation and induction of AML involves cooperation between mutations that dysregulate protein tyrosine kinase signaling and those that disrupt hematopoietic gene transcription.

Acute Disease↗
PubMed1997

Expression of adhesion molecules and functional stimulation in human neutrophils: modulation by GM-CSF and role of the Bcr gene.

Although devoid of proliferative capacity, polymorphonuclear neutrophils (PMN) express receptors for haemopoietic growth factors and need growth factors for survival and functional stimulation. This study showed that in vitro treatment of human PMN with GM-CSF for up to 48 h increases cell surface expression of the beta 2-integrin molecules CD11b/CD18 and CD11c/CD18 and of the receptor for the chemotactic peptide fMLP. Such modifications are usually expression of PMN activation. PMN treated with GM-CSF also displayed increased phagocytosis of latex particles and enhanced oxidative burst and superoxide anion release. Since integrins mediate PMN adhesion to endothelium, homotypic adhesion, chemotaxis/phagocytosis and the triggering of respiratory burst, our results suggested that functional stimulation of PMN persisted following prolonged exposure of PMN to growth factors and that it was not a temporary phenomenon which lasted only for the first 12-24 h of treatment. We also used oligonucleotides antisense to the Bcr gene mRNA to inhibit expression of the gene and evaluate its function in PMN, following the recent observation that PMN from Bcr-null mutant mice produced increased amounts of reactive oxygen metabolites upon activation. The antisense oligonucleotides had no effect on the parameters investigated. This may indicate that increased production of O2 by neutrophils in which the Bcr gene is not expressed requires either that gene expression is absent in the earlier stages of myeloid differentiation/maturation, so that when inhibition occurs in the terminally differentiated neutrophils their functional status is no longer influenced, or that the residual low-level expression of the gene which may be present in the antisense-treated cells is sufficient to provide a normal response to stimulation.

Antibodies, Monoclonal↗
PubMed2000

Novel BCR-ABL transcript containing an intronic sequence insert in a patient with Philadelphia-positive acute lymphoblastic leukaemia.

In a patient with Philadelphia chromosome-positive acute lymphoblastic leukaemia (ALL), a novel variant of the chimaeric BCR-ABL mRNA transcript was detected by reverse transcription polymerase chain reaction (RT-PCR). Sequencing revealed the novel transcript to be a chimaeric mRNA produced by fusion of the BCR exon 14 (b3) to the ABL exon a2 with a 49-base pair (bp) insertion of an ABL intron 1b sequence between them. The insertion of the 49 bp introduced a stop codon. These data show that this variant of the chimaeric mRNA would not be translated into the p210 BCR-ABL protein. This could be one of the explanations as to why clinically the patient has responded well to therapy and continues to follow a mild clinical course.

Aged↗
PubMed2001

E1A3 as a unique, naturally occurring BCR-ABL transcript in an indolent case of chronic myeloid leukaemia.

A woman with Ph-positive chronic myeloid leukaemia (CML) with an atypical e1a3 BCR-ABL hybrid gene is described. To our knowledge, this is the first report of this transcript type as a unique naturally occurring BCR-ABL fusion in a CML patient. This case was characterized by a low leucocyte count and a very indolent course without treatment. Because the deletion of ABL exon 2 sequences results in deletion of an essential part of the ABL SH3 domain, our case suggests that this ABL SH3 domain is not absolutely necessary for efficient induction of a myeloproliferative disease in the context of BCR-ABL/p190.

Aged↗
PubMed2001

Targeting neighbouring poly(purine.pyrimidine) sequences located in the human bcr promoter by triplex-forming oligonucleotides.

Most poly(purine.pyrimidine) [poly(R.Y)] sequences in eukaryotic genomes are interrupted by one or more base pair inversions. When the inversions are centrally located, the poly(R.Y) sequences can be regarded as the sum of two abutting sites, each potentially capable of forming a triple helix. Employing band-shift, footprinting and modeling methods we examined the formation of triple helices at a critical 27 bp poly(R.Y) sequence interrupted by two adjacent CG inversions, and located in the promoter of the human bcr gene at transcription initiation. We designed several 13-mer and 14-mer triplex-forming oligonucleotides (TFOs) capable of binding the bcr abutting sites, thereby generating different base juxtapositions at the triple helical junction, to examine whether triplex formation occurs in a cooperative manner. It is found that in 50 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 2 mM spermine, 37 degrees C, the 13-and the 14-mer TFOs bind to one half of the bcr site with Delta G between -30 and -35 kJ x mol-1. However, when different 13-mer/14-mer combinations of TFOs were directed against the abutting poly(R x Y) sites, triplex formation has been found to be enhanced only for the triple helical junction formed by the 5'-A-T-3' base juxtaposition, in keeping with a partial stacking suggested from modeling analysis. On the other hand, a longer 24-mer TFO, binding noncooperatively to the same abutting sites, forms a much more stable triplex (Delta G = -51 kJ x mol-1), notwithstanding the two T x CG triads in the middle. Modeling investigations reveal that there is no continuity or propagation of base stacking involving adjacent bases of the third strand at the site of base inversion as well as on the 5' side. The data indicate that the entropy penalty of forming a triplex with two oligonucleotides is much higher than the energy gained from base stacking interactions at the triplex junction formed between the two TFOs.

Base Sequence↗
PubMed2005

Comparative analysis of two clinically active BCR-ABL kinase inhibitors reveals the role of conformation-specific binding in resistance.

Structural studies suggest that most point mutations in the BCR-ABL kinase domain cause resistance to the ABL kinase inhibitor imatinib by impairing the flexibility of the kinase domain, restricting its ability to adopt the inactive conformation required for optimal imatinib binding, rather than by directly interfering with drug contact residues. BMS-354825, currently in clinical development for imatinib-resistant chronic myelogenous leukemia, is a dual SRC/ABL kinase inhibitor that binds ABL in both the active and inactive conformation. To examine the potential role of conformational binding properties in drug resistance, we mapped the mutations in BCR-ABL capable of conferring resistance to BMS-354825. Through saturation mutagenesis, we identified 10 such BCR-ABL mutations, 8 of which occurred at drug contact residues. Some mutants were unique to BMS-354825, whereas others also conferred imatinib resistance. Remarkably, the identity of the amino acid substitution at either of two contact residues differentially affects sensitivity to imatinib or BMS-354825. The combination of imatinib plus BMS-354825 greatly reduced the recovery of drug-resistant clones. Our findings provide further rationale for considering kinase conformation in the design of kinase inhibitors against cancer targets.

Base Sequence↗
PubMed2003

Cytokines and BCR-ABL mediate suppression of TRAIL-induced apoptosis through inhibition of forkhead FOXO3a transcription factor.

Cytokine-provided survival signals are known to suppress apoptosis through inhibition of mitochondrial pathways that involve Bcl-2 family members. Here we show that in hematopoietic cells, cytokines also regulate death receptor-mediated pathways. We demonstrate that hematopoietic cytokines such as IL-3 and erythropoietin in normal cells, as well as BCR-ABL oncoprotein in transformed cells, inhibit transcription of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using small interfering RNAs, we show that the inhibition of TRAIL function is sufficient to partially rescue cytokine-deprived cells from apoptosis. Finally, we demonstrate that cytokine and BCR-ABL suppression of TRAIL transcription is mediated through phosphorylation and inhibition of the forkhead FOXO3a transcription factor. BCR-ABL-induced inhibition of TRAIL transcription in hematopoietic cells may provide a novel mechanism for tumorigenicity in chronic myeloid leukemia.

Apoptosis↗
PubMed1989

Localization of preferential sites of rearrangement within the BCR gene in Philadelphia chromosome-positive acute lymphoblastic leukemia.

The Philadelphia chromosome associated with acute lymphoblastic leukemia (ALL) has been linked to a hybrid BCR/ABL protein product that differs from that found in chronic myelogenous leukemia. This implies that the molecular structures of the two chromosomal translocations also differ. Localization of translocation breakpoints in Philadelphia chromosome-positive ALL has been impeded due to the only partial characterization of the BCR locus. We have isolated the entire 130-kilobase BCR genomic locus from a human cosmid library. A series of five single-copy genomic probes from the 70-kilobase first intron of BCR were used to localize rearrangements in 8 of 10 Philadelphia chromosome-positive ALLs. We have demonstrated that these breakpoints are all located at the 3' end of the intron around an unusual restriction fragment length polymorphism caused by deletion of a 1-kilobase fragment containing Alu family reiterated sequences. This clustering is unexpected in light of previous theories of rearrangement in Philadelphia chromosome-positive chronic myelogenous leukemia that would have predicted a random dispersion of breakpoints in the first intron in Philadelphia chromosome-positive ALL. The proximity of the translocation breakpoints to this constitutive deletion may indicate shared mechanisms of rearrangement or that such polymorphisms mark areas of the genome prone to recombination.

Base Sequence↗
PubMed2001

The reciprocal role of Egr-1 and Sp family proteins in regulation of the PTP1B promoter in response to the p210 Bcr-Abl oncoprotein-tyrosine kinase.

Protein-tyrosine phosphatase 1B (PTP1B) is an important regulator of protein-tyrosine kinase-dependent signaling pathways. Changes in expression and activity of PTP1B have been associated with various human diseases; however, the mechanisms by which PTP1B expression is regulated have yet to be characterized. Previously, we have shown that the expression of PTP1B is enhanced by p210 Bcr-Abl and that PTP1B is a specific antagonist of transformation induced by this oncoprotein protein-tyrosine kinase. Here we have characterized the PTP1B promoter and demonstrate that a motif with features of a stress-response element acts as a p210 Bcr-Abl-responsive sequence, termed PRS. We have shown that three C(2)H(2) zinc finger proteins, namely Sp1, Sp3, and Egr-1, bind to PRS. Whereas binding of either Sp1 or Sp3 induced promoter function, Egr-1 repressed Sp3-mediated PTP1B promoter activation. The binding of Egr-1 to PRS is suppressed by p210 Bcr-Abl due to the inhibition of Egr-1 expression, resulting in the enhancement of PTP1B promoter activity. Our data indicate that Egr-1 and Sp family proteins play a reciprocal role in the control of expression from the PTP1B promoter.

Base Sequence↗
PubMed2001

The Dbl homology domain of BCR is not a simple spacer in P210BCR-ABL of the Philadelphia chromosome.

The Dbl homology (DH) domain of BCR in P210BCR-ABL (P210/WT) has been thought to have a negative effect on the activation of BCR-ABL because P185BCR-ABL, in which this region is physically deleted, has stronger biochemical and biological activities. To study the role of the DH domain of BCR in the background of P210/WT, the region was replaced with homologous sequences derived from Dbl (P210/Dbl) or CDC24 (P210/CDC24) or with irrelevant sequences from LacZ (P210/LacZ) or luciferase (P210/Luci). Surprisingly, the abilities to transform Rat1 cells or mouse bone marrow cells and induce growth factor independence in interleukin 3-dependent mouse Ba/F3 cells were retained only in P210/Dbl. However, even P210/Dbl could not achieve the wild type level of surviving potential against genotoxins in Rat1 cells and in Ba/F3 cells. Activation of Akt correlated with the biological changes in Rat1 cells but did not correlate with the biological changes in Ba/F3 cells. The DH domain was not tyrosine-phosphorylated in vitro, nor could we find any differences in peptide mapping between in vitro phosphorylated P210/WT and P210/Dbl. Although functions of the DH domain remain to be discovered, we propose that the DH domain makes positive contributions to P210BCR-ABL.

Animals↗
PubMed1988

The first BCR gene intron contains breakpoints in Philadelphia chromosome positive leukemia.

The hallmark of chronic myelogenous leukemia (CML) is a translocation between chromosomes 9 and 22 - the Philadelphia (Ph') translocation. The translocation is also found in acute lymphocytic leukemia (ALL) albeit in a lower percentage of patients. The breakpoint on chromosome 22 is located within the BCR gene: in CML, breakpoints are clustered within 5.8 kb of DNA, the major breakpoint cluster region (Mbcr). In ALL, breakpoints have been reported within the Mbcr but also in more 5' regions encompassing the BCR gene. To characterize the latter breakpoints, we have molecularly cloned and mapped the entire gene, which encompasses approximately 130 kb of DNA. Mbcr negative, Ph'-positive ALL breakpoints were not distributed at random within the gene but rather were found exclusively within the 3' half of the first BCR gene intron. In contrast to the Mbcr, which is limited to a region of 5.8 kb, this part of the intron has a size of 35 kb. Translocation breakpoints in this region appear to be specific for ALL, since it was not rearranged in clinically well-defined CML specimens nor in any other tumor DNA samples examined.

Base Sequence↗
PubMed1996

Quantification of Bcr-Abl transcripts in chronic myelogenous leukemia (CML) using standardized, internally controlled, competitive differential PCR (CD-PCR).

The quantification of Bcr-Abl transcript numbers in chronic myelogenous leukemia (CML) patients described here uses simultaneous competitive PCR amplification of the target gene (Bcr-Abl) and a reference gene (porphobilinogen deaminase; Pbgd) together with a single composite competitor molecule for both targets based on heterologous sequences. Using this technique, Bcr-Abl transcript numbers could be reproducibly determined even in clinical samples known to harbour poor quality RNA.

DNA Primers↗
PubMed1989

Clonal B lymphocytes lack bcr rearrangement in Ph-positive chronic myelogenous leukaemia.

Philadelphia (Ph) chromosome-positive chronic myelogenous leukaemia (CML) was studied in a subject heterozygous for the X chromosome-linked alloenzyme system of glucose-6-phosphate dehydrogenase (G6PD). Determination of G6PD mosaicism showed homogeneous expression in granulocytes, erythrocytes and platelets. Cytogenetic studies showed the typical Ph translocation in all metaphases from bone marrow and peripheral blood myeloid cells, bcr rearrangement was detected in bone marrow and in granulocytes. B cells were stimulated with Epstein-Barr virus (EBV) in order to evaluate involvement of lymphocytes, EBV-transformed lymphoblastoid cells expressed a single G6PD phenotype and therefore probably derived from the leukaemic stem cell. However they had a normal karyotype and a constitutional bcr restriction pattern. Molecular analysis in this case of CML clarifies the differentiative potential of cells belonging to the leukaemic clone, by demonstrating that clonal Ph-negative B cells maintain normal differentiative capacity and have a bcr gene sequence which is not rearranged.

B-Lymphocytes↗
PubMed1989

Clinical stage of chronic granulocytic leukaemia and BCR breakpoint location in South-East Asian patients.

Forty-six Malaysian patients with chronic granulocytic leukaemia were found to be rearranged in the breakpoint cluster region (BCR) of chromosome 22, molecular evidence of Philadelphia chromosome (t9.22) translocation. Through the use of a 1.2 kb 3' BCR probe and two restriction enzyme digests, patients' breakpoints could be localized either to 5' or 3' regions of the BCR. Breakpoint site localization at the time of DNA sampling did not show any positive statistical association to clinical status defined as chronic phase, chronic phase with less than 6 months to blast crisis, accelerated phase and blast crisis. This was in contrast to earlier reports which indicated that patients with breakpoint at 3' site were at a higher biologic risk for entering blast crisis.

Adolescent↗
PubMed1995

PCR-monitoring of minimal residual leukaemia after conventional chemotherapy and bone marrow transplantation in BCR-ABL-positive acute lymphoblastic leukaemia.

We report the results of consecutive tests in nine BCR-ABL-positive ALL patients by one-step and two-step (nested primer) reverse transcriptase-polymerase chain reaction (RT-PCR). Six patients could be tested in complete haematological remission (CHR). One patient remained one-step positive; four patients became one-step negative, but remained two-step positive; only one patient became two-step negative. In five patients the haematological relapse was preceded by one-step positivity by 1.5-5 weeks. In two patients who received autologous BMT in CHR, BCR-ABL was still detectable by two-step PCR, whereas allogeneic BMT was able to transiently reduce BCR-ABL below the two-step detection level. Our results show that one-step combined with two-step RT-PCR analysis gives valuable information about the efficacy of treatment and the dynamics of the leukaemic clone.

Adult↗
PubMed1999

Correlation of cytogenetics, BCR-ABL PCR studies and fluorescence in situ hybridisation (FISH) in adult acute lymphoblastic leukaemia.

BACKGROUND: Philadelphia positive (Ph+) acute lymphoblastic leukaemia (ALL) accounts for 11-29% of adult ALL. Reverse transcriptase polymerase chain reaction (RT-PCR) for the BCR-ABL fusion mRNA has identified patients with the fusion mRNA without cytogenetic evidence of the 9;22 translocation. The reason for discrepancies between cytogenetic and molecular diagnoses is unclear. AIM: Our aim was to study cases of ALL with discordant cytogenetic and RT-PCR results and identify any reasons for such discrepancies. METHODS: Laboratory records were scanned for cases of ALL tested by both RT-PCR and cytogenetics and positive by either for the 9;22 translocation. Fluorescence in situ hybridisation (FISH) was used to study discordant results where a specimen was available. RESULTS: We identified 15 patients with ALL who had both cytogenetic and RT-PCR studies for BCR-ABL. Seven had discordant results; five patients had positive RT-PCR studies with normal (four/five) or abnormal but Ph negative cytogenetics (one/five), and two were Ph+ but RT-PCR negative. FISH, using Vysis LSI bcr/abl translocation probes, showed fused signals in 12% interphase cells but not in metaphase cells in one specimen with normal cytogenetics, and 6% interphase cells in the Ph negative patient with abnormal cytogenetics. This second patient subsequently relapsed with a minor Ph+ cell line derived from the Ph negative line. CONCLUSIONS: These results confirmed the need for both cytogenetics and RT-PCR to identify Ph+ ALL. FISH did not show sub-microscopic rearrangements of BCR-ABL in normal metaphases. Failure to identify the Philadelphia chromosome cytogenetically appeared due rather to Ph+ cells failing to divide.

Adolescent↗
PubMed1989

Molecular analysis of the bcr rearrangement in a case of Ph'-negative blastic crisis of Ph'-positive chronic myelogenous leukemia.

We describe here a patient with Ph'-positive chronic myelogenous leukemia (CML) who developed a Ph'-negative blastic crisis. The blast DNA was analyzed on two different occasions, at the beginning of the blastic phase and at the end, shortly before the patient's death. Although cells from both samples had no Ph' chromosome marker (not even a masked one) we could detect a rearrangement of the bcr gene in the second DNA sample, using a '3'-bcr' probe. The same probe and a '5'-bcr' probe failed to detect any rearranged band in the first DNA sample. No rearrangement was identified at the c-myc and N-ras loci, while a slight c-myc amplification was evident in both DNA samples tested.

Blast Crisis↗
PubMed1992

Initiation of deregulated growth of multipotent progenitor cells by bcr-abl in vitro.

Expression of the bcr-abl oncogene in multipotent progenitor cells (MPPCs) is implicated as a key event in the development of chronic myelogenous leukemia. Bone marrow enriched for MPPCs was infected with a retrovirus that carried bcr-abl. The mixed-lineage colonies that resulted were responsive to growth factors and could differentiate. These cells later became growth factor-independent but, when injected into severe combined immunodeficient mice, were not leukemogenic. Thus, the presence of bcr-abl alone does not cause growth factor independence, although it initiates a stepwise process. This system may prove useful in the study of other oncogenes that cause leukemia.

Animals↗