Abstract C112: ACR-2316 is a novel, differentiated, clinical-stage WEE1/PKMYT1 inhibitor designed by Acrivon’s Generative Phosphoproteomics AP3 Platform for optimal pro-apoptotic pathway effects in tumor cells resulting in superior preclinical activi

Abstract Introduction: ACR-2316 is an internally discovered, clinical stage, potentially first- and best-in-class WEE1/PKMYT1 (MYT1) inhibitor specifically designed using Acrivon’s Predictive Precision Proteomics (AP3) platform. Here, we delineate the differentiated profile by which ACR-2316 achieves superior preclinical efficacy compared to benchmark WEE1 or MYT1 inhibitors. Methods: Intracellular global pathway activity modulation, kinase activity, and target engagement (TE) were assessed by mass-spectrometry based AP3, AlphaLISA, immunofluorescence, and Western blot (WB). Cell viability was assessed by CellTiter Glo (CTG). MYT1 stably overexpressing cells were generated by selection following transfection of MYT1 cDNA. Replication stress, DNA damage, and cell death were assessed by flow cytometry, CTG, and live cell imaging. In vivo efficacy and TE were evaluated in a human cancer cell line-derived xenograft (CDX) model. Results: Through AP3 profiling, we previously uncovered WEE1 inhibitor-induced resistance mechanisms that were quenched by MYT1 inhibition. ACR-2316 was rationally designed using AP3 to suppress this resistance mechanism alongside potent WEE1 inhibition and robust activation of CDK1, CDK2, and PLK1, to induce potent tumor cell death. In cellular TE assays, ACR-2316 displayed more potent WEE1 TE than all benchmark WEE1 inhibitors (azenosertib, adavosertib, Debio0123), while simultaneously targeting MYT1. The impact of MYT1 TE was evidenced by a >3X loss of sensitivity induced by MYT1 overexpression, which was restored by co-treatment with the MYT1 inhibitor lunresertib. AP3 profiling in tumor cells and WB in human keratinocytes confirmed that ACR-2316, in contrast to lunresertib, does not modulate the canonical BRAF-MAPK pathway components, which may be involved in the dose-limiting skin rash observed clinically for lunresertib. Further differentiating from benchmark WEE1 or MYT1 inhibitors, ACR-2316 more potently induced DNA damage, premature mitotic entry, and cell death. Accordingly, ACR-2316 exhibited superior potency in cell viability compared to all benchmark inhibitors, including the WEE1/MYT1 inhibitor SGR-3515 (based on previously reported data), across a broad range of human cancer cell lines. In human CDX models, azenosertib and lunresertib resulted in only modest growth inhibition at their maximum tolerated/formulable doses, while ACR-2316 demonstrated complete tumor regression at doses associated with potent WEE1 and balanced MYT1 inhibition, highlighting the importance of dual targeting. Conclusions: WEE1 inhibitor-induced MYT1 activation constitutes a resistance mechanism that may limit the clinical efficacy of WEE1 inhibition. ACR-2316 is a potent, selective WEE1/MYT1 inhibitor that displays superior preclinical efficacy via its differentiated profile optimized by AP3 pathway-based structure-activity relationships in the intact cell. Acrivon’s ongoing Phase 1 ACR-2316 monotherapy trial in solid tumors has already demonstrated clinical activity during dose escalation prior to reaching Recommended Phase 2 Dose. Citation Format: Portia Lombardo, Anna-Maria Alves, Martina Pasetto, Mohamed Eldeeb, Zachary Best, Reina Improgo, Nina Lipjankić, Subodh Kumar, Ruban Cornelius, Kate Rappard, Uthira Muralitharan, Valentina Siino, Ignacio Arribas Diez, William Dahlberg, Shahrzad Rafiei, Kailash Singh, Ayesha Murshid, Joelle Baddour-Sousounis, Magnus E. Jakobsson, Michail Shipitsin, Helén Nilsson, Caroline Wigerup, Lei Shi, David Proia, Kristina Masson, Peter Blume-Jensen. ACR-2316 is a novel, differentiated, clinical-stage WEE1/PKMYT1 inhibitor designed by Acrivon’s Generative Phosphoproteomics AP3 Platform for optimal pro-apoptotic pathway effects in tumor cells resulting in superior preclinical activity [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 C112.