Abstract 357: Detailed mechanistic understanding of ACR-2316, a novel, clinical-stage WEE1/PKMYT1 inhibitor, rationally designed for superior single-agent activity through potent activation of CDK1, CDK2, and PLK1 using Acrivon’s machine learning-dri

Abstract Introduction: WEE1 and PKMYT1 are essential kinases that safeguard genomic integrity by regulating CDK1 and CDK2. ACR-2316 is a potent, selective WEE1/PKMYT1 inhibitor rationally designed using Acrivon’s Predictive Precision Proteomics (AP3) platform. ACR-2316 was uniquely designed, optimized, and advanced from initial lead to first-in-human dosing in < 15 months by AP3 to deliver complete tumor regression and pro-apoptotic tumor cell death through potent activation of CDK1, CDK2, and, importantly, PLK1. Here, we leveraged AP3-based global differential phosphoproteomic profiling and computational analyses to map and quantify the ACR-2316-regulated CDK1/2 and PLK1 substrates for unbiased understanding of drug action. Results: AP3-based data-independent acquisition mass spectrometry was conducted using human cancer cells treated with vehicle or ACR-2316, -/+ CDK1 or CDK2 inhibition. Kinase activity inference analyzed by AP3 machine learning leveraging curated kinase-substrate relationships revealed that ACR-2316 strongly activated CDK1 and CDK2 followed by mitotic and DNA damage response kinases. We quantified hundreds of CDK1 and CDK2 substrates, regulated by ACR-2316, associated with S-phase DNA replication and replicative stress (CDK2) and with G2/M phase mitotic chromosome re-organization and segregation (CDK1). Multiparametric flow cytometry confirmed that ACR-2316 triggers DNA damage, premature mitosis, and S/G2-M cell cycle arrest. CellTiter-Glo and live cell imaging assays demonstrated robust inhibition of cell proliferation and potent cell death across human cancer cell lines. Inhibition of CDK1 or CDK2 partially rescued these phenotypes, confirming on-target mechanism of action identified through AP3. Chromatin immunoprecipitation sequencing showed unique intronic regions were susceptible to ACR-2316-induced double strand breaks. Finally, multiplexed immunofluorescence for alpha-tubulin and DNA revealed a large ACR-2316-induced increase in the number of mitotic aberrations in human cancer cells compared to vehicle control, and inhibition of only WEE1 or PKMYT1. Conclusions: The unique capabilities of our generative AI-driven AP3 platform used to rationally design ACR-2316 enabled a comprehensive analysis of ACR-2316-regulated CDK1/2-and PLK1-induced pathways underlying its differentiated, potent anticancer activity, as demonstrated in head:head preclinical studies against clinical benchmark inhibitors. ACR-2316 has advanced into the clinic ahead of schedule uniquely enabled by AP3 and is currently in a Phase 1 clinical trial in subjects with AP3-selected advanced solid tumors. Acrivon anticipates reporting initial ACR-2316 clinical Phase 1 data second half of 2025. Citation Format: Lei Shi, Martina Pasetto, Mohamed Eldeeb, Reina Improgo, Shahrzad Rafiei, Maria Rodriguez Zabala, Calvin Yang, Ahmed Youssef, Georgia Mitsa, Nina Lipjankić, Anna-Maria Alves, Portia Lombardo, Jessica Hopkins, Bryan Thornlow, William Dahlberg, Everett Hay, Ignacio Arribas Díez, Marc Isaksson, Zachary Best, Chris S. Balagtas, Corey Xu, Magnus E. Jakobsson, Helén Nilsson, Joon Jung, Caroline Wigerup, David A. Proia, Kristina Masson, Peter Blume-Jensen. Detailed mechanistic understanding of ACR-2316, a novel, clinical-stage WEE1/PKMYT1 inhibitor, rationally designed for superior single-agent activity through potent activation of CDK1, CDK2, and PLK1 using Acrivon’s machine learning-driven AP3 platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 357.