Discovery of an Endonuclease G-Inhibitory Ku80 Peptide Protecting Against Leukemogenic Rearrangements at the MLL Breakpoint Cluster

Abstract:
Chromosomal rearrangements at the Mixed-Lineage Leukemia (MLL) breakpoint cluster region are frequently implicated in the pathogenesis of acute leukemias. Here, we report the identification of a peptide derived from Ku80 that selectively inhibits Endonuclease G (EndoG) activity. Functional assays demonstrate that this Ku80-derived peptide prevents EndoG-mediated DNA cleavage at the MLL breakpoint cluster, thereby reducing the incidence of leukemogenic translocations. These findings suggest that the Ku80 peptide may serve as a molecular tool for mitigating genome instability and a potential therapeutic candidate for preventing MLL-associated leukemia.

Introduction:
Genomic instability is a hallmark of leukemogenesis, with chromosomal rearrangements at the MLL locus contributing to aggressive leukemia subtypes. Endonuclease G (EndoG), a mitochondrial nuclease, has been implicated in DNA cleavage events that facilitate these chromosomal translocations. The Ku80 protein, a key component of the non-homologous end joining (NHEJ) DNA repair pathway, has been hypothesized to interact with EndoG, modulating its activity at specific genomic loci. In this study, we investigate a Ku80-derived peptide as a selective inhibitor of EndoG, aiming to prevent pathological rearrangements at the MLL breakpoint cluster.

Methods:
A peptide fragment corresponding to the DNA-binding domain of Ku80 was synthesized and tested for its ability to inhibit EndoG activity in vitro. Electrophoretic mobility shift assays (EMSA) and in vitro cleavage assays were performed to evaluate peptide-EndoG interactions. The protective effect against MLL locus rearrangements was assessed in cultured hematopoietic progenitor cells using targeted DNA damage induction and subsequent genomic analysis.

Results:
The Ku80-derived peptide effectively binds EndoG, reducing its nuclease activity in a dose-dependent manner. Cells treated with the peptide showed a significant decrease in DNA breaks at the MLL breakpoint cluster and a corresponding reduction in aberrant chromosomal translocations. No cytotoxic effects were observed at concentrations effective for EndoG inhibition, suggesting a high therapeutic index.

Discussion:
These results reveal a novel function of Ku80 in safeguarding genomic integrity beyond its canonical role in NHEJ. By selectively inhibiting EndoG activity at the MLL breakpoint cluster, the Ku80 peptide provides a targeted approach to prevent leukemogenic rearrangements. Further optimization and in vivo studies could establish this peptide as a promising therapeutic candidate for high-risk leukemia.

Conclusion:
The discovery of an EndoG-inhibitory Ku80 peptide provides a novel molecular strategy to protect against MLL-associated chromosomal rearrangements. This approach underscores the potential of targeted peptide inhibitors in mitigating genome instability and preventing leukemia initiation.

Keywords:
Ku80 peptide, Endonuclease G, MLL breakpoint cluster, leukemia, chromosomal rearrangements, genomic stability