Targeted degradation of DNA ligase IV through a double-stranded DNA-based PROTAC for precision radiosensitization

DNA ligase IV (LIG4), the terminal ligase of the non-homologous end-joining (NHEJ) pathway, represents one of the most potent yet least druggable radiosensitization targets in double-strand break repair. To overcome this challenge, we designed a first-in-class, double-stranded DNA-based proteolysis-targeting chimera (PROTAC; termed as NHEJ-P) that couples a short 32-bp duplex “DNA bait” to a cereblon-binding E3 ligase ligand, enabling selective proteasomal degradation of LIG4. NHEJ-P achieves near-complete loss of endogenous LIG4 at ~10 nM, verified by western blot and rescued by MG132, confirming proteasome dependence. In vitro, NHEJ-P induces sustained gammaH2AX and 53BP1 foci with no effects on RAD51 foci, disrupts end-joining kinetics, and enhances radiation-induced cytotoxicity in U2OS and HeLa cells but not in non-transformed RPE1 or HEK293T models, defining a favorable tumor-to-normal therapeutic index. These results suggest that genomically unstable cancer cells are uniquely dependent on LIG4 for survival under replication stress. Building on this foundation, we are conjugating NHEJ-P to trastuzumab using a site-directed enzymatic approach to generate t-NHEJ-P, a HER2-directed antibody-oligonucleotide conjugate (AOC) for tumor-restricted delivery. Planned studies will assess pharmacokinetics, tissue-selective LIG4 degradation, and radiosensitization in HER2⁺ xenografts, along with prospective safety in gastrointestinal, cardiac, pulmonary, and skin radiation-toxicity models. Together, these findings establish targeted LIG4 degradation as a new paradigm for DNA-damage-response modulation and lay the groundwork for next-generation, bystander-limited radiosensitizers that integrate molecular precision with translational safety.