HDAC8 Disrupts DNA Damage Response By Deacetylation of U2AF1 in Inv(16) Acute Myeloid Leukemia

Genomic instability due to defective DNA damage response (DDR) and DNA repair increases the probability of acquiring mutations, which may contribute to the pathogenesis of acute myeloid leukemia (AML). Inv(16) AML [inv(16)(p13q22) or t(16;16)(p13.1;q22)] creates a fusion gene CBFb-MYH11 (CM) by fusing core binding factor b (CBFb) with MYH11 which encodes smooth muscle myosin heavy chain (SMMHC). Our previous studies using a conditional Cbfb-MYH11 knock-in (KI) mouse model showed that preleukemic progenitors are prone to leukemia initiation, however, the underlying mechanism is not clear. Here, we show increased DNA double-strand breaks measured by comet assay and γH2AX staining in CM preleukemic progenitors compared to control counterparts. RNA-seq using sorted LSK (Lin-Sca1+Kit+) cells isolated from BM of CM KI mice (n = 5) and control (n = 4) identified a total of 1019 genes differentially expressed (526 up; 493 down; > 1.5-fold change; p < 0.01). Gene set enrichment analysis revealed that G2/M checkpoint, E2F targets, and mitotic spindle pathways were differentially downregulated in CM LSK, suggesting a possible contribution of cell cycle checkpoint dysregulation. To further explore the mechanism underlying CM-induced DNA damage, we expressed CM in 32D cells and subjected them to irradiation (IR; 3.5 Gy) followed by γH2AX and RAD51 staining. Significantly more γH2AX foci per nucleus were observed in CM cells (CM 35.03 vs Ctrl 24.71 at 0.5h, p<0.0001; CM 14.79 vs Ctrl 11.67 at 3h, p=0.0186), while fewer RAD51 foci per nucleus were seen in CM cells (CM 4.932 vs Ctrl 9.6 at 3h, p<0.0001), suggesting that CM may impair homologous recombination (HR)-mediated DNA repair. We reported that CM interacts with HDAC8 and enhances its activity, thus we examined whether HDAC8 may mediate the impact on DDR and HR activity. HDAC8 overexpression (OE) in 32D cells resulted in significantly more γH2AX foci per nucleus after IR (3.5 Gy) (HDAC8-OE 42.20 vs Ctrl 32.17 at 0.5h, p<0.0001; HDAC8-OE 15.45 vs Ctrl 10.63 at 3h, p<0.0001). Similar to CM cells, fewer RAD51 foci per nucleus were seen in HDAC8-OE cells (HDAC8-OE 11.63 vs Ctrl 17.39 at 3h, p<0.0001). To verify HDAC8 function in HR repair, we used the I-SceI and DR-GFP (homology-directed repair) or EJ5-GFP (end-joining-directed repair) DNA repair reporter (Gunn A et al, Methods Mol Biol, 2012). The frequency of repaired GFP+ population in the HR-directed repair reporter was reduced in HDAC8-OE cells compared to control (HDAC8-OE 7.807% vs Ctrl 11.34%, p=0.0169), while no difference was observed in the NHEJ-directed repair reporter. These results indicate that high HDAC8 expression disturbs HR DNA repair. The U2 small nuclear RNA auxiliary factor 1 (U2AF1) has been reported as a component of BRCA1-mRNA splicing machinery that regulates pre-mRNA splicing of critical genes involved in HR DNA repair. Here, we show that U2AF1 is subjected to post-translational acetylation and that U2AF1 interacts with HDAC8. We show that U2AF1 acetylation and its interaction with HDAC8 were increased following DNA damage and that HDAC8 is critical for deacetylating U2AF1. Site-directed mutagenesis for reported putative acetylation lysine sites (K15, K23, K175) (Elia AE et al, Mol Cell, 2015) revealed that the acetylation level of U2AF1-K23R decreased after IR and corresponded to reduced interaction with HDAC8 and the BRCA1/BCLAF1 complex. Notably, the interaction with BRCA1/BCLAF1 complex was rescued by mutating lysine to the acetylation mimic glutamine (Q). These results indicate that the acetylation state of U2AF1 is essential for the assembly of BRCA1-mRNA splicing complex and suggest that HDAC8 may regulate HR DNA repair by modulating the U2AF1 acetylation state. Similar to HDAC8 OE, CM expression reduced U2AF1 acetylation and impaired its interaction with BRCA1/BCLAF1 complex. Collectively, our results suggest that the increased DNA damage induced by CM may be due to the reduced acetylation of U2AF1 resulting from high HDAC8 activity. These results highlight a direct impact of inv(16) fusion protein and high HDAC8 activity on increased genomic instability in AML.

Marcucci:Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.