BCR-ABL Chimeric Gene Research Articles

The mRNA Expression of PTEN, LEF1, JAK3, LC3 and p62/SQSTM1 Genes in Patients with Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a progressive myeloproliferative disorder resulting from forming a chimeric BCR-ABL gene. The proteins derived from this gene can affect some genes from various signaling pathways such as PI3K/AKT/Wnt/catenin/JAK/Stat involved in proliferation, differentiation, cell death, and genes related to autophagy. Imatinib is the first-line treatment for CML patients, with durable and proper responses in Iranian children and adult CML patients. Hence, we aimed to evaluate the mRNA expression of some selected key genes from those pathways in patients with CML before and under treatment. In the case-control study, the mRNA expression of PTEN, LEF1, JAK3, LC3 and p62 genes were measured in 51 CML patients (6 patients before treatment and 45 patients under treatment with imatinib mesylate) and 40 healthy controls using the Real-time PCR method. The mRNA expression of PTEN and P62 were significantly higher in newly diagnosed patients than in controls (P<0.0001 and P = 0.0183, respectively), while the expression of the LC3 gene was significantly lower in the untreated newly diagnosed group than in control subjects (P = 0.0191). The expression level of PTEN, LEF1, JAK3 and P62 genes were significantly decreased in patients under treatment than in the group before treatment (P = 0.0172, P = 0.0002, P = 0.0047 and P = 0.0038, respectively). A positive correlation was seen between the gene expression of P62 and BCR-ABL in the patients under treatment (r 0529, P = 0.016). Our findings showed that the changes in expression of these genes were related to the patient's treatment. Due to the key role of these genes in proliferation, differentiation and tumor suppression, it is proposed that these genes may be helpful for follow-up of treatment in CML patients.

Medium-mass Molecules in Plasma of Patients at Different Stages of Chronic Myeloid Leukemia Progression

Chronic myeloid leukemia (CML) is a clonal tumor disease caused by malignant transformation of hematopoietic stem cells, in which a chimeric BCR-ABL gene is formed as a result of specific translocation of areas between the 9th and 22nd chromosomes. This genetic «defect» ensures the uncontrolled proliferation of myeloid cells. The objective: to analyse the content of medium-mass molecules (MMM) in peripheral venous blood plasma of patients with CML at different stages of the disease and theoretical justification of approaches to the correction of detected changes. Materials and methods. The study enrolled 77 patients with CML, who were divided into three groups according to the stage of disease. The control group included 20 people who were primary donors of the Kyiv City Blood Center. The main parameters characterizing the state of protein and carbohydrate metabolism were analyzed. All patients groups were similar in terms of age, sex, anthropometric parameters, and duration of the disease. All patients during hospitalization were examined by clinical, laboratory, instrumental and special research methods, and if necessary, consulted by specialists. Statistical processing of the results was done by methods of variational statistics, using the Microsoft Excel XP computer program. Results. Today, there are known factors that determine the development of CML, namely: chromosomal abnormalities, ionizing radiation, hematopoietic system diseases, toxic effects of drugs and polluted environment, etc. Changes in the content of MMM in plasma of patients with CML compared to the control group were determined. As the disease progresses, the imbalance of MMM metabolism deepens. The severity of plasma MMM content violations of CML patients is accompanied by endogenous body intoxication, since MMM are universal markers of endogenous metabolic intoxication. Conclusions. Changes in the content of medium-mass molecules in the plasma of patients with chronic myeloid leukemia dictate the need for further in-depth research to determine role in the pathophysiology of the disease for further treatment and reduction of the manifestations of endogenous intoxication. Effective treatment of patients with CML is based on modern diagnostics, timely prescription of drugs and subsequent monitoring of the course of the disease.

Effective Downregulation of BCR-ABL Tumorigenicity by RNA Targeted CRISPR-Cas13a.

To induce BCR-ABL gene silencing using CRISPR Cas13a. CML is a clonal myeloproliferative disorder of pluripotent stem cells driven by a reciprocal translocation between chromosomes 9 and 22 forming a BCR-ABL fusion gene. Tyrosine- kinase inhibitor drugs like imatinib are the mainstay of treatment and cases resistant to these drugs have a poor prognosis in the absence of a compatible stem-cell donor. However with rapid advancements in gene-editing technologies most studies are now focusing on developing a translational model targeting single-gene disorders with a prospective permanent cure. To explore the potential application of the RNA targeting CRISPR-Cas13a system for effective knockdown of BCR-ABL fusion transcript in a CML cell line K562. CRISPR Cas13a crRNA was designed specific to the chimeric BCR-ABL gene and the system was transfected as a two-plasmid system into a CML cell line K562. The effects were enumerated by evaluating the expression levels of downstream genes dependent on the expression of the BCR-ABL gene. Also next-generation sequencing was used to ascertain the effects of CRISPR on the gene. The CRISPR system was successfully able to lower the expression of downstream genes [pCRKL and pCRK] dependent on the activated BCR-ABL kinase signal by up-to 4.3 folds. The viability of the CRISPR-treated cells was also significantly lowered by 373.83-fold [p-value= 0.000891196]. The time-dependent kinetics also highlighted the significant in-vitro suppressive activity to last up to 8 weeks [p-value: 0.025]. As per the cDNA sequencing data from the Oxford MinION next-generation sequencer the CRISPR treated cells show 62.37% suspected cleaved reads. These preliminary results highlight an excellent potential application of RNA targeting CRISPRs in Haematological neoplasms like CML and should pave the way for further research in this direction.

Efficacy and safety of autologous peripheral blood stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia: A study protocol for a multicenter exploratory prospective study (Auto-Ph17 study).

Introduction:The prognosis of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL) has been dramatically improved since the introduction of tyrosine kinase inhibitors (TKIs). Although allogeneic hematopoietic cell transplantation (allo-HCT) is a major treatment option, the role of autologous peripheral blood stem cell transplantation (auto-PBSCT) has been reconsidered, especially in patients who achieved early molecular remission.Methods and analysis:This is a multicenter exploratory study for Ph + ALL patients aged between 55 and 70 years who achieved complete molecular remission within 3 cycles of chemotherapy. The target sample size is 5, and the registration period is 2 years. The primary endpoint is Day100- mortality after transplantation, and the secondary endpoints are survival, relapse rate, nonrelapse mortality, and adverse events.This study is divided into 3 phases: peripheral blood stem cell harvest, transplantation, and maintenance. Chemomobilization is performed using a combination of cyclophosphamide (CPM), doxorubicin, vincristine (VCR), and prednisolone (PSL). As a preparative regimen, the LEED regimen is used, which consists of melphalan, CPM, etoposide, and dexamethasone. Twelve cycles of maintenance therapy using a combination of VCR, PSL, and dasatinib are performed.In association with relapse, the minimal residual disease (MRD) of BCR-ABL chimeric gene and T-cell subsets are analyzed both before and after auto-PBSCT.Ethics and dissemination:The protocol was approved by the institutional review board of Nagoya University Hospital and all the participating hospitals. Written informed consent was obtained from all patients before registration, in accordance with the Declaration of Helsinki. Results of the study will be disseminated via publications in peer-reviewed journals.Trial registration:Trial registration number UMIN000026445.

Evaluation of a new flow cytometry based method for detection of BCR-ABL1 fusion protein in chronic myeloid leukemia.

BackgroundPhiladelphia chromosome, a hallmark of chronic myeloid leukemia (CML), plays a key role in disease pathogenesis. It reflects a balanced reciprocal translocation between long arms of chromosomes 9 and 22 involving BCR and ABL1 genes, respectively. An accurate and reliable detection of BCR-ABL fusion gene is necessary for the diagnosis and monitoring of CML. Previously, many technologies, most of which are laborious and time consuming, have been developed to detect BCR-ABL chimeric gene or chromosome.MethodsA new flow cytometric immunobead assay was used for detection of BCR-ABL fusion proteins and applicability, sensitivity, reliability, efficacy and rapidity of this method was evaluated.ResultsFrom February 2009 to January 2014, a total 648 CML patients were investigated for the status of BCR-ABL1 protein. Among them, 83 patients were enrolled for comparative study of BCR-ABL1 positivity by three routinely used procedures like karyotyping, and quantitative real time PCR (RT-PCR) as well as immunobead flow cytometry assay. BCR-ABL protein analysis was found consistent, more sensitive (17% greater sensitivity) and reliable than the conventional cytogenetics, as flow cytometry showed 95% concordance rate to RT-PCR.ConclusionBCR-ABL fusion protein assay using a new flow cytometric immunobead might be useful in the diagnosis and monitoring CML patients.

Significance of bcr-abl molecular detection in chronic myeloid leukemia patients

Important advances in the understanding of the molecular basis of chronic myeloid leukemia have resulted in the development of new therapies and changed the paradigm for managing this myeloproliferative disease. The reciprocal translocation of the abl (Abelson murine leukemia) proto-oncogene on chromosome 9 to the bcr (breakpoint cluster region) gene on chromosome 22 creates a transcriptionally active, chimeric bcr-abl gene and gives rise to the Philadelphia chromosome. 1&#x0D; The genetic hallmark of chronic myeloid leukemia (CML) is the Philadelphia chromosome (Ph), which occurs in at least 95% of CML cases as well as some cases of acute lymphocytic leukemia (ALL; approximately 5% for children, 20% for adults). It also has been reported in some other hematologic disorders, albeit rarely. 2-4 JMS 2016; 19(2):95-96

CD117 (c-kit) Expression on CD34+ Cells Participates in the Cytogenetic Response to Imatinib in Patients with Chronic Myeloid Leukemia in the First Chronic Phase

Background: Chronic myeloid leukemia (CML) biology seemed to be perfectly explored especially at the beginning of the tyrosine kinase inhibitors era. Later years with imatinib and second-generation tyrosine kinase inhibitors showed a variety of resistance mechanisms and it became obvious that the bcr-abl chimeric gene is not the only enemy to fight. Some studies assumed the decreased rate of programmed cell death (apoptotic) to be the primary mechanism by which BCR-ABL affects expansion of the leukemic clone in CML. Therefore, the aim of this study was to investigate the role of c-kit inhibition in treatment response. Methods: Cytogenetic analysis, real-time quantitative reverse-transcriptase polymerase chain reaction, flow-cytometric analysis and imatinib serum level quantification were applied. Results: The percentage of CD34+ cells expressing c-kit (CD117) isolated from bone marrow samples of 54 CML patients treated with standard-dose imatinib was significantly lower among imatinib responders. The fraction of apoptotic CD34+CD117+ cells in this patient group was significantly higher than in nonresponders. Conclusion: To achieve optimal treatment response in CML patients, the elimination of CD34+CD117+ may be necessary through an apoptotic pathway.

CD117 (c-kit) Expression On CD34+ Cells Participates In The Cytogenetic Response To Imatinib In CML Patients In First Chronic Phase

BackgroundChronic myeloid leukemia (CML) biology seemed to be perfectly explored especially at the beginning of tyrosine kinase inhibitors era (TKI). Later years with imatinib and second generation TKI showed variety of resistance mechanisms and it became obvious bcr-abl chimeric gene is not the only enemy to fight. Some studies assumed the decreased rate of programmed cell death (apoptotic) to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML. Other studies showed the important role of patients adherence in achieving best possible response for imatinib. Imatinib plasma level was recognized as a useful tool for adherence evaluation. In this study we evaluate the expression of apoptotic marker, Annexin V in CD34+CD117+ cells of CML patients in first chronic phase treated with imatinib and try to find the correlation between this expression and cytogenetic response or imatinib plasma level. PatientsThe group of 54 CML patients (F/M = 30/24, median age, 50.5) in first chronic phase treated with imatinib (400 mg daily) was analyzed. The median time of treatment was 17 months (min. 12 months, max. 24 months. Only patients with the conventional translocation (Philadelphia chromosome) without additional chromosomal aberrations or clonal evolution during the treatment period were included in the study. Patients were categorized according to ELN criteria for cytogenetic response: complete cytogenetic response (CCyR) vs. no cytogenetic response (NCyR, defined as everything less than CCyR), and for molecular response: major molecular response (MMR) vs. no molecular response (NMR). ResultsIn the cohort of 54 patients, 39 achieved CCyR in a median time of 12 months. Among these patients, 30 achieved MMR within the same time. Bone marrow CD34 positive cells were assessed for expression of CD117 and Annexin V in all groups of patients (CCyR with MMR, CCyR with NMR, and NCyR). The mean percentage of CD34+CD117+ cells was significantly higher in the NCyR group (7.67±5.62) in comparison with the CCyR group (2.27±1,78; p=0.002). The difference between MMR and NMR subgroups was not significant. While analyzing the CD34+CD117+ population, we found a significantly higher percentage of apoptotic cells (Annexin V positive) in the CCyR group (4.65±4.55) than in the NCyR group (1.67±1.22; p=0.004). Once again this difference was not significant between MMR and NMR subgroups. Serum imatinib levels were quantified in both CCyR and NCyR groups. We found higher values in the CCyR group (1244 μg/l±599) than in the NCyR group (1192 μg/l±593) but this difference was insignificant. ConclusionsIt was recently reported that c-kit must be inhibited to allow apoptosis of CML cells. Our results also correspond with these data. Not only was a lower percentage of CD34+CD117+ cells found in the CCyR group, but the fraction of these cells that were apoptotic was significantly greater compared with the NCyR group. Although other studies have indicated that trough plasma concentration of imatinib reflects clinical response in chronic phase of CML, we did not observe this. Our results showed higher imatinib levels in CCyR, but these data were insignificant. In conclusion, our results indicate that to achieve optimal treatment response in CML patients, c-kit kinase inhibition may be a requirement for successful proapoptotic activity of imatinib. Disclosures:No relevant conflicts of interest to declare.

Improving the Quality of Quantitative Real-Time Polymerase Chain Reaction Laboratory Reporting in Chronic Myeloid Leukemia

Molecular pathologists play a central role in monitoring disease response in patients with chronic myeloid leukemia (CML). Imatinib, nilotinib, and dasatinib regimens have been shown to be highly effective therapies in the management of this disease. Optimal benefit from these agents depends on regular molecular monitoring of disease burden, achieved through measurements of transcript levels of the BCR-ABL chimeric gene. Major molecular response is a key milestone that correlates with improved long-term outcomes and has recently been shown to predict increased survival. Because of the clinical importance of accurate BCR-ABL monitoring, laboratory reports of BCR-ABL levels must be accurate, relevant, and readily understood by physicians. We discuss herein the key areas for optimizing BCR-ABL reports and emerging technologies for standardization and automation of these processes. * CML : chronic myeloid leukemia ABL1 : c-abl oncogene 1 BCR : breakpoint cluster gene Ph : Philadelphia chromosome NCCN : National Comprehensive Cancer Network ELN : European LeukemiaNet qRT-PCR : quantitative real-time polymerase chain reaction MMR : major molecular response IRIS : International Randomized Study of Interferon and STI571 CMR : complete molecular response EAC : Europe Against Cancer ABL : Abelson B2M : beta-2-microglobulin GUSB : beta-glucuronidase IS : International Scale CAP : College of American Pathologists TKI : tyrosine kinase inhibitor LOD : limit of detection NCN : normalized copy number

Molecular interactions of c-ABL mutants in complex with imatinib/nilotinib: a computational study using linear interaction energy (LIE) calculations

In spite of the effectiveness of Imatinib for chronic myeloid leukemia (CML) treatment, resistance has repeatedly been reported and is associated with point mutations in the BCR-ABL chimeric gene. To overcome this resistance, several inhibitors of BCR-ABL tyrosine kinase activity were developed. In this context, computational simulations have become a powerful tool for understanding drug-protein interactions. Herein, we report a comparative molecular dynamics analysis of the interaction between two tyrosine kinase inhibitors (imatinib or nilotinib) against wild type c-ABL protein and 12 mutants, using the semi-empirical linear interaction energy (LIE) method, to assess the feasibility of this approach for studying resistance against the inhibitory activity of these drugs. In addition, to understand the structural changes that are associated with resistance, we describe the behavior of water molecules that interact simultaneously with specific residues (Glu286, Lys271 and Asp381) of c-ABL (wild type or mutant) and their relationship with drug resistance. Experimental IC50 values for the interaction between imatinib, wild type c-ABL, and 12 mutants were used to obtain the proper LIE coefficients (α, β and γ) to estimate the free energy of the binding of imatinib with wild-type and mutant proteins, and values were extrapolated for the analysis of the nilotinib/c-ABL interaction. Our results indicate that LIE was suitable to predict the superior inhibitory activity of nilotinib and the resistance to inhibition that was observed in c-ABL mutants. Additionally, for c-ABL mutants, the observed number of water molecules being turned over while interacting with amino acids Glu286, Lys271 and Asp381 was associated with resistance to imatinib, resulting in a less effective inhibition of the kinase activity.

Expression of CAMKIIγ, HSP70 and HSP90 Genes Are Differentially Associated with the Mutational Status of Tirosine Kinase Domain in Chronic Myeloid Leukemia Resistant Patients

Abstract 4877Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by the reciprocal chromosomal translocation between chromosome 9 and 22, t(9;22)(q34;q11), known as Philadelphia chromosome. At molecular level, this cytogenetic aberration associates with a chimeric BCR-ABL fusion gene responsible for the pathogenesis of the disease. Targeting BCR-ABL with tyrosine kinase inhibitors (TKIs) has led to a rapid clinical response in most CML cases; however, harboring of point mutations within the kinase domain (KD) of BCR-ABL is the most common mechanism responsible for acquired resistance to therapy.The objective of this work was to evaluate the expression profiles of 4 different genes involved in proliferation, differentiation or cell survival, in order to study their association with acquired missense mutations within the KD of BCR-ABL.Due to its clear involvement in cell proliferation and apotosis the following genes were analyzed. CAMKIIγ (Ca2+ /calmodulin dependent protein kinase IIγ) is a critical regulator of multiple signalling networks regulating the proliferation in myeloid leukaemia. HSP70 (Heat Shock protein 70) is a chaperone protein that protects cells from apoptotic and necrotic stimuli. HSP90 (Heat Shock protein 90) is a chaperone that facilitates folding of client proteins such as BCR-ABL. Ki-67 is a nuclear antigen associated with cellular proliferation and ribosomal RNA transcription.Total RNA was extracted from 50 TKI-resistant CML-patient’s PBMC samples; by quantitative real time-PCR (QRT-PCR) (SybrGreen method, melting analysis and β2-microglobulin as an endogenous control reference gene) we measured the gene expression of CaMKIIγ, Ki67, HSP70, HSP90 and BCR-ABL. All patients were treated with TKIs (26 imatinib, 13 nilotinib, 11 dasatinib) and showed primary lack or loss of haematological and/or cytogenetic response according to the ELN (European Leukaemia Net) criteria.To characterize TKI-resistant mutations within the KD of the chimeric BCR-ABL gene, ABL exons 4, 5, 6 and 7 were subjected to automated DNA sequencing following a nested- BCR-ABL-specific PCR. Mutations were observed in 27/50 cases at 15 different residues: 1 L248V, 1 G250E, 1 Y253H, 5 E255K/V, 1 E279K, 1 V289F, 4 T315I, 2 F317L, 1 L348M, 3 M351T, 1 E355G, 1 N358S, 2 F359V/C, 2 L387M and 1 L389V. Remaining 23 CML cases did not show mutations (detection limit 10–20%).Our results showed a significant increase in the expression of CaMKIIγ and HSP70 and decrease of HSP90 in mutated patients (MT) with respect to cases without mutations (WT) (p<0.01). On the contrary, transcript levels of Ki67 and BCR-ABL did not show significant differences between MT and WT, likely due to the resistant status of both groups.Taking into account these results we design a score (TKI-MT) to estimate the likelihoods for a patient to harbor TKI-resistance mutations using the transcript normalised expression of CaMKIIγ, HSP70 and HSP90 as follows: [Log10(CaMKIIγ) + Log10(HSP70) – Log10(HSP90)]. TKI-MT scores from 27 and 23 patients from the MT and WT groups respectively were analysed by use of ROC curves in order to find an optimal cut-off value to classify new unstudied cases. We found that patients with TKI-MT scores over a cut-off value of −0.79 showed 4.8 times more probabilities to present TKI-resistance mutations than those below −0.79 (OR 4.8, CI95% 1.3–17.6, P<0.02).We concluded that the expression of CaMKIIγ, HSP70 and HSP90 allowed prediction of mutations in the ABL tyrosine kinase domain with 82% of specificity in CML patients treated with TKIs and associated with lack or loss of response. Disclosures:No relevant conflicts of interest to declare.

A Bead-Based Activity Screen for Small-Molecule Inhibitors of Signal Transduction in Chronic Myelogenous Leukemia Cells

Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system in which molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by nonlinear regression from well-specific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing.

Targeting the BCR–ABL tyrosine kinase in chronic myeloid leukemia as a model of rational drug design in cancer

Many biological and clinical features of chronic myeloid leukemia make it as a paradigm of rational drug design in human cancer. Chronic myeloid leukemia was the first malignancy to be linked to a clear genetic anomaly, the Philadelphia chromosome and, at present, it is probably the best understood of all human malignancies. Studies of the disease pathology revealed that the molecular consequence of the Philadelphia translocation is a BCR–ABL chimeric gene, which encodes a constitutively active tyrosine kinase with wholesale range of biological activities. Animal models have validated the direct role of the BCR–ABL protein in malignant transformation and subsequent research confirmed that its enhanced tyrosine kinase activity is essential and sufficient for the leukemogenesis. The very existence of a single genetic abnormality, presented in essentially all patients with the disease, made it an effective target for molecularly designed therapeutic approaches for the disease. The advent of tyrosine kinase inhibitors, designed specifically to inhibit the tyrosine kinase activity of the BCR–ABL oncoprotein, represents one of the major innovations in cancer therapy and may serve as a model for, how discoveries of disease pathogenesis may be translated into the development of successful targeted therapies in cancer medicine.

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

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

Suppression of bcr-abl mRNA by Chemically Modified siRNA

Synthesis of 21 nt siRNAs bearing chemically modified dangling ends with a novel nucleobase was achieved. Evaluation of gene silencing of bcr-abl chimeric gene derived from Philadelphia chromosome by thus obtained chemically modified siRNAs was performed using human leukaemia cell line K-562 and resulted in efficient suppression of the targeted gene. siRNAs whose sense strands were modified with a novel base were found to be more effective than a native siRNA and that siRNAs whose antisense strands were modified with a novel base showed largely decreased silencing effects on the contrary.

Functional genomics of hTERT gene in leukemic myelopoiesis

Keeping in view the fact that a single acquired genetic abnormality "Bcr-Abl chimeric gene" accompanied by elevated telomerase activity has been widely recognized to be responsible for the leukemic myelopoiesis observed in chronic myeloid leukemia (CML), the present study was addressed to understand as to how selective and specific knock-down of human telomerase reverse transcriptase (hTERT) gene within mononuclear cells derived from untreated CML subjects could influence the apoptotic, genotypic (such as Bcr-Abl; C-myc; Bcl-2; IL-6; GMCSF; IL-3; and acetylated H(3) and H(4)), and phenotypic (such as CD34 and CD89) characteristics of these cells. Based upon these results, we propose that hTERT gene-based drug design may be useful in the treatment of leukemic myelopoiesis.

Dasatinib: a new step in molecular target therapy

The chimeric BCR-ABL gene, originated by the Philadelphia chromosome, encodes a fusion protein, BCR-ABL, bearing unregulated tyrosine kinase activity, the pivotal pathogenetic step of chronic myeloid leukemia (CML). Imatinib, an inhibitor of the BCR-ABL tyrosine kinase, significantly improves the outcome of patients with CML. Although the majority of CML patients are responsive to imatinib, a subset of patients loses the response and some progress to accelerated- or blast-phase CML. The understanding of mechanisms of imatinib resistance has led to the development of novel BCR-ABL inhibitors; among these, dasatinib emerged as the most promising, being approximately 300-fold more potent than imatinib; it also inhibits SRC family kinases. Preliminary data, after the introduction of dasatinib in clinical trials, in patients with CML, suggest that this drug is safe and well tolerated; furthermore, the majority of patients with imatinib-resistant disease achieved objective responses, although the durability of responses remains to be defined. Recently, dasatinib emerged as a potent inhibitor of imatinib-resistant protein tyrosin kinase (KIT) activation loop mutants and it is able to induce apoptosis in mast cell and leukemic cell lines expressing these mutations. The preclinical data concerning its activity on several human solid tumor lines widen new opportunities for their use outside CML.

Reduction of Bcr‐Abl Function Leads to Erythroid Differentiation of K562 Cells via Downregulation of ERK

The chimeric bcr-abl gene encodes a constitutively active tyrosine kinase that leads to abnormal transduction of growth and survival signals leading to chronic myeloid leukemia (CML). According to our previous observations, in vitro differentiation of several erythroid cell lines is accompanied by the downregulation of extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase (MAPK) activities. In this work we investigated whether ERKs have a decisive role in either the erythroid differentiation process or apoptosis of bcr-abl+ K562 cells by means of direct (MEK1/2 inhibitor UO126) and indirect (reduced Bcr-Abl function) inhibition of their activities. We found that both Gleevec and UO126 induced hemoglobin expression. Gleevec treatment reduced the phosphorylation of Bcr-Abl, ERK and STAT-5 for up to 24 h, decreased Bcl-XL levels, and induced caspase-3-dependent apoptosis. In contrast, UO126 treatment resulted in only a transient decrease of ERK activity and did not induce cell death. For studying the effect of reduced Bcr-Abl function on erythroid differentiation at the level of the bcr-abl transcript, we applied the siRNA approach. Stable degradation of bcr-abl mRNA was achieved by using a retroviral vector with enhanced green fluorescent protein (EGFP) reporter. Despite a high (>90%) transduction efficiency we detected only a transient decrease in Bcr-Abl protein and in phosphorylated ERK1/2 levels. This transient change in Bcr-Abl signaling was sufficient to induce hemoglobin expression without significant cell death. These results suggest that by transiently reducing Bcr-Abl function it is possible to overcome the differentiation blockade without evoking apoptosis in CML cells and that reduced ERK activity may have a crucial role in this process.

Galectin-1 Is Overexpressed in Myeloid Cells and There Is a Significant Different Overexpression in Cells Carrying the BCR-ABL Chimeric Gene.

The galectins are a family of lectins, beta-galactoside sugar chain binding proteins, that have been identified as mediator of cell adhesion, cell growth regulators, and as triggers or inhibitors of apoptosis. Their expression pattern is deregulated during inflammation and in breast, colon, prostate, and thyroid cancers and their overexpression correlates to metastatic potential. Galectin-1 is a pleiotropic dimeric protein participating in cancer progression. The gene encoding galectin-1 is located in an 11-kb region on chromosome 22q13.1. Induction of apoptosis in activated T lymphocytes is a well-known function of this galectin. The association of accumulation of galectin-1 in the stroma of malignant tissue and aggressiveness of the tumor suggested a role for this lectin in the acquisition of the invasive phenotype and may act as an immunological shield by inducing activated T-cell apoptosis. To be susceptible to galectin-1 induced apoptosis, the T cell must express specific glycoprotein receptors, such as CD7, that bear the specific oligosaccharides recognized by galectin-1. The sensitivity to galectin-1 is associated with repression of the endogenous galectin-1 gene whereas non-sensitive cells express high levels of galectin-1. The current analysis has been performed in myeloid cells negative or positive for BCR-ABL, a chimeric gene derived by the t(9;22), that is present in chronic myeloid leukemia patients. The BCR-ABL gene, the expression of which was governed by the tetracycline inducible promoter, has been introduced by transfection in a myeloid cell line. Total RNA for microarray analysis, in presence or absence of BCR-ABL gene expression, has been isolated. The comparison involved ~5300 transcripts. A comprehensive galectin fingerprinting in these cells by microarray gene expression analysis to define the pattern in BCR-ABL positive and negative cells has been done. The results showed in the table clearly demonstrated that myeloid cells express more mRNA species for galectin-1 than for galectin-9 while none of the other lectins belonging to this family are expressed in either BCR-ABL positive or negative cells. A significant statistical difference has been found between the levels of expression of galectin-1 compared to galectin-9. A higher significant statistical difference has been found in the levels of galectin-1 overexpression in BCR-ABL positive cells. To better understand the preliminary data, a time course after the addition of tetracycline to the 32Dtetp210Bcr-Abl cells has been carried out. This last experiment demonstrated that galectin expression levels decrease over 18 hours and the expression of galectin-9 is already absent after 6 hours from the addition of tetracycline to the culture medium while the expression of galectin-1 is still present after 12 hours but at a level comparable to normal cells. These results might suggest a role for the modulation of galectin-1 activity as a new strategy for molecular treatment of BCR-ABL positive leukemia.Galectins gene expression profile in myeloid cellsBCR-ABL negativeBCR-ABL positiverawt-test p-valueflagsrawt-test p-valueflagsGal97760,238P520,20,347PGal715,80,874A13,20,849AGal639,60,928A61,90,962AGal871,21A51,20,937AGal360,853A4,60,83AGal15.652,800,0315P12.087,300,0142P

Leucémie myéloïde chronique

Chronic myelogenous leukaemia (CML) is a rare myeloproliferative disorder (5 to 15% of all cases of leukaemia). Nevertheless, CML was the first model to study leukemogenesis because leukaemic cells are characterised by a small chromosome 22 called the Philadelphia chromosome. This chromosome results from the reciprocal chromosome translocation t(9 ;22). This cytogenetic abnormality leads to the chimeric BCR-ABL gene which is the molecular counterpart of the t(9;22). In vitro and in vivo studies have demonstrated that BCR-ABL protein drives the leukaemic process. Usually, without any treatment, CML remains in a chronic phase for 3 to 5 years and then turns out in acute leukaemia leading quickly to death. Treatments such as busulfan or hydroxydurea do not prolong the survival. Allogenic stem cell transplantation (SCT) may cure the disease but is available only for few patients. Recombinant alpha interferon improves the survival in responding patients but the proportion of these patients is very low. Imatinib mesylate is now the gold standard for CML treatment. It is also the first inhibitor of tyrosine kinase developed in haematology. Imatinib mesylate is used as a model for targeted therapies. Long-term follow up of patients treated by imatinib mesylate is necessary to redefine the place of allogenic SCT in the future.