Intragenic ERG Deletions Do Not Explain the Biology of ERG-Related Acute Lymphoblastic Leukemia

Intragenic ERG deletions occur in 3–5% of B-cell precursor acute lymphoblastic leukemia, specifically in B-other subtype lacking the classifying genetic lesions. They represent the only genetic lesion described so far present in the majority of cases clustering into a subgroup of B-other subtype characterized by a unique gene expression profile, probably sharing a common, however, not yet fully described, biological background. We aimed to elucidate whether ERG deletions could drive the specific biology of this ERG-related leukemia subgroup through expression of aberrant or decreased expression of wild type ERG isoforms. We showed that leukemic cells with endogenous ERG deletion express an aberrant transcript translated into two proteins in transfected cell lines and that one of these proteins colocalizes with wild type ERG. However, we did not confirm expression of the proteins in acute lymphoblastic leukemia cases with endogenous ERG deletion. ERG deletions resulted in significantly lower expression of wild type ERG transcripts compared to B-other cases without ERG deletion. However, cases with subclonal ERG deletion, clustering to the same ERG deletion associated subgroup, presented similar levels of wild type ERG as cases without ERG deletion. In conclusion, our data suggest that neither the expression of aberrant proteins from internally deleted allele nor the reduced expression of wild type ERG seem to provide a plausible explanation of the specific biology of ERG -related leukemia subgroup.


In vitro transcription and translation assay (T/T assay)
Two separate segments of ERGaber coding sequence encoding ERGaberN and ERGaberC were amplified from ERGaber-pcDNA3.1 plasmid by PCR using primers listed in Table B. The synthesized proteins were analyzed by western blot.

Transfection of HeLa and HEK293T cells
HeLa and HEK293T cells were seeded on 6 well plate at densities of 480,000 or 600,000 cells per well, respectively, 24 hours before the transfection. Adherent cultures were transfected using Lipofectamine2000 (Thermo Fisher Scientific) reagent according to manufacturer's instructions: 6,4μg plasmid DNA with 8μl of Lipofectamine2000 in 2ml of serum-free medium per well.

Western blot
Protein concentration of nuclear and cytoplasmic protein lysates was determined by Lowry method using DC™ Protein Assay (Bio-Rad, California, USA). Protein lysates and proteins synthesized by T/T assay were diluted in Bolt® LDS Sample Buffer with Bolt® Sample Reducing Agent (Thermo Fisher Scientific) and incubated at 70°C for 10min. Proteins were separated by electrophoresis on Bolt™ 4-12% Bis-Tris Plus Gels (Thermo Fisher Scientific), transferred to nitrocellulose membrane (Bio-Rad) and blocked by Blotting-Grade Blocker (Bio-Rad). Membranes were probed with primary antibodies overnight. Membrane-bound primary antibodies were detected using appropriate secondary antibodies conjugated with horseradish peroxidase (primary and secondary antibodies are listed in Table C). Antibody complexes were visualized using Clarity™ ECL Western Blotting Substrate Kit (Bio-Rad), SuperSignal™ West Pico Chemiluminescent Substrate Kit and/or SuperSignal™ West Femto Maximum Sensitivity Substrate Kit (Thermo Fisher Scientific) followed by exposition to X-ray films.

Confocal microscopy
HeLa and HEK293T cells were seeded on sterile cover slips placed inside 6 well plates at densities of 240,000 or 300,000 cells per well, respectively, 24 hours before transfection. The transfection with pcDNA3.1 based ERG constructs was performed as described above. Forty-eight hours after transfection cells were fixed by 4% paraformaldehyde (30min), blocked by Normal Goat Serum (Cell Signalling, Massachusetts, USA; 15min), incubated with a primary antibody (60min) and a secondary antibody conjugated with Alexa Fluor® 488 (30min; for antibodies see Table C). Finally, cells were stained by DAPI (Thermo Fisher Scientific) and placed on a microscope slide covered with ProLong® Gold Antifade Reagent (Thermo Fisher Scientific). Microscope slides were inspected using Leica DMi8 inverted microscope equipped with TCS SP8 confocal system and Leica Application Suite X software (Leica Microsystems, Germany). Alexa Fluor® 488 was excited by the 488nm laser and detected in the range of 520-547nm and DAPI was excited by the 405nm laser and detected in the range of 410-452nm.

Quantification of physiological ERG isoforms by PCR
All measurements were performed on 2720 Thermal Cycler (Applied Biosystems, USA). Forward primer annealing to exons 9/10 junction and reverse primer annealing to exon 12 were used to quantify expression of ERG isoforms containing ERG exon 10, while forward primer annealing to exons 9/11 junction and reverse primer annealing to exon 13 were used to quantify expression of ERG isoforms lacking ERG exon 10. The amplification was carried out in TaqMan® Universal PCR Master Mix (Applied Biosystems, USA) supplemented with primers and probe. Annealing temperature was 63°C for both ERG detection systems. Measurements were performed in duplicate. For graphical presentation all expression data were normalized to the lowest expression value within the dataset which was set to 1. Mann-Whitney U test was used to analyze differences in expression between two subgroups.

Figure B: Analysis of subcellular localization of ERG isoforms by confocal microscopy
HeLa (A) and HEK293T (B) cells were transiently transfected by ERG3, ERG3var and ERGaber isoforms in pcDNA3.1 vector or by empty vector. Forty-eight hours after transfection the presence and subcellular localisation of ERG isoforms was analyzed by confocal microscopy using Ab-N antibody. Scale bars represent 10μm.

Figure C: ERG protein expression in NALM6 and ALL samples -full scans
Individual lanes (highlighted by black arrows) of these scans (X-ray films from western blot analyses) were cut and grouped and are presented in Figure 5. Remaining lanes were excluded from the analysis because of protein degradation 1,15,6,16,4,9,8) or subclonality of ERGdel (ERGdel positive by PCR but negative by SNParray; ALL-13 and 17).