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Targeting FLT3-ITD in AML
ent experiments. Statistical analysis was done on GraphPad Prism software using two-tailed t-tests (version 7, GraphPad software, LaJolla, CA, USA). Data are depicted in column bar graphs plotting mean with SD values.
Antibodies and cytometry
Staining for apoptosis was done using AnnexinV-CF488A (Biotium, Germany) in AnnexinV buffer and Hoechst 33342 (10 mg/ml) for 15 min. at 37C, followed by several washes. Propidium iodide was added shortly before imaging on the Nucleocounter NC-3000 (ChemoMetec, Allerod, Denmark). For cell cycle analysis cells were incubated in lysis buffer with DAPI (10 mg/ml) for 5 min. at 37°C and analyzed on NC-3000 imager.
Results
Sensitivity of AML cell lines to MDM2 and FLT3 inhibitors
To determine the sensitivity of AML cells to MDM2 and FLT3 inhibitors, AML cell lines were treated with three MDM2- and three FLT3-inhibitors for 24 hours in dose escalation experiments before cell viability assessment. The AML cell lines covered the major morphologic and molecular subtypes including particularly FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, as well as TP53 wild type, mutant, hemizygous and null cells (Table I). The two FLT3-ITD cell lines MV4-11 and MOLM-13 had high allelic ratios of FLT3-ITD and chromosomal
AB
CD
EF
Figure 2. Variable responses of AML blast cells to midostaurin and HDM201. Cell viability was determined in AML patient cells treated with midostaurin (PKC412) (A) or NVP-HDM201 (B). AML cells were grouped according to major molecular subtypes (FLT3/TP53/NPM1). Cell viability measurements are depicted in dose response curves (A, B) or with 50nM single compound (C, D), as well as combination treatment of midostaurin with conventional induction therapy (E) or midostaurin with NVP-HDM201 (F). PBM are peripheral blood monocytes of normal controls. AML patient samples were analyzed in groups of at least four individual samples (Online Supplementary Table S1) using GraphPad prism software. Significance is denoted for P<0.05 (*); P<0.005 (**); P<0.0005 (***); P<0.0001 (****); P>0.05 (ns).
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