The HDAC8–U2AF1 Axis Dysregulates DNA Damage–Responsive RNA Splicing and Creates Targetable Vulnerabilities in Acute Myeloid Leukemia

Despite recent treatment advances using targeted therapies, acute myeloid leukemia (AML) remains a highly lethal disease. Mechanism-based biomarker-informed therapies could expand therapeutic options and enable rational combination strategies. Here, we demonstrated that HDAC8 impairs DNA repair and confers therapeutic vulnerabilities in AML. Analysis of AML patient datasets revealed that HDAC8 is overexpressed in more than half of cases and is associated with dysregulated DNA damage response (DDR) gene expression. Elevated HDAC8 expression induced DNA damage phenotypes and compromised homologous recombination (HR)-mediated repair. Mechanistically, HDAC8 regulated acetylation of the splicing factor U2AF1, which blocked DNA repair by regulating RNA splicing. In response to DNA damage, the acetyltransferase TIP60 acetylated U2AF1 at lysines 23 and 175, modifications required for the interaction of U2AF1 with BRCA1 and BCLAF1 within a DDR-associated spliceosomal complex. HDAC8 promoted the removal of these acetyl groups, disrupting U2AF1-BRCA1-BCLAF1 complex formation and altering DDR-associated RNA splicing. Consequently, HDAC8 hyperactivity drove widespread RNA splicing dysregulation, defective HR repair, and increased sensitivity to both splicing modulators and PARP inhibitors. In preclinical models, including patient-derived xenografts, the splicing modulator H3B-8800 and the PARP inhibitor talazoparib reduced leukemia burden and leukemia stem cell activity, with enhanced efficacy observed in combination. In inv(16)(p13q22) AML, the CBFβ::SMMHC fusion protein exploited the HDAC8-U2AF1 axis to promote genomic instability. These findings identify the HDAC8-U2AF1 axis as a mechanistic driver of DDR dysfunction and genomic instability and uncover actionable, biomarker-informed therapeutic vulnerabilities in HDAC8-high AML.