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.
Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.
Julio
J
Castaño
Hematology Department, University Hospital of Salamanca, IBSAL, IBMCC (USAL-CSIC), Salamanca, Spain.
Ana B
AB
Herrero
Institute Pharmaceutical Biology, Goethe-University, Frankfurt/Main, Germany.
Aldeheid
A
Bursen
IBEC - Institute for Bioengineering of Catalonia, Barcelona, Spain.
Federico
F
González
Institute Pharmaceutical Biology, Goethe-University, Frankfurt/Main, Germany.
Rolf
R
Marschalek
Hematology Department, University Hospital of Salamanca, IBSAL, IBMCC (USAL-CSIC), Salamanca, Spain.
Norma C
NC
Gutiérrez
Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.
Pablo
P
Menendez
D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D059766:DNA End-Joining Repair; D005047:Etoposide / Q000494:pharmacology; D015870:Gene Expression; D057809:HEK293 Cells; D006657:Histones / Q000378:metabolism; D059765:Homologous Recombination; D006801:Humans; D007223:Infant; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics* / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011839:Radiation, Ionizing
C468783:H2AX protein, human; D006657:Histones; C113855:MLL-AF4 fusion protein, human; D015514:Oncogene Proteins, Fusion; D051788:Myeloid-Lymphoid Leukemia Protein; D005047:Etoposide
AF4.MLL; DSB; MLL.AF4; t(4;11)