The leukemia-associated RUNX1/ETO oncoprotein confers a mutator phenotype

t(8;21) is a frequent chromosomal translocation in acute myeloid leukemia (AML) and is also reported in lymphoid and biphenotypic acute leukemia.1, 2 t(8;21) fuses the RUNX1 gene (AML1) on chromosome 21 to the ETO gene (RUNX1T1) on chromosome 8, encoding the RUNX1/ETO chimeric transcription factor that represses expression of RUNX1 target genes, promoting self-renewal and blocking myeloid differentiation.3, 4, 5, 6 t(8;21) is insufficient for leukemogenesis and additional co-operating mutations are required for transformation,7 including point mutations that activate and/or over express c-KIT.8 The mechanisms driving the acquisition of co-operating mutations remain unclear, although there is evidence that initiating lesions such as RUNX1/ETO may promote mutagenesis.9, 10 For example, ectopic expression of RUNX1/ETO downregulates several DNA-repair proteins (BRCA2, OGG1 and ATM) and increases the level of phosphorylated TP53 and γH2AX, indicating elevated DNA damage and a possible pro-mutagenic phenotype.10, 11

initial transduction by ultracentrifugation. Target cells were transduced by spinfection at 1500g in 6-well tissue culture dishes for 2 hours at 32°C with 4μg/ml polybrene (Sigma Aldrich, Poole, UK). Cells were initially assessed for successful transduction by flow cytometry for EGFP and later real-time qRT-PCR and western blotting.

Selection of successfully transduced cells
Three days after transduction with lentivirus, TK6 were transferred to 96-well plates at a mean density of 2 cells per well in 200μl of culture media and incubated for 10-14 days to isolate individual clonal populations. TK6 clones were assessed for successful lentiviral transduction initially by flow cytometry for EGFP and subsequently for RUNX1/ETO transcript and protein by real-time quantitative PCR and Western analysis, respectively.

Assessment of cell proliferation
Cells were seeded at 2 x 10 4 cells/ml and growth was assessed every 24 hours by cell counting using trypan blue exclusion with an improved Neubauer haemocytometer (VWR International Limited, Leicestershire, UK). A minimum of 2 separate preparations were counted for each cell sample and the average was taken to calculate cell concentration.

Real-time quantitative PCR
RNA was isolated using the RNeasy Mini Kit according to manufacturer's instructions (Qiagen Ltd, Crawley, UK) and 2μg was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit according to manufacturer's instructions (Applied Biosystems, Warrington, UK). Quantitative RT-PCR (qRT-PCR) was performed using SYBR Green Master Mix on a 7900HT Fast Real-Time PCR system (Applied Biosystems) and analysed with SDS version 2.3 software (Applied Biosystems). All primers for qRT-PCR applications were synthesised by Sigma Aldrich.

Western analysis
Whole cell protein extracts were generated from 10 6 cells using sodium dodecyl sulphate (SDS) extraction. Briefly, cell pellets were prepared by centrifugation at 300g before washing twice in PBS to remove protein contamination from media. Pellets were then resuspended in SDS sample buffer (62.5mM Tris-HCl pH 6.8, 2% (w/v) SDS, 20% (v/v) glycerol), followed by boiling samples at 100°C for 5 minutes. Samples were then centrifuged at 14,000g to pellet cell debris and protein-containing supernatants were stored at -80°C. Protein samples were quantified using the Pierce® BCA Assay (Fisher Scientific UK Ltd, Leicestershire, UK) on a Spectromax®250 Microplate Spectrophotometer System (Molecular Devices Corporation, Crawley, UK). 20μg protein was electrophoresed on a Mini-PROTEAN® TGX TM 4-20% Trisglycine gel (BioRad, Hemel Hempsted, UK) using a Mini PROTEAN Tetra system (BioRad) and transferred to PVDF membrane (BioRad). After hybridisation of antibodies, detection was performed with ECL plus Western Blotting Detection Kit (GE Healthcare, Amersham, UK). Primary antibodies used were goat anti-ETO (Santa Cruz, Heidelberg, Germany) and mouse anti-β-actin (Dako, Ely, UK). RUNX1/ETO protein expression was determined using semi-quantitative densitometry on a Fuji LAS-3000 Luminescent Image Analyzer System (model LAS-3000), and is expressed as a percentage relative to Kasumi-1 following background subtraction and normalisation to actin.

Irradiation of cells
Cell suspensions were irradiated in a 25cm 3 tissue culture flasks using a D3300 X-Ray system (Gulmay Medical Ltd, Surrey, UK) at a dose rate of 2.4Gy/min.

Cytotoxicity assays
Cells in logarithmic phase were irradiated or treated with drug (or vehicle control) and growth inhibition was calculated using trypan blue exclusion with an improved Neubauer haemocytometer (VWR International Limited) to count cells at multiple time points after exposure.

Electroporation of cell lines with siRNA
10 7 cells were pelleted by centrifugation at 300g for 5 minutes and resuspended in fresh media. The cell suspension was transferred to an electroporation cuvette with 4mm electrode gap (PEQLAB, Southampton, UK). The required amount of 20μM siRNA stock solution to give a final concentration of between 10-500nM was added to the cuvette. The cuvette was electroporated at 330V (Kasumi-1) or 350V (SKNO-1) for 10ms using an EPI 2500 electroporator (Fischer, Heidelberg, Germany).

Determination of mutation frequency (Mf) at the thymidine kinase (TK) locus
Prior to experimental use, cell populations were purged of pre-existing TK mutants by 48hour culture in CHAT medium (standard growth medium supplemented with 10μM 2deoxycycline, 17.5μM thymidine, 200μM hypoxanthine and 0.2μM aminopterin; all chemicals were from Sigma Aldrich) followed by 72 hours in THC medium (as CHAT, but without aminopterin). For assessment of drug-induced Mf, exponentially growing cell populations were dosed by supplementing drug (or vehicle control) into standard growth medium.
Following 4 hours exposure to doxorubicin at a final concentration of 100nM in 10ml cell culture medium in 25cm 3 flasks, cells were washed in PBS and Mf was determined according to the method of Liber and Thilly (1982). The frequency of mutations attributable to doxorubicin or radiation (treatment-induced Mf) was calculated by subtracting the Mf in mock-treated cells from the Mf in doxorubicin or radiation-treated cells.

Determination of surface CD55 and CD59 status by flow cytometry
Mutation using loss of CD55 and CD59 as a surrogate for loss of PIGA was performed as previously described (Chen et al, 2001;Krüger et al, 2014). Briefly, expression of two GPIanchored proteins (CD55 and CD59) was determined on TK6 cells using PE conjugated antibodies. Cells were also assessed for expression of CD19 B-cell marker and exclusion of propidium iodide (PI) as a cell viability stain. CD55-PE (clone IA10), CD59-PE (clone p282 (H19)) and CD19-APC (clone SJ25C1) conjugated antibodies were all purchased from BD Biosciences (Oxford, UK). Base substitutions and small insertions/deletions in the PIGA gene are the predominant mechanism leading to loss of surface CD55/CD59 expression in vivo (diagnostic for paroxysmal nocturnal hemoglobinuria (Shen et al, 2014)) and cell line studies in vitro (Chen et al, 2001). However, we cannot exclude the possibility that other mechanisms of PIGA inactivation (such as large gene deletions and gene silencing) or mutation in other genes may also lead to loss of CD55/CD59 expression. PIGA -ve cells were defined as those which were positive for CD19, negative for CD55 and CD59 and did not stain with PI. A four-colour FACSCalibur instrument (Becton Dickinson, Oxford, UK) with CellQuestPro software (Becton Dickinson) was used for analysis. PIGA Mf was determined using the following equation: PIGA Mf = number of PIGA -ve cells / total number of cells meeting the inclusion criteria (positive for CD19 and exclusion of propidium iodide).