Human samples and cell lines

The human AML cell line MV4-11 (homozygous FLT3-ITD) and MOLM-13 (heterozygous FLT3-ITD) were used for in vitro studies. Midostaurin-resistant cell lines were developed as follows: (1) 1 × 10⁶/ml MV4-11 and MOLM-13 cells were plated in 24-well plates in 2 ml cell culture medium and treated with 10 nM Midostaurin, (2) every 7 days, leukemia cells were adjusted to 1 × 10⁶/ml in fresh medium and 2 ml of this cell suspension plated per well in 24-well plates, and (3) MV4-11 and MOLM-13 cells were initially exposed to 10 nM Midostaurin and concentration was increased serially (increased by 10 nM weekly) up to 50 nM. MV4-11 and MOLM-13 parental cell lines were purchased from ATCC and DSMZ, respectively. They were maintained in culture in RPMI-1640 medium supplemented with 10% fetal bovine serum with 1% penicillin-streptomicin and 1% L-Glutamine at 37 °C in a humid atmosphere containing 5% CO₂ at a concentration of 2 × 10⁵ to 1 × 10⁶. HS-5 MSC line was kindly provided by Patricia Maiso laboratory and were maintained in DMEM medium supplemented with 10% fetal bovine serum with 1% penicillin-streptomicin 1% L-Glutamine at 37 °C in a humid atmosphere containing 5% CO₂ at a concentration of 5 × 10⁵ to 1 × 10⁶. The cell lines were tested for mycoplasma contamination monthly. BM mononuclear cells were obtained from refractory FLT3-mutated AML patients after an informed consent was signed by the patients or the patient’s guardians, in accordance with the Declaration of Helsinki. Clinical characteristics of relapsed FLT3-mutated AML patients used in this study are explained in Table S1. To test the toxicity of the compounds that were used in this work, we used PBMCs obtained from three healthy donors.