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Although ABL tyrosine kinase inhibitors (TKIs) such as imatinib have demonstrated the potency against Philadelphia chromosome (Ph)-positive leukemia patients, resistance to ABL TKI can develop in chronic myeloid leukemia (CML) patients. Therefore, new approach against ABL TKI resistant cells may improve the outcome of Ph-positive leukemia patients. It has already reported that ABL kinase domain mutations have been implicated in the pathogenesis of ABL TKI resistance, however, it is fully not known the molecular mechanism of drug resistance including second (nilotinib and dasatinib ) and third generation (ponatinib) ABL TKIs.

As leukemia is a genetic disease driven by heritable or somatic mutations, we hypothesized that ABL TKI resistance may often happen due to additional somatic mutations in the oncogene.

We established several TKI-resistant in vitro cell line models. We also investigated model to evaluate the next-generation sequencing (NGS) panel, NGS platform to screen mutational hotspots in 50 leukemia-related genes.

We established ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R, Ba/F3 T315I and Ba/F3 ponatinib-R) in this study. We conducted fluorescence in situ hybridization (FISH) analysis on parental K562 and ABL TKI resistant K562 cells. BCR-ABL expression levels were not increased in ABL TKI resistant K562 cells compared to parental K562. We next investigated the BCR-ABL point mutation by direct sequence analysis. We could not detect the BCR-ABL point mutation in ABL TKI resistant K562 cells. However, the exon 4 deletion in the BCR-ABL gene was found in K562 ponatinib-R cells. In contrast, compound mutations in BCR-ABL were found in Ba/F3 ponatinib-R cells. K562 ponatinib-R cells were also highly resistant to imatinib, nilotinib and dasatinib. We examined the intracellular signaling of ABL TKI resistant K562 cells. Phosphorylation of BCR-ABL and Crk-L was reduced in K562 dasatinib-R cells, however, MAPK activity was increased. In K562 ponatinib-R cells, MAPK activity was reduced. We next evaluated the NGS panel (GeneRead DNAseq Targeted Panels V2) to investigate the mutation. We found that several somatic mutations in TET2, FLT3, RB1, TP53, SETBP1, ASXL1, and BCORL1 in parental K562 cells. We also found that additional somatic mutations in K562 imatinib-R (IDH1 and KRAS), K562 dasatinib-R (IDH1) and K562 ponatinib-R (SF3A1). We could not detect additional mutation in K562 nilotinib-R cells. We next investigated the MEK inhibitor and IDH1 inhibitor activity against K562 imatinib-R and K562 dasatinib-R cells. MEK inhibitor or IDH1 inhibitor did not induce cell growth inhibition directly. However, combined treatment of ABL TKI resistant K562 with imatinib or dasatinib and MEK inhibitor or IDH1 inhibitor caused more cytotoxicity than each drug alone. Because aberrant activation of PI3K signaling pathway and deregulation of HDAC activity may be a cause of malignant disease in humans, we examined the PI3K and HDAC inhibitor in ABL TKI resistant cells. We found 72 h treatment of oral inhibitor of class I PI3K as well as class I and II HDAC enzymes, CUDC-907 exhibits cell growth inhibition ABL TKI resistant K562 cells and Ba/F3 ponatinib-R cells in a dose dependent manner. In the mouse study, a dose of 20 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of CUDC-907 inhibited tumor growth of T315I mutant cells compared with control mice and induced apoptosis in tumor samples.

Our study indicated that leukemia cells have acquired resistance through somatic mutation or exon 4 deletion in the BCR-ABL gene, suggested that individual based investigations may be important to evaluate the ABL TKI resistance. We also provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.