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Martin Ruthardt - One of the best experts on this subject based on the ideXlab platform.
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the functional interplay between the t 9 22 associated fusion proteins bcr abl and abl bcr in philadelphia chromosome positive acute Lymphatic Leukemia
PLOS Genetics, 2015Co-Authors: Anahita Rafiei, Afsar Ali Mian, Anna Metodieva, Oliver G Ottmann, Claudia Doring, Claudia Oancea, Frederic B Thalheimer, Martinleo Hansmann, Martin RuthardtAbstract:The hallmark of Philadelphia chromosome positive (Ph+) Leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph+ Leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid Leukemia (CML) and 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. The co-expression of p96ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185BCR/ABL. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and Ph+ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96ABL/BCR and p185BCR/ABL in hematopoietic stem cells. Co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph+ ALL.
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the functional interplay between the t 9 22 associated fusion proteins bcr abl and abl bcr in philadelphia chromosome positive acute Lymphatic Leukemia
Blood, 2014Co-Authors: Afsar Ali Mian, Oliver G Ottmann, Anahita Rafiei, Claudia Oancea, Martin RuthardtAbstract:The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) Leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid Leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL. Disclosures No relevant conflicts of interest to declare.
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prominin 1 cd133 is involved in the lineage decision as well as a in the sensitivity to molecular therapy in ph acute Lymphatic Leukemia
Blood, 2012Co-Authors: Anna Metodieva, Afsar Ali Mian, Oliver G Ottmann, Heike Pfeifer, Anja Vogel, Hubert Serve, Martin RuthardtAbstract:Abstract 2579 Chronic myelogenous Leukemia (CML) and Philadephia chromosome-positive acute Lymphatic Leukemia (Ph+ ALL) is caused by the t(9;22) which fuses the BCR to ABL resulting in a deregulated tyrosine kinase activity. ABL Kinase inhibitiors (AKI) such as imatinib, are effective in the early stage CML, but in advanced stages patients relapse as a result of point mutations within the BCR/ABL. However a certain group of resistant patients do not have point mutations, which can explain the resistance. The mechanisms of resistance remain often unknown. CD133 (PROMININ-1 or PROM-1; in the mouse: Prom-1) is a novel hematopoietic stem/progenitor cell (HSPC) marker, but its biological function is nearly unknown. Its expression is found on various leukemic cells. It has been shown that PROM-1-negative colon cancer-initiating cells were more aggressive than PROM-1-positive cells, suggesting that cancer-initiating cells are in fact PROM-1-negative. We examined the role of PROM-1 in the normal and BCR/ABL-induced malignant haematopoiesis in Prom-1−/− in comparison to Prom+/+ murine HSPCs. Our results suggest that PROM-1 plays an important role in the induction of the BCR/ABL related leukemic phenotype. BCR/ABL induced a significantly higher colony number in Prom−/− as compared to Prom+/+ HSPCs in factor independent CFU-Assays, which did not respond to 1μM Imatinib. In fact in comparison to Prom +/+ HSPCs Prom −/− HSPCs exhibited in presence as well as in absence of BCR/ABL a different response to Imatinib characterized by a significantly increase of immature c-Kit and Sca-1-positive cells. Furthermore in a transduction/transplantation model, BCR/ABL induced a significantly higher rate of ALLs (50%) in the Prom−/− than in Prom+/+ background, where only CML-like diseases were seen. Based on these results we studied the role of PROM-1 for the non-mutational resistance in Ph+ ALL. We investigated the expression of PROM-1 in different non mutational resistance models of Ph+ ALL among the patient derived cell lines (SupB15 and Tom-1) as well as 7 primary long term cultures derived from patient with Ph+ ALL (PDLTCs). These PDLTCs exhibit several grades of resistance against established ABL-kinase inhibitors (AKI). Furthermore there is a cross resistance against other molecular therapy approaches, such as allosteric inhibition by GNF-2 or oligomerization inhibition by competitive peptides. In these cells there was a direct relationship between PROM-1 expression and response to AKIs. In fact sensitivity to AKIs increased with the expression of PROM-1. In these PDLTCs targeting PROM-1 by siRNA reduced and the over expression of PROM-1 increased the response to AKIs. Interestingly, also the only PDLTC harboring the BCR/ABL-T315I was PROM-1 negative. Furthermore the induction of resistance by increasing concentrations of either Imatinib or Nilotinib in the Ph+ ALL line SupB15 was accompanied by a progressive reduction of PROM-1 expression on the surface (CD133). In summary, our data show for the first time the importance of PROM-1/CD133 for the determination of the leukemic phenotype and as a potential marker for resistance to AKIs in Ph+ ALL, which is actually under examination in cohorts of Ph+ ALL patients with non mutational resistance. Disclosures: No relevant conflicts of interest to declare.
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the reciprocal t 9 22 p96abl bcr fusion protein eenhances the transformation potential of bcr abl and increases therapy resistance in models of ph acute Lymphatic Leukemia
Blood, 2012Co-Authors: Anahita Rafiei, Afsar Ali Mian, Anna Metodieva, Oliver G Ottmann, Claudia Oancea, Heike Pfeifer, Hubert Serve, Martin RuthardtAbstract:Abstract 3539 The t(9;22)(q34;q11) is a balanced translocation. The cytogenetic correlate of der22 is the so-called Philadelphia chromosome (Ph). Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210 BCR/ABL and the m-bcr, encoding for the 185 BCR/ABL fusion proteins, respectively. BCR/ABL is a constitutively activated kinase which induces the leukemic phenotype by the aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The BCR/ABL kinase activity is efficiently targeted by tyrosin-Kinase inhibitors such Imatinib, Nilotinib, or Dasatinib. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40 ABL/BCR , present in 65% of patients suffering from chronic myeloid Leukemia (CML) and the p96 ABL/BCR , detectable in 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). In our previous studies we have shown the leukemogenic potential of the ABL/BCR fusion proteins. To further disclose the role of ABL/BCR proteins, mainly p96 ABL/BCR , in the transformation process induced by BCR/ABL and the leukemogenesis of Ph+ ALL, we co-expressed p96 ABL/BCR and p185 BCR/ABL retrovirally in the IL-3 dependent murine Ba/F3 pro-lymphocytic cell line. p96 ABL/BCR and p185 BCR/ABL were expressed from P2A peptide-linked multicistronic retroviral vectors, which allow the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The effect of p96 ABL/BCR on the kinase activity of p185 BCR/ABL , was assessed by the rate of autophosphorylation at Y245 and Y412, the BCR/ABL-dependent substrate phosphorylation (CrkL, Bcr) and by the activation of down-stream signaling pathway (Stat, Erk,) determined by Western blotting. Proliferation of the cells was assessed by growth curve and XTT assays upon withdrawal of IL-3. As classical transformation assays we performed focus formation assays (loss of contact inhibition) and colony formation in semi-solid medium (support independent growth) in untransformed Rat-1 fibroblasts. The p96 ABL/BCR expression in primary Ph+ ALL patient derived long term cultures (PDLTCs) was targeted by retrovirally transduced shRNA. The efficient targeting of p96 ABL/BCR was confirmed by western blotting. Here we report that p96ABL/BCR i.) p96 ABL/BCR enhanced not only the autophosphorylation of p185 BCR/ABL at Y245, but also the activation of all the downstream signaling pathways; ii.) p96 ABL/BCR by itself did not transform Rat-1 cells but impressively increased the number of colonies and foci induced by p185 BCR/ABL in Rat-1 cells; iii.) p96 ABL/BCR increased the proliferation of p185 BCR/ABL -positive Ba/F3 cells; iv.)p96 ABL/BCR reduced the responsiveness to TKI in p185 BCR/ABL positive Ba/F3 cells; v.) targeting the p96 ABL/BCR by shRNA decreased the proliferation of Ph+PDLTCs by the induction of apoptosis and increased their sensitivity towards kinase inhibitors (Imatinib, Nilotinib) and the allosteric inhibition by GNF-2 directed against p185 BCR/ABL . Taken together these data suggest that p96 ABL/BCR plays an important role in the determination of the leukemic phenotype and the therapy resistance of Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.
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p185 bcr abl has a lower sensitivity than p210 bcr abl to the allosteric inhibitor gnf 2 in philadelphia chromosome positive acute Lymphatic Leukemia
Haematologica, 2012Co-Authors: Afsar Ali Mian, Anna Metodieva, Yousef Najajreh, Oliver G Ottmann, Jamal Mahajna, Martin RuthardtAbstract:Background The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 ( BCR/ABL ) fusion gene on der22, the Philadelphia chromosome. The p185BCR/ABL or the p210BCR/ABL fusion proteins are encoded as a result of the translocation, depending on whether a “minor” or “major” breakpoint occurs, respectively. Both p185BCR/ABL and p210BCR/ABL exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185BCR/ABL and p210BCR/ABL “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185BCR/ABL and p210BCR/ABL regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute Lymphatic Leukemia. Design and Methods We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute Lymphatic Leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent Lymphatic Ba/F3 cells expressing either p185BCR/ABL or p210BCR/ABL and their resistance mutants. Results The inhibitory effects of GNF-2 differed constantly between p185BCR/ABL and p210BCR/ABL expressing cells. In all three Philadelphia chromosome-positive acute Lymphatic Leukemia models, p210BCR/ABL-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185BCR/ABL and the p210BCR/ABL harboring resistance mutations. Conclusions Our data provide the first evidence of a differential response of p185BCR/ABL- and p210BCR/ABL- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute Lymphatic Leukemia.
Richard Greil - One of the best experts on this subject based on the ideXlab platform.
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drug induced apoptosis is associated with enhanced fas apo 1 cd95 ligand expression but occurs independently of fas apo 1 cd95 signaling in human t acute Lymphatic Leukemia cells
Cancer Research, 1997Co-Authors: Andreas Villunger, Alexander Egle, Marion Kos, Bernd L Hartmann, Stephan Geley, Reinhard Kofler, Richard GreilAbstract:Induction of apoptosis is considered to be the underlying mechanism that accounts for the efficiency of chemotherapeutic drugs. It has recently been proposed that induction of Fas ligand (FasL) expression with subsequent autocrine and/or paracrine induction of cell death through binding to the Fas (Apo-1/CD95) membrane accounts for chemotherapy-associated apoptosis. In the present study, we analyzed the significance of FasL expression in the mediation of drug-induced apoptosis in the T-acute Lymphatic Leukemia model CEM. In particular, we examined the potential of the tumor drugs fludarabine, doxorubicin, and cisplatin to induce FasL expression. We also raised the question of whether apoptosis induced by these drugs occurs through the Fas pathway and hence can be blocked by the cowpox virus protein CrmA, a specific inhibitor of this pathway. All tumor drugs examined led to an increase in FasL protein. However, overexpression of CrmA had no effect on drug-induced apoptosis. Moreover, neither incubation with inhibitory monoclonal antibodies against Fas that completely prevented Fas-induced apoptosis in these cells nor pretreatment with a monoclonal antibody to FasL affected drug-induced cell death. Our observations suggest a Fas/FasL-independent mechanism for drug-induced apoptosis and exclude the involvement of caspase 1 and caspase 8 in this process in T-acute Lymphatic Leukemia cells.
Afsar Ali Mian - One of the best experts on this subject based on the ideXlab platform.
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the functional interplay between the t 9 22 associated fusion proteins bcr abl and abl bcr in philadelphia chromosome positive acute Lymphatic Leukemia
PLOS Genetics, 2015Co-Authors: Anahita Rafiei, Afsar Ali Mian, Anna Metodieva, Oliver G Ottmann, Claudia Doring, Claudia Oancea, Frederic B Thalheimer, Martinleo Hansmann, Martin RuthardtAbstract:The hallmark of Philadelphia chromosome positive (Ph+) Leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph+ Leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid Leukemia (CML) and 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. The co-expression of p96ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185BCR/ABL. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and Ph+ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96ABL/BCR and p185BCR/ABL in hematopoietic stem cells. Co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph+ ALL.
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the functional interplay between the t 9 22 associated fusion proteins bcr abl and abl bcr in philadelphia chromosome positive acute Lymphatic Leukemia
Blood, 2014Co-Authors: Afsar Ali Mian, Oliver G Ottmann, Anahita Rafiei, Claudia Oancea, Martin RuthardtAbstract:The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) Leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid Leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL. Disclosures No relevant conflicts of interest to declare.
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prominin 1 cd133 is involved in the lineage decision as well as a in the sensitivity to molecular therapy in ph acute Lymphatic Leukemia
Blood, 2012Co-Authors: Anna Metodieva, Afsar Ali Mian, Oliver G Ottmann, Heike Pfeifer, Anja Vogel, Hubert Serve, Martin RuthardtAbstract:Abstract 2579 Chronic myelogenous Leukemia (CML) and Philadephia chromosome-positive acute Lymphatic Leukemia (Ph+ ALL) is caused by the t(9;22) which fuses the BCR to ABL resulting in a deregulated tyrosine kinase activity. ABL Kinase inhibitiors (AKI) such as imatinib, are effective in the early stage CML, but in advanced stages patients relapse as a result of point mutations within the BCR/ABL. However a certain group of resistant patients do not have point mutations, which can explain the resistance. The mechanisms of resistance remain often unknown. CD133 (PROMININ-1 or PROM-1; in the mouse: Prom-1) is a novel hematopoietic stem/progenitor cell (HSPC) marker, but its biological function is nearly unknown. Its expression is found on various leukemic cells. It has been shown that PROM-1-negative colon cancer-initiating cells were more aggressive than PROM-1-positive cells, suggesting that cancer-initiating cells are in fact PROM-1-negative. We examined the role of PROM-1 in the normal and BCR/ABL-induced malignant haematopoiesis in Prom-1−/− in comparison to Prom+/+ murine HSPCs. Our results suggest that PROM-1 plays an important role in the induction of the BCR/ABL related leukemic phenotype. BCR/ABL induced a significantly higher colony number in Prom−/− as compared to Prom+/+ HSPCs in factor independent CFU-Assays, which did not respond to 1μM Imatinib. In fact in comparison to Prom +/+ HSPCs Prom −/− HSPCs exhibited in presence as well as in absence of BCR/ABL a different response to Imatinib characterized by a significantly increase of immature c-Kit and Sca-1-positive cells. Furthermore in a transduction/transplantation model, BCR/ABL induced a significantly higher rate of ALLs (50%) in the Prom−/− than in Prom+/+ background, where only CML-like diseases were seen. Based on these results we studied the role of PROM-1 for the non-mutational resistance in Ph+ ALL. We investigated the expression of PROM-1 in different non mutational resistance models of Ph+ ALL among the patient derived cell lines (SupB15 and Tom-1) as well as 7 primary long term cultures derived from patient with Ph+ ALL (PDLTCs). These PDLTCs exhibit several grades of resistance against established ABL-kinase inhibitors (AKI). Furthermore there is a cross resistance against other molecular therapy approaches, such as allosteric inhibition by GNF-2 or oligomerization inhibition by competitive peptides. In these cells there was a direct relationship between PROM-1 expression and response to AKIs. In fact sensitivity to AKIs increased with the expression of PROM-1. In these PDLTCs targeting PROM-1 by siRNA reduced and the over expression of PROM-1 increased the response to AKIs. Interestingly, also the only PDLTC harboring the BCR/ABL-T315I was PROM-1 negative. Furthermore the induction of resistance by increasing concentrations of either Imatinib or Nilotinib in the Ph+ ALL line SupB15 was accompanied by a progressive reduction of PROM-1 expression on the surface (CD133). In summary, our data show for the first time the importance of PROM-1/CD133 for the determination of the leukemic phenotype and as a potential marker for resistance to AKIs in Ph+ ALL, which is actually under examination in cohorts of Ph+ ALL patients with non mutational resistance. Disclosures: No relevant conflicts of interest to declare.
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the reciprocal t 9 22 p96abl bcr fusion protein eenhances the transformation potential of bcr abl and increases therapy resistance in models of ph acute Lymphatic Leukemia
Blood, 2012Co-Authors: Anahita Rafiei, Afsar Ali Mian, Anna Metodieva, Oliver G Ottmann, Claudia Oancea, Heike Pfeifer, Hubert Serve, Martin RuthardtAbstract:Abstract 3539 The t(9;22)(q34;q11) is a balanced translocation. The cytogenetic correlate of der22 is the so-called Philadelphia chromosome (Ph). Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210 BCR/ABL and the m-bcr, encoding for the 185 BCR/ABL fusion proteins, respectively. BCR/ABL is a constitutively activated kinase which induces the leukemic phenotype by the aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The BCR/ABL kinase activity is efficiently targeted by tyrosin-Kinase inhibitors such Imatinib, Nilotinib, or Dasatinib. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40 ABL/BCR , present in 65% of patients suffering from chronic myeloid Leukemia (CML) and the p96 ABL/BCR , detectable in 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). In our previous studies we have shown the leukemogenic potential of the ABL/BCR fusion proteins. To further disclose the role of ABL/BCR proteins, mainly p96 ABL/BCR , in the transformation process induced by BCR/ABL and the leukemogenesis of Ph+ ALL, we co-expressed p96 ABL/BCR and p185 BCR/ABL retrovirally in the IL-3 dependent murine Ba/F3 pro-lymphocytic cell line. p96 ABL/BCR and p185 BCR/ABL were expressed from P2A peptide-linked multicistronic retroviral vectors, which allow the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The effect of p96 ABL/BCR on the kinase activity of p185 BCR/ABL , was assessed by the rate of autophosphorylation at Y245 and Y412, the BCR/ABL-dependent substrate phosphorylation (CrkL, Bcr) and by the activation of down-stream signaling pathway (Stat, Erk,) determined by Western blotting. Proliferation of the cells was assessed by growth curve and XTT assays upon withdrawal of IL-3. As classical transformation assays we performed focus formation assays (loss of contact inhibition) and colony formation in semi-solid medium (support independent growth) in untransformed Rat-1 fibroblasts. The p96 ABL/BCR expression in primary Ph+ ALL patient derived long term cultures (PDLTCs) was targeted by retrovirally transduced shRNA. The efficient targeting of p96 ABL/BCR was confirmed by western blotting. Here we report that p96ABL/BCR i.) p96 ABL/BCR enhanced not only the autophosphorylation of p185 BCR/ABL at Y245, but also the activation of all the downstream signaling pathways; ii.) p96 ABL/BCR by itself did not transform Rat-1 cells but impressively increased the number of colonies and foci induced by p185 BCR/ABL in Rat-1 cells; iii.) p96 ABL/BCR increased the proliferation of p185 BCR/ABL -positive Ba/F3 cells; iv.)p96 ABL/BCR reduced the responsiveness to TKI in p185 BCR/ABL positive Ba/F3 cells; v.) targeting the p96 ABL/BCR by shRNA decreased the proliferation of Ph+PDLTCs by the induction of apoptosis and increased their sensitivity towards kinase inhibitors (Imatinib, Nilotinib) and the allosteric inhibition by GNF-2 directed against p185 BCR/ABL . Taken together these data suggest that p96 ABL/BCR plays an important role in the determination of the leukemic phenotype and the therapy resistance of Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.
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p185 bcr abl has a lower sensitivity than p210 bcr abl to the allosteric inhibitor gnf 2 in philadelphia chromosome positive acute Lymphatic Leukemia
Haematologica, 2012Co-Authors: Afsar Ali Mian, Anna Metodieva, Yousef Najajreh, Oliver G Ottmann, Jamal Mahajna, Martin RuthardtAbstract:Background The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 ( BCR/ABL ) fusion gene on der22, the Philadelphia chromosome. The p185BCR/ABL or the p210BCR/ABL fusion proteins are encoded as a result of the translocation, depending on whether a “minor” or “major” breakpoint occurs, respectively. Both p185BCR/ABL and p210BCR/ABL exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185BCR/ABL and p210BCR/ABL “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185BCR/ABL and p210BCR/ABL regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute Lymphatic Leukemia. Design and Methods We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute Lymphatic Leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent Lymphatic Ba/F3 cells expressing either p185BCR/ABL or p210BCR/ABL and their resistance mutants. Results The inhibitory effects of GNF-2 differed constantly between p185BCR/ABL and p210BCR/ABL expressing cells. In all three Philadelphia chromosome-positive acute Lymphatic Leukemia models, p210BCR/ABL-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185BCR/ABL and the p210BCR/ABL harboring resistance mutations. Conclusions Our data provide the first evidence of a differential response of p185BCR/ABL- and p210BCR/ABL- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute Lymphatic Leukemia.
Joachim L Schultze - One of the best experts on this subject based on the ideXlab platform.
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increased frequency of ctla4 tgfβ cd4 cd25 t cells in peripheral blood of patients with chronic Lymphatic Leukemia and multiple myeloma
Cancer Cell International, 2004Co-Authors: Marc Beyer, M Kochanek, M Von Bergweltbaildon, Alexey Popov, J Wolf, Joachim L SchultzeAbstract:A globally suppressed T cell function has been described for cancer patients including patients with chronic Lymphatic Leukemia (CLL) or multiple myeloma (MM). This has been mainly associated with inhibitory factors released by the tumor cells, while the role of recently characterized regulatory immune cells is not understood. Several different regulatory T cells such as CD4+ CD25+ T cells (Treg), TGFβ producing TH3 or IL-10 producing TR1 cells have been described in murine models, and Treg cells have also been implicated in the control of graft versus host disease after allogeneic transplantation in humans. In contrast, little is known about frequencies and function of regulatory cells in Leukemias and lymphomas. To address this issue we have analyzed 82 peripheral blood samples from 24 CLL patients, 18 MM patients and 26 healthy individuals. By assessing CD4+ CD25+ T cells we established a strongly significant increase of this subpopulation in both CLL (13 ± 7% mean ± SD, p < 0.01) and MM (13 ± 9%, p < 0.01) when compared to healthy individuals (3.5 ± 1.5%). While CDC4+ CD25+ T cells could also comprise previously activated T cells, the expression of CTLA4 has been associated with Treg cells. In fact, 80± 14% respectively 67± 26% of CD4+ CD25+ T cells in CLL resp. MM were also CTLA4+, while only 30 ± 22% were found to be positive in healthy individuals strongly suggesting that these cells are mainly Treg cells. In contrast, using the BDCA-4 specific antibody for Neuropilin-1, we were unable to detect this molecule recently described on murine Treg cells on any of our samples. Interestingly, a significant proportion of CD4+ CD25+ T cells in CLL 28 ± 13% and MM 22 ± 12% also expressed intracellular TGFβ, which was only found in 6 ± 4% of these T cells in healthy individuals. Whether TGFβ production reflects a particular activation status of Treg cells or whether these cells are a defined subpopulation requires further investigation. By analyzing co-expression of CCR7 and CD45RA we established that the fraction of CD4+ CD25+ T cells was particularly increased in the naive and the central memory pool in peripheral blood of CLL and MM patients. Next we assessed T cell activation as a function of Treg cells. As expected, there was already a significantly decreased proliferative response of CD4+ T cells in many CLL patients even when CD25+ cells were depleted. These findings are most likely explained by chronic exposure to inhibitory cytokines as well as Treg cells. However, even under these conditions, coculture experiments of CD4+ CD25and CD4+ CD25+ Treg cells supported the inhibitory role of Treg cells in CLL. We therefore propose that immunotherapy in any malignancy including CLL and MM characterized by an increase of regulatory factors and cells will significantly benefit from strategies inhibiting immune repression. from Association for Immunotherapy of Cancer: Cancer Immunotherapy – 2nd Annual Meeting Mainz, Germany, 6–7 May 2004
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minimal residual disease detection after myeloablative chemotherapy in chronic Lymphatic Leukemia
Journal of Molecular Medicine, 1999Co-Authors: Joachim L Schultze, John W Donovan, John G GribbenAbstract:Detection of clonal tumor cells in Leukemias and lymphomas by PCR in minimal residual disease (MRD) has been shown to be a valuable parameter for identifying patients who may require further treatment. Here we introduce the studies underway in our own and other institutions addressing the value of PCR technology in detecting residual CLL cells either in the autologous stem cell product or after induction of MRD in patients after autologous or allogeneic stem cell transplant. The PCR technology used for these questions and the results are discussed.
Oliver G Ottmann - One of the best experts on this subject based on the ideXlab platform.
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the functional interplay between the t 9 22 associated fusion proteins bcr abl and abl bcr in philadelphia chromosome positive acute Lymphatic Leukemia
PLOS Genetics, 2015Co-Authors: Anahita Rafiei, Afsar Ali Mian, Anna Metodieva, Oliver G Ottmann, Claudia Doring, Claudia Oancea, Frederic B Thalheimer, Martinleo Hansmann, Martin RuthardtAbstract:The hallmark of Philadelphia chromosome positive (Ph+) Leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph+ Leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid Leukemia (CML) and 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. The co-expression of p96ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185BCR/ABL. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and Ph+ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96ABL/BCR and p185BCR/ABL in hematopoietic stem cells. Co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph+ ALL.
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the functional interplay between the t 9 22 associated fusion proteins bcr abl and abl bcr in philadelphia chromosome positive acute Lymphatic Leukemia
Blood, 2014Co-Authors: Afsar Ali Mian, Oliver G Ottmann, Anahita Rafiei, Claudia Oancea, Martin RuthardtAbstract:The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) Leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid Leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL. Disclosures No relevant conflicts of interest to declare.
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prominin 1 cd133 is involved in the lineage decision as well as a in the sensitivity to molecular therapy in ph acute Lymphatic Leukemia
Blood, 2012Co-Authors: Anna Metodieva, Afsar Ali Mian, Oliver G Ottmann, Heike Pfeifer, Anja Vogel, Hubert Serve, Martin RuthardtAbstract:Abstract 2579 Chronic myelogenous Leukemia (CML) and Philadephia chromosome-positive acute Lymphatic Leukemia (Ph+ ALL) is caused by the t(9;22) which fuses the BCR to ABL resulting in a deregulated tyrosine kinase activity. ABL Kinase inhibitiors (AKI) such as imatinib, are effective in the early stage CML, but in advanced stages patients relapse as a result of point mutations within the BCR/ABL. However a certain group of resistant patients do not have point mutations, which can explain the resistance. The mechanisms of resistance remain often unknown. CD133 (PROMININ-1 or PROM-1; in the mouse: Prom-1) is a novel hematopoietic stem/progenitor cell (HSPC) marker, but its biological function is nearly unknown. Its expression is found on various leukemic cells. It has been shown that PROM-1-negative colon cancer-initiating cells were more aggressive than PROM-1-positive cells, suggesting that cancer-initiating cells are in fact PROM-1-negative. We examined the role of PROM-1 in the normal and BCR/ABL-induced malignant haematopoiesis in Prom-1−/− in comparison to Prom+/+ murine HSPCs. Our results suggest that PROM-1 plays an important role in the induction of the BCR/ABL related leukemic phenotype. BCR/ABL induced a significantly higher colony number in Prom−/− as compared to Prom+/+ HSPCs in factor independent CFU-Assays, which did not respond to 1μM Imatinib. In fact in comparison to Prom +/+ HSPCs Prom −/− HSPCs exhibited in presence as well as in absence of BCR/ABL a different response to Imatinib characterized by a significantly increase of immature c-Kit and Sca-1-positive cells. Furthermore in a transduction/transplantation model, BCR/ABL induced a significantly higher rate of ALLs (50%) in the Prom−/− than in Prom+/+ background, where only CML-like diseases were seen. Based on these results we studied the role of PROM-1 for the non-mutational resistance in Ph+ ALL. We investigated the expression of PROM-1 in different non mutational resistance models of Ph+ ALL among the patient derived cell lines (SupB15 and Tom-1) as well as 7 primary long term cultures derived from patient with Ph+ ALL (PDLTCs). These PDLTCs exhibit several grades of resistance against established ABL-kinase inhibitors (AKI). Furthermore there is a cross resistance against other molecular therapy approaches, such as allosteric inhibition by GNF-2 or oligomerization inhibition by competitive peptides. In these cells there was a direct relationship between PROM-1 expression and response to AKIs. In fact sensitivity to AKIs increased with the expression of PROM-1. In these PDLTCs targeting PROM-1 by siRNA reduced and the over expression of PROM-1 increased the response to AKIs. Interestingly, also the only PDLTC harboring the BCR/ABL-T315I was PROM-1 negative. Furthermore the induction of resistance by increasing concentrations of either Imatinib or Nilotinib in the Ph+ ALL line SupB15 was accompanied by a progressive reduction of PROM-1 expression on the surface (CD133). In summary, our data show for the first time the importance of PROM-1/CD133 for the determination of the leukemic phenotype and as a potential marker for resistance to AKIs in Ph+ ALL, which is actually under examination in cohorts of Ph+ ALL patients with non mutational resistance. Disclosures: No relevant conflicts of interest to declare.
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the reciprocal t 9 22 p96abl bcr fusion protein eenhances the transformation potential of bcr abl and increases therapy resistance in models of ph acute Lymphatic Leukemia
Blood, 2012Co-Authors: Anahita Rafiei, Afsar Ali Mian, Anna Metodieva, Oliver G Ottmann, Claudia Oancea, Heike Pfeifer, Hubert Serve, Martin RuthardtAbstract:Abstract 3539 The t(9;22)(q34;q11) is a balanced translocation. The cytogenetic correlate of der22 is the so-called Philadelphia chromosome (Ph). Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210 BCR/ABL and the m-bcr, encoding for the 185 BCR/ABL fusion proteins, respectively. BCR/ABL is a constitutively activated kinase which induces the leukemic phenotype by the aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The BCR/ABL kinase activity is efficiently targeted by tyrosin-Kinase inhibitors such Imatinib, Nilotinib, or Dasatinib. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40 ABL/BCR , present in 65% of patients suffering from chronic myeloid Leukemia (CML) and the p96 ABL/BCR , detectable in 100% of patients with Ph+ acute Lymphatic Leukemia (ALL). In our previous studies we have shown the leukemogenic potential of the ABL/BCR fusion proteins. To further disclose the role of ABL/BCR proteins, mainly p96 ABL/BCR , in the transformation process induced by BCR/ABL and the leukemogenesis of Ph+ ALL, we co-expressed p96 ABL/BCR and p185 BCR/ABL retrovirally in the IL-3 dependent murine Ba/F3 pro-lymphocytic cell line. p96 ABL/BCR and p185 BCR/ABL were expressed from P2A peptide-linked multicistronic retroviral vectors, which allow the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The effect of p96 ABL/BCR on the kinase activity of p185 BCR/ABL , was assessed by the rate of autophosphorylation at Y245 and Y412, the BCR/ABL-dependent substrate phosphorylation (CrkL, Bcr) and by the activation of down-stream signaling pathway (Stat, Erk,) determined by Western blotting. Proliferation of the cells was assessed by growth curve and XTT assays upon withdrawal of IL-3. As classical transformation assays we performed focus formation assays (loss of contact inhibition) and colony formation in semi-solid medium (support independent growth) in untransformed Rat-1 fibroblasts. The p96 ABL/BCR expression in primary Ph+ ALL patient derived long term cultures (PDLTCs) was targeted by retrovirally transduced shRNA. The efficient targeting of p96 ABL/BCR was confirmed by western blotting. Here we report that p96ABL/BCR i.) p96 ABL/BCR enhanced not only the autophosphorylation of p185 BCR/ABL at Y245, but also the activation of all the downstream signaling pathways; ii.) p96 ABL/BCR by itself did not transform Rat-1 cells but impressively increased the number of colonies and foci induced by p185 BCR/ABL in Rat-1 cells; iii.) p96 ABL/BCR increased the proliferation of p185 BCR/ABL -positive Ba/F3 cells; iv.)p96 ABL/BCR reduced the responsiveness to TKI in p185 BCR/ABL positive Ba/F3 cells; v.) targeting the p96 ABL/BCR by shRNA decreased the proliferation of Ph+PDLTCs by the induction of apoptosis and increased their sensitivity towards kinase inhibitors (Imatinib, Nilotinib) and the allosteric inhibition by GNF-2 directed against p185 BCR/ABL . Taken together these data suggest that p96 ABL/BCR plays an important role in the determination of the leukemic phenotype and the therapy resistance of Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.
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p185 bcr abl has a lower sensitivity than p210 bcr abl to the allosteric inhibitor gnf 2 in philadelphia chromosome positive acute Lymphatic Leukemia
Haematologica, 2012Co-Authors: Afsar Ali Mian, Anna Metodieva, Yousef Najajreh, Oliver G Ottmann, Jamal Mahajna, Martin RuthardtAbstract:Background The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 ( BCR/ABL ) fusion gene on der22, the Philadelphia chromosome. The p185BCR/ABL or the p210BCR/ABL fusion proteins are encoded as a result of the translocation, depending on whether a “minor” or “major” breakpoint occurs, respectively. Both p185BCR/ABL and p210BCR/ABL exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185BCR/ABL and p210BCR/ABL “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185BCR/ABL and p210BCR/ABL regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute Lymphatic Leukemia. Design and Methods We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute Lymphatic Leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent Lymphatic Ba/F3 cells expressing either p185BCR/ABL or p210BCR/ABL and their resistance mutants. Results The inhibitory effects of GNF-2 differed constantly between p185BCR/ABL and p210BCR/ABL expressing cells. In all three Philadelphia chromosome-positive acute Lymphatic Leukemia models, p210BCR/ABL-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185BCR/ABL and the p210BCR/ABL harboring resistance mutations. Conclusions Our data provide the first evidence of a differential response of p185BCR/ABL- and p210BCR/ABL- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute Lymphatic Leukemia.
