Inhibition of
Knockdown of
Presence of
The use of tyrosine kinase inhibitors, such as imatinib, against the chronic myeloid leukemia (CML)-causing kinase BCR::ABL1 has become the model for successful targeted therapy. Nevertheless, drug resistance remains a clinical problem. Analysis of genome-wide expression and genetic aberrations of an
The use of tyrosine kinase inhibitors (TKIs), i.e., imatinib, in the hematological malignancy chronic myeloid leukemia (CML) is the most prominent model for successful targeted therapy. This drug targets the disease-causing, constitutively active BCR::ABL1 (breakpoint cluster region/Abelson tyrosine kinase 1) kinase, which results from the reciprocal t(9;22)(q34;q11) translocation forming the so-called Philadelphia chromosome and the
First identified in loss-of-function mutations causing X-linked agammaglobulinemia (XLA)/Bruton syndrome, BTK is central for B lymphocyte development (
Consequently, BTK became a potential druggable candidate for targeted therapy, leading to the introduction of the selective and FDA-approved BTK inhibitor (BTKi) ibrutinib, which is used as a single agent or combinational treatment in various cancers (
The aim of the current study was to investigate the role of
All chemicals and reagents not indicated differently, were purchased from Sigma-Aldrich or Carl Roth. Imatinib and ibrutinib were obtained from Sigma-Aldrich; Merck KGaA. Imatinib was diluted in 10 mM aqueous stock solutions, ibrutinib in 100 mM stock solutions in DMSO, both stored at −20°C.
K-562 cells (RRID: CVCL 0004), retrieved from the pleural effusion of a 53-year old woman (
Mutagenesis of an
Total RNA isolation was performed using 1×106 K-562 cells and PeqGOLD TriFast (VWR) according to the manufacturer's recommendation. Cell line DNA was purified using Gentra Puregene Cell Kit (Qiagen) according to the manufacturer's protocol.
Generation of PCR products was performed using MyTaq Polymerase (Bioline), DNA of treatment-naïve, 0.5 µM and 2 µM imatinib-resistant cells, and the primers BTK_Intron_F 5′-TGACACTCTTGTGACCGTGC-3′ and BTK_Intron_R 5′-ACAGTAAGCACTCCCCAAGG-3′ with annealing at 54°C. PCR products were purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Inc.). After PCR amplicon preparation with the Nextera XT Sequencing Kit (Illumina), Next Generation Sequencing (NGS) SBS technology with Illumina MiSeq was performed as described elsewhere (
According to the manufacturer's protocol, 1 µg total RNA was reversely transcribed using random hexamer primers and the High Capacity cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.) for 10 min at 25°C, 120 min at 37°C and 5 min at 85°C. cDNA was stored at −80°C prior further usage. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) of target genes was performed in triplicates using the TaqMan assays (Thermo Fischer Scientific, Inc.)
Whole cell lysates and immunoblotting were performed as described elsewhere (
Plasmid and siRNA transfection were performed using the Amaxa nucleofector Kit V and nucleofector 2 b device (Lonza). For plasmid transfection, 2×106 cells were transfected with 10 µg of the respective plasmid. 24 h after transfection, cells were seeded onto cell plates for subsequent experiments. For siRNA transfection, K-562 cells were transfected with 200 nM negative control stealth (1295300) and stealth RNAi Pre-designed
Cell numbers were obtained by trypan blue staining and performed after 48 h imatinib incubation as described elsewhere (
Pathogenicity prediction of
In a previous study, an
As previously reported, the novel
Since a putative pathogenic
To investigate whether loss of
Vice versa, to analyze whether high
Since
As the
The Bruton's tyrosine kinase (BTK) was initially associated with X-linked agammaglobulinemia (XLA), a primary immunodeficiency disorder characterized by a severe blockade of B cell development in the bone marrow resulting in a lack of B cells and serum antibodies due to (
BTK is a 77 kDa protein composed of 659 amino acids and five domains: A plectrin homology (PH), a Tec homology (TH), an SH3 and SH2 domain, and the C-terminal kinase domain (
BCR::ABL1 TKIs are tremendously successful in CML treatment. However, therapy resistance remains a clinical problem with a need for TKI alternatives for 20–25% of CML patients within five years after therapy onset (
Due to its function as a kinase and its role as an oncogene, it is generally assumed that
In conclusion, the present data show that
The authors would like to thank Ms. Irina Naujoks, Ms. Kerstin Viertmann and Ms. Anna Jürgensen (Institute of Experimental and Clinical Pharmacology, Kiel, Germany) and Ms. Claudia Becher (Institute of Human Genetics, Kiel, Germany), for their technical assistance. The authors would like to also thank Dr. D. Langfeldt, Ms. Manuela Pendziwiat and Dr. B. Löscher (Institute of Clinical Molecular Biology, Kiel Germany) for their technical support. The authors would like to acknowledge the Institute of Clinical Molecular Biology in Kiel (Kiel, Germany) for providing Sanger sequencing as supported in part by the DFG Clusters of Excellence ‘Precision Medicine in Chronic Inflammation’ and ‘ROOTS’.
The data generated in the present study may be requested re from the corresponding author. The exome sequencing and gene expression data in the present study may be found in the European Nucleotide Archive (ENA) repository under the accession number (PRJEB60565;
MK and IN conceptualized the study. MK designed the research. LS performed the experiments. LS, MK and IV analyzed the data. LS, IC, MK interpreted the data. LS and MK wrote the original draft. All authors read and approved the final version of the manuscript. MK and LS confirmed the authenticity of all the raw data.
Not applicable.
Not applicable.
All authors declare that they have no competing interests.
Dr Inga Nagel,
Inhibition of
Knockdown of
Presence of