Proper checkpoint activation is necessary to temporarily halt cell cycle progression when cells are in nutrient-deprived conditions or encounter insults. These safeguard mechanisms are universally lost in cancer cells; nevertheless, some causes for the checkpoint perturbation in cancer remain to be investigated. In addition, it remains a challenge to translate our knowledge in this field into practical cancer therapy. The main goal of this project is to develop novel therapeutics aiming at cancer checkpoint perturbation. In this proposal, first we will study how certain hotspot mutations of tumor suppressor p53 actively inhibit the checkpoint response. Second, we will determine whether some mutant p53 directly promotes DNA replication, and if so, by what mechanism(s). Third, we will identify small molecule inhibitors targeting these molecular events. Lastly, we propose a “synthetic targeted therapy” strategy and leverage our new findings with the existing knowledge for cancer therapy. In our preliminary study, we have identified several lead inhibitors targeting this event. With the aid of one lead inhibitor we have obtained proof-of-concept to support feasibility of the proposed concept. In short, the proposed study will elucidate novel mechanisms of checkpoint perturbation and translate our discoveries into new therapeutics for a broad range of cancers.