Investigation of resistance mechanisms against tyrosine kinase inhibitors in chronic myeloid leukemia cell line

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease characterized by the t(9;22)(q34;q11) translocation resulting in the chimeric BCR-ABL oncogene. Despite the significant success of tyrosine kinase inhibitors (such as imatinib) inhibiting Bcr-Abl oncoprotein function in the treatment of CML, drug resistance is still a serious and maybe the most important problem affecting 20-30% of patients. Our in vitro studies are designed using a TKI resistant K562 subclone (K562-Ir) which does not harbour any BCR-ABL amplification or kinase domain mutation. The K562-Ir subclone was developed in our laboratory by natural selection under imatinib pressure, to understand the underlying causes of drug resistance. We performed immunofluorescence, Western blot and mRNA microarray (transcriptome) techniques for gene expression analyses; three-dimensional cell culture studies; cell differentiation experiments; different TKIs applications with cell death analysis; flow cytometric cell membrane marker screening; proliferation assays; senescence assays; Q-PCR for amplification and expression confirmation studies. We observed that unlike K562 wild type cells, K562-Ir cells were adherent and resistant to nilotinib, dasatinib, bosutinib and ponatinib in addition to imatinib. K562-Ir cells are more resistant to cell death and proliferate slowly. They are capable of forming tumor spheroids in three-dimensional cell culture studies. We also showed a significant differences in the expression of genes that regulate cell-tissue-organ differentiation and development processes in K562-Ir cells. There is an increase in the expression of embryonic and cancer stem cell surface markers. There is also an increase in the expression of microRNAs that regulate the expression of genes involving cell differentiation, drug resistance, proliferation and apoptosis resistance. In conclusion, we present a new TKI resistance model suggesting that under imatinib stress a sub-clone of the leukemic cells develop transcriptional instability resulting in a Bcr-Abl-independent/aggressive population with phenotypic adaptation capacity