Ex vivo functional drug screens were performed on freshly isolated mononuclear cells from AML samples. Briefly, 10,000 cells per well were arrayed into three, 384-well plates containing 122 small-molecule inhibitors. This panel contained graded concentrations of a drugs with activity against two-thirds of the tyrosine kinome as well as other non-tyrosine kinase pathways, including mitogen activated protein kinases (MAPKs), phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT serine/threonine kinase 1/mechanistic target of rapamycin kinase (PIK3C/AKT/MTOR), protein kinase AMP-activated (AMPK/PRKAA), ATM serine/threonine kinase (ATM), Aurora kinases, calcium/calmodulin dependent protein kinases (CAMKs), cyclin-dependent kinases (CDKs), serine/threonine protein kinase 3 (GSK3), I-kappaB kinase (IKK), cAMP dependent protein kinase (PKA), protein kinase C (PKC), polo-like kinase 1 (PLK1), and RAF proto-oncogene serine/threonine kinase (RAF). In addition, the library contained small molecule inhibitors with activity against the BCL2 family, bromodomain containing 4 (BRD4), Hedgehog, heat shock protein 90 (HSP90), NOTCH/gamma-secretase, proteasome, survivin, signal transducer and activator of transcription 3 (STAT3), histone deacetylase (HDAC), and WNT/beta-catenin. Drug plates were created using inhibitors purchased from LC Laboratories and Selleck Chemicals and master stocks were reconstituted in dimethyl sulfoxide (DMSO) and stored at −80 °C. Master plates were created by distributing a single agent per well in a seven-point concentration series, created from three-fold dilutions of the most concentrated stock resulting in a range pf 10 μM to 0.0137 μM for each drug (except dasatinib, ponatinib, sunitinib, and YM-155 which were plated at a concentration range of 1 μM to 0.00137 μM). DMSO control wells and positive control wells containing a drug combination of Flavopiridol, Staurosporine and Velcade were placed on each plate, with the final concentration of DMSO ≤0.1% in all wells. Daughter plates were created using a V&P Scientific 384-well pin tool head operated by the Caliper Sciclone ALH 3000 and equipped with 0.457mm diameter, 30 nanoliter, slotted stainless steel pins (cat num: FP1NS30). Daughter and destination plates were sealed with pealable thermal seals using a PlateLoc thermal sealer. Destination plates were stored at −20 °C for no more than three months and thawed immediately before use. Primary mononuclear cells were plated across single-agent inhibitor panels within 24 h of collection. Cells were seeded into 384-well assay plates at 10,000 cells per well in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with fetal bovine serum (FBS) (10%), l-glutamine, penicillin/streptomycin, and β-mercaptoethanol (10−4 M). After 3 d of culture at 37 °C in 5% CO2, MTS reagent (CellTiter96 AQueous One; Promega) was added, optical density was measured at 490 nm, and raw absorbance values were adjusted to a reference blank value and then used to determine cell viability (normalized to untreated control wells).