Dasatinib Is An Effective Inhibitor of Proliferation and Inducer of Apoptosis in the KASUMI Cell Line Bearing the T(8;21)(q22;q22) and the N822K KIT Mutation

Abstract

Introduction. Within the group of core binding factor (CBF) AML, the presence of the t(8;21)(q22;q22) confers a favorable prognosis based on high complete remission rates and high survival probabilities. However within this subgroup the presence of KIT mutations and in some studies specifically mutations at codon 816 in exon 17 have been associated with inferior event free survival, relapse free survival, cumulative incidence of relapse and overall survival. Dasatinib a dual SRC/ABL kinase inhibitor is an active agent already approved for the treatment of imatinib resistant or intolerant chronic myelogenous leukemia which has shown in vitro activity against KIT exon 17 mutations including the D816 imatinib resistant mutation. The aim of the present study was the investigation of the activity of dasatinib on cell proliferation and apoptosis of leukemic cell lines with or without KIT mutations. Materials and methods. The leukemic cell lines ME-1, NB4 and KASUMI were cultured in RPMI. Following RNA extraction RT-PCR was performed for the amplification of the extracellular (exon 8,9), transmembrane/juxtamembrane (exon 10,11) and tyrosine kinase 2 domains (exon 17,18) of c-Kit.Following sequencing only the KASUMI cell line derived from a t(8;21)(q22;q22) AML was found to bear the N822K KIT mutation at exon 17, also described in patients’samples. The KASUMI, the K562 cell line bearing the t(9;22) used as a positive control and the NB4 cell line without KIT mutations used as a negative control, were subsequently cultured under the presence of dasatinib at the concentrations of 1nM, 10nM, 100nM, 500 nM. Cell proliferation, was determined at 24, 48, 72 h using the Cell Proliferation Elisa, BrDU protocol and apoptosis was determined by the method of annexin using flow cytometry at the same time points. Results The BrDU value of K562 cells at 48h without the drug was 1.046 significantly higher compared to those of cells cultured under the presence of Dasatinib at 1nM, 10nM, 100nM, 500 nM (0.6485, 0,5647, 0,4770, 0.4755 respectively) (p<0.001). Similarly the BrDU value of K562 cells without the drug at 72h was 1.320 significantly higher to those under the presence of the drug at 10, 100, 500 nM (0.8137, 0.7292, 0.6637 respectively) (p<0.001). The level of apoptosis was significantly induced by the drug at all concentrations at 24h(p<0.001) and at the concentrations of 10nM, 100nM, 500 nM at 48h (p<0.001) but not at 72h.Ôhere was no effect of the drug on the proliferation and apoptosis of the NB4 cell line. In the KASUMI cells there was a significant reduction of the BrDU values by the presence of dasatinib at the concentrations of 10nM, 100nM, 500nM at 48h (0.9517 vs 0.6462, 0.5653, 0.3467, p=0.038, 0.011, 0.002 respectively). The same was true at the concentrations of 100nM and 500nM at 72h (0.9538 vs 0.2412, 0.1907, p=0.002, 0.004 respectively). Dasatinib significantly increased the level of apoptosis of the KASUMI cells at 24h at 1nM, 10nM, 100nM (2.45 vs 1.41, 1.71, 2.18, p<0.001, <0.001, 0.026 respectively) At 48h dasatinib significantly increased the level of apoptosis at the concentrations of 1nM, 10nM, 100nM (0.84vs 1.03, 1.49, 2.81, p=0.02, p<0.001, p<0.001 respectively). At 72h there was a significant induction of apoptosis by the drug at all concentrations (0.16 vs 1.11, 1.94, 2.93, 1.88 p<0.001) Conclusion. Dasatinib is an effective suppressor of proliferation and inducer of apoptosis of the KASUMI cell line with the t(8;21)(q22;q22) and the N822K KIT mutation. These encouraging results need to be confirmed on patients’ cells with the view to integrate the drug in conventional chemotherapy regimens in future clinical trials.

Transitory dasatinib-resistant states in KITmut t(8;21) acute myeloid leukemia cells correlate with altered KIT expression

“…Consistent with prior reports [17,18,25], acute inhibition of KIT by dasatinib, similar to KIT knockdown, disrupted downstream signaling and induced apoptosis in a time and dose dependent manner (Fig. 1).…”

“…1). For long-term exposure, concentrations were chosen that exert sufficient inhibitory action [17,18] and are in the range of clinically achieved levels [33]. Despite initial treatment-induced growth inhibition, cells early on regained ability to grow in the presence of dasatinib within a few weeks (not shown).…”

“…Up to 50% of patients with t(8;21) AML harbor constitutively activating KIT-kinase domain mutations that are associated with higher relapse rates, shorter overall survival, or both [16]. Treatment of KIT mut t(8;21) AML cells with dasatinib resulted in growth inhibition and induction of apoptosis in vitro [17,18], as well as prolonged survival in a corresponding mouse model [19]. Accordingly, the clinical relevance is evaluated in ongoing trials (e.g., NCT00850382; www.clinicaltrials.gov).…”

“…Drawing conclusions on the effects of dasatinib in AML from CML can be misleading, however. Although the acute effects of dasatinib on KIT mut AML cells have been subject to multiple studies [17,18,[25][26][27], data on continuous longterm exposure of KIT mut AML cells to dasatinib are, to our knowledge, lacking and its effects on dasatinib-sensitivity are unexplored.…”

“…In this study, we used the well-established dasatinib-sensitive KIT mut t(8;21) AML model cell line Kasumi-1 [17,18] to study cellular and molecular effects on prolonged dasatinib-exposure with clinically relevant concentrations and treatment periods in a confined and controllable way. Interestingly, we delineated transitory drug-resistant states in dasatinib-treated KIT mut AML cells that can be readily reversed by treatment intermission.…”