48 results on '"Schemionek M"'
Search Results
2. Mtss1 is a critical epigenetically regulated tumor suppressor in CML
- Author
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Schemionek, M, Herrmann, O, Reher, M M, Chatain, N, Schubert, C, Costa, I G, Hänzelmann, S, Gusmao, E G, Kintsler, S, Braunschweig, T, Hamilton, A, Helgason, G V, Copland, M, Schwab, A, Müller-Tidow, C, Li, S, Holyoake, T L, Brümmendorf, T H, and Koschmieder, S
- Published
- 2016
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3. Rare FLT3 deletion mutants may provide additional treatment options to patients with AML: an approach to individualized medicine
- Author
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Chatain, N, Perera, R C, Rossetti, G, Rossa, J, Carloni, P, Schemionek, M, Haferlach, T, Brümmendorf, T H, Schnittger, S, and Koschmieder, S
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- 2015
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4. Comparison of flow-FISH and MM-qPCR telomere length assessment techniques for the screening of telomeropathies
- Author
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Ferreira, M.S.V., Kirschner, M., Halfmeyer, I., Estrada, Natalia, Xicoy, Blanca, Isfort, S., Vieri, M., Zamora, Lurdes, Abels, A., Bouillon, A.S., Begemann, M., Schemionek, M., Maurer, A., Koschmieder, S., Wilop, S., Panse, J., Brümmendorf, T.H., Beier, F., and Universitat Autònoma de Barcelona
- Subjects
0301 basic medicine ,Male ,Lymphocyte ,Gastroenterology ,law.invention ,Cohort Studies ,0302 clinical medicine ,law ,Polymerase chain reaction ,In Situ Hybridization, Fluorescence ,Telomere Shortening ,medicine.diagnostic_test ,General Neuroscience ,Flow-FISH ,Telomeropathy ,Middle Aged ,Telomere ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,Dyskeratosis congenita ,Adult ,medicine.medical_specialty ,Real-Time Polymerase Chain Reaction ,General Biochemistry, Genetics and Molecular Biology ,Dyskeratosis Congenita ,03 medical and health sciences ,Young Adult ,History and Philosophy of Science ,Internal medicine ,medicine ,Humans ,Gene ,Genetic testing ,Aged ,Telomere length ,business.industry ,Genetic Diseases, Inborn ,Telomere Homeostasis ,Bone Marrow Failure Disorders ,medicine.disease ,030104 developmental biology ,Case-Control Studies ,MM-qPCR ,business ,Multiplex Polymerase Chain Reaction ,Fluorescence in situ hybridization - Abstract
Assessment of telomere length (TL) in peripheral blood leukocytes is part of the diagnostic algorithm applied to patients with acquired bone marrow failure syndromes (BMFSs) and dyskeratosis congenita (DKC). Monochrome multiplex-quantitative polymerase chain reaction (MM-qPCR) and fluorescence in situ hybridization (flow-FISH) are methodologies available for TL screening. Dependent on TL expressed in relation to percentiles of healthy controls, further genetic testing for inherited mutations in telomere maintenance genes is recommended. However, the correct threshold to trigger this genetic workup is still under debate. Here, we prospectively compared MM-qPCR and flow-FISH regarding their capacity for accurate identification of DKC patients. All patients (n = 105) underwent genetic testing by next-generation sequencing and in 16 patients, mutations in DKC-relevant genes were identified. Whole leukocyte TL of patients measured by MM-qPCR was found to be moderately correlated with lymphocyte TL measured by flow-FISH (r² = 0.34; P < 0.0001). The sensitivity of both methods was high, but the specificity of MM-qPCR (29%) was significantly lower compared with flow-FISH (58%). These results suggest that MM-qPCR of peripheral blood cells is inferior to flow-FISH for clinical routine screening for suspected DKC in adult patients with BMFS due to lower specificity and a higher rate of false-positive results.
- Published
- 2021
5. Leukemic spleen cells are more potent than bone marrow-derived cells in a transgenic mouse model of CML
- Author
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Schemionek, M, Spieker, T, Kerstiens, L, Elling, C, Essers, M, Trumpp, A, Berdel, W E, Müller-Tidow, C, and Koschmieder, S
- Published
- 2012
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6. NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR–ABL-positive mice
- Author
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Chen, C I-U, Koschmieder, S, Kerstiens, L, Schemionek, M, Altvater, B, Pscherer, S, Gerss, J, Maecker, H T, Berdel, W E, Juergens, H, Lee, P P, and Rossig, C
- Published
- 2012
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7. Epimutations mimic genomic mutations of DNMT3A in acute myeloid leukemia
- Author
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Jost, E, Lin, Q, Weidner, C I, Wilop, S, Hoffmann, M, Walenda, T, Schemionek, M, Herrmann, O, Zenke, M, Brümmendorf, T H, Koschmieder, S, and Wagner, W
- Published
- 2014
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8. Comparison of flow-FISH and MM-qPCR telomere length assessment techniques for the screening of telomeropathies
- Author
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Ferreira MSV, Kirschner M, Halfmeyer I, Estrada N, Xicoy B, Isfort S, Vieri M, Zamora L, Abels A, Bouillon AS, Begemann M, Schemionek M, Maurer A, Koschmieder S, Wilop S, Panse J, Brümmendorf TH, and Beier F
- Subjects
MM-qPCR, dyskeratosis congenita, flow-FISH, telomere length, telomeropathy - Abstract
Assessment of telomere length (TL) in peripheral blood leukocytes is part of the diagnostic algorithm applied to patients with acquired bone marrow failure syndromes (BMFSs) and dyskeratosis congenita (DKC). Monochrome multiplex-quantitative polymerase chain reaction (MM-qPCR) and fluorescence in situ hybridization (flow-FISH) are methodologies available for TL screening. Dependent on TL expressed in relation to percentiles of healthy controls, further genetic testing for inherited mutations in telomere maintenance genes is recommended. However, the correct threshold to trigger this genetic workup is still under debate. Here, we prospectively compared MM-qPCR and flow-FISH regarding their capacity for accurate identification of DKC patients. All patients (n = 105) underwent genetic testing by next-generation sequencing and in 16 patients, mutations in DKC-relevant genes were identified. Whole leukocyte TL of patients measured by MM-qPCR was found to be moderately correlated with lymphocyte TL measured by flow-FISH (r² = 0.34; P < 0.0001). The sensitivity of both methods was high, but the specificity of MM-qPCR (29%) was significantly lower compared with flow-FISH (58%). These results suggest that MM-qPCR of peripheral blood cells is inferior to flow-FISH for clinical routine screening for suspected DKC in adult patients with BMFS due to lower specificity and a higher rate of false-positive results.
- Published
- 2020
9. Novel PDGFRA point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia in vitro and in vivo: V759
- Author
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Elling, C., Erben, P., Walz, C., Frickenhaus, M., Schemionek, M., Stehling, M., Serve, H., Cross, N. C.P., Hochhaus, A., Hofmann, W.-K., Berdel, W. E., Müller-Tidow, C., Reiter, A., and Koschmieder, S.
- Published
- 2010
10. BCR-ABL leads to differentiation of long-term repopulating hematopoietic stem cells: V79
- Author
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Schemionek, M., Elling, C., Bäumer, N., Hamilton, A., Spieker, T., Göthert, J., Stehling, M., Wagers, A., Huettner, C. S., Tenen, D. G., Tickenbrock, L., Berdel, W., Serve, H., Holyoake, L., Müller-Tidow, C., and Koschmieder, S.
- Published
- 2009
11. PF663 TRANSCRIPTION ALTERATION OF DNA REPAIR GENES IN PHILADELPHIA CHROMOSOME NEGATIVE MYELOPROLIFERATIVE NEOPLASMS
- Author
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Kirschner, M., primary, Bornemann, A., additional, Schubert, C., additional, Gezer, D., additional, Kricheldorf, K., additional, Isfort, S., additional, Brümmendorf, T.H., additional, Schemionek, M., additional, Chatain, N., additional, Skorski, T., additional, and Koschmieder, S., additional
- Published
- 2019
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12. PF152 XBP1 PROMOTES PRE‐B CELL ACUTE LYMPHOBLASTIC LEUKEMIA THROUGH OF IL‐7 RECEPTOR SIGNALING IN MICE
- Author
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Salimi, A., primary, Vieri, M., additional, Schemionek, M., additional, Huber, M., additional, Brümmendorf, T.H., additional, Masouleh, B. Kharabi, additional, and Appelmann, I., additional
- Published
- 2019
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13. Mtss1 is a critical epigenetically regulated tumor suppressor in CML
- Author
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Schemionek, M, primary, Herrmann, O, additional, Reher, M M, additional, Chatain, N, additional, Schubert, C, additional, Costa, I G, additional, Hänzelmann, S, additional, Gusmao, E G, additional, Kintsler, S, additional, Braunschweig, T, additional, Hamilton, A, additional, Helgason, G V, additional, Copland, M, additional, Schwab, A, additional, Müller-Tidow, C, additional, Li, S, additional, Holyoake, T L, additional, Brümmendorf, T H, additional, and Koschmieder, S, additional
- Published
- 2015
- Full Text
- View/download PDF
14. Epimutations mimic genomic mutations of DNMT3A in acute myeloid leukemia
- Author
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Jost, E, primary, Lin, Q, additional, Weidner, C I, additional, Wilop, S, additional, Hoffmann, M, additional, Walenda, T, additional, Schemionek, M, additional, Herrmann, O, additional, Zenke, M, additional, Brümmendorf, T H, additional, Koschmieder, S, additional, and Wagner, W, additional
- Published
- 2013
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15. Leukemic spleen cells are more potent than bone marrow-derived cells in a transgenic mouse model of CML
- Author
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Schemionek, M, primary, Spieker, T, additional, Kerstiens, L, additional, Elling, C, additional, Essers, M, additional, Trumpp, A, additional, Berdel, W E, additional, Müller-Tidow, C, additional, and Koschmieder, S, additional
- Published
- 2011
- Full Text
- View/download PDF
16. NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR–ABL-positive mice
- Author
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Chen, C I-U, primary, Koschmieder, S, additional, Kerstiens, L, additional, Schemionek, M, additional, Altvater, B, additional, Pscherer, S, additional, Gerss, J, additional, Maecker, H T, additional, Berdel, W E, additional, Juergens, H, additional, Lee, P P, additional, and Rossig, C, additional
- Published
- 2011
- Full Text
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17. Mtss1(CSC156)mutant mice fail to display efficient Mtss1 protein depletion
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Fahrenkamp, D, Herrmann, O, Koschmieder, S, Brümmendorf, T H, and Schemionek, M
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- 2017
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18. BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells
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Schemionek, M., Elling, C., Steidl, U., Baumer, N., Hamilton, A., Spieker, T., Gothert, J. R., Stehling, M., Wagers, Amy Jo, Huettner, Claudia Sonja, Tenen, Daniel Geoffrey, Tickenbrock, L., Berdel, W. E., Serve, H., Holyoake, T. L., Muller-Tidow, C., and Koschmieder, S.
- Abstract
In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity., Stem Cell and Regenerative Biology
- Published
- 2010
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19. Myelofibrosis at diagnosis is associated with the failure of treatment-free remission in CML patients.
- Author
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Jacobi H, Vieri M, Bütow M, Namasu CY, Flüter L, Costa IG, Maié T, Lindemann-Docter K, Chatain N, Beier F, Huber M, Wagner W, Crysandt M, Brümmendorf TH, and Schemionek M
- Abstract
The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence., Competing Interests: TB served as a consultant or speaker at satellite symposia for AstraZeneca, Janssen, Merck, Novartis and Pfizer and received research funding form Novartis and Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jacobi, Vieri, Bütow, Namasu, Flüter, Costa, Maié, Lindemann-Docter, Chatain, Beier, Huber, Wagner, Crysandt, Brümmendorf and Schemionek.)
- Published
- 2023
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20. A phase I first-in-man study to investigate the pharmacokinetics and safety of liposomal dexamethasone in patients with progressive multiple myeloma.
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Metselaar J, Lammers T, Boquoi A, Fenk R, Testaquadra F, Schemionek M, Kiessling F, Isfort S, Wilop S, and Crysandt M
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- Humans, Antineoplastic Combined Chemotherapy Protocols, Dexamethasone adverse effects, Liposomes therapeutic use, Treatment Outcome, Multiple Myeloma drug therapy, Multiple Myeloma etiology, Multiple Myeloma pathology
- Abstract
Despite the introduction of multiple new drugs and combination therapies, conventional dexamethasone remains a cornerstone in the treatment of multiple myeloma (MM). Its application is, however, limited by frequent adverse effects of which the increased infection rate may have the strongest clinical impact. The efficacy-safety ratio of dexamethasone in MM may be increased by encapsulation in long-circulating PEG-liposomes, thereby both enhancing drug delivery to MM lesions and reducing systemic corticosteroid exposure. We evaluated the preliminary safety and feasibility of a single intravenous (i.v.) infusion of pegylated liposomal dexamethasone phosphate (Dex-PL) in heavily pretreated relapsing or progressive symptomatic MM patients within a phase I open-label non-comparative interventional trial at two dose levels. In the 7 patients that were enrolled (prior to having to close the study prematurely due to slow recruitment), Dex-PL was found to be well tolerated and, as compared to conventional dexamethasone, no new or unexpected adverse events were detected. Pharmacokinetic analysis showed high and persisting concentrations of dexamethasone in the circulation for over a week after i.v. administration, likely caused by the long-circulation half-life of the liposomes that retain dexamethasone as the inactive phosphate prodrug form, something which could significantly limit systemic exposure to the active parent drug. Thus, despite the limitations of this small first-in-man trial, Dex-PL seems safe and well tolerated without severe side effects. Follow-up studies are needed to confirm this in a larger patient cohort and to evaluate if i.v. Dex-PL can provide a safer and more efficacious dexamethasone treatment option for MM., (© 2023. The Author(s).)
- Published
- 2023
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21. Targeting cytokine-induced leukemic stem cell persistence in chronic myeloid leukemia by IKK2-inhibition.
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Bütow M, Testaquadra FJ, Baumeister J, Maié T, Chatain N, Jaquet T, Tillmann S, Crysandt M, Costa IG, Brümmendorf TH, and Schemionek M
- Subjects
- Humans, Chronic Disease, Cytokines, Neoplastic Stem Cells, Stem Cells, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Leukemia, Myeloid, Leukemia, Myeloid, Acute
- Published
- 2023
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22. Combined inhibition of BCR-ABL1 and the proteasome as a potential novel therapeutic approach in BCR-ABL positive acute lymphoblastic leukemia.
- Author
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Maletzke S, Salimi A, Vieri M, Schroeder KM, Schemionek M, Masouleh BK, Brümmendorf TH, Koschmieder S, and Appelmann I
- Subjects
- Adult, Aged, Boron Compounds, Bortezomib pharmacology, Bortezomib therapeutic use, Dasatinib pharmacology, Dasatinib therapeutic use, Drug Resistance, Neoplasm genetics, Fusion Proteins, bcr-abl metabolism, Glycine analogs & derivatives, Humans, Imatinib Mesylate therapeutic use, Proteasome Endopeptidase Complex, Proteasome Inhibitors pharmacology, Proteasome Inhibitors therapeutic use, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma drug therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics
- Abstract
Acute lymphoblastic leukemia (ALL) is a disease of lymphoid progenitor cells with an often aggressive course and is commonly caused by the BCR-ABL fusion gene t(9;22) in adults. This fusion gene encodes a constitutively active tyrosine kinase that can be effectively inhibited by tyrosine kinase inhibitors (TKIs), with imatinib being the paradigmatic agent of this class. However, BCR-ABL+ ALL cells rapidly develop mutations against many of the available TKIs, and consecutive disease relapse still results in an overall unfavorable prognosis for patients with this disease. To date, allogeneic stem cell transplantation is the only known curative therapeutic option for the mostly elderly patients with BCR-ABL+ ALL. The discrepancy between the limited therapeutic armamentarium and the growing therapeutic need in an aging population is therefore a reason to test drug combinations against BCR-ABL+ ALL. In this study, we demonstrate that the combination of TKIs with proteasome inhibitors efficiently and under certain conditions synergistically exerts cytotoxic effects in BCR-ABL+ ALL cells in vitro with respect to the induction of apoptosis. Both sole and combined treatment of BCR-ABL+ ALL with the proteasome inhibitors bortezomib and ixazomib, respectively, and TKI causes a significantly greater reduction in cell viability than TKI treatment alone in both BCR-ABL+ cell lines TOM-1 and BV-173. In BV-173 cells, we observed a significant reduction in cell viability to only 1.26%±0.46% with bortezomib treatment and 1.57±0.7% with combination treatment, whereas cells treated with dasatinib alone still had a viable percentage of 40.58±2.6%. Similar results were obtained when ixazomib was applied to both cell lines, and apoptosis was induced in both cases (93.36%±2.7% apoptotic BV-173 cells when treated with ixazomib and TKI). The combination of TKI and proteasome inhibitor is efficient in vitro, potentially expanding the spectrum of therapeutic options for patients with BCR-ABL+ ALL., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: IA received funding from Novartis® and Takeda Pharmaceuticals®. This as well as the adapted competing interests statement does not alter authors’ adherence to PLOS ONE policies on sharing data and materials.
- Published
- 2022
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23. The Unfolded Protein Response Is a Major Driver of LCN2 Expression in BCR-ABL- and JAK2V617F-Positive MPN.
- Author
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Tillmann S, Olschok K, Schröder SK, Bütow M, Baumeister J, Kalmer M, Preußger V, Weinbergerova B, Kricheldorf K, Mayer J, Kubesova B, Racil Z, Wessiepe M, Eschweiler J, Isfort S, Brümmendorf TH, Becker W, Schemionek M, Weiskirchen R, Koschmieder S, and Chatain N
- Abstract
Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N -terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1-JNK-NF-κB-C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.
- Published
- 2021
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24. Targeting of BCR-ABL1 and IRE1α induces synthetic lethality in Philadelphia-positive acute lymphoblastic leukemia.
- Author
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Vieri M, Preisinger C, Schemionek M, Salimi A, Patterson JB, Samali A, Brümmendorf TH, Appelmann I, and Kharabi Masouleh B
- Subjects
- Animals, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Benzamides pharmacology, Benzopyrans pharmacology, Benzopyrans therapeutic use, Cell Line, Tumor, Cell Survival drug effects, Cell Survival genetics, Disease Models, Animal, Drug Synergism, Fusion Proteins, bcr-abl genetics, Humans, JNK Mitogen-Activated Protein Kinases antagonists & inhibitors, JNK Mitogen-Activated Protein Kinases metabolism, MAP Kinase Signaling System drug effects, MAP Kinase Signaling System genetics, Mice, Transgenic, Morpholines pharmacology, Morpholines therapeutic use, Naphthalenes pharmacology, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics, Precursor Cell Lymphoblastic Leukemia-Lymphoma pathology, Primary Cell Culture, Pyrazoles pharmacology, Pyridines pharmacology, Pyrimidines pharmacology, Pyrimidines therapeutic use, X-Box Binding Protein 1 genetics, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, p38 Mitogen-Activated Protein Kinases metabolism, Antineoplastic Combined Chemotherapy Protocols pharmacology, Endoribonucleases antagonists & inhibitors, Fusion Proteins, bcr-abl antagonists & inhibitors, Precursor Cell Lymphoblastic Leukemia-Lymphoma drug therapy, Protein Serine-Threonine Kinases antagonists & inhibitors, Synthetic Lethal Mutations drug effects
- Abstract
BCR-ABL1-positive acute lymphoblastic leukemia (ALL) cell survival is dependent on the inositol-requiring enzyme 1 alpha (IRE1α) branch of the unfolded protein response. In the current study, we have focused on exploring the efficacy of a simultaneous pharmacological inhibition of BCR-ABL1 and IRE1α in Philadelphia-positive (Ph+) ALL using tyrosine kinase inhibitor (TKI) nilotinib and the IRE1α inhibitor MKC-8866. The combination of 0.5 µM nilotinib and 30 µM MKC-8866 in Ph+ ALL cell lines led to a synergistic effect on cell viability. To mimic this dual inhibition on a genetic level, pre-B-cells from conditional Xbp1+/fl mice were transduced with a BCR-ABL1 construct and with either tamoxifen-inducible cre or empty vector. Cells showed a significant sensitization to the effect of TKIs after the induction of the heterozygous deletion. Finally, we performed a phosphoproteomic analysis on Ph+ ALL cell lines treated with the combination of nilotinib and MKC-8866 to identify potential targets involved in their synergistic effect. An enhanced activation of p38 mitogen-activated protein kinase α (p38α MAPK) was identified. In line with this findings, p38 MAPK and, another important endoplasmic reticulum-stress-related kinase, c-Jun N-terminal kinase (JNK) were found to mediate the potentiated cytotoxic effect induced by the combination of MKC-8866 and nilotinib since the targeting of p38 MAPK with its specific inhibitor BIRB-796 or JNK with JNK-in-8 hindered the synergistic effect observed upon treatment with nilotinib and MKC-8866. In conclusion, the identified combined action of nilotinib and MKC-8866 might represent a successful therapeutic strategy in high-risk Ph+ ALL., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2021
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25. Therapeutic inhibition of FcγRIIb signaling targets leukemic stem cells in chronic myeloid leukemia.
- Author
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Parting O, Langer S, Kuepper MK, Wessling C, Li S, Braunschweig T, Chatain N, Maié T, Costa IG, Crysandt M, Huber M, Brümmendorf TH, Koschmieder S, and Schemionek M
- Subjects
- Animals, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Mice, Mice, Inbred C57BL, Receptors, IgG genetics, Agammaglobulinaemia Tyrosine Kinase antagonists & inhibitors, Antineoplastic Agents pharmacology, Fusion Proteins, bcr-abl antagonists & inhibitors, Leukemia, Myelogenous, Chronic, BCR-ABL Positive metabolism, Neoplastic Stem Cells metabolism, Protein Kinase Inhibitors pharmacology, Receptors, IgG antagonists & inhibitors, Signal Transduction drug effects
- Abstract
Despite the successes achieved with molecular targeted inhibition of the oncogenic driver Bcr-Abl in chronic myeloid leukemia (CML), the majority of patients still require lifelong tyrosine kinase inhibitor (TKI) therapy. This is primarily caused by resisting leukemic stem cells (LSCs), which prevent achievement of treatment-free remission in all patients. Here we describe the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc gamma receptor IIb (FcγRIIb, CD32b) for being critical in LSC resistance and show that targeting FcγRIIb downstream signaling, by using a Food and Drug Administration-approved BTK inhibitor, provides a successful therapeutic approach. First, we identified FcγRIIb upregulation in primary CML stem cells. FcγRIIb depletion caused reduced serial re-plaiting efficiency and cell proliferation in malignant cells. FcγRIIb targeting in both a transgenic and retroviral CML mouse model provided in vivo evidence for successful LSC reduction. Subsequently, we identified BTK as a main downstream mediator and targeting the Bcr-Abl-FcγRIIb-BTK axis in primary CML CD34
+ cells using ibrutinib, in combination with standard TKI therapy, significantly increased apoptosis in quiescent CML stem cells thereby contributing to the eradication of LSCs.. As a potential curative therapeutic approach, we therefore suggest combining Bcr-Abl TKI therapy along with BTK inhibition.- Published
- 2020
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26. LCP1 triggers mTORC2/AKT activity and is pharmacologically targeted by enzastaurin in hypereosinophilia.
- Author
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Ma G, Gezer D, Herrmann O, Feldberg K, Schemionek M, Jawhar M, Reiter A, Brümmendorf TH, Koschmieder S, and Chatain N
- Subjects
- Adult, Aged, Cell Line, Cell Line, Tumor, Cell Proliferation drug effects, Cells, Cultured, Female, Humans, Hypereosinophilic Syndrome metabolism, Indoles therapeutic use, Male, Middle Aged, Protein Kinase Inhibitors therapeutic use, Hypereosinophilic Syndrome drug therapy, Indoles pharmacology, Mechanistic Target of Rapamycin Complex 2 metabolism, Microfilament Proteins metabolism, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-akt metabolism
- Abstract
Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Only a small proportion of HE cases are clonal malignancies, and one of these, the group of eosinophilia-associated tyrosine kinase fusion-driven neoplasms, is sensitive to tyrosine kinase inhibitors, while most subtypes lack specific treatment. Eosinophil functions are highly dependent on actin polymerization, promoting priming, shape change, and infiltration of inflamed tissues. Therefore, we investigated the role of the actin-binding protein lymphocyte cytosolic protein 1 (LCP1) in malignant and nonmalignant eosinophil differentiation. We use the protein kinase C-β (PKCβ) selective inhibitor enzastaurin (Enza) to dephosphorylate and inactivate LCP1 in FIP1L1-platelet-derived growth factor receptor α (PDGFRA)-positive Eol-1 cells, and this was associated with reduced proliferation, metabolic activity, and colony formation as well as enhanced apoptosis and impaired migration. While Enza did not alter FIP1L1-PDGFRA-induced signal transducer and activator of transcription 3 (STAT3), STAT5, and ERK1/2 phosphorylation, it inhibited STAT1
Tyr701 and AKTSer473 (but not AKTThr308 ) phosphorylation, and short hairpin RNA knockdown experiments confirmed that this process was mediated by LCP1 and associated mammalian target of rapamycin complex 2 (mTORC2) activity loss. Homeobox protein HoxB8 immortalized murine bone marrow cells showed impaired eosinophilic differentiation upon Enza treatment or LCP1 knockdown. Furthermore, Enza treatment of primary HE samples reduced eosinophil differentiation and survival. In conclusion, our data show that HE involves active LCP1, which interacts with mTOR and triggers mTORC2 activity, and that the PKCβ inhibitor Enza as well as targeting of LCP1 may provide a novel treatment approach to hypereosinophilic disorders., (© 2019 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.)- Published
- 2020
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27. Transcriptional alteration of DNA repair genes in Philadelphia chromosome negative myeloproliferative neoplasms.
- Author
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Kirschner M, Bornemann A, Schubert C, Gezer D, Kricheldorf K, Isfort S, Brümmendorf TH, Schemionek M, Chatain N, Skorski T, and Koschmieder S
- Subjects
- Adult, Female, Humans, Male, Middle Aged, Mutation, Philadelphia Chromosome, DNA Repair, DNA, Neoplasm genetics, DNA, Neoplasm metabolism, Hematologic Neoplasms genetics, Hematologic Neoplasms metabolism, Hematologic Neoplasms pathology, Myeloproliferative Disorders genetics, Myeloproliferative Disorders metabolism, Myeloproliferative Disorders pathology, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Transcription, Genetic
- Abstract
Philadelphia negative (Ph-neg) myeloproliferative neoplasms (MPN) are a heterogenous group of clonal stem cell disorders. Approved treatment options include hydroxyurea, anagrelide, and ruxolitinib, which are not curative. The concept of synthetic lethality may become an additional therapeutic strategy in these diseases. In our study, we show that DNA repair is altered in classical Ph-neg MPN, as analyzed by gene expression analysis of 11 genes involved in the homologous recombination repair pathway (HRR), the non-homologous end-joining pathway (NHEJ), and the single-strand break repair pathway (SSB). Altogether, peripheral blood-derived cells from 57 patients with classical Ph-neg MPN and 13 healthy controls were analyzed. LIG3 as an essential part of the SSB was significantly lower expressed compared to controls in all three entities (essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF)). In addition, while genes of other DNA-repair pathways showed-possibly compensatory-increased expression in ET (HRR, NHEJ) and PV (NHEJ), MF samples displayed downregulation of all genes involved in NHEJ. With regard to the JAK2 mutational status (analyzed in ET and MF only), no upregulation of the HRR was detected. Though further studies are needed, based on these findings, we conclude that synthetic lethality may become a promising strategy in treating patients with Ph-neg MPN.
- Published
- 2019
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28. Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling.
- Author
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Kuepper MK, Bütow M, Herrmann O, Ziemons J, Chatain N, Maurer A, Kirschner M, Maié T, Costa IG, Eschweiler J, Koschmieder S, Brümmendorf TH, Müller-Newen G, and Schemionek M
- Subjects
- Animals, Apoptosis, Cell Line, Tumor, Fusion Proteins, bcr-abl antagonists & inhibitors, Fusion Proteins, bcr-abl physiology, Humans, Janus Kinase 1 antagonists & inhibitors, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Mice, Janus Kinase 1 physiology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Neoplastic Stem Cells pathology, STAT3 Transcription Factor physiology, Signal Transduction physiology
- Abstract
Tyrosine kinase inhibitor (TKI) therapy effectively blocks oncogenic Bcr-Abl signaling and induces molecular remission in the majority of CML patients. However, the disease-driving stem cell population is not fully targeted by TKI therapy in the majority of patients, and leukemic stem cells (LSCs) capable of re-inducing the disease can persist. In TKI-resistant CML, STAT3 inhibition was previously shown to reduce malignant cell survival. Here, we show therapy-resistant cell-extrinsic STAT3 activation in TKI-sensitive CML cells, using cell lines, HoxB8-immortalized murine BM cells, and primary human stem cells. Moreover, we identified JAK1 but not JAK2 as the STAT3-activating kinase by applying JAK1/2 selective inhibitors and genetic inactivation. Employing an IL-6-blocking peptide, we identified IL-6 as a mediator of STAT3 activation. Combined inhibition of Bcr-Abl and JAK1 further reduced CFUs from murine CML BM, human CML MNCs, as well as CD34
+ CML cells, and similarly decreased LT-HSCs in a transgenic CML mouse model. In line with these observations, proliferation of human CML CD34+ cells was strongly reduced upon combined Bcr-Abl and JAK1 inhibition. Remarkably, the combinatory therapy significantly induced apoptosis even in quiescent LSCs. Our findings suggest JAK1 as a potential therapeutic target for curative CML therapies.- Published
- 2019
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29. Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia.
- Author
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Herrmann O, Kuepper MK, Bütow M, Costa IG, Appelmann I, Beier F, Luedde T, Braunschweig T, Koschmieder S, Brümmendorf TH, and Schemionek M
- Subjects
- Animals, Apoptosis drug effects, Cell Line, Tumor, Cell Proliferation drug effects, Disease Models, Animal, Drug Resistance, Neoplasm drug effects, Drug Synergism, Drug Therapy, Combination, Fusion Proteins, bcr-abl antagonists & inhibitors, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells metabolism, Humans, Infliximab pharmacology, Mice, Mice, Transgenic, Neoplastic Stem Cells metabolism, Protein Kinase Inhibitors pharmacology, Transduction, Genetic, Tumor Necrosis Factor-alpha antagonists & inhibitors, Tumor Necrosis Factor-alpha immunology, Xenograft Model Antitumor Assays, Anti-Inflammatory Agents therapeutic use, Infliximab therapeutic use, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Neoplastic Stem Cells drug effects, Protein Kinase Inhibitors therapeutic use, Pyrimidines therapeutic use, Tumor Necrosis Factor-alpha metabolism
- Abstract
Background: Expression of Bcr-Abl in hematopoietic stem cells is sufficient to cause chronic myeloid leukemia (CML) and tyrosine kinase inhibitors (TKI) induce molecular remission in the majority of CML patients. However, the disease driving stem cell population is not fully targeted by TKI therapy, and leukemic stem cells (LSC) capable of re-inducing the disease can persist. Single-cell RNA-sequencing technology recently identified an enriched inflammatory gene signature with TNFα and TGFβ being activated in TKI persisting quiescent LSC. Here, we studied the effects of human TNFα antibody infliximab (IFX), which has been shown to induce anti-inflammatory effects in mice, combined with TKI treatment on LSC function., Methods: We first performed GSEA-pathway analysis using our microarray data of murine LSK cells (lin
- ; Sca-1+ ; c-kit+ ) from the SCLtTA/Bcr-Abl CML transgenic mouse model. Bcr-Abl positive cell lines were generated by retroviral transduction. Clonogenic potential was assessed by CFU (colony forming unit). CML mice were treated with nilotinib or nilotinib plus infliximab, and serial transplantation experiments were performed., Results: Likewise to human CML, TNFα signaling was specifically active in murine CML stem cells, and ectopic expression of Bcr-Abl in murine and human progenitor cell lines induced TNFα expression. In vitro exposure to human (IFX) or murine (MP6-XT22) TNFα antibody reduced clonogenic growth of CML cells. Interestingly, TNFα antibody treatment enhanced TKI-induced effects on immature cells in vitro. Additionally, in transplant and serial transplant experiments, using our transgenic CML mouse model, we could subsequently show that IFX therapy boosted TKI-induced effects and further reduced the proportion of malignant stem cells in vivo., Conclusion: TNFα signaling is induced in CML stem cells, and anti-inflammatory therapy enhances TKI-induced decline of LSC, confirming that successful targeting of persisting CML stem cells can be enhanced by addressing their malignant microenvironment simultaneously.- Published
- 2019
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30. Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha.
- Author
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Schubert C, Allhoff M, Tillmann S, Maié T, Costa IG, Lipka DB, Schemionek M, Feldberg K, Baumeister J, Brümmendorf TH, Chatain N, and Koschmieder S
- Subjects
- Apoptosis, Cell Line, Tumor, Cell Proliferation, Humans, Fusion Proteins, bcr-abl metabolism, Interferon-alpha metabolism, Janus Kinase 2 genetics, STAT1 Transcription Factor genetics, STAT2 Transcription Factor genetics
- Abstract
Background: Interferon alpha (IFNa) monotherapy is recommended as the standard therapy in polycythemia vera (PV) but not in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa efficacy in JAK2V617F- vs. BCR-ABL-positive cells., Methods: Gene expression microarrays and RT-qPCR of PV vs. CML patient PBMCs and CD34+ cells and of the murine cell line 32D expressing JAK2V617F or BCR-ABL were used to analyze and compare interferon-stimulated gene (ISG) expression. Furthermore, using CRISPR/Cas9n technology, targeted disruption of STAT1 or STAT2, respectively, was performed in 32D-BCR-ABL and 32D-JAK2V617F cells to evaluate the role of these transcription factors for IFNa efficacy. The knockout cell lines were reconstituted with STAT1, STAT2, STAT1Y701F, or STAT2Y689F to analyze the importance of wild-type and phosphomutant STATs for the IFNa response. ChIP-seq and ChIP were performed to correlate histone marks with ISG expression., Results: Microarray analysis and RT-qPCR revealed significant upregulation of ISGs in 32D-JAK2V617F but downregulation in 32D-BCR-ABL cells, and these effects were reversed by tyrosine kinase inhibitor (TKI) treatment. Similar expression patterns were confirmed in human cell lines, primary PV and CML patient PBMCs and CD34+ cells, demonstrating that these effects are operational in patients. IFNa treatment increased Stat1, Stat2, and Irf9 mRNA as well as pY-STAT1 in all cell lines; however, viability was specifically decreased in 32D-JAK2V617F. STAT1 or STAT2 knockout and reconstitution with wild-type or phospho-deficient STAT mutants demonstrated the necessity of STAT2 for IFNa-induced STAT1 phosphorylation in BCR-ABL- but not in JAK2V617F-expressing cells. STAT1 was essential for IFNa activity in both BCR-ABL- and JAK2V617F-positive cells. Furthermore, ChIP experiments demonstrate higher repressive and lower active chromatin marks at the promoters of ISGs in BCR-ABL-expressing cells., Conclusions: JAK2V617F but not BCR-ABL sensitizes MPN cells to interferon, and this effect was dependent on STAT1. Moreover, STAT2 is a survival factor in BCR-ABL- and JAK2V617F-positive cells but an IFNa-sensitizing factor solely in 32D-JAK2V617F cells by upregulation of STAT1 expression.
- Published
- 2019
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31. The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy.
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Bilich T, Nelde A, Bichmann L, Roerden M, Salih HR, Kowalewski DJ, Schuster H, Tsou CC, Marcu A, Neidert MC, Lübke M, Rieth J, Schemionek M, Brümmendorf TH, Vucinic V, Niederwieser D, Bauer J, Märklin M, Peper JK, Klein R, Kohlbacher O, Kanz L, Rammensee HG, Stevanović S, and Walz JS
- Subjects
- Antigens, Neoplasm metabolism, Epitopes, T-Lymphocyte metabolism, HLA Antigens metabolism, Humans, Immunotherapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive metabolism, Ligands, Antigens, Neoplasm immunology, CD4-Positive T-Lymphocytes immunology, Epitopes, T-Lymphocyte immunology, Fusion Proteins, bcr-abl immunology, HLA Antigens immunology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell-based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients., (© 2019 by The American Society of Hematology.)
- Published
- 2019
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32. Recurrent somatic mutations are rare in patients with cryptic dyskeratosis congenita.
- Author
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Kirschner M, Maurer A, Wlodarski MW, Ventura Ferreira MS, Bouillon AS, Halfmeyer I, Blau W, Kreuter M, Rosewich M, Corbacioglu S, Beck J, Schwarz M, Bittenbring J, Radsak MP, Wilk CM, Koschmieder S, Begemann M, Kurth I, Schemionek M, Brümmendorf TH, and Beier F
- Subjects
- Adult, Dyskeratosis Congenita complications, Female, Follow-Up Studies, Germany epidemiology, Humans, Incidence, Leukemia, Myeloid, Acute epidemiology, Male, Middle Aged, Myelodysplastic Syndromes epidemiology, Prognosis, Young Adult, Biomarkers, Tumor genetics, Dyskeratosis Congenita genetics, Leukemia, Myeloid, Acute genetics, Mutation, Myelodysplastic Syndromes genetics, Telomere Shortening genetics
- Abstract
Dyskeratosis congenita (DKC) is a paradigmatic telomere disorder characterized by substantial and premature telomere shortening, bone marrow failure, and a dramatically increased risk of developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). DKC can occur as a late-onset, so-called cryptic form, with first manifestation in adults. Somatic MDS-related mutations are found in up to 35% of patients with acquired aplastic anemia (AA), especially in patients with short telomeres. The aim of our study was to investigate whether cryptic DKC is associated with an increased incidence of MDS-related somatic mutations, thereby linking the accelerated telomere shortening with the increased risk of MDS/AML. Samples from 15 adult patients (median age: 42 years, range: 23-60 years) with molecularly confirmed cryptic DKC were screened using next-generation gene panel sequencing to detect MDS-related somatic variants. Only one of the 15 patients (7%) demonstrated a clinically relevant MDS-related somatic variant. This incidence was dramatically lower than formerly described in acquired AA. Based on our data, we conclude that clonal evolution of subclones carrying MDS-related mutations is not the predominant mechanism for MDS/AML initiation in adult cryptic DKC patients.
- Published
- 2018
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33. Telomere shortening correlates with leukemic stem cell burden at diagnosis of chronic myeloid leukemia.
- Author
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Bouillon AS, Ventura Ferreira MS, Awad SA, Richter J, Hochhaus A, Kunzmann V, Dengler J, Janssen J, Ossenkoppele G, Westerweel PE, Te Boekhorst PAW, Mahon FX, Hjorth-Hansen H, Isfort S, Fioretos T, Hummel S, Schemionek M, Wilop S, Koschmieder S, Saußele S, Mustjoki S, Beier F, and Brümmendorf TH
- Subjects
- Adult, Aged, Female, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive diagnosis, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Male, Middle Aged, Protein Kinase Inhibitors administration & dosage, Hematopoietic Stem Cells metabolism, Leukemia, Myelogenous, Chronic, BCR-ABL Positive metabolism, Telomere Homeostasis
- Abstract
Telomere length (TL) in peripheral blood (PB) cells of patients with chronic myeloid leukemia (CML) has been shown to correlate with disease stage, prognostic scores, response to therapy, and disease progression. However, due to considerable genetic interindividual variability, TL varies substantially between individuals, limiting its use as a robust prognostic marker in individual patients. Here, we compared TL of BCR-ABL
- , nonleukemic CD34+ CD38- hematopoietic stem cells (HSC) in the bone marrow of CML patients at diagnosis to their individual BCR-ABL+ leukemic stem cell (LSC) counterparts. We observed significantly accelerated telomere shortening in LSC compared with nonleukemic HSC. Interestingly, the degree of LSC telomere shortening was found to correlate significantly with the leukemic clone size. To validate the diagnostic value of nonleukemic cells as internal controls and to rule out effects of tyrosine kinase inhibitor (TKI) treatment on these nontarget cells, we prospectively assessed TL in 134 PB samples collected in deep molecular remission after TKI treatment within the EURO-SKI study (NCT01596114). Here, no significant telomere shortening was observed in granulocytes compared with an age-adjusted control cohort. In conclusion, this study provides proof of principle for accelerated telomere shortening in LSC as opposed to HSC in CML patients at diagnosis. The fact that the degree of telomere shortening correlates with leukemic clone's size supports the use of TL in leukemic cells as a prognostic parameter pending prospective validation. TL in nonleukemic myeloid cells seems unaffected even by long-term TKI treatment arguing against a reduction of telomere-mediated replicative reserve in normal hematopoiesis under TKI treatment., (© 2018 by The American Society of Hematology.)- Published
- 2018
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34. Evidence for a pre-existing telomere deficit in non-clonal hematopoietic stem cells in patients with acute myeloid leukemia.
- Author
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Ventura Ferreira MS, Crysandt M, Ziegler P, Hummel S, Wilop S, Kirschner M, Schemionek M, Jost E, Wagner W, Brümmendorf TH, and Beier F
- Subjects
- Adult, Age Factors, Aged, Aged, 80 and over, Female, Follow-Up Studies, Humans, Leukemia, Myeloid, Acute diagnosis, Leukemia, Myeloid, Acute therapy, Male, Middle Aged, Polymerase Chain Reaction, Hematopoietic Stem Cells metabolism, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute metabolism, Telomere genetics, Telomere metabolism, Telomere Homeostasis genetics
- Abstract
Telomere shortening represents an established mechanism connecting aging and cancer development. We sequentially analyzed telomere length (TL) of 49 acute myeloid leukemia (AML) patients at diagnosis (n = 24), once they achieved complete cytological remission (CCR) and/or during refractory disease or relapse and after 1-year follow-up, with all patients having at least two sequential samples. TL was analyzed by monochrome multiplex quantitative polymerase chain reaction. We have observed substantially shortened TL in the cells of patients at diagnosis compared to age-adjusted controls. In patients reaching CCR after chemotherapy, telomere shortening was less pronounced than in persistence or relapse but still significantly shortened compared to controls. We estimate patients harboring approximately 20 years of premature telomere loss compared to healthy aged-matched subjects at the time of AML onset. Our data indicate a pre-existing telomere deficit in non-clonal hematopoiesis of AML patients providing a link between age and AML development.
- Published
- 2017
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35. A meta-analysis of HLA peptidome composition in different hematological entities: entity-specific dividing lines and "pan-leukemia" antigens.
- Author
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Backert L, Kowalewski DJ, Walz S, Schuster H, Berlin C, Neidert MC, Schemionek M, Brümmendorf TH, Vucinic V, Niederwieser D, Kanz L, Salih HR, Kohlbacher O, Weisel K, Rammensee HG, Stevanovic S, and Walz JS
- Subjects
- Antigens, Neoplasm immunology, Cell Line, Tumor, Cluster Analysis, Epitopes chemistry, Epitopes metabolism, HLA Antigens chemistry, HLA Antigens metabolism, Hematologic Neoplasms immunology, Hematologic Neoplasms metabolism, Hematologic Neoplasms therapy, Humans, Immunotherapy, Leukemia immunology, Leukemia metabolism, Leukemia therapy, Ligands, Peptides chemistry, Peptides metabolism, Epitope Mapping methods, Epitopes immunology, HLA Antigens immunology, Peptides immunology
- Abstract
Hematological malignancies (HM) are highly amenable targets for immunotherapeutic intervention and may be effectively treated by antigen-specific T-cell based treatment. Recent studies demonstrate that physiologically occurring anti-cancer T-cell responses in certain HM entities target broadly presented non-mutated epitopes. HLA ligands are thus implied as prime targets for broadly applicable and antigen-specific off-the-shelf compounds. With the aim of assessing the presence of common targets shared among different HM which may enable addressing a larger patient collective we conducted a meta-analysis of 83 mass spectrometry-based HLA peptidome datasets (comprising 40,361 unique peptide identifications) across four major HM (19 AML, 16 CML, 35 CLL, and 13 MM/MCL samples) and investigated similarities and differences within the HLA presented antigenic landscape. We found the cancer HLA peptidome datasets to cluster specifically along entity and lineage lines, suggesting that the immunopeptidome directly reflects the differences in the underlying (tumor-)biology. In line with these findings, we only detected a small set of entity-spanning antigens, which were predominantly characterized by low presentation frequencies within the different patient cohorts. These findings suggest that design of T-cell immunotherapies for the treatment of HM should ideally be conducted in an entity-specific fashion.
- Published
- 2017
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36. The SCLtTAxBCR-ABL transgenic mouse model closely reflects the differential effects of dasatinib on normal and malignant hematopoiesis in chronic phase-CML patients.
- Author
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Schubert C, Chatain N, Braunschweig T, Schemionek M, Feldberg K, Hoffmann M, Dufva O, Mustjoki S, Brümmendorf TH, and Koschmieder S
- Subjects
- Animals, Dasatinib pharmacology, Erythrocytes drug effects, Granulocytes drug effects, Hematopoiesis drug effects, Humans, Killer Cells, Natural drug effects, Leukemia, Myeloid, Chronic-Phase genetics, Mice, Mice, Transgenic, Protein Kinase Inhibitors pharmacology, Dasatinib administration & dosage, Disease Models, Animal, Fusion Proteins, bcr-abl genetics, Leukemia, Myeloid, Chronic-Phase drug therapy, Protein Kinase Inhibitors administration & dosage
- Abstract
The second generation tyrosine kinase inhibitor (TKI) dasatinib is a clinically approved drug for chronic myeloid leukemia (CML) as well as Ph+ acute lymphoblastic leukemia. In addition to its antileukemic effects, dasatinib was shown to impact on normal hematopoiesis and cells of the immune system.Due to the fact that the murine in vivo studies so far have not been performed in a chronic-phase CML model under steady-state conditions, our aim was to study the hematopoietic effects of dasatinib (20 mg/kg p.o.) in BCR-ABL expressing SCLtTAxBCR-ABL double transgenic (dtg) mice. Dasatinib robustly antagonized the CML phenotype in vivo in our transgenic mouse model, and this effect included both mature and immature cell populations. However, similar to patients with CML, the fraction of LinnegSca-1+KIT+CD48negCD150+ hematopoietic stem cells was not reduced by dasatinib treatment, suggesting that these cells are not oncogene-addicted. Moreover, we observed differential effects of dasatinib in these animals as compared to wild-type (wt) animals: while granulocytes were significantly reduced in dtg animals, they were increased in wt mice. And Ter119+ erythrocytic and B220+ B cells were increased in dtg mice but decreased in wt mice. Finally, while dasatinib induced a shift from CD49b/NK1.1 positive NK cells from the bone marrow to the spleen in wt animals, there was no change in dtg mice. In conclusion, the present mouse model provides a useful tool to study mechanisms of TKI resistance and dasatinib-associated beneficial effects and adverse events.
- Published
- 2017
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37. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion.
- Author
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Han L, Schubert C, Köhler J, Schemionek M, Isfort S, Brümmendorf TH, Koschmieder S, and Chatain N
- Subjects
- Animals, Calreticulin physiology, Cell Death, Cell Line, Cell Line, Tumor, Frameshift Mutation, Humans, Mice, Myeloproliferative Disorders genetics, NF-E2 Transcription Factor, p45 Subunit analysis, Platelet Membrane Glycoprotein IIb analysis, Proteolysis, Receptors, Thrombopoietin analysis, Calreticulin genetics, Cell Transformation, Neoplastic genetics, Golgi Apparatus metabolism, Megakaryocytes metabolism, Mutant Proteins physiology, Signal Transduction
- Abstract
Background: Somatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated., Methods: The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment., Results: The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation., Conclusions: These findings demonstrate the potency of CALR mutants to drive expression of megakaryocytic differentiation markers such as NF-E2 and CD41 as well as Mpl. Furthermore, CALR mutants undergo accelerated protein degradation that involves the secretory pathway and/or protein glycosylation.
- Published
- 2016
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38. Tumor Suppressor Analysis in CML.
- Author
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Herrmann O and Schemionek M
- Subjects
- Animals, Bone Marrow Transplantation, Disease Models, Animal, Genes, Tumor Suppressor, Genetic Vectors administration & dosage, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Mice, Mice, Knockout, Fusion Proteins, bcr-abl genetics, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Retroviridae genetics
- Abstract
Retroviral models have tremendously contributed to our understanding of CML development and have been indispensable for preclinical drug testing which facilitated the implementation of a targeted therapy. The retroviral insertion of Bcr-Abl into mice that are genetically depleted for a potential tumor suppressor is a tool to test for a specific gene function in Bcr-Abl disease. Here we describe how to generate a Bcr-Abl retrovirus that is subsequently used for infection of primary murine BM cells, which are genetically depleted for a potential tumor suppressor gene. We will suggest control experiments and outline further methods that are required to allow for assessment of disease development upon tumor suppressor knockout in CML.
- Published
- 2016
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39. Identification of the Adapter Molecule MTSS1 as a Potential Oncogene-Specific Tumor Suppressor in Acute Myeloid Leukemia.
- Author
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Schemionek M, Kharabi Masouleh B, Klaile Y, Krug U, Hebestreit K, Schubert C, Dugas M, Büchner T, Wörmann B, Hiddemann W, Berdel WE, Brümmendorf TH, Müller-Tidow C, and Koschmieder S
- Subjects
- Cell Line, Tumor, Chromosome Aberrations, Cluster Analysis, Core Binding Factor Alpha 2 Subunit genetics, Gene Expression, Gene Expression Profiling, Gene Expression Regulation, Leukemic drug effects, Humans, Karyotype, Leukemia, Myeloid, Acute metabolism, Leukemia, Myeloid, Acute mortality, Microfilament Proteins metabolism, Neoplasm Proteins metabolism, Oncogene Proteins, Fusion genetics, Prognosis, Protein Kinase Inhibitors pharmacology, RUNX1 Translocation Partner 1 Protein, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, fms-Like Tyrosine Kinase 3 genetics, Leukemia, Myeloid, Acute genetics, Microfilament Proteins genetics, Neoplasm Proteins genetics
- Abstract
The adapter protein metastasis suppressor 1 (MTSS1) is implicated as a tumor suppressor or tumor promoter, depending on the type of solid cancer. Here, we identified Mtss1 expression to be increased in AML subsets with favorable outcome, while suppressed in high risk AML patients. High expression of MTSS1 predicted better clinical outcome of patients with normal-karyotype AML. Mechanistically, MTSS1 expression was negatively regulated by FLT3-ITD signaling but enhanced by the AML1-ETO fusion protein. DNMT3B, a negative regulator of MTSS1, showed strong binding to the MTSS1 promoter in PML-RARA positive but not AML1-ETO positive cells, suggesting that AML1-ETO leads to derepression of MTSS1. Pharmacological treatment of AML cell lines carrying the FLT3-ITD mutation with the specific FLT3 inhibitor PKC-412 caused upregulation of MTSS1. Moreover, treatment of acute promyelocytic cells (APL) with all-trans retinoic acid (ATRA) increased MTSS1 mRNA levels. Taken together, our findings suggest that MTSS1 might have a context-dependent function and could act as a tumor suppressor, which is pharmacologically targetable in AML patients.
- Published
- 2015
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40. Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing.
- Author
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Kirschner MM, Schemionek M, Schubert C, Chatain N, Sontag S, Isfort S, Ortiz-Brüchle N, Schmitt K, Krüger L, Zerres K, Zenke M, Brümmendorf TH, and Koschmieder S
- Subjects
- Alleles, Cell Line, Tumor, Follow-Up Studies, Humans, Janus Kinase 2 genetics, Mutation genetics, Polymorphism, Single Nucleotide genetics, Quality Control, Bone Marrow Neoplasms genetics, Genome, Human, High-Throughput Nucleotide Sequencing methods, Multiplex Polymerase Chain Reaction methods, Myeloproliferative Disorders genetics
- Abstract
In order to assess the feasibility of amplicon-based parallel next generation sequencing (NGS) for the diagnosis of myeloproliferative neoplasms (MPN), we investigated multiplex-PCR of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes. Samples from 64 patients with MPN and five controls as well as seven (myeloid) cell lines were analyzed. Healthy donor and reactive erythrocytosis samples showed several frequent single-nucleotide polymorphisms (SNPs) but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of the known mutations. In the patient samples, JAK2 V617F was present in all PV, 4 of 10 ET, and 16 of 19 MF patients. The JAK2 V617F allele burden was different in the three groups (ET, 33+/-22%; PV 48+/-28% and MF 68+/- 29%). Further analysis detected both previously described and undescribed mutations (i.e., G12V NRAS, IDH1 R132H, E255G ABL, and V125G IDH1 mutations). One patient with lymphoid BC/Ph+ ALL who harbored a T315I ABL mutation and was treated with ponatinib was found to have developed a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation might have caused ponatinib resistance in this patient. In conclusion, amplicon-sequencing-based NGS allows simultaneous analysis of multiple MPN associated genes for diagnosis and during treatment and measurement of the mutant allele burden.
- Published
- 2015
- Full Text
- View/download PDF
41. Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells.
- Author
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Bolton-Gillespie E, Schemionek M, Klein HU, Flis S, Hoser G, Lange T, Nieborowska-Skorska M, Maier J, Kerstiens L, Koptyra M, Müller MC, Modi H, Stoklosa T, Seferynska I, Bhatia R, Holyoake TL, Koschmieder S, and Skorski T
- Subjects
- Animals, Cells, Cultured, DNA Damage drug effects, Humans, Imatinib Mesylate, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Mice, Mice, Transgenic, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology, Oxidative Stress drug effects, Oxidative Stress genetics, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Reactive Oxygen Species metabolism, Antineoplastic Agents therapeutic use, Benzamides therapeutic use, Drug Resistance, Neoplasm genetics, Genomic Instability drug effects, Genomic Instability physiology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Neoplastic Stem Cells physiology, Piperazines therapeutic use, Pyrimidines therapeutic use
- Abstract
Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.
- Published
- 2013
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42. Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival.
- Author
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Hamilton A, Helgason GV, Schemionek M, Zhang B, Myssina S, Allan EK, Nicolini FE, Müller-Tidow C, Bhatia R, Brunton VG, Koschmieder S, and Holyoake TL
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Animals, Antigens, CD34 metabolism, Apoptosis drug effects, Blotting, Western, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, Dasatinib, Dose-Response Relationship, Drug, Flow Cytometry, Fusion Proteins, bcr-abl antagonists & inhibitors, Fusion Proteins, bcr-abl genetics, Gene Expression Regulation, Leukemic, Gene Knockout Techniques, HEK293 Cells, Humans, K562 Cells, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Mice, Mice, Transgenic, Neoplastic Stem Cells pathology, Nuclear Proteins metabolism, Protein Kinase Inhibitors pharmacology, Pyrimidines pharmacology, Reverse Transcriptase Polymerase Chain Reaction, STAT5 Transcription Factor metabolism, Thiazoles pharmacology, Fusion Proteins, bcr-abl metabolism, Leukemia, Myelogenous, Chronic, BCR-ABL Positive metabolism, Neoplastic Stem Cells metabolism
- Abstract
Recent evidence suggests chronic myeloid leukemia (CML) stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, after retransplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off were able to persist in vivo and reinitiate leukemia in secondary recipients on Bcr-Abl reexpression. Bcr-Abl knockdown in human CD34(+) CML cells cultured for 12 days in physiologic growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%; however, the surviving fraction was enriched for primitive leukemic cells capable of growth in a long-term culture-initiating cell assay and expansion on removal of dasatinib and addition of growth factors. Together, these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance.
- Published
- 2012
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43. Intracellular retention of ABL kinase inhibitors determines commitment to apoptosis in CML cells.
- Author
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Lipka DB, Wagner MC, Dziadosz M, Schnöder T, Heidel F, Schemionek M, Melo JV, Kindler T, Müller-Tidow C, Koschmieder S, and Fischer T
- Subjects
- ATP-Binding Cassette Transporters metabolism, Annexin A5 metabolism, Benzamides, Caspase 3 metabolism, Cell Line, Tumor, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, Humans, Imatinib Mesylate, Intracellular Space drug effects, Kinetics, Piperazines pharmacology, Proto-Oncogene Proteins c-abl metabolism, Pyrimidines pharmacology, Signal Transduction drug effects, Time Factors, Apoptosis drug effects, Intracellular Space metabolism, Leukemia, Myelogenous, Chronic, BCR-ABL Positive enzymology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-abl antagonists & inhibitors
- Abstract
Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs.
- Published
- 2012
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44. Novel imatinib-sensitive PDGFRA-activating point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia-like disease.
- Author
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Elling C, Erben P, Walz C, Frickenhaus M, Schemionek M, Stehling M, Serve H, Cross NC, Hochhaus A, Hofmann WK, Berdel WE, Müller-Tidow C, Reiter A, and Koschmieder S
- Subjects
- Animals, Benzamides, Blotting, Western, Cell Separation, Female, Flow Cytometry, Humans, Imatinib Mesylate, Leukemia drug therapy, Male, Mice, Reverse Transcriptase Polymerase Chain Reaction, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Cell Transformation, Neoplastic genetics, Hypereosinophilic Syndrome genetics, Leukemia genetics, Piperazines pharmacology, Point Mutation, Pyrimidines pharmacology, Receptor, Platelet-Derived Growth Factor alpha genetics
- Abstract
The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients.
- Published
- 2011
- Full Text
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45. Mouse models as tools to understand and study BCR-ABL1 diseases.
- Author
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Koschmieder S and Schemionek M
- Abstract
Mouse models of human malignancy have greatly enhanced our understanding of disease pathophysiology and have led to novel therapeutic approaches, some with extraordinary success, one such example being inhibition of the BCR-ABL1 oncogene in chronic myeloid leukaemia (CML). Here, we review aspects of the biology of CML that have been uncovered at least in part through the generation and analysis of retroviral and transgenic mouse models of BCR-ABL1 disease. It can be expected that these models will also serve as important tools in the future, especially in the rational design of strategies to eradicate leukemic stem cells and potentially cure CML as well as other cancers.
- Published
- 2011
46. Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells.
- Author
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Myssina S, Helgason GV, Serrels A, Jørgensen HG, Bhatia R, Modi H, Baird JW, Mountford JC, Hamilton A, Schemionek M, Koschmieder S, Brunton VG, and Holyoake TL
- Subjects
- Annexin A5 biosynthesis, Annexin A5 genetics, Apoptosis genetics, Blotting, Western, Caspase 3 biosynthesis, Caspase 3 genetics, Dasatinib, Dose-Response Relationship, Drug, Flow Cytometry, Fusion Proteins, bcr-abl genetics, Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Enzymologic genetics, Gene Expression Regulation, Leukemic drug effects, Gene Expression Regulation, Leukemic genetics, Humans, K562 Cells, RNA genetics, RNA metabolism, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Apoptosis drug effects, Fusion Proteins, bcr-abl antagonists & inhibitors, Fusion Proteins, bcr-abl metabolism, Lentivirus, Philadelphia Chromosome, Protein Kinase Inhibitors pharmacology, Pyrimidines pharmacology, Thiazoles pharmacology
- Abstract
Objective: This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph(+)) cells., Materials and Methods: shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining., Results: Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral delivery and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0-10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells., Conclusion: These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment.
- Published
- 2009
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47. PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa.
- Author
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Campisano A, Schroeder C, Schemionek M, Overhage J, and Rehm BH
- Subjects
- Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins genetics, Microscopy, Fluorescence, Mutation, Periplasm metabolism, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism, Bacterial Outer Membrane Proteins metabolism, Biofilms growth & development, Gene Expression Regulation, Bacterial, Pseudomonas aeruginosa growth & development
- Abstract
The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.
- Published
- 2006
- Full Text
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48. Expression of the psl operon in Pseudomonas aeruginosa PAO1 biofilms: PslA performs an essential function in biofilm formation.
- Author
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Overhage J, Schemionek M, Webb JS, and Rehm BH
- Subjects
- Bacterial Proteins genetics, Base Sequence, Carrier Proteins genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Lac Operon genetics, Lac Operon physiology, Molecular Sequence Data, Operon, Promoter Regions, Genetic, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Uridine Diphosphate Glucose metabolism, Bacterial Proteins metabolism, Biofilms growth & development, Carrier Proteins metabolism, Gene Expression Regulation, Bacterial, Polysaccharides, Bacterial biosynthesis, Pseudomonas aeruginosa growth & development
- Abstract
The psl gene cluster, comprising 15 cotranscribed genes from Pseudomonas aeruginosa, was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the psl gene cluster and the function of the first gene in this cluster, the pslA gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free pslA deletion mutant of P. aeruginosa PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the pslA gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid P. aeruginosa. The promoter region of the psl gene cluster, which encodes PslA-PslO, was identified by rapid amplification of cDNA 5' ends. Promoter assays using transcriptional fusions to lacZ and gfp indicated a constitutive expression of the psl cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the psl cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the psl operon enables efficient attachment to surfaces and that regulated localized psl operon expression is required for biofilm differentiation.
- Published
- 2005
- Full Text
- View/download PDF
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