Effects of First and Next-Generation Tyrosine Kinase Inhibitors on Telomere-Mediated Chromosomal Instability in Chronic Myeloid Leukemia Cells

Introduction of tyrosine kinase inhibitors (TKI) to the therapy of chronic myeloid leukemia (CML) remains one of the most remarkable achievements in oncology. Five TKI are currently approved to treat patients with CML. While imatinib revolutionized the treatment of BCR/ABL1-positive leukemias and represents first-in-class selective inhibitor of ABL1 kinase, the place of next generation TKI in CML therapy is being a matter of an intensive debate. Dasatinib and nilotinib, both 2^nd generations TKI, are approved as first or second line treatment, while bosutinib (2^nd generation TKI) and ponatinib (3^rdgeneration TKI) are restricted for patients resistant to the former ones. Recent reports, demonstrating unexpected side effects, including serious cardiovascular events, caused by next-generation TKIs, underscored that mechanism of TKI action is not fully understood and require further studies.

The aim of the present study was to examine the possible role of different TKI in telomere-mediated chromosomal instability in CML cells. We employed human BCR-ABL1-positive cells (K562), and BCR-ABL1-negative cells (HL60), as well as murine myeloid 32Dcl3 cells with 32Dcl3 BCR-ABL1-positive counterparts. Additionally, CD34+ primary cells isolated from peripheral blood leukocytes of CML patients at various stages of the disease (chronic and blastic phase) were used. Blood samples were taken after informed consent. TKI concentrations were ranging from 1nM to 1uM for each TKI used in this study (imatinib, dasatinib, nilotinib, bosutinib and ponatinib), relevant to concentrations observed in patients in clinics. Upon incubation with different TKI, cells were subjected to micronuclei (MN) generation analysis by fluorescent in-situ hybridization (FISH) with pan-human centromere probe. FISH analysis was applied to centromere visualization in nuclei and micronuclei to reveal the generation of micronuclei (MN) and to determine if centromere fragments are present within MN. Analysis was performed at 24 and 48h time points. In BCR-ABL1-positive cells, highest (3-fold) change in MN frequency was observed after longer (48h) incubation with 100 nM imatinib, as compared to the control growth conditions. Less pronounced increase in MN generation with centromeric signals was observed in cells treated with next-generation TKI (dasatinib, nilotinib, bosutinib and ponatinib), which may indicate that different TKIs may exert different aneugenic effects. Such effects may be BCR-ABL1 kinase-independent, since we have also observed an increase in the frequency of MN in HL60 cells treated with 100 nM imatinib, as compared to control ones.

It is widely accepted that genomic instability may be provoked, when the length of telomeres is affected, which, in turn, may induce the generation of micronuclei leading to aneuploidy or chromothripsis. To verify, if observed increase in MN generation after some TKI is caused by dysfunction of telomeres, maintenance of telomeres in BCR-ABL1-positive and negative cells treated with TKI was investigated. Enzymatic activity of telomerase was measured immunoenzymatically, length of telomeres was determined by Southern blotting and expression of TERT and TERC, the subunits of telomerase, were examined by qPCR. No significant changes in the length of telomeres and enzymatic activity of telomerase were observed upon treatment with different TKIs in K562 and HL60 cells as well as in murine leukemic cells. Neither expression of TERT nor TERC was affected by TKI.

In conclusion, we postulate that BCR-ABL1- positive cells may activate the alternative lengthening of telomeres pathway and this might be affected by different TKI in a different manner by mechanisms which require additional studies.