Human leukemic cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). HL-60 and Kasumi-1 were cultured in RPMI 1640 (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany), 1% penicillin-streptomycin (PAA laboratories, Pasching, Austria) and 1% L-Glutamine (Sigma-Aldrich). MV4-11 and HEK293T were cultured in IMDM (Invitrogen, Carlsbad, CA, USA), also supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% L-Glutamine. All cell lines were regularly tested for mycoplasma contamination.
Primary AML cells
Primary cells were obtained from peripheral blood at the time of relapse with informed consent of the donor according to protocols approved by the institutional review boards (Ethik-Kommision der Ärztekammer Westfalen-Lippe und der Medizinischen Fakultät der Westfälischen Wilhelms Universität Münster, AZ 2007-390-f-S) and in accordance with the Declaration of Helsinki. Cells were grown in RPMI, supplemented with 20% fetal bovine serum and 5% giant cell tumor conditioned medium (Irvine Scientific, Santa Ana, CA, USA).
Cytarabine, azacytidine and pevonedistat (MLN4924) were purchased from Sigma-Aldrich. All drugs were stored at -20°C.
Barcoded negative-selection RNAi screen
The Decipher lentiviral shRNA library module 1 (Cellecta, Mountain View, CA, USA), covering 5,043 genes, contained 27,500 shRNAs in a lentiviral backbone with appropriate positive and negative controls. The library was designed to target each gene with 5 different shRNAs.
For production of lentiviral particles were produced by HEK293T cells. HEK293T cells were transfected with 6µg of lentiviral plasmid and ViraPower™ helper plasmids (Life Technologies, Darmstadt, Germany) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Supernatants containing viral particles were harvested after 48 hours and afterwards filtered through sterile syringe filters (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -80°C.
For lentiviral transduction dishes were coated with RetroNectin® (Fisher Scientific, Waltham, MA, USA). To guarantee sufficient library representation total number of 30 million cells were used for transduction and at least a quantity of 20 million was maintained during the experiment. 72h after transduction, infected cells were selected by cell sorting using a BD FACSAria™ III (BD Biosciences, San Jose, CA, USA) and subsequently expanded for approximately further 72h until required cell number was reached. At this time point, 20 million cells were harvested (day 0) and frozen at -20°C. Remaining cells were split into three treatment groups and exposed to either 100nM azacytidine, 100nM cytarabine or to DMSO for 72h. After treatment cells were kept in culture for further 48 hours and harvested on day 5. Genomic DNA from samples harvested at day 0 and day 5 was extracted using the DNeasy Blood&Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA was further concentrated by ethanol precipitation. Next, two rounds of PCR for barcode amplification followed as described elsewhere (Fredebohm et al. 2013). Before sequencing analysis, samples were marked with a specific barcode and multiplexed with Encore TM 384 Multiplex System (NuGen Technologies, Leek, NL). A total of 12pmol gDNA was used. Decipher barcode abundance was analyzed by next-generation sequencing with a HiScanTMSQ System (Illumina, San Diego, USA).
Quantitive Real-Time PCR (qPCR)
To analyze expression of mRNA HEK293T cells were transduced with highest scoring shRNA for PSMD11, NEDD8 and RBX1. RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantitive PCR was performed according to manufacturer’s instructions with SYBR green. All signals were normalized to levels of Gapdh and results were calculated by the ΔCt method.
Western blot analyisis
Positively transduced cells were lysed using the radioimmunoprecipitation assay (RIPA) buffer. SDS-PAGE and western blotting were performed as already described (Tickenbrock et al. 2006). Antibodies against NEDD8 and RBX1 were obtained from Cell Signaling Technology (Danvers, MA, USA), an antibody against PSMD11 was obtained from Novus Biologicals (Littleton, CO, USA).
Cell profliferation assays were performed by cell counting or by MTS assays. All assays were done in triplicate. MTS assays (Promega Corp., Fitvhburg, WI USA) were used for the determination of IC50. HL-60, Kasumi-1 and MV4-11 were treated with azacytidine or cytarabine from 5nm to 1000nm. Cell proliferation was measured after 48 hours with a Benchmark microplate reader (Bio-Rad, Hercules, CA, USA).
In vitro shRNA Proliferation assays were performed with HL-60, Kasumi-1, and MV4-11. Transduced cells were selected as described above and exposed to 100nM azacytidine for 72 hours. For inhibitor testing, cells were exposed to 25nM pevonedistat in combination with either 100nM, 500nM azacytidine or 25nM cytarabine for 72 hours. Subsequently, cells were transferred to fresh medium. Viable cells were counted at day 5 using the TC20TM Automated Cell Counter (Bio-Rad, Hercules, CA USA), dead cells were excluded by Trypan Blue staining. Relative proliferation rates were calculated by normalizing to mock-treated cells.
Methylcellulose Colony-Forming Assay
HL-60 cells were exposed to pevonedistat in combination with azacytidine or cytarabine in a 3-day drug treatment scheme; medium was exchanged every 24 hours. An equal number of cells (500 per plate) were plated in triplicates on to methylcellulose M0512 (Sigma-Aldrich) supplemented with fetal bovine serum and L-Glutamine. Colonies containing more than 40 cells were scored after 7 days.
Raw data of sequencing analysis was first exported using the software Illumina CASAVA Software Version 1.8.2. Next sequences were analyzed by the “Barcode Deconvoluter“ software provided by Cellecta. Further analysis was performed in BRB ArrayTools, an R-based software with Excel-front end.
Statistical differences were analyzed by Student’s t-test using GraphPad Prism software. A p-value < 0.05 was defined as a significant difference.