Background: Chronic myeloid leukemia (CML) is one of the most extensively studied hematopoietic disorders. Caused by reciprocal translocation t(9;22) and formation of the Philadelphia chromosome, it leads to constitutive expression of the BCR-ABL kinase and malignant transformation. Targeted therapy using tyrosine kinase inhibitors (TKI), such as imatinib or nilotinib, led to tremendous success in treatment. However, resistances are an uprising problem in patient cure. Besides BCR-ABL-dependent mechanisms (i.e. BCR-ABL amplification, mutations), cellular alterations of gene expression and its regulation are involved. Therefore, we performed an in vitro-study in K-562 CML cells to investigate changes of gene expression and epigenetics, as well as occurrence of mutations during the course of imatinib or nilotinib resistance.
Methods: We analyzed mRNA expression, DNA-methylation and mutation status of treatment-naïve and TKI-resistant sublines (0.5 µM, 2 µM imatinib; 100 nM nilotinib) of the in vitro-cell line model (K-562). mRNA expression was analyzed using Affymetrix Human Gene 2.0 ST arrays and DNA-methylation by Infinium MethylationEPIC BeadChips (Illumina); exome sequencing was performed at GATC, Konstanz, Germany. Target verification was performed using qRT-PCR, pyrosequencing or Sanger sequencing. Effects on TKI-susceptibility were analyzed using transfection experiments with analyses of cell viability (WST-1 assay) and apoptosis (Caspase Glo 9 assay).
Results: Cell resistant to low imatinib doses exhibited differentially expression of 485 mRNAs compared to treatment-naïve cells, whereas at high dose-resistance 166 mRNAs differed. 151 genes showed expression changes independent from the imatinib dose. In addition, we found 45 genes with expression changes during the course of imatinib resistance, i.e. the drug efflux transporter ABCG2, which was significantly upregulated at low and downregulated at high imatinib doses. A total of 254 genes was associated with nilotinib resistance, while 71 genes were differentially expressed in all resistant sublines. Regarding methylation and occurrence of mutations in imatinib resistance, we observed distinct variations pointing to genetic changes, especially at high-dose resistance, i.e. in the promoter region of microRNA-212, a proven regulator of ABCG2. In nilotinib resistance, we found 125 genes differentially methylated during development of resistance.
Conclusions: Our data indicate that development of resistance to tyrosine kinase inhibitors is based on complex expression profile changes that can in part be explained by epigenetic alteration e.g. in drug efflux transporters. Besides, the mechanisms underlying resistances to imatinib or nilotinib seem to be dissimilar. After verification in clinical studies, these in vitro findings could be relevant in attempts to overcome TKI-resistances.
Citation Format: Meike Kaehler, Inga Nagel, Henrike Bruckmueller, Ruwen Boehm, Ole Ammerpohl, Ingolf Cascorbi. Drug resistance in chronic myeloid leukemia: Impact of methylation on gene expression in imatinib and nilotinib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5846.