Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

Endonuclease G (EndoG) is an evolutionarily conserved enzyme that cleaves the Mixed Lineage Leukemia breakpoint cluster region (MLLbcr) under sublethal chemotherapeutic treatment conditions, causing leukemogenic chromosomal rearrangements. While endogenous inhibitors (EndoGI) control EndoG in lower organisms, no such EndoGI has been identified in mammalian cells. Due to the structural similarity of EndoGI from Drosophila melanogaster to the C-terminus (Ct) of human Ku80, we perform immunoprecipitation, surface plasmon resonance analysis and 3D molecular modeling, revealing binding of human EndoG to Ku80-Ct putatively between amino acid 110-184. Docking modeling predicts EndoGI-like peptides clustering around residues 686-707 of Ku80. Our experimental studies provide evidence that Ku80-Ct and 28-mer peptide Ku3 reduce MLLbcr breakage after doxorubicin treatment independently of DNA-PK activity. Proximity ligation and single molecule tracking studies show that Ku3 antagonizes Ku80-EndoG association and modulates chromatin-binding of EndoG. Such MLLbcr protection blocks EndoG´s pro-tumorigenic functions without limiting cytotoxicity, pursued for co-treatments that reduce secondary leukemia, a severe side effect of chemotherapy.