Abl Oncogene Research Articles

Molecular Pathogenesis and Treatment Strategies of Chronic Myeloid Leukemia (CML)

Chronic Myeloid Leukemia (CML) is a myeloproliferative disease diagnosed in bone marrow, arising from a chromosomal translocation between chromosomes 9 and 22, resulting in the formation of fusion oncogene BCR–ABL. The product of this fusion oncogene is a new oncoprotein bcr–abl which possesses abnormal tyrosine kinase activity. In response to this, abnormal signaling pathway activation occurs, leading to cell transformation. BCR–ABL oncogene could be targeted by tyrosine kinase inhibitors (TKIs) to delay or inhibit the disease progression. Imatinib is the first drug designed against CML but resistance to this has led to the development of the second- and third generations of inhibitors that are active against many types of BCR–ABL gene mutations. However, somehow, due to disease progression, TKIs do not remain as effective. There are three well-characterized phases of CML: The chronic phase (CP), the accelerated phase, and the terminal stage which is the blast crisis (BC) stage. In the CP of CML, mature granulocytes and myeloid precursors become aggregated majorly in the bone marrow and peripheral blood. The accelerated phase is marked by increased disease severity and an increase in progenitor/precursor cell number. In the BC stage, undifferentiated blast cells grow in number. Many patients with CML are diagnosed during the CP of the disease, so the survival rate of CML is high. However, 20% of CML patients proceed to advanced stages that result in drug resistance, intolerance, and mortality. So, for proper CML treatment, drugs are needed to target multiple BCR– ABL mutations, delay or stop disease progression, and overcome resistance caused by BCR–ABL independent mechanisms, especially during advanced phases of CML. Moreover, drugs could be developed to eradicate the stem cells of CML. These targets could be achieved by understanding mechanisms of disease progression, disease relapse, and drug resistance by utilizing high throughput molecular genetics, cell biology and immunology techniques.

Thymoquinone Induces Downregulation of BCR-ABL/JAK/STAT Pathway and Apoptosis in K562 Leukemia Cells.

Objective:BCR ABL oncogene encodes the BCR-ABL chimeric protein, which is a constitutively activated non-receptor tyrosine kinase. The BCR-ABL oncoprotein is a key molecular basis for the pathogenesis of chronic myeloid leukemia (CML) via activation of several downstream signaling pathways including JAK/STAT pathway. Development of leukemia involves constitutive activation of signaling molecules including, JAK2, STAT3, STAT5A and STAT5B. Thymoquinone (TQ) is a bioactive constituent of Nigella sativa that has shown anticancer properties in various cancers. The present study aimed to evaluate the effect of TQ on the expression of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes and their consequences on the cell proliferation and apoptosis in K562 CML cells. Methods:BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis. Results:TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001). Conclusion:our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.

Abstract 3571: Identification of a novel imatinib upregulated lncRNA family that is required for efficient cellular transformation by Abl oncogene

Abstract Bcr-Abl oncogene is generated by a reciprocal translocation between chromosome 9 and 22 in human genome, giving Bcr-Abl protein with constitutive tyrosine kinase activity that causes chronic myeloid leukemia (CML) . Few acute lymphoblastic leukemia (ALL) patients also contain Bcr-Abl oncogene. Owing to the development of imatinib, over 90% of CML patients can be cured in recent years. However, the functional relevance of lncRNAs in Bcr-Abl-mediated leukemia remains obscure. Here, we identified a conserved, imatinib-upregulated lncRNA (IUR) family, named lncRNA-IUR37. Upregulation of lncRNA-IUR37 has been detected in both human and mouse Abl-transformed cell lines after imatinib treatment. Interestingly, lncRNA-IUR37 expression levels were significantly lower in leukemic cells derived from Bcr-Abl-positive ALL patients than those in normal control group. Furthermore, altering lncRNA-IUR37 expression remarkably affected survival of Abl-transformed leukemic cells, and tumorigenesis induced by these leukemic cells in xenograft mouse model. Knockdown of lncRNA-IUR37 in transgenic mice significantly promoted Bcr-Abl-mediated primary bone marrow transformation, and leukemia development in leukemia mouse model. These results indicate that lncRNA-IUR37 functions as a suppressor gene in Bcr-Abl-induced tumorigenesis. In addition, we demonstrated that lncRNA-IUR37 directly bound and affected the phosphorylation of STAT5, thereby impacting expression of transferrin receptor protein (TfR or CD71) to regulate leukemic cell survival. Together, our observations suggest that lncRNA-IUR37 is critically involved in Bcr-Abl-mediated leukemogenesis, and provide novel insights into complicated mechanisms underlying cellular transformation by Bcr-Abl oncogene. Citation Format: Jilong Chen, Xuefei Wang. Identification of a novel imatinib upregulated lncRNA family that is required for efficient cellular transformation by Abl oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3571.

1002 - The ABL Oncogene and Kinase Targeted Therapies

1002 - The ABL Oncogene and Kinase Targeted Therapies

Complex three-way Philadelphia translocation t(6;9;22) (p23;q34;q11.2) along with unusual t(1;4): A chronic myeloid leukemia case report

Chronic myeloid leukemia (CML) is a myeloproliferative disorder and genetically characterized by the presence of the Philadelphia (Ph) chromosome, resulting from a balanced reciprocal translocation between chromosome 9 and 22 at bands 9q34 and 22q11.2 [t(9;22)(q34;q11.2)]. In the formation of the Ph chromosome, the specific region of the ABL oncogene is transposed from 9q34 to the specific region of the BCR gene on chromosome 22 to form a fusion gene BCR‑ABL, which encodes a constitutively active protein, with tyrosine kinase activity. Vast majority of CML patients show classical Ph translocation, t(9;22) and 5–10% of cases are observed with variant Ph translocation. In variant Ph translocation, generally, the third chromosome involved with chromosome 9 and 22. The segment of the third chromosome usually translocates to the chromosome 9 at band 9q34. The formation of variant or complex Ph translocation is controversial topic. A wide array of additional chromosome involved in translocation with the t(9;22) has been described previously in CML patients. In this report, we present a unique and complex Ph translocation involving three chromosomes (6;9;22) in a 25-year-old female 66who was clinically diagnosed as CML. Chromosome analysis using 24 h and 48 h unstimulated culture with GTG-banding was performed according to standard protocol. A total of 20 metaphases analyzed from the bone marrow culture of the patient and karyotyping was performed on the basis of International System for Human Cytogenetic Nomenclature (2016). The FISH analysis was performed according to the standard protocol from sample for confirmation of BCR/ABL gene using dual color fusion FISH probe (Zytovision Probe). All cell’s images were captured using Olympus fluorescent microscope BX-61 equipped with a CCD camera and analyzed using Bioview FISH software.

Gene expression profile of circulating CD34+ cells and granulocytes in chronic myeloid leukemia

PurposeWe compared the gene expression profile of peripheral blood CD34+ cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR–ABL oncogene. MethodsThe microarray analyses have been performed in circulating CD34+ cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR–ABL positive CML patients were in chronic phase, with a mean value of 2012±SD of CD34+cells/μl in peripheral blood. ResultsThe gene expression profile was more prominent in CML CD34+ cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34+ cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR–ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34+ cells. Among genes linked to the inhibition of cellular proliferation by BCR–ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. ConclusionThe presence of BCR–ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34+ cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.

Abstract 2846: Ultrasensitive detection of actionable subclones in chronic myeloid leukemia

Abstract Targeted Abl inhibitors such as imatinib have vastly improved survival in chronic myeloid leukemia (CML). However resistance occurs in many patients, most frequently due to point mutations in the Abl kinase. At the time of diagnosis, resistance mutations may already exist in a small fraction of the malignant cells. Second-generation inhibitors have been developed which can overcome specific resistance mutations. Identification of pre-existing resistance mutations would thus have important therapeutic implications, as it could inform which therapy would result in the most durable response in a given patient. Prior searches for pre-existing Abl mutations in CML have used methods such as allele-specific PCR or next-generation DNA sequencing; these methods are prone to background errors resulting in frequent false positive mutations. For example, next-generation sequencing has an error rate of 0.1%-1%, which results in a large number of erroneous mutations that obscure true variants. Enrichment of the Abl gene is also a major limitation. Nearly all prior reports have used PCR amplification for dozens of cycles to enrich the Abl gene prior to mutation detection; this process introduces thousands of PCR errors which make differentiation of true mutations from artifactual mutations challenging. We have developed a protocol for DNA sequencing, Duplex Sequencing, that separately tags and sequences each of the two strands of single DNA molecules. True mutations are present at the same position in both strands and are complementary, while PCR or sequencing errors occur in only one of the strands and are not scored. Duplex Sequencing has a calculated error rate of less than one artifactual mutation per billion nucleotides sequenced. To apply Duplex Sequencing to the Abl oncogene, we established a new, extremely efficient targeted capture approach that utilizes sequential rounds of capture with biotinylated DNA probes. This protocol results in &amp;gt;1,000,000 fold enrichment, with up to 99.8% of reads corresponding to the Abl gene. Duplex Sequencing was carried out at high depth (&amp;gt;100,000 fold), resulting in an average of 2,000 duplex reads per nucleotide position. The estimated accuracy for mutation identification was 99.9999%. We are in the process of sequencing samples from CML at the time of diagnosis and relapse to assay for drug resistance mutations below the limit of detection of conventional assays. Our approach could be extended to any targeted molecular agent for which mutation of the target confers resistance to the drug. Citation Format: Michael W. Schmitt, Bella Aminov, Jerald P. Radich, Lawrence A. Loeb. Ultrasensitive detection of actionable subclones in chronic myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2015-2846

Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition

AbstractC-abl oncogene 1, nonreceptor tyrosine kinase (ABL1) kinase inhibitors such as imatinib mesylate (imatinib) are effective in managing chronic myeloid leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinase-independent pathways support LSC survival. Given that the bone marrow (BM) hypoxic microenvironment supports hematopoietic stem cells, we investigated whether hypoxia similarly contributes to LSC persistence. Importantly, we found that although breakpoint cluster region (BCR)-ABL1 kinase remained effectively inhibited by imatinib under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of imatinib. Hypoxia-inducible factor 1 α (HIF1-α), which is the master regulator of the hypoxia transcriptional response, is expressed in the BM specimens of CML individuals. In vitro, HIF1-α is stabilized during hypoxia, and its expression and transcriptional activity can be partially attenuated by concurrent imatinib treatment. Expression analysis demonstrates at the whole-transcriptome level that hypoxia and imatinib regulate distinct subsets of genes. Functionally, knockdown of HIF1-α abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1-α signaling supports LSC persistence independent of BCR-ABL1 kinase activity. Thus, targeting HIF1-α and its pathway components may be therapeutically important for the complete eradication of LSCs.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Fas-associated phosphatase 1 mediates Fas resistance in myeloid progenitor cells expressing the Bcr–abl oncogene

The interferon consensus sequence binding protein (Icsbp) is a transcription factor that influences multiple aspects of myelopoiesis. Expression of Icsbp is decreased in the bone marrow of human subjects with chronic myeloid leukemia (CML), and studies in murine models suggest that Icsbp functions as an anti-oncogene for CML. We previously identified a set of Icsbp target genes that may contribute to this anti-oncogene effect. The set includes PTPN13, the gene encoding Fas-associated phosphatase 1 (Fap1, a Fas antagonist). We previously demonstrated that myeloid progenitor cells from Icsbp-knockout mice exhibit Fap1-dependent Fas resistance. In the present study, we determined that the Fas resistance of Bcr–abl+cells is Icsbp- and Fap1-dependent. We also found that treatment of Bcr–abl bone marrow cells with a Fap1-blocking peptide prevents in vitro selection of a tyrosine kinase inhibitor (TKI)-resistant population. Therefore, these results have implications for therapeutic targeting of the Fas-resistant leukemia stem cell population and addressing TKI resistance in CML.

Abstract 11: A requirement for SOCS-1 and SOCS-3 phosphorylation in BCR-ABL-mediated transformation

Abstract Previous studies have demonstrated that constitutive activation of JAK/STAT signaling is required for efficient transformation by the Abelson murine leukemia virus (A-MuLV), which expresses v-Abl oncoprotein. Importantly, there is increasing evidence that deregulation of the JAK or STAT activity plays a critical role in BCR-ABL-induced malignant transformation. SOCS-1 and SOCS-3 are inhibitors of JAK-STAT pathway and as such are involved in a negative feedback loop of cytokine signaling. However, the mechanism by which Abl oncogene bypasses SOCS inhibitory regulation remains poorly defined. Here, we found that co-expression of BCR-ABL with SOCS-1 or SOCS-3 resulted in tyrosine phosphorylation of these SOCS proteins. Interestingly, tyrosine-phosphorylated SOCS-1 was detected in three of five primary CML samples which express BCR-ABL. BCR-ABL-dependent phosphorylation of SOCS-1 and SOCS-3 occurred mainly on Tyr 155 and Tyr 204 residues of SOCS-1, and Tyr 221 residue of SOCS-3. Phosphorylation of these SOCS proteins was associated with its binding to BCR-ABL, and this interaction was disrupted by mutation of the sites of SOCS phosphorylation. We demonstrated that BCR-ABL-dependent phosphorylation of SOCS-1 and SOCS-3 alters their inhibitory effects on the activation of JAK1 and JAK2 and thereby enhances JAK-STAT signaling. Moreover, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 promotes K562 leukemic cells to undergo apoptosis and blocks tumor formation in mouse model. Together, these results reveal a requirement for tyrosine phosphorylation of SOCS-1 and SCOCS-3 in BCR-ABL-mediated transformation in the presence of these SOCS proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 11. doi:1538-7445.AM2012-11

Mechanisms and novel approaches in overriding tyrosine kinase inhibitor resistance in chronic myeloid leukemia

Chronic myeloid leukemia is a stem cell-initiated but progenitor-driven disease induced by the BCR–ABL oncogene. Tyrosine kinase inhibitors (TKIs) were introduced in the late 1990s and have revolutionized the management of chronic myeloid leukemia in chronic phase. The majority of patients can now expect to live a normal life as long as they continue to comply with TKI treatment. However, in a significant proportion of cases TKI resistance develops over time, requiring a switch of therapy. The most frequent mechanism for drug resistance is the development of kinase domain mutations that reduce or completely ablate drug efficacy. Fortunately, the last 10 years have seen an impressive array of new drugs, some modeled on the mechanism of action of imatinib, others employing more novel approaches, for these patients.

Fully reversible pulmonary arterial hypertension associated with dasatinib treatment for chronic myeloid leukaemia

To the Editors: Pulmonary arterial hypertension (PAH) is a devastating disease that is characterised by a progressive increase in pulmonary vascular resistance (PVR), chronic right heart failure and premature death. Pulmonary hypertension (PH) is defined by a mean pulmonary artery pressure ( P pa) ≥25 mmHg at rest, and the haemodynamic definition discerns pre- and post-capillary PH [1]. Furthermore, PAH is distinguished from other forms of PH that may occur secondary to various underlying causes, including left heart disease, chronic lung disorders and thromboembolic disease [1]. Although the development of targeted PAH therapies, such as endothelin receptor antagonists, phosphodiesterase type-5 inhibitors (PDE5i) and prostanoids, has markedly improved the clinical situation and outcome of affected patients, the current medical treatment of PAH is not satisfactory. Recently, tyrosine kinase inhibitors (TKIs), particularly imatinib, have been proposed as a potential novel treatment option in PAH [2–7]. Imatinib was the first TKI that achieved regulatory approval for the treatment of chronic myeloid leukaemia (CML) and has become the standard of care for patients with this disease, as it inhibits the hybrid BCR–ABL oncogene that is pathogenic in the development of CML. Imatinib inhibits several tyrosine kinases, including BCR–ABL, the platelet-derived growth factor receptor (PDGFR) and c-Kit. More recently, additional TKIs, including dasatinib and nilotinib, have been introduced for the treatment of CML in patients who are intolerant or unresponsive to imatinib. The potential efficacy of imatinib in PAH is attributable to its inhibitory effect on the PDGFR. Platelet-derived growth factor (PDGF) is thought to play a pivotal role in the pathobiology of PAH by initiating and maintaining the underlying pulmonary vascular remodelling [7, 8]. Consistent with this, inhibition of PDGFR signalling by imatinib was recently shown to reverse PAH in animal models, and to …

Abstract 4013: Functional analysis of Bcr-Abl-dependent phosphorylation of SOCSs

Abstract Previous studies have demonstrated that constitutive activation of JAK/STAT (Janus tyrosine kinase/signal transducers and activators of transcription) signaling is required for efficient transformation by the Abelson murine leukemia virus (A-MuLV), which results from the expression of the v-Abl oncogene. Although the activation of JAK/STAT signaling pathway in cells expressing BCR-ABL still remains controversial, there is increasing evidence that deregulation of the JAK or STAT activity plays an important role in BCR-ABL malignant transformation. The suppressors of cytokine signaling (SOCS) are inhibitors of Jak kinases and as such are critically involved in a negative feedback loop of Jak/STAT signal transduction pathway. The mechanism by which Abl oncogene bypasses SOCS negative regulation remains poorly defined. Here, we found that co-expression of BCR-ABL with some SOCS proteins resulted in phosphorylation of SOCSs. Moreover, we observed that BCR-ABL-dependent phosphorylation of SOCSs altered SOCS function at least at two levels. One involves alteration of direct interaction of SOCSs with JAKs, which maintains JAK kinase activity. The other level involves inhibition of interaction of SOCS box with the Elongin BC complex, which disrupted the formation of E3 ubiquitin ligase complex that mediates proteosomal degradation of JAK kinases. Together, these results suggest that the BCR-ABL may regulate the inhibitory function of SOCSs through mediating phosphorylation of SOCS proteins. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4013.

Detection of the BCR-ABL Fusion protein2 by Using the Abbott Cell Dyn Sapphire.a Routine Blood Hematology Analyser.

Abstract 4699 ObjectivesWe have evaluated the possibility to use the new BD Cytometric Bead Array to detect the presence of the BCR-ABL fusion protein2 in peripheral blood samples with a routine blood hematology analyser, the Abbott Cell-Dyn Sapphire, for a quick identification of those positive samples, both at diagnosis and at follow up to detect the MRD. This pilot study was carried out on 5 samples with different level of BCR-ABL fusion protein2 plus 2 control samples positive and negative respectively: results were compared with those obtained with the BD FACScanto and the standard FISH analysis reference method. MethodsFlow cytometry allows for the discrimination of particles on the basis of attributes such as size and fluorescence. BD Cytometric Bead Array (CBA) systems provide a way of coupling a soluble analyte or set of analytes with beads of known size and fluorescence, making it possible to detect analytes through flow cytometry.1 The BD” BCR-ABL Protein Kit uses this technology to detect BCR-ABL fusion proteins2 in human blood research samples.Cell-Dyn Sapphire is a routine automated hematology analyzer for full blood count. Moreover the system employs immuno-fluorescence analysis technology, similar to that used on a dedicated fluorescence flow cytometer, for analysis of MAb applications.BD FacScant is a routine dedicated flow cytometer using 6 fluorescence channels.FISH (Fluorescence In Situ Hybridation) is a cytogenetic technique using fluorescent probes that bind to only those part of the chromosome with which they show a high degree of sequence similarity in interphase nuclei and on metaphase chromosome.FISH detects and localizes the presence, the absence or the translocation of specific DNA sequences on chromosomes. In this patients' category, LSI bcr/abl dual fusion DNA probe hybridises to chromosome 22q11.2 (breakpoint cluster region SpectrumGreen) and to chromosome 9q34 (abl oncogene SpectrumOrange). In interphase nuclei of normal cells, the probe signals generally appear as two distinct signals of each colour. In the pathological cells with bcr/abl translocation, individual orange and green signals from the normal 9 and 22 chromosome and two orange/green fusion signals (one each from the derivative 9 and 22 chromosomes) is observed. We have analysed 200 nuclei in fluorescence microscopy for each sample.Statistical analyses were performed using the MedCalc program comparing the median fluorescence values obtained by the 2 cytometers and with the standard diagnostic procedure. ResultsCorrelation of median fluorescence data between the BD FACScanto and the CD Sapphire is excellent with a Correlation coefficient r = 1,0000 and a Significance level P<0,0001 (Fig 1).The Box –and –whisker Multiple comparison graph of the CD method versus the FISH reference one is represented in Figure 2.The results of our pilot study demonstrate the robustness of this new diagnostic tool in detecting the presence of the BCR-ABL fusion protein2 in the routine Laboratory of Hematology. The main limit to use this tool is now represented by the high cost of the BD Cytometric Bead Array. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.2 to 2 μM induced DNA damage in human leukemic K562 and BV173 cells expressing the BCR/ ABL oncogene, whereas it had no effect in normal human lymphocytes and leukemic CCRF-CEM cells without the expression of BCR/ABL. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the circular plasmid relaxation assay. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. K562 cells were unable to repair H 2O 2-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with Vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Therefore, the mechanism of the anti-leukemic action of STI571 may involve not only the inhibition of BCR/ABL, but also DNA damage in the cells expressing this fusion protein. DNA damage induced by STI571 may follow from oxidative and alkylative base modifications.

Phosphoinositide 3-kinase signaling is essential for ABL oncogene–mediated transformation of B-lineage cells

AbstractBCR-ABL and v-ABL are oncogenic forms of the Abl tyrosine kinase that can cause leukemias in mice and humans. ABL oncogenes trigger multiple signaling pathways whose contribution to transformation varies among cell types. Activation of phosphoinositide 3-kinase (PI3K) is essential for ABL-dependent proliferation and survival in some cell types, and global PI3K inhibitors can enhance the antileukemia effects of the Abl kinase inhibitor imatinib. Although a significant fraction of BCR-ABL-induced human leukemias are of B-cell origin, little is known about PI3K signaling mechanisms in B-lineage cells transformed by ABL oncogenes. Here we show that activation of class IA PI3K and downstream inactivation of FOXO transcription factors are essential for survival of murine pro/pre-B cells transformed by v-ABL or BCR-ABL. In addition, analysis of mice lacking individual PI3K genes indicates that products of the Pik3r1 gene contribute to transformation efficiency by BCR-ABL. These findings establish a role for PI3K signaling in B-lineage transformation by ABL oncogenes. (Blood. 2004;103:4268-4275)

Contribution of fluorescence in situ hybridization analyses to the characterization of masked and complex Philadelphia chromosome translocations in chronic myelocytic leukemia

Bone marrow samples from 112 patients with chronic myelocytic leukemia were investigated using cytogenetic methods. Fluorescent in situ hybridization (FISH) with whole-chromosome paints and BCR-ABL probes was used to confirm and/or complete the banding findings when a variant or a masked Philadelphia chromosome (Ph) translocation was found. Eight variant Ph translocations were identified. Three-way Ph translocations were found in seven patients. Chromosome 4 was involved in two of these cases and chromosomes 3, 11, 14, 17, and 16 in one case each; in the patient with chromosome 16 involvement, a ring of the translocated chromosome 9 was identified, that is r(9)t(9;16;22). The eighth patient had a five-way Ph translocation: t(2;9;16;22;22). The BCR-ABL fusion gene was detected on the Ph chromosome in all eight cases; two cases presented also a deletion of the 5′ ABL region on the derivative chromosome 9. In the five-way translocation, the 3′ DNA sequence of the ABL oncogene was fused with the 5′ DNA sequence of the BCR gene on the Ph chromosome and the 5′ end of ABL was inserted into the other chromosome 22. A masked Ph chromosome was identified in one of the 112 patients; it involved the insertion of the 3′ ABL into BCR on an apparently normal chromosome 22, resulting in the BCR-ABL fusion gene. In conclusion, FISH analyses allowed not only a more accurate characterization of complex Ph translocations with subtle abnormalities and the identification of cryptic rearrangements, but also the recognition of deletion of the 5′ ABL region, which could carry with it a poor prognosis.

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA–enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 μM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ ABL-transformed cells. Vitamin C at 10 and 50 μM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N- tert-butyl-α-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.

Détection du chromosome Philadelphie chez les patients atteints de leucémie myéloïde chronique: Places respectives de la cytogénétique de l'hybridation in situ en fluorescence et de l'analyse moléculaire par RT-PCR

Chronic myelogenous leukemia (CML) is characterized by a translocation t(9;22)(q34;q11) that can be cytogenetically observed in hematopoietic cells of 95 % of leukemic patients. The fusion of the abl oncogene with the bcr gene which results from this translocation can also be detected by molecular analysis. Fluorescence in situ hybridization (FISH) is a specific and sensitive method for detection of the bcr/abl fusion gene in samples from CML patients. A good correlation is observed between cytogenetic and FISH results in patients at diagnosis or under treatment. However dyscrepancies between the results of these two techniques must be considered. FISH is usefull for detecting cryptic or variant translocations, or for assessing the remission rate when cytogenetics is unsuccesfull. Reverse transcriptase polymerase chain reaction (RT-PCR) is more sensitive than cytogenetics. The need of quantitative methods is evident, but technical problems in standardization limits its application for residual disease evaluation.