Expression of MLL-AF4 or AF4-MLL fusions does not impact the efficiency of DNA damage repair

The most frequent rearrangement of the human MLL gene fuses MLL to AF4 resulting in high-risk infant B-cell acute lymphoblastic leukemia (B-ALL). MLL fusions are also hallmark oncogenic events in secondary acute myeloid leukemia. They are a direct consequence of mis-repaired DNA double strand breaks (DNA-DSBs) due to defects in the DNA damage response associated with exposure to topoisomerase- II poisons such as etoposide. It has been suggested that MLL fusions render cells susceptible to additional chromosomal damage upon exposure to etoposide. Conversely, the genome-wide mutational landscape in MLL-rearranged infant B-ALL has been reported silent. Thus, whether MLL fusions compromise the recognition and/or repair of DNA damage remains unanswered. Here, the fusion proteins MLL-AF4 (MA4) and AF4-MLL (A4M) were CRISPR/Cas9-genome edited in the AAVS1 locus of HEK293 cells as a model to study MLL fusion-mediated DNA-DSB formation/repair. Repair kinetics of etoposide- and ionizing radiation-induced DSBs was identical in WT, MA4- and A4M-expressing cells, as revealed by flow cytometry, by immunoblot for γH2AX and by comet assay. Accordingly, no differences were observed between WT, MA4- and A4M-expressing cells in the presence of master proteins involved in non-homologous end-joining (NHEJ; i.e.KU86, KU70), alternative-NHEJ (Alt-NHEJ; i.e.LigIIIa, WRN and PARP1), and homologous recombination (HR, i.e.RAD51). Moreover, functional assays revealed identical NHEJ and HR efficiency irrespective of the genotype. Treatment with etoposide consistently induced cell cycle arrest in S/G2/M independent of MA4/A4M expression, revealing a proper activation of the DNA damage checkpoints. Collectively, expression of MA4 or A4M does neither influence DNA signaling nor DNA-DSB repair.
This work was supported by the European Research Council to P.M (ERC-2014-CoG-646903), MINECO (SAF2013-43065 to P.M), The Foundation Inocente Inocente and the Spanish Association of Cancer Research (AECC) to P.M and the Deutsche José Carreras Leukämie Stiftung to R.M/P.M. P.M also acknowledges the financial support from The Obra Social La Caixa-Fundaciò Josep Carreras and The Generalitat de Catalunya (SGR330). F.G. is supported by a Ramón y Cajal Grant (RYC-2014-16751) from the Ministry of Economy and Competitiveness (MINECO), Spain. N.C.G and A.B.H acknowledge financial support from The Cooperative Research Thematic Networks (RTICC) (RD12/0036/0058) and the INNOCAMPUS Program (CEI10-1-0010).