Anthracyclines represent established components of current treatment regimes for pediatric AML, while most of the other identified drug classes either were or are currently under clinical investigation for the treatment of AML , , , . Gibco, Uxbridge, UK) containing 20% FCS (GE healthcare), 100IU/ml penicillin-streptomycin (ThermoFisher), 0.125?g/ml amphotericin B (ThermoFisher), 0.2?mg/ml gentamycin (ThermoFisher), 5?mg/ml insulin, 5?mg/ml transferrin, 5?ng/ml sodium selenite (ITS; Sigma-aldrich) and 2?mM l-glutamine (ThermoFisher). Note, that sensitivity of cells were not influenced by cryopreservation . Cells were washed in culturing medium twice at 1500?rpm for 5?min at RT. Thereafter, resuspended cells were diluted in culturing medium to a concentration of 1C1.5??106 cells/ml and incubated at 37?C in humified air containing 5% CO2 until further use. The leukemic samples used in this study contained >80% leukemic blasts before and after culture, and the two non-leukemic bone marrow controls contained <1% blasts, as determined by May-Grnwald Giemsa (Merck) stained cytospins. Cell lines Human AML VPC 23019 cell lines HEL, KASUMI-1, ME-1, MKPL-1 were all purchased from DSMZ-German collection of microorganisms and cell cultures. The wildtype AML cell line CHRF-288C11 was kindly provided by Dr. Gruber (St. Jude Children's Research Hospital, Memphis, TN, USA). Cell line VPC 23019 characteristics are listed in Supplemental Table 2. All cell lines were cultured in RPMI-1640 with GlutaMAX (ThermoFisher), 10%?20% fetal calf serum (GE healthcare), 100IU/ml penicillin streptomycin (ThermoFisher), and 0.125?g/ml amphotericin B (ThermoFisher), at 37?C in humified air containing 5% CO2. Cell line characteristics are shown in Supplemental Table 2. Mycoplasma testing and DNA fingerprinting were regularly performed as quality control. High-throughput drug screening and drug libraries Cells were seeded semi-automatically in 384-well plates (Corning) using a Multidrop dispenser (Thermo Fisher Scientific) Patient-derived cells were seeded at a concentration of 1C1.5??106 cells/ml, the seeding density of the cell lines are provided in Supplemental Table 2. On the same day, drugs were added to a final concentration of 10?nM, 100?nM or 1000?nM, using the Caliper SciClone ALH3000 liquid handling robot. The following commercially available drug libraries were utilized: Prestwick Chemical library (Prestwick Chemical, France), anti-neoplastic sequoia library (Sequoia Research Products, United Kingdom), Epigenetics library (Enzo Life Sciences, Belgium), Epigenetics screening library (Cayman Chemical, MI, USA), Spectrum collection (Microsource, CT, USA), the Cell cycle/DNA Damage compound library (MedChemExpress, Sweden), and VPC 23019 some additional compounds (Supplemental Table 3). Upon drug exposure, cell viability was assessed by a 4-day thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich) assay . Compounds and viability assays Pyrvinium pamoate was purchased from MedChemExpress. Venetoclax, navitoclax, etoposide, cytarabine and daunorubicin were purchased from Selleckchem. All compounds were dissolved in DMSO and subsequently diluted in medium when used for viability assays. For the validation of the most interesting hits from our drug screen and for drug combination studies, expanded dose response curves were made using the Tecan D300 Digital Dispenser (Tecan, Switzerland) to dispense PRKCA the drug. The drug response on the cell viability was assessed by a 4-day MTT assay as described elsewhere . Briefly, after 4 days of incubation, 5L/well of 5?mg/ml MTT was added to 40L of drug-exposed cells for 6?h. Thereafter, the reaction was stopped by adding 40L/well of a 10% SDS/0.01?M HCL solution. The next day, the absorbance of the cells was measured at wavelengths 570?nm and 720?nm using the microplate reader (Versamax). The data of 720?nm served as background noise and was subtracted from the 570?nm data. MTT data was normalized to DMSO control, tolerating a maximum concentration of 0.5% (v/v). The value of optical density for the controls were at least 0.07 to ensure high viability of the primary cells when assessing the effect of the drug. Western blotting Cell lysates were prepared in RIPA buffer (#89,901, Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor cocktail (#78,440, Thermo Fisher Scientific). Lysates were quantified using the Pierce BCA Protein Assay Kit (#23,223 for reagent A and #23,224 for reagent B, Thermo Fisher Scientific). 25?g lysate was loaded on pre-cast SDS-polyacrylamide gels (TGX, Bio-Rad) and transferred onto a nitrocellulose membrane using the Transblot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% milk (Elk, Campina). Proteins of interest were detected.