Abstract 6160: Design and preclinical evaluation of a first-in-class covalent reversible inhibitor of IRAK4 with immunomodulatory properties and selective anti-lymphoma effect

Abstract Oncogenic activating mutations in the myeloid differentiation primary response gene 88 (MyD88) and the consequent activation of the Toll-like receptor pathway are hallmarks of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). This cascade includes phosphorylation of interleukin-1 receptor-associated kinase 4 (IRAK4), triggering downstream NF-κB activation and driving tumor survival, proliferation, and chemotherapeutic resistance. Although several IRAK4 small-molecule inhibitors (IRAK4-i) are undergoing clinical evaluation for various cancers, most are non-covalent inhibitors with limited activity and specificity.To address this, we employed computer-aided drug design to create a library of reversible covalent IRAK4-i based on a pyrido[2, 3-d]pyrimidine scaffold with lysine-targeting warheads. From this library, the ten most promising candidates were synthesized using organometallic reactions and microwave-assisted techniques. Subsequent multikinase screening identified four potential IRAK4-specific inhibitors, with GGP314 emerging as the most potent. GGP314 exhibited IC50 values of 6.6-7.3 μM at 24 hours and 1.8-2.8 μM at 72 hours in MyD88mut ABC-DLBCL cell lines, while showing minimal activity against non-ABC-DLBCL cells and normal peripheral blood mononuclear cells (PBMCs). Mechanistically, GGP314 reduced IRAK4 phosphorylation and impaired NF-κB signaling, as evidenced by the downregulation of several target genes. The compound demonstrated antitumor activity in 3D spheroid cultures of ABC-DLBCL cells, achieving a 50-60% reduction in Ki67 levels. However, the cytostatic effect was partially attenuated in the presence of follicular dendritic or mesenchymal stromal cells, though not with M2-polarized macrophages. Single-cell RNA sequencing (scRNA-seq) analysis of 3D spheroids revealed that GGP314 preferentially targets tumor-associated macrophages, shifting the tumor microenvironment towards a pro-inflammatory state via upregulation of interferon-gamma response pathways. Finally, GGP314 efficacy was validated in vivo in an immunocompetent chicken embryo chorioallantoic membrane (CAM) model of MYD88-mutant ABC-DLBCL. Twice-weekly dosing resulted in up to 55% tumor growth inhibition in a dose-dependent manner without systemic toxicity. In conclusion, we report here the development and preclinical validation of GGP314, a first-in-class reversible covalent IRAK4 inhibitor. GGP314 exhibits significant antitumor activity, high specificity, and the ability to modulate both tumor-intrinsic and microenvironment-dependent signals, supporting its potential as a therapeutic candidate for ABC-DLBCL. Citation Format: Gema Gorjon de Pablo, Nuria Profitos-Peleja, Miek Jacobs, Aitor Gonzalez-Herrero, Jeronimo Parra, Elisabetta Mereu, Paula Cifuentes, Melissa Orphali, Jordi Teixido, Manel Esteller, Jose I. Borrell, Roger Estrada-Tejedor, Gael Roue. Design and preclinical evaluation of a first-in-class covalent reversible inhibitor of IRAK4 with immunomodulatory properties and selective anti-lymphoma effect [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 6160.