Acute Myeloid Leukemia SUPPLEMENTARY APPENDIX Impact of MLL5 expression on decitabine efficacy and DNA methylation in acute myeloid leukemia Haiyang Yun,1 Frederik Damm,2 Damian Yap,3,4 Adrian Schwarzer,5 Anuhar Chaturvedi,1 Nidhi Jyotsana,1 Michael Lübbert,6 Lars Bullinger,7 Konstanze Döhner,7 Robert Geffers,8 Samuel Aparicio,3,4 R. Keith Humphries,9,10 Arnold Ganser,1 and Michael Heuser1 1Department of Hematology, Hemostasis, Oncology and Stem cell Transplantation, Hannover Medical School, Germany; 2Department of Hematology, Oncology, and Tumor Immunology, Charité, Berlin, Germany; 3Department of Molecular Oncology, British Columbia Cancer Agency, Vancouver, BC, Canada; 4Department of Pathology and Laboratory Medi- cine, University of British Columbia, Vancouver, BC, Canada; 5Institute of Experimental Hematology, Hannover Medical School, Germany; 6Division of Hematology and Oncology, University of Freiburg Medical Center, Germany; 7Department of Internal Medicine III, University Hospital of Ulm, Germany; 8Department of Cell Biology and Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany; 9Terry Fox Laboratory, British Columbia Cancer Agency, Vancou- ver, BC, Canada; and 10Department of Medicine, University of British Columbia, Vancouver, BC, Canada ©2014 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2013.101386 *These authors contributed equally to this work. †These authors contributed equally to this work. Manuscript received on November 19, 2013. Manuscript accepted on May 23, 2014. Correspondence: [email protected] Impact of MLL5 expression on decitabine efficacy and DNA methylation in acute myeloid leukemia Haiyang Yun1, Frederik Damm2, Damian Yap3,4, Adrian Schwarzer5, Anuhar Chaturvedi1, Nidhi Jyotsana1, Michael Lübbert6, Lars Bullinger7, Konstanze Döhner7, Robert Geffers8, Samuel Aparicio3,4, R. Keith Humphries9,10, Arnold Ganser1, Michael Heuser1 Supplementary Data Supplementary Methods Cytogenetic and molecular analysis Cytogenetic analysis was performed centrally by G- and R-banding analysis. Mononuclear cells from patients’ bone marrow or peripheral blood samples were enriched by Ficoll density gradient centrifugation and genomic DNA was extracted using the All Prep DNA/RNA Kit (Qiagen, Hilden, Germany). Mutation analyses were performed for DNMT3A,1 NPM12 and FLT33 as previously reported. Quantification of MLL5, HOXA9, and Cebpa transcript levels Mononuclear cells from diagnostic bone marrow or blood specimens were enriched by Ficoll density gradient centrifugation. Total RNA was isolated using the All Prep DNA/RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Random hexamer priming and M-MLV reverse transcriptase (Life Technologies, Darmstadt, Germany) were used to generate cDNA. MLL5 expression levels were quantified in mononuclear cells from bone marrow (n=50, median blast percentage 48%) or peripheral blood (n=7, median blast percentage 54%) using the MLL5 TaqMan Gene Expression Assay (Applied Biosystems, Assay ID: Hs00218773_m1) and the ABL FusionQuant Standard Kit as an endogenous control (Ipsogen, Marseille, France) as previously reported.4 HOXA9 quantification was performed in 35 out of 57 samples using HOXA9 TaqMan Gene Expression Assay (Applied Biosystems, Assay ID: Hs00266821_m1) and the same ABL quantification kit as an endogenous control. Total RNAs 1 and cDNAs from KG1a, HL60 and 293T cell lines were similarly prepared. Real-time reverse- transcriptase-polymerase chain reaction (RT-PCR) was carried out on a StepOne Plus real-time PCR system (Life Technologies). All reactions were quantified in duplicate. The data were analyzed using the StepOne Plus Software v2.0 (Life Technologies). To obtain a standard curve for MLL5, quantitative PCR reactions were carried out with 5-fold dilutions covering the expected detection range with a reference cDNA (KG1a cell line). To quantify the transcript level of Cebpa, total RNA was isolated and cDNA was prepared from corresponding mouse cell lines as described above. Cebpa mRNA expression levels were quantified using QuantiTect SYBR Green PCR Kits (Qiagen) with specific primer set targeting Cebpa and Hprt mRNA expression was measured as endogenous control. Real-time RT-PCR was carried out and analysed on a StepOne Plus real-time PCR system and its software. All reactions were quantified in duplicate. Primer sequences are listed below (F, forward; R, reverse). Cebpa: F, 5’-CAAGAACAGCAACGAGTACCG-3’; R, 5’- GTCACTGGTCAACTCCAGCAC-3’; Hprt: F, 5’-GATTAGCGATGATGAACCAGGTT-3’; R, 5’- CCTCCCATCTCCTTCATGACA-3’; Immunoblotting Western blot was performed as previously described.5 In brief, 107 cells were collected for KG1a, HL60 and 293T cells and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycolate, 0.1% SDS, 100 mM NaF; 10 mM EDTA; 1 mM PMSF; 1 mM Na2VO4) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), transferred to PVDF membrane (Whatman GmbH, Dassel, Germany), and immunoblotted with polyclonal rabbit anti-MLL5 antibody (Acris Antibodies, Herford, Germany) and monoclonal mouse anti-ß-actin (AC-15) (Sigma-Aldrich, Hannover, Germany) in 5% BSA overnight at 4°C. Secondary horseradish peroxidase-conjugated antibodies used were donkey anti-rabbit IgG (Santa Cruz Biotechnology, Heidelberg, Germany) and goat anti-mouse IgG (Beckman Coulter, Fullerton, CA, USA). Chemoluminescence was used for visualization using the ECL Western blotting detection reagents (Amersham Biosciences-GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. 2 Retroviral infection of murine bone marrow cells Wildtype and transgenic mice with homozygous loss of Mll5 in 129S6/SvEv genetic background have been described previously.6 Primary mouse bone marrow cells were harvested from stock mice at 6-8 weeks of age and stimulated for 48 hours in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (Stemcell Technologies, Vancouver, Canada), 10 ng/mL of human interleukin-6 (hIL-6), 6 ng/mL of murine interleukin-3 (mIL-3), and 20 ng/mL of murine stem cell factor (mSCF; all from Peprotech, Hamburg, Germany). Generation of HOXA9- or HOXA9/MEIS1-transduced cells was performed as described.7 Briefly, MSCV-HOXA9-PGKneomycin and MSCV-MEIS1-IRESYFP vectors were used for generating recombinant ecotropic retrovirus-producing GP+E86 cells. Pre-stimulated cells were co-cultured with irradiated GP+E86 cells for 48 hours in the presence of 5 μg/mL protamine sulfate (Sigma-Aldrich) and later were selected by geneticin (Gibco, Darmstadt, Germany), or were sorted for yellow fluorescent protein (YFP) expression by fluorescence-activated cell sorting (FACS). Fluorescence-activated cell sorting (FACS) analysis, cell cycle analysis, and apoptosis measurement Cells were harvested after 3 days of decitabine treatment at the indicated concentrations, and were stained using the following antibodies for differentiation analysis: Phycoerythrin- (PE) labeled Gr-1-PE (clone-RB6-8CS) from BD Biosciences (Heidelberg, Germany) and Allophycocyanin- (APC) labeled CD11b-APC (clone-M 1/70) from eBiosciences (Frankfurt, Germany). For cell cycle analysis, cells were first fixed with 2.5 volumes of ethanol for 15 minutes on ice, then were treated with 50 µg/mL propidium iodide (PI; BD Biosciences) for staining along with 0.1 mg/mL RNase A and 0.05% Triton X-100 (all from Sigma-Aldrich) for 40 minutes incubation at 37°C, afterwards the samples were resuspended in phosphate buffered saline (PBS; PAA Laboratories, Cölbe, Germany) for flow cytometry measurement. For apoptosis analysis, cells were stained with Annexin V-FITC and PI according to the manufacturer’s protocol (BD Biosciences), and measured by FACS. Data acquisition was 3 performed on a BD FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany). All FACS analyses were performed with FlowJo 7.2.5 software (Tree Star, Ashland, USA). Colony forming cell (CFC) assay Cells were treated with decitabine in vitro as described above. Then, 1,000 viable cells were plated in duplicate in methylcellulose medium (Methocult M3234; Stemcell Technologies) supplemented with 10 ng/mL murine IL3, 10 ng/mL human IL6, 50 ng/mL SCF, and 3 U/mL erythropoetin (EPO; Peprotech). Plates were incubated at 37 °C for 10 days, and colonies were evaluated microscopically by standard criteria (minimum of 30 cells per colony). Morphology was captured using an Olympus CKX41 (Olympus, Tokyo, Japan) microscope equipped with a Camedia C-5060 wide zoom digital compact camera and a 40x/0.75 numerical aperture objective. Methylated DNA immunoprecipitation combined with microarray (MeDIP-chip) or real- time PCR (MeDIP-PCR) MeDIP was performed with genomic DNA from cells untreated or treated with 20nM decitabine for three days using the MagMeDIP kit (Diagenode, Liège, Belgium), following the manufacturer’s instructions. For each cell type, three independent treatment and immunoprecipitation were prepared, and MeDIP-enriched DNA was amplified with the GenomePlex Complete Whole Genome Amplification (WGA) Kit (Sigma-Aldrich). DNA from three immunoprecipitations (IP) and was pooled for subsequent microarray analysis. Labeling, hybridization and scanning was performed in the department for genome analytics at the Helmholtz Center for Infection Research in Braunschweig, Germany. The enriched DNA and corresponding input