Abstract Loss of function mutations of the TP53 gene are the most common genetic defects across all human cancers. These mutations have long been intractable to drug development. The p53 Y220C mutation is common, occurring in ∼1% of cancers, leading to destabilization, aggregation and loss of p53 protein function. Application of the FrontierTM platform, which integrates chemoproteomics, machine-learning, and covalent fragment-based drug discovery, enabled the discovery of FMC-220, a potential first-in-class covalent activator of p53 Y220C, which is highly potent and selective in restoring p53 tumor suppressor function. The covalent mechanism of action of FMC-220 delivers potent and durable induction of p53 transcriptional response that includes upregulation of known regulators of cell cycle arrest, senescence and cell death. ChIPseq analysis demonstrated that FMC-220 delivers far more durable activation and ​persistent engagement of target gene promoters than noncovalent strategies such as rezatapopt (PC14586). FMC-220 inhibits tumor cell viability with ∼100-fold increased potency relative to rezatapopt across a panel of Y220C mutant cell lines including in tumor cells with a KRAS co-mutation and irrespective of histology. This translates into potent pharmacodynamic activity and tumor regression in Y220C mutant CDX and PDX models in vivo. Regression, including sustained complete regression (CR), is achieved with single agent FMC-220C treatment. In addition, FMC-220 combines effectively with other therapeutics including MDM2 inhibitors or DNA damaging agents. Together, these data demonstrate the promise of FMC-220, a first-in-class covalent activator of p53 Y220C, with the potential to deliver improved outcomes for patients with Y220C mutation positive tumors. Citation Format: Kevin R. Webster, Yan Wang, Shayna Simonstein, Truc Nguyen, John-Paul Upton, Vikram Narayan, Karsten Krug, Tiep Le, Sarah Gilfillan, Shefali Sabhlok, Irina Dotsenko, Alessandra Ianari, Lata Jayaraman. FMC-220, a highly potent and selective covalent activator of p53 Y220C, is broadly active in p53 Y220C containing PDX models across cancer indications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B123.