Transposon-mediated generation of <i>BCR-ABL1</i>-expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors

Byrgazov, Konstantin; Lucini, Chantal; Berkowitsch, Bettina; Koenig, Margit; Haas, Oskar A.; Hoermann, Gregor; Valent, Peter; Lion, Thomas

doi:10.18632/oncotarget.12943

Cited by 10 publications

(5 citation statements)

References 46 publications

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“…In addition, we employed untransfected (BCR-ABL1-negative) Ba/F3 cells, Ba/F3 cells harboring wild type (WT) BCR-ABL1 (Ba/F3p210 WT ) or BCR-ABL1 T315I (Ba/F3p210 T315I ) [43]. Ba/F3 cells expressing T315I-based compound mutations (BCR-ABL1 T315I/E255K , BCR-ABL1 T315I/F311L , BCR-ABL1 T315I/F359V , BCR-ABL1 T315I/G250E ) were generated as described recently [44]. Primary PB and BM mononuclear cells (MNC) were kept in culture as reported [45].…”

Section: Methodsmentioning

confidence: 99%

CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML

Schneeweiss-Gleixner

Byrgazov²,

Stefanzl

et al. 2019

EBioMedicine

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Ponatinib is the only approved tyrosine kinase inhibitor (TKI) suppressing BCR-ABL1 T315I-mutated cells in chronic myeloid leukemia (CML). However, due to side effects and resistance, BCR-ABL1 T315I-mutated CML remains a clinical challenge. Hydroxyurea (HU) has been used for cytoreduction in CML for decades. We found that HU suppresses or even eliminates BCR-ABL1 T315I + sub-clones in heavily pretreated CML patients. Based on this observation, we investigated the effects of HU on TKI-resistant CML cells in vitro. Methods: Viability, apoptosis and proliferation of drug-exposed primary CML cells and BCR-ABL1+ cell lines were examined by flow cytometry and 3 H-thymidine-uptake. Expression of drug targets was analyzed by qPCR and Western blotting. Findings: HU was more effective in inhibiting the proliferation of leukemic cells harboring BCR-ABL1 T315I or T315I-including compound-mutations compared to cells expressing wildtype BCR-ABL1. Moreover, HU synergized with ponatinib and ABL001 in inducing growth inhibition in CML cells. Furthermore, HU blocked cell cycle progression in leukemic cells, which was accompanied by decreased expression of CDK4 and CDK6. Palbociclib, a more specific CDK4/CDK6-inhibitor, was also found to suppress proliferation in primary CML cells and to synergize with ponatinib in producing growth inhibition in BCR-ABL1 T315I + cells, suggesting that suppression of CDK4/CDK6 may be a promising concept to overcome BCR-ABL1 T315I-associated TKI resistance. Interpretation: HU and the CDK4/CDK6-blocker palbociclib inhibit growth of CML clones expressing BCR-ABL1 T315I or complex T315I-including compound-mutations. Clinical studies are required to confirm single drug effects and the efficacy of`ponatinib+HUandponatinib+palbociclibcombinations in advanced CML. Funding: This project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625.

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Section: Methodsmentioning

confidence: 99%

CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML

Schneeweiss-Gleixner

Byrgazov²,

Stefanzl

et al. 2019

EBioMedicine

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“…Such differences might explain the fact that the data provided by different heatmaps do not overlap in all instances. In fact, a direct comparison of the predicted TKI responses for individual mutations may reveal major differences between various heatmaps [123]. Moreover, in some instances, detection of a BCR-ABL1 KD mutation in a patient may merely identify a specific leukemic subclone in which TKI resistance is not driven by the KD mutation detected, but potentially by other unidentified genetic changes in the affected cells.…”

Section: Main Textmentioning

confidence: 99%

Treatment and monitoring of Philadelphia chromosome-positive leukemia patients: recent advances and remaining challenges

2019

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The Philadelphia (Ph) chromosome, resulting from the t(9;22)(q34;q11) translocation, can be found in chronic myeloid leukemia (CML) as well as in a subset of acute lymphoblastic leukemias (ALL). The deregulated BCR-ABL1 tyrosine kinase encoded by the fusion gene resulting from the translocation is considered the pathogenetic driver and can be therapeutically targeted. In both CML and Ph-positive (Ph+) ALL, tyrosine kinase inhibitors (TKIs) have significantly improved outcomes. In the TKI era, testing for BCR-ABL1 transcript levels by real-time quantitative polymerase chain reaction (RQ-PCR) has become the gold standard to monitor patient response, anticipate relapse, and guide therapeutic decisions. In CML, key molecular response milestones have been defined that draw the ideal trajectory towards optimal long-term outcomes. Treatment discontinuation (treatment-free remission, TFR) has proven feasible in a proportion of patients, and clinical efforts are now focused on how to increase this proportion and how to best select TFR candidates. In Ph+ ALL, results of trials with second- and third-generation TKIs are challenging the role of intensive chemotherapy and even that of allogeneic stem cell transplantation. Additional weapons are offered by the recently introduced monoclonal antibodies. In patients harboring mutations in the BCR-ABL1 kinase domain, prompt therapeutic reassessment and individualization based on mutation status are important to regain response and prevent disease progression. Next-generation sequencing is likely to become a precious tool for mutation testing because of the greater sensitivity and the possibility to discriminate between compound and polyclonal mutations. In this review, we discuss the latest advances in treatment and monitoring of CML and Ph+ ALL and the issues that still need to be addressed to make the best use of the therapeutic armamentarium and molecular testing technologies currently at our disposal.

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“…10 We have therefore established a transposon-based approach to rapid and efficient transfection of mutant BCR-ABL1 constructs into cells, and implemented a flow cytometryassisted selection method facilitating targeted enrichment of cells carrying single gene construct insertions in the genome. This protocol was demonstrated to provide BCR-ABL1 expression levels similar to those observed in patient specimens and to permit unbiased testing of TKI sensitivity in vitro.…”

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confidence: 99%

BCR-ABL1 compound mutants display differential and dose-dependent responses to ponatinib

et al. 2017

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Transposon-mediated generation of BCR-ABL1-expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors

Cited by 10 publications

References 46 publications

CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML

CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML

Treatment and monitoring of Philadelphia chromosome-positive leukemia patients: recent advances and remaining challenges

BCR-ABL1 compound mutants display differential and dose-dependent responses to ponatinib

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