Flavopiridol

For plate-based small molecule screens, translations were scaled to provide necessary reagents for duplicate reactions in 96-well plates. Each Cfluc-Kinase was translated separately along with Fos-Nfluc using the conditions listed above. After translation and incubation at 30 °C for 90 min, several 24 μL aliquots of lysate were supplemented with 1 μL of buffer A and set aside as negative controls. The remaining lysate was treated with 2 to a final concentration of 125 nM. 24 μL aliquots of lysate treated with 2 were added to each well of a 96-well Lumitrac 200 plate (Grenier Bio-one) containing 1 μL DMSO (for positive controls) or 250 μM inhibitor (final concentration of 10 μM). 76 of the inhibitors used on each plate were from the Tocris Kinase Inhibitor Toolbox (Tocris Bioscience). Four inhibitors, PKC-412 (LC Labs), Sunitinib (LC Labs), Flavopiridol (Alexis), and Roscovitine (LC Labs), were also included. A list of all compounds tested, references, and their location on each plate can be found in the Supplementary Information (Table S2). The final concentrations of 2 and inhibitor were 120 nM and 10 μM respectively. Plates were covered with foil and equilibrated for 1 hour at room temperature. The Fos-Nfluc with Cfluc-Jun control was prepared in parallel under identical conditions, excluding the addition of 2. Using a Centro XS LB 960 plate reader (Berthold Technologies), 80 μL of luciferin assay reagent was injected into each well containing sample and luminescence immediately measured with a 1 sec integration time.

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).

Automated microscopy with reflection-based laser autofocus was performed on a Molecular Devices ImageXpress Micro screening microscope equipped with a ×10, 0.5 N.A. S Fluor dry objective (Nikon), controlled by Metamorph macros developed in-house²¹ . Cells were maintained in a microscope stage incubator at 37 °C in a humidified atmosphere of 5% CO₂ throughout the experiment. Illumination was adjusted so that the cell death rate was below 5% in untreated control cells. Confocal microscopy was performed on a customized Zeiss LSM 510 Axiovert microscope using a ×20, 0.8 N.A. Plan-Apochromat dry objective, a ×40, 1.3 N.A. oil DIC EC Plan-Neofluar objective, or a ×63, 1.4 N.A. oil Plan-Apochromat objective (Zeiss). The microscope was equipped with piezo focus drives (piezosystem jena), custom-designed filters (Chroma) and an EMBL incubation chamber (European Molecular Biology Laboratory), which provided a humidified atmosphere at 37 °C with 5% CO₂.
For imaging chemically induced mitotic exit, cells were seeded in chambered LabTek coverslips overnight and then transfected with the indicated siRNAs. After transfection (52 h), the medium was replaced with imaging medium containing 30 μM MG132 (proteasome inhibitor; Sigma). The chambered coverslips were placed into the microscope stage incubator, which was maintained at 37 °C in a humidified atmosphere of 5% CO₂, for 45 min, when imaging locations with metaphase-arrested cells were selected. These cells were imaged for 10–15 min, before mitotic exit was induced with 20 μM flavopiridol (Cdk inhibitor)^{25 (link)}.