Dysregulated Alternative Splicing in Inv(16) Acute Myeloid Leukemia Via U2AF1 Deacetylation during DNA Damage Response

Inversion of chromosome 16, inv(16) [inv(16)(p13q22) or t(16;16)(p13.1;q22)], is a recurrent chromosomal translocation observed in 5-8% of acute myeloid leukemia (AML) cases. Inv(16) creates a fusion gene CBFb-MYH11 (CM) which impairs hematopoietic differentiation and creates abnormal progenitor populations prone to leukemic transformation. We reported that CM interacts with HDAC8 and enhances its activity, and high HDAC8 activity impaired DNA damage response (DDR) in CM knock-in (KI) hematopoietic stem and progenitor cells (HSPCs) (Zhang, L et al, ASH meeting 2022, https://doi.org/10.1182/blood-2022-160280). The recruitment of BRCA1-mRNA splicing machinery is critical for efficient DDR and the U2 small nuclear RNA auxiliary factor 1 (U2AF1) is required for accurate 3'-splice site selection. Here, we show how U2AF1 is post-translationally regulated following DNA damage and its impact on alternative splicing in CM KI HSPCs upon DNA damage. First, we identified TIP60 as the histone acetyltransferase (HAT) responsible for the increased U2AF1 acetylation induced by ionizing radiation (IR). We detected enhanced interaction between U2AF1 and TIP60 but not with other HATs (GCN5, PCAF, CBP) along with the increased U2AF1 acetylation in response to IR. In addition, TIP60 knockdown (KD) reduced U2AF1 acetylation, while TIP60 expression increased U2AF1 acetylation, indicating that TIP60 mediates the acetylation of U2AF1. To pinpoint the specific lysine residue subjected to TIP60 catalysis, we introduced lysine site-specific mutants U2AF1-HA-K15R, K23R, K39R, K175R, or 4 lysine sites mutant (U2AF1-HA-4mut) with TIP60. Co-immunoprecipitation (anti-HA) followed by western blotting (anti-acetyl-lysine) showed undetectable acetylation of U2AF1-HA-K23R and K175R mutants (similar to U2AF1-HA-4mut), suggesting that K23 and K175 residues are essential for TIP60-mediated acetylation. To investigate the impact of K23 or K175 acetylation on the assembly of the BRCA1-mRNA splicing complex and DDR, we subjected U2AF1-K23R or K175R expressing 32D cells to IR (3.5 Gy). Both U2AF1-K23R and K175R mutants exhibited markedly reduced acetylation levels after IR, confirming that K23 and K175 sites are the acetylation sites responding to DNA damage. Furthermore, the interaction of U2AF1-K23R and K175R with BRCA1 and BCLAF1 was impaired. We observed increased apoptosis in U2AF1-K23R and K175R cells compared to wild-type (WT) U2AF1 cells (K23R vs WT, 51.975±7.867% vs 6.055±0.634%, P=0.0011; K175R vs WT,22.645±3.037% vs 6.055±0.634%, P=0.0017) after KD of endogenous U2AF1 and subjected to IR (3.5 Gy). These results indicate that U2AF1 is acetylated by TIP60 upon DNA damage on K23 and K175 which is critical for cell survival and assembly of the BRCA1-mRNA splicing complex in DDR.

Our studies revealed that HDAC8 interacts with U2AF1 and modulates U2AF1 acetylation following DNA damage and acetylation on U2AF1-K23 is important for HDAC8 binding. Consistent with the enhanced HDAC8 activity induced by CM, we show that CM also diminished U2AF1 acetylation and impaired the assembly of the BRCA1-mRNA splicing complex after IR. To examine the consequences in alternative splicing events upon IR, we sorted CM KI (n=5) or control (n=3) Lin-Sca1+Kit+ (LSK) cells and exposed them to IR (2.0 Gy) followed by RNA-seq. We identified a total of 829 splicing events in WT-IR vs WT-NIR (NIR: no IR) and 643 splicing events in CM-IR vs CM-NIR. Specifically, we found that skipped exon (SE) and retained intron (RI) events are selectively reduced in CM vs WT (SE events: 496 vs 653; RI events: 2 vs 26). Gene set enrichment analysis revealed striking differential enrichment in 38 BRCA1-related pathways, including regulation of response to DNA damage stimulus (NES: -1.0816 vs 0.8707), regulation of DNA repair (NES: -1.1557 vs 0.7729), cell cycle checkpoint signaling (NES: -0.9719 vs 0.7895), and recombinational repair (NES: -0.9215 vs 0.7299). Altogether, these studies reveal mechanistic insights into DNA damage induced regulation of post-translational acetylation of U2AF1 splicing factor and provide a mechanistic link to the dysregulated alternative splicing and impaired DNA damage response in inv(16) AML.