Although DNA damage-based chemotherapy has been well investigated for clinical oncology, their effectiveness is greatly hindered by the DNA damage defenses including DNA damage repair and cell cycle arrest. Herein, we propose hybrid prodrug nanoassemblies using core-matched strategy and self-assembly technology to facilitate a synthetic lethal relationship between DNA damage and DNA damage defense. Hydroxycamptothecin (HCPT), a topoisomerase I inhibitor, is chosen as the DNA toxic chemotherapeutic. Norcantharidin (NCTD), a PP2A inhibitor, serves to abrogate homologous recombination repair and G/M cell cycle arrest initiated by HCPT. Hybrid prodrug nanoassemblies efficiently co-deliver the two drugs with notably different physicochemical properties and lay solid foundation for their synthetic lethal synergy. The developed synthetic lethal synergy potently damages DNA and successfully attenuates DNA damage defense. More intriguingly, such synthetic lethality synergy induces tumor apoptosis by the cell death mechanism of mitotic catastrophe. In both breast tumor and colon tumor mouse models, hybrid prodrug nanoassemblies remarkably suppresses tumor proliferation and improves the safety of chemotherapy. Our results provide a promising strategy to potentiate DNA damage-based translational medicine.