Abstract B074: Evaluation of a small molecule, AP-232, as a splicing factor inhibitor of U2 auxiliary factor 1 (U2AF1) in leukemia cells

Abstract

Mutations in splicing factors (SF3B1, SRSF2, and U2AF1) are recurrent in myelodysplastic syndromes (MDS) and related hematologic malignancies, where they occur in a mutually exclusive manner that create a vulnerability when a second wild-type splicing factor is therapeutically targeted. However, the feasibility of this approach and mechanisms underlying selective toxicity of splicing factor–mutant cells remain incompletely defined. We developed AP-232, a small-molecule inhibitor of U2AF1, and investigated its mechanism of action in K562 cells (wild-type splicing factors) and HNT-34 cells, an MDS-derived AML model harboring the SF3B1K700E mutation. We found that AP-232 induces two temporally distinct and mechanistically opposing cellular states. Cell viability assays showed that at 24 hours, it drives acute cytotoxicity (up to ∼83% for K562 and ∼100% for HNT-34), while at later timepoints it shifts to sustained cytostatic effects. The cytotoxic effect of AP-232 diminished to ∼10% in K562 cells (IC50 of ∼17 uM) but remained ∼40% in HNT-34 cells (IC50 of ∼7 uM) at 72h, indicating greater sensitivity to U2AF1 inhibition in HNT-34 cells. Mechanistically, early responses in K562 are characterized by inflammatory, non-apoptotic cell death. LDH release, pyroptosis-associated markers, and Necrostatin-1 co-treatment implicate the involvement of necrosis or non-canonical pyroptosis. RNA-seq analysis at 48 h revealed strong induction of inflammatory pathways, which may result from inflammatory cell death. We previously showed that AP-232 induces transient autophagy inhibition, suggesting the contribution of lysosomal dysfunction and impaired autophagic flux to this early inflammatory cytotoxicity. Western blot analysis at 24 h demonstrated that K562 cells undergo replication stress, as indicated by increased levels of PCNA, Chk1, PARP, and c-Myc. In contrast, at 48 hours, replication collapses. All four markers are markedly reduced, and cells undergo G1 arrest accompanied by suppression of proliferative programs. Transcriptomic analysis revealed downregulation of c-Myc and E2F target genes, which was confirmed at the protein level by decreased expression of c-Myc, PCNA, Cdc6, MCM7, CDC20, and PLK1. Despite reduced checkpoint signaling at 48 hours, no DNA damage was detected by comet assay or immunoblotting for γ-H2AX, a marker of double-strand breaks, indicating that this state exhibits diminished replication activity without genotoxic stress. Collectively, AP-232 exerts a dual mechanism of action: an early inflammatory, non-apoptotic cell death program linked to autophagy disruption and replication stress, followed by sustained cytostatic repression of c-Myc/E2F-driven cell cycle programs. Enhanced activity in SF3B1-mutant cells suggests that simultaneous targeting of splicing and autophagy dependencies may represent a therapeutic strategy for SF3B1–mutant hematological malignancies, potentially driven by increased reliance on autophagy as a survival mechanism in this genetic context. Mona Kazemi Sabzvar, Amol D. Patil, Eun Bee Cho, Arda Durmaz, Daniel M. Collier, Jianxiong Jiang, Valeria Visconte, Chao-Yie Yang. Evaluation of a small molecule, AP-232, as a splicing factor inhibitor of U2 auxiliary factor 1 (U2AF1) in leukemia cells [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B074.