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Hagop M Kantarjian - One of the best experts on this subject based on the ideXlab platform.
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triptolide induces cell death independent of cellular responses to imatinib in blast crisis chronic myelogenous leukemia cells including quiescent cd34 primitive progenitor cells
Molecular Cancer Therapeutics, 2009Co-Authors: Duncan H Mak, Hagop M Kantarjian, Michael Andreeff, Jorge E Cortes, Wendy D Schober, Wenjing Chen, Marina Konopleva, Bing Z CarterAbstract:The advent of BCR-ABL tyrosine kinase inhibitors (TKI) has revolutionized the treatment of chronic myelogenous leukemia (CML). However, resistance evolves due to BCR-ABL mutations and other mechanisms. Furthermore, patients with blast crisis CML are less responsive and quiescent CML stem cells are insensitive to these inhibitors. We found that triptolide, a diterpenoid, at nanomolar concentrations, promoted equally significant death of KBM5 cells, a cell line derived from a BCR-ABL–bearing blast crisis CML patient and KBM5STI571 cells, an imatinib-resistant KBM5 subline bearing the T315I mutation. Similarly, Ba/F3 cells harboring mutated BCR-ABL were as sensitive as Ba/F3BCR-ABLp210wt cells to triptolide. Importantly, triptolide induced apoptosis in primary samples from blast crisis CML patients, who showed resistance to BCR-ABL TKIs in vivo , with less toxicity to normal cells. Triptolide decreased X-linked inhibitor of apoptosis Protein, Mcl-1, and BCR-ABL Protein levels in K562, KBM5, and KBM5STI571 cells and in cells from blast crisis CML patients. It sensitized KBM5, but not KBM5STI571, cells to imatinib. More importantly, triptolide also induced death of quiescent CD34+ CML progenitor cells, a major problem in the therapy of CML with TKIs. Collectively, these results suggest that triptolide potently induces blast crisis CML cell death independent of the cellular responses to BCR-ABL TKIs, suggesting that triptolide could eradicate residual quiescent CML progenitor cells in TKI-treated patients and benefit TKI-resistant blast crisis CML patients. [Mol Cancer Ther 2009;8(9):2509–16]
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an immunological method for the detection of BCR ABL fusion Protein and monitoring its activation
Leukemia Research, 2008Co-Authors: Iman Jilani, Hagop M Kantarjian, Homan Faraji, Jorge E Cortes, Oliver G Ottmann, Kapil Bhalla, Susan Obrien, Francis J Giles, M Gorre, Maher AlbitarAbstract:Abstract We have developed a simplified sandwich immunoassay to measure free circulating total and phosphorylated fusion BCR-ABL Protein in patients with the t(9;22)(q34;q11) chromosomal translocation. The assay is based on immunoprecipitating BCR-ABL Protein using beads coated with anti-BCR antibody and detecting the fusion Protein with anti-ABL antibody and flow cytometry. We show that this method allows the quantification of this Protein in the plasma and may allow the measurement of tumor load. This method also allows the measurement of the level of phosphorylation of the immunoprecipitated BCR-ABL using antibodies against phosphorylated ABL Protein, which can be used for monitoring of therapy with kinase inhibitors. The sensitivity of this immunoassay was comparABLe to the sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) assay. This technique is useful in monitoring patients with chronic myeloid leukemia (CML) or acute lymphoblastic leukemia (ALL), but the same approach can be used in other translocations and has the potential of multiplexing.
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measurement of free circulating BCR ABL fusion Protein and its phosphorylation in patients with chronic myeloid leukemia
Blood, 2005Co-Authors: Iman Jilani, Mercedes E Gorre, Hagop M Kantarjian, Homan Faraji, Jorge E Cortes, Oliver G Ottmann, Kapil Bhalla, Susan Obrien, Francis J Giles, M AlbitarAbstract:Diagnosis and monitoring of therapy in chronic myeloid leukemia (CML) depend on cytogenetic, FISH, and PCR assays for detecting the fusion BCR-ABL gene. However, the there is an inherent variability and difficulty in standardizing quantitative PCR-based assays. We assessed the utility of a simplified immunoassay for measuring levels of BCR-ABL Protein and phosphorylated BCR-ABL for diagnosis of CML and detection of minimal residual disease (MRD). BCR-ABL Protein was immunoprecipitated on beads with anti-BCR antibody, and the fusion Protein was detected with anti-ABL antibody. Phosphorylation of the immunoprecipitated BCR-ABL was measured using antibodies against phosphorylated ABL Protein at tyr245 and thr735. Because the relatively high turnover of leukemic cells enriches plasma with leukemic DNA, RNA, and Protein, as we previously reported, this assay can use cell lysate as well as PB plasma. In vitro studies with denatured K562 cells in plasma showed reliABLe detection at levels as low as 10 cells/mL plasma. After exposure of cell cultures to 0.5 μM imatinib, K562 cell lysates exhibited a decreased ratio of phosphorylated BCR-ABL Protein:total BCR-ABL Protein with both the tyr245 (from 0.53 to 0.11) and the thr735 (from 0.36 to 0.08) antibodies. The assay was validated by demonstrating negativity (specificity) in 116 normal control subjects (or patients with other translocations) and positivity in all 380 positive samples, as confirmed by FISH or cytogenetics. The CV for inter- and intra-assay variation was 0.05) had significantly more relative phosphorylated BCR-ABL Protein than those with low disease activity ( P =0.05). In conclusion, these findings demonstrate that BCR-ABL and phosphorylated BCR-ABL are detectABLe at significant levels in the plasma of CML patients and that these levels can be used to monitor MRD. This free circulating BCR-ABL Protein is most likely complexed with other Proteins. Unlike PCR-based assays, our ELISA-like assay can be standardized for use in various laboratories. More importantly, the CV of the immunoassay (<6%) is much lower than that typically reported for RT-PCR (up to 30%). We speculate that the use of plasma rather than cells in this assay allows more accurate quantitative measurement of the disease in the whole body, without potential bias due to sampling errors.
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imatinib mesylate resistance through BCR ABL independence in chronic myelogenous leukemia
Cancer Research, 2004Co-Authors: Nicholas J Donato, Ralph B Arlinghaus, Hagop M Kantarjian, Kimberly Hayes, Jonathan Stapley, Hui Lin, Bharat B Aggarwal, Shishir Shishodin, Maher Albitar, Moshe TalpazAbstract:Imatinib mesylate (IM) binds to the BCR-ABL Protein, inhibiting its kinase activity and effectively controlling diseases driven by this kinase. IM resistance has been associated with kinase mutations or increased BCR-ABL expression. However, disease progression may be mediated by other mechanisms that render tumor cells independent of BCR-ABL. To demonstrate this potential, IM-resistant cells were found in chronic myelogenous leukemia patients with continuous BCR-ABL gene expression but undetectABLe BCR-ABL Protein expression. These cells were unresponsive to IM and acquired BCR-ABL-independent signaling characteristics. IM resistance in some patients may be mediated through loss of kinase target dependence.
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philadelphia chromosome positive leukemias from basic mechanisms to molecular therapeutics
Annals of Internal Medicine, 2003Co-Authors: Razelle Kurzrock, Hagop M Kantarjian, Brian J Druker, Moshe TalpazAbstract:The Philadelphia chromosome translocation (t(9;22)) results in the molecular juxtaposition of two genes, BCR and ABL, to form an aberrant BCR-ABL gene on chromosome 22. BCR-ABL is critical to the pathogenesis of chronic myelogenous leukemia and a subset of acute leukemias. The chimeric BCR-ABL Protein has constitutively elevated tyrosine phosphokinase activity. This abnormal enzymatic activation is critical to the oncogenic potential of BCR-ABL. Initially, Protein kinases were thought to be poor therapeutic targets because of their ubiquitous nature and crucial role in many normal physiologic processes. However, the advent of imatinib mesylate (Gleevec, Novartis Pharmaceuticals, Basel, Switzerland), formerly known as STI571 and CGP57148B, demonstrated that designer kinase inhibitors could be specific. This agent has shown striking activity in chronic myelogenous leukemia. It also inhibits phosphorylation of Kit (stem-cell factor receptor) and platelet-derived growth factor receptor. In addition, it has shown similar impressive responses, with little host toxicity, in gastrointestinal stromal tumors, which harbor activating Kit mutations, and in tumors with activated platelet-derived growth factor receptor. The studies of imatinib mesylate provide proof-of-principle for using aberrant kinases as a therapeutic target and are a model for the promise of molecular therapeutics. This paper reviews the current knowledge on the function of BCR-ABL and its normal counterparts (BCR and ABL), as well as the impact of this knowledge on the development of a remarkABLy successful targeted therapy approach.
Moshe Talpaz - One of the best experts on this subject based on the ideXlab platform.
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degrasyn a novel tyrophostin impacts BCR ABL Protein level and is cytotoxic to chronic myeloid leukemia early progenitors
Blood, 2008Co-Authors: Luke F Peterson, Hanshi Sun, William G Bornmann, Zhenghong Peng, David Maxwell, Nicholas J Donato, Moshe TalpazAbstract:Abstract Despite the potent anti-proliferative and apoptotic effects of tyrosine kinase inhibitors (imatinib, dasatinib) on mature CML cells these compounds have limited activity on Chronic Myeloid Leukemia (CML) early progenitors (Lin-/CD38−/CD34+). Mechanisms of resistance to these agents in the early progenitor population are poorly understood but are distinct from BCR-ABL mutation induced resistance. Degrasyn (WP1130) was previously shown by our laboratory to inhibit CML cell growth and colony formation. Further structure-activity relationship studies led to the development of a more potent compound, CP2005 (CP). CP displays enhanced activity in suppressing growth and activating apoptosis in various CML and ALL cell lines (IC50 ~1 microM). Here we show that total CD34+ from CML patients are more effectively growth inhibited by CP2005 compared to WP1130. An anti-leukemic effect was also noted in primitive progenitors and was associated with the induction of apoptosis. BaF3 cells expressing an EGFP-tagged BCR-ABL Protein treated with CP or WP1130 show that both facilitate translocation of BCR-ABL from the cytoplasm to a detergent insoluble compartment which subsequently leads to degradation of BCR-ABL. We observed that the Akt/mTOR pathway is activated by CP which could be partially suppressed by mTOR or PI3K/Akt inhibition (rapamycin and wortmannin, respectively). CP-induced BCR-ABL translocation was associated with a reduction in the level of tyrosine-phosphorylated Stat3 and Stat5. However, although there is activation of survival signals associated with Akt-signaling 2 microM CP is ABLe to promote significant apoptosis (67%) as a single agent in CML early progenitors. Although imatinib was minimally effective in reducing the early progenitor cell survival, co-incubation of imatinib with CP2005 resulted in near complete induction of apoptosis in early progenitor cells (~ 93%), suggesting that the combined effects of both CP and imatinib on BCR-ABL Protein degradation and kinase inhibition markedly impair the survival of CML early progenitors. The combination of BCR-ABL degradation and kinase inhibition may be effective in engaging pro-apoptotic signaling in CML early progenitor cells.
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imatinib mesylate resistance through BCR ABL independence in chronic myelogenous leukemia
Cancer Research, 2004Co-Authors: Nicholas J Donato, Ralph B Arlinghaus, Hagop M Kantarjian, Kimberly Hayes, Jonathan Stapley, Hui Lin, Bharat B Aggarwal, Shishir Shishodin, Maher Albitar, Moshe TalpazAbstract:Imatinib mesylate (IM) binds to the BCR-ABL Protein, inhibiting its kinase activity and effectively controlling diseases driven by this kinase. IM resistance has been associated with kinase mutations or increased BCR-ABL expression. However, disease progression may be mediated by other mechanisms that render tumor cells independent of BCR-ABL. To demonstrate this potential, IM-resistant cells were found in chronic myelogenous leukemia patients with continuous BCR-ABL gene expression but undetectABLe BCR-ABL Protein expression. These cells were unresponsive to IM and acquired BCR-ABL-independent signaling characteristics. IM resistance in some patients may be mediated through loss of kinase target dependence.
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Imatinib mesylate causes hypopigmentation in the skin
Cancer, 2003Co-Authors: Anne S. Tsao, Hagop Kantarjian, Jorge Cortes, Susan O'brien, Moshe TalpazAbstract:BACKGROUND Imatinib mesylate is a tyrosine kinase inhibitor that targets the BCR-ABL Protein in CML, c-kit (KIT) and platelet-derived growth factor receptors. In clinical trials with imatinib mesylate, common side effects of nausea, emesis, diarrhea, periorbital edema, fluid retention, and myelosuppression have been documented. METHODS In this case series, the authors describe unique clinical findings of skin hypopigmentation in six patients with CML who were treated with imatinib mesylate. RESULTS Most patients developed onset of skin hypopigmentation within the first month of treatment and all of the patients experienced additional drug toxicity. Despite patient susceptibility to toxicity, the presence of hypopigmentation did not appear to predict leukemic cell response or clinical outcome. All six patients estABLished a hematologic response but only two patients had a complete cytogenetic response. Imatinib mesylate induced hypopigmentation also appeared to be reversible and potentially dose related. CONCLUSION Skin hypopigmentation is a benign side effect from imatinib mesylate treatment that appears to be reversible upon discontinuation or dose reduction. Several lines of evidence have previously reported that KIT and its ligand stem cell factor (SCF) have a regulatory role in melanocyte development and survival, suggesting a rational mechanism of action for imatinib mesylate in the pathogenesis of hypopigmentation. The signal transduction mechanism currently is believed to involve SCF ligand binding of KIT and downstream activation of MAP kinase (Erk-2). Microphthalmia (Mi), a basic helix-loop-helix leucine zipper (bHLHZip) transcription factor, is phosphorylated by MAP kinase at a serine residue (S73). Once phosphorylated, Mi transactivates the tyrosine pigmentation gene promoter and affects pigment production. Cancer 2003. © 2003 American Cancer Society.
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philadelphia chromosome positive leukemias from basic mechanisms to molecular therapeutics
Annals of Internal Medicine, 2003Co-Authors: Razelle Kurzrock, Hagop M Kantarjian, Brian J Druker, Moshe TalpazAbstract:The Philadelphia chromosome translocation (t(9;22)) results in the molecular juxtaposition of two genes, BCR and ABL, to form an aberrant BCR-ABL gene on chromosome 22. BCR-ABL is critical to the pathogenesis of chronic myelogenous leukemia and a subset of acute leukemias. The chimeric BCR-ABL Protein has constitutively elevated tyrosine phosphokinase activity. This abnormal enzymatic activation is critical to the oncogenic potential of BCR-ABL. Initially, Protein kinases were thought to be poor therapeutic targets because of their ubiquitous nature and crucial role in many normal physiologic processes. However, the advent of imatinib mesylate (Gleevec, Novartis Pharmaceuticals, Basel, Switzerland), formerly known as STI571 and CGP57148B, demonstrated that designer kinase inhibitors could be specific. This agent has shown striking activity in chronic myelogenous leukemia. It also inhibits phosphorylation of Kit (stem-cell factor receptor) and platelet-derived growth factor receptor. In addition, it has shown similar impressive responses, with little host toxicity, in gastrointestinal stromal tumors, which harbor activating Kit mutations, and in tumors with activated platelet-derived growth factor receptor. The studies of imatinib mesylate provide proof-of-principle for using aberrant kinases as a therapeutic target and are a model for the promise of molecular therapeutics. This paper reviews the current knowledge on the function of BCR-ABL and its normal counterparts (BCR and ABL), as well as the impact of this knowledge on the development of a remarkABLy successful targeted therapy approach.
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selective inhibition of cell proliferation and BCR ABL phosphorylation in acute lymphoblastic leukemia cells expressing mr 190 000 BCR ABL Protein by a tyrosine kinase inhibitor cgp 57148
Clinical Cancer Research, 1998Co-Authors: Miloslav Beran, Moshe Talpaz, Ralph B Arlinghaus, Susan Obrien, Xiaobo Cao, Zeev Estrov, Sima Jeha, Guozhong Jin, Nicholas B Lydon, Hagop M KantarjianAbstract:The excessive proliferation of the myeloid marrow compartment in Philadelphia chromosome (Ph)-positive acute and chronic leukemias has been largely attributed to a hyperactive and autonomously acting hybrid tyrosine kinase BCR-ABL, a product of the fusion between the second exon of the c-ABL proto-oncogene and 5' portions of the BCR gene on chromosome 22. This specific molecular event, amenABLe to attack with specifically designed inhibitors, has recently been successfully influenced by the drug CGP-57148 in mammalian cells transfected with full-length BCR-ABL gene and expressing full-length p210BCR-ABL Protein, as well as in primary human leukemic cells expressing p210BCR-ABL fusion Protein. In view of the heterogeneity of BCR-ABL transcripts associated with various phenotypes, we investigated the effect of CGP-57148 on p190BCR-ABL- and p210BCR-ABL-expressing, patient-derived cell lines and primary intact blast cells. In particular, we were interested in whether the variations in molecular events and/or the phenotype of Ph-positive cells would affect their susceptibility to the specific tyrosine kinase inhibitor CGP-57148. We have demonstrated that the sensitivity of human cells with lymphoblastic immunophenotype expressing p190BCR-ABL Protein is comparABLe to that for leukemic myeloid cells expressing p210BCR-ABL Protein. After documenting profound and phenotype-independent suppression of both autophosphorylation and cell growth, we explored the importance of time and dose of exposure on the manifestation and stability of the induced events. Although there were variations between target cells, in vitro exposure for 24-48 h induced extensive and apparently irreversible apoptosis in BCR-ABL-expressing but not other normal or BCR-ABL-negative leukemic cells. These findings support the potential use of CGP-57148 to purge Ph-positive cells from autologous bone marrow in vitro. Another important finding was the comparABLe suppressive effect of temporary CGP-57148 exposure on both clonogenic KBM-5 cells and the whole cell population. Exposure time and dose appeared to be important variABLes among various cell types. Moreover, effective doses appeared uniformly harmless to cells lacking BCR-ABL Protein functioning as tyrosine kinase. Thus, the continuous exposure of target cells, at least during the initial period of 24-48 h, may prove to be an important variABLe in the design of in vitro and in vivo therapy using tyrosine kinase inhibitors.
Ralph B Arlinghaus - One of the best experts on this subject based on the ideXlab platform.
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imatinib mesylate resistance through BCR ABL independence in chronic myelogenous leukemia
Cancer Research, 2004Co-Authors: Nicholas J Donato, Ralph B Arlinghaus, Hagop M Kantarjian, Kimberly Hayes, Jonathan Stapley, Hui Lin, Bharat B Aggarwal, Shishir Shishodin, Maher Albitar, Moshe TalpazAbstract:Imatinib mesylate (IM) binds to the BCR-ABL Protein, inhibiting its kinase activity and effectively controlling diseases driven by this kinase. IM resistance has been associated with kinase mutations or increased BCR-ABL expression. However, disease progression may be mediated by other mechanisms that render tumor cells independent of BCR-ABL. To demonstrate this potential, IM-resistant cells were found in chronic myelogenous leukemia patients with continuous BCR-ABL gene expression but undetectABLe BCR-ABL Protein expression. These cells were unresponsive to IM and acquired BCR-ABL-independent signaling characteristics. IM resistance in some patients may be mediated through loss of kinase target dependence.
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selective inhibition of cell proliferation and BCR ABL phosphorylation in acute lymphoblastic leukemia cells expressing mr 190 000 BCR ABL Protein by a tyrosine kinase inhibitor cgp 57148
Clinical Cancer Research, 1998Co-Authors: Miloslav Beran, Moshe Talpaz, Ralph B Arlinghaus, Susan Obrien, Xiaobo Cao, Zeev Estrov, Sima Jeha, Guozhong Jin, Nicholas B Lydon, Hagop M KantarjianAbstract:The excessive proliferation of the myeloid marrow compartment in Philadelphia chromosome (Ph)-positive acute and chronic leukemias has been largely attributed to a hyperactive and autonomously acting hybrid tyrosine kinase BCR-ABL, a product of the fusion between the second exon of the c-ABL proto-oncogene and 5' portions of the BCR gene on chromosome 22. This specific molecular event, amenABLe to attack with specifically designed inhibitors, has recently been successfully influenced by the drug CGP-57148 in mammalian cells transfected with full-length BCR-ABL gene and expressing full-length p210BCR-ABL Protein, as well as in primary human leukemic cells expressing p210BCR-ABL fusion Protein. In view of the heterogeneity of BCR-ABL transcripts associated with various phenotypes, we investigated the effect of CGP-57148 on p190BCR-ABL- and p210BCR-ABL-expressing, patient-derived cell lines and primary intact blast cells. In particular, we were interested in whether the variations in molecular events and/or the phenotype of Ph-positive cells would affect their susceptibility to the specific tyrosine kinase inhibitor CGP-57148. We have demonstrated that the sensitivity of human cells with lymphoblastic immunophenotype expressing p190BCR-ABL Protein is comparABLe to that for leukemic myeloid cells expressing p210BCR-ABL Protein. After documenting profound and phenotype-independent suppression of both autophosphorylation and cell growth, we explored the importance of time and dose of exposure on the manifestation and stability of the induced events. Although there were variations between target cells, in vitro exposure for 24-48 h induced extensive and apparently irreversible apoptosis in BCR-ABL-expressing but not other normal or BCR-ABL-negative leukemic cells. These findings support the potential use of CGP-57148 to purge Ph-positive cells from autologous bone marrow in vitro. Another important finding was the comparABLe suppressive effect of temporary CGP-57148 exposure on both clonogenic KBM-5 cells and the whole cell population. Exposure time and dose appeared to be important variABLes among various cell types. Moreover, effective doses appeared uniformly harmless to cells lacking BCR-ABL Protein functioning as tyrosine kinase. Thus, the continuous exposure of target cells, at least during the initial period of 24-48 h, may prove to be an important variABLe in the design of in vitro and in vivo therapy using tyrosine kinase inhibitors.
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comparison of BCR ABL Protein expression and philadelphia chromosome analyses in chronic myelogenous leukemia patients
American Journal of Clinical Pathology, 1996Co-Authors: J Q Guo, Moshe Talpaz, Jin Ying Lian, Albert B Deisseroth, Armand B Glassman, Hagop M Kantarjian, Ralph B ArlinghausAbstract:The Philadelphia chromosome (Ph) is found in most chronic myelogenous leukemia (CML) patients. The BCR-ABL oncoProtein (P210 or PI85), the product of the fused BCR-ABL gene produced by the Ph, is known to be the major factor in initiation and maintenance of the leukemic state in these types of leukemias. The authors have devised a Western blot test to detect and quantitate the BCR-ABL oncoProtein in blood and bone marrow cells. The authors analyzed 1,155 peripheral blood samples from CML patients, for which there were same day Ph data, to determine if there was a correlation between BCR-ABL Protein levels and the percentage of Ph. A ratio of BCR-ABL Protein (P210 or P185) to normal ABL Protein (P145), the latter is ubiquitously expressed in all blood cells, has been estABLished as a criterion for quantitating BCR-ABL levels. The BCR-ABL / ABL Protein ratio from 399 patient who were 100% Ph-positive had a mean of 2.47 (standard error [SE] of ±0.05); no false negatives were detected. A decreasing ratio was found in patients with de-creasing percentages of Ph. The relationship between the ratio of BCR-ABL / ABL Proteins to the percentage of Ph-positive cells was nearly linear in 392 patients with Ph percentages between 5% to 95% (r = 0.97, P <.001). For patients in remission with no detectABLe Ph, the BCR-ABL / ABL ratio had a mean of 0.01 (SE = 0 ± 0.00). The level of BCR-ABL expression in samples from peripheral blood and bone marrow also were compared. The results indicated that the amount of BCR-ABL Protein within peripheral blood is usually similar to that in marrow. In conclusion, quantitation of the BCR-ABL oncoProtein in peripheral blood cells or marrow cells as measured by a Western blot assay provides reliABLe data for both diagnosis and monitoring patients with Ph-chromosomc positive leukemia.
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BCR ABL Protein expression in peripheral blood cells of chronic myelogenous leukemia patients undergoing therapy
Blood, 1994Co-Authors: Jie Qiang Guo, Moshe Talpaz, Jin Ying Lian, Yong Ming Xian, Ming Sheng Lee, Albert B Deisseroth, Sanford A Stass, Richard E Champlin, Jean Y J Wang, Ralph B ArlinghausAbstract:Chronic myelogenous leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome (Ph1) in more than 95% of these patients. The Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. In CML, the product of the fused BCR-ABL gene is typically a Protein of approximately 2,000 amino acids termed P210 BCR-ABL. We have developed an assay for the BCR-ABL Protein involving Western blotting of circulating white blood cells (WBC) with an anti-ABL monoclonal antibody that can detect P210 BCR-ABL and P145 ABL in peripheral blood cells from chronic phase Ph1-positive leukemia patients. This assay was used to analyze the BCR-ABL Protein content of circulating WBC from CML patients before and after various treatments. In parallel to changes in percentages of Ph1-positive blood cells as determined by cytogenetic analyses of bone marrow samples, BCR-ABL Protein expression in blood cells decreased or increased as patients entered remission or underwent relapse. Of interest, six Ph1-negative CML patients were BCR-ABL Protein-positive. All except one had a rearrangement in the major breakpoint cluster region and that patient expressed P185 BCR-ABL and not P210. Our results indicate that the BCR-ABL Western blotting assay has clinical applications for both diagnosis and prospective evaluation of Ph1-positive and Ph1-negative CML patients.
Giuseppe Saglio - One of the best experts on this subject based on the ideXlab platform.
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Protein kinase ck2 a targetABLe BCR ABL partner in philadelphia positive leukemias
Advances in Hematology, 2015Co-Authors: Alessandro Morotti, Giovanna Carra, Cristina Panuzzo, Sabrina Crivellaro, Riccardo Taulli, Angelo Guerrasio, Giuseppe SaglioAbstract:BCR-ABL-mediated leukemias, either Chronic Myeloid Leukemia (CML) or Philadelphia positive Acute Lymphoblastic Leukemia (ALL), are the paradigm of targeted molecular therapy of cancer due to the impressive clinical responses obtained with BCR-ABL specific tyrosine kinase inhibitors (TKIs). However, BCR-ABL TKIs do not allow completely eradicating both CML and ALL. Furthermore, ALL therapy is associated with much worse responses to TKIs than those observed in CML. The identification of additional pathways that mediate BCR-ABL leukemogenesis is indeed mandatory to achieve synthetic lethality together with TKI. Here, we review the role of BCR-ABL/Protein kinase CK2 interaction in BCR-ABL leukemias, with potentially relevant implications for therapy.
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BCR ABL mrna and the p210 BCR ABL Protein are downmodulated by interferon alpha in chronic myeloid leukemia patients
Blood, 1999Co-Authors: Fabrizio Pane, Giuseppe Saglio, I Mostarda, Carmine Selleri, Rossella Salzano, Anna Maria Raiola, Luigia Luciano, Bruno Rotoli, F SalvatoreAbstract:The BCR/ABL hybrid gene plays a central role in the pathogenesis of the chronic phase of chronic myeloid leukemia (CML). We used a very sensitive quantitative reverse transcriptase-polymerase chain reaction to investigate the levels of hybrid BCR/ABL mRNA in bone marrow cells of 20 patients with Philadelphia positive (Ph(+)) CML treated with interferon-alpha (IFN-alpha) as a single agent. Bone marrow samples were collected at diagnosis and at hematologic remission induced by IFN-alpha, or by hydroxyurea in case of resistance to IFN-alpha. The mean levels of BCR/ABL transcripts in bone marrow mononuclear cells of patients who showed a complete hematologic response to IFN-alpha were significantly reduced with respect to those at diagnosis (48 x 10(3) v 168 x 10(3); P <.001), whereas no difference was detected between the values at diagnosis and at hematologic remission in patients resistant to IFN-alpha. In cell culture experiments, IFN-alpha priming significantly reduced the levels of BCR/ABL hybrid transcripts in a dose-dependent manner in Ph+ bone marrow precursors obtained at diagnosis from patients who subsequently responded to IFN-alpha treatment (P < .005). No downmodulation was observed in bone marrow precursors from patients who subsequently proved to be IFN-resistant. These results indicate that downmodulation of BCR/ABL gene expression could be one of the mechanisms involved in the response of CML patients to IFN-alpha treatment.
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p230 BCR ABL Protein may be associated with an acute leukaemia phenotype
British Journal of Haematology, 1998Co-Authors: Cedrik Haskovec, Carola Ponzetto, Jaroslav Polak, Diego Maritano, Zuzana Zemanova, A Serra, Kyra Michalova, Hana Klamova, Jaroslav Cermak, Giuseppe SaglioAbstract:The BCR/ABL rearrangement, the molecular hallmark of chronic myelogenous leukaemia (CML), is rare in acute myeloid leukaemia (AML), being detected in approximately 1% of cases. In the vast majority of CML cases the breakpoint on chromosome 22 falls in the so-called major breakpoint cluster region of the BCR gene. Only a few cases of CML with breakpoint in the minor or in the micro BCR region have so far been reported. The micro breakpoint position has been associated mainly with a mild form of CML, defined as Philadelphia chromosome-positive chronic neutrophilic leukaemia (Ph-positive CNL). Using reverse transcription-polymerase chain reaction (RT-PCR) we report a patient with an acute myeloid leukaemia phenotype at diagnosis who showed a BCR/ABL rearrangement with a breakpoint located in the micro BCR region (e19a2 junction). Cytogenetic analysis showed a progression of the malignant clone, finally leading to cells with two Ph chromosomes, trisomy 8, isochromosome 17q and deletion of the long arms of chromosome 7. The findings of chromosomal changes point to a possibility of blast crisis of CML with a clinically silent chronic phase. Immunoprecipitation and auto-phosphorylation assay revealed the expression, by the patient's blast cells, of an abnormal P230 BCR/ABL Protein, which showed for the first time that this Protein was constitutively activated in primary cells from patients. This finding may contribute to the understanding of the role of the BCR/ABL rearrangements in determining different leukaemia phenotypes ranging from acute lymphoid and myeloid leukaemias to mild chronic neutrophilic leukaemias.
Anthony D Whetton - One of the best experts on this subject based on the ideXlab platform.
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RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
2016Co-Authors: Anthony D Whetton, Ltb Leukotriene BAbstract:Chronic Myeloid Leukaemia (CML) is a hematopoietic stem cell disease, the hallmark of which is the BCR-ABL Protein tyrosine kinase (PTK). Without intervention the disease progresses from a benign chronic phase to a rapidly fatal blast crisis. To identify the molecular mechanisms underlying disease progression we employed 2-dimensional gel electrophoresis on a model we have previously described using the expression of a conditional mutant of BCR-ABL PTK in a multipotent stem cell line, FDCP-Mix. Long-term exposure of FDCP-Mix cells to BCR-ABL mimics disease progression in CML. Four major differences were observed as a consequence of long-term exposure to the BCR-ABL PTK, compared to cells exposed short-term. The Proteins were identified using MALDI-ToF MS generated peptide mass fingerprint data and liquid chromatography-tandem MS generated sequence information. Leukotriene A4 hydrolase, an enzyme known to be deregulated in CML, was found to be upregulated. Annexin VI, vacuolar ATP synthase catalytic subunit A and mortalin were found to be downregulated. PolyA PCR cDNA analysi
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jak2 stat5 inhibition by nilotinib with ruxolitinib contributes to the elimination of cml cd34 cells in vitro and in vivo
Blood, 2014Co-Authors: Paolo Gallipoli, Anthony D Whetton, Amy Cook, Susan Rhodes, Lisa E M Hopcroft, Helen Wheadon, Heather G Jorgensen, Ravi Bhatia, Tessa L HolyoakeAbstract:Chronic myeloid leukemia (CML) stem cell survival is not dependent on BCR-ABL Protein kinase and treatment with ABL tyrosine kinase inhibitors cures only a minority of CML patients, thus highlighting the need for novel therapeutic targets. The Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)5 pathway has recently been explored for providing putative survival signals to CML stem/progenitor cells (SPCs) with contradictory results. We investigated the role of this pathway using the JAK2 inhibitor, ruxolitinib (RUX). We demonstrated that the combination of RUX, at clinically achievABLe concentrations, with the specific and potent tyrosine kinase inhibitor nilotinib, reduced the activity of the JAK2/STAT5 pathway in vitro relative to either single agent alone. These effects correlated with increased apoptosis of CML SPCs in vitro and a reduction in primitive quiescent CML stem cells, including NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice repopulating cells, induced by combination treatment. A degree of toxicity toward normal SPCs was observed with the combination treatment, although this related to mature B-cell engraftment in NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice with minimal effects on primitive CD34(+) cells. These results support the JAK2/STAT5 pathway as a relevant therapeutic target in CML SPCs and endorse the current use of nilotinib in combination with RUX in clinical trials to eradicate persistent disease in CML patients.
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assessment of downstream effectors of BCR ABL Protein tyrosine kinase using combined proteomic approaches
Proteomics, 2010Co-Authors: Chia Fang Lee, Andrew Pierce, Stephen D Griffiths, Eva Rodriguezsuarez, Richard D Unwin, Ewa Jaworska, Caroline A Evans, Simon J Gaskell, Anthony D WhettonAbstract:Leukaemic transformation is frequently associated with the aberrant activity of a Protein tyrosine kinase (PTK). As such it is of clinical relevance to be ABLe to map the effects of these leukaemogenic PTKs on haemopoietic cells at the level of phosphorylation modulation. In this paradigm study we have employed a range of proteomic approaches to analyse the effects of one such PTK, BCR/ABL. We have employed phosphoproteome enrichment techniques allied to peptide and Protein quantification to identify Proteins and pathways involved in cellular transformation. Amongst the Proteins shown to be regulated at the post-translational level were cofilin, an actin-severing Protein thus linked to altered motility and Cbl an E3 ubiquitin ligase integrally linked to the control of tyrosine kinase signalling (regulated by 5 and 6 PTKs respectively). The major class of Proteins identified however were molecular chaperones. We also showed that HSP90 phosphorylation is altered by BCR/ABL action and that HSP90 plays a crucial role in oncogene stability. Further investigation with another six leukaemogenic PTKs demonstrates that this HSP90 role in oncogene stability appears to be a common phenomenon in a range of leukaemias. This opens up the potential opportunity to treat different leukaemias with HSP90 inhibitors.
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BCR ABL Protein tyrosine kinase activity induces a loss of p53 Protein that mediates a delay in myeloid differentiation
Oncogene, 2000Co-Authors: Andrew Pierce, Elaine Spooncer, Sarah Wooley, Caroline Dive, Julia M Francis, Jaleel A Miyan, Jane P Owenlynch, Michael T Dexter, Anthony D WhettonAbstract:Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the BCR-ABL Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive BCR-ABL PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the BCR-ABL PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to BCR-ABL PTK results in interleukin-3 independent growth and decreased p53 Protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 Protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged BCR-ABL PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.
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p210 BCR ABL expression in a primitive multipotent haematopoietic cell line models the development of chronic myeloid leukaemia
Oncogene, 1998Co-Authors: Andrew Pierce, Elaine Spooncer, P J Owenlynch, T M Dexter, Anthony D WhettonAbstract:Chronic myeloid leukaemia (CML) is a clonal disorder of the pluripotent haemopoietic stem cell, the hallmark of which is the constitutively activated BCR-ABL Protein tyrosine kinase. During the initial chronic phase of CML the primitive multipotent leukaemic progenitor cells remain growth factor dependent and are capABLe of producing terminally differentiated cells. Although the availABLe evidence suggests that BCR-ABL directly affects signalling pathways involved in controlling the development of primitive haemopoietic progenitors the identification of the specific biological consequences of BCR-ABL activity in these progenitors has been hampered by the lack of suitABLe systems modelling CML. By transfecting the multipotent haemopoietic cell line FDCP-Mix with a temperature sensitive mutant of BCR-ABL we have developed the first working model that mirrors the chronic phase of CML. FDCP-Mix cells expressing BCR-ABL tyrosine kinase activity remain growth factor dependent and retain their ability to differentiate. Normal neutrophilic cells are formed in response to G-CSF and GM-CSF. In addition, the transfected FDCP-Mix cells grown at the permissive temperature for BCR-ABL tyrosine kinase activity display enhanced survival and proliferation in low concentrations of growth factor. These findings are consistent with the initial subtle changes seen in CML progenitor cells during the chronic phase and confirm that BCR-ABL effects are context specific, i.e. they depend on the origin and developmental potential of the transfected cells. This questions the significance of studies in non-haemopoietic and differentiation blocked haemopoietic cells.