KAT7 is a genetic vulnerability of acute myeloid leukemias driven by MLL rearrangements

Abstract

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.

Figure 1. Loss of KAT7 exhibits anti-leukemic effects in vitro and in vivo

A) Proliferation of KAT7 KO using two sgRNAs or empty control in MLL-X AML cell lines. Percentages of BFP-positive (KAT7-KO) cells were assayed at the indicated time point and were normalized to day 4. Data are shown as mean ± SD (n= 3). B) CD11b staining in WT (empty gRNA) and KAT7 KO (gKAT7-A10) cells 7 days post transduction. C) Annexin V/PI staining of WT (Empty) and KAT7 KO (gKAT7-A10) cells 9 days post transduction. Data are shown as mean ± SD (n= 3). Two-tailed t-test was performed (N.S., not significant; **, P < 0.01; ***, P < 0.001). D) Xenograft analysis of KAT7-KO MOLM-13. Kaplan-Meier survival analysis were performed (n= 5 in each arm).

Figure 2. Catalytic activity of KAT7 is required for leukemic maintenance.

A) Western blot analysis of potential KAT7 acetylation sites on H3 and H4 in MOLM-13 cells. B) Western blot analysis of the KAT7 acetylation sites in MOLM-13 expressing exogenous WT-KAT7 or HAT-dead KAT7 (E508Q) with or without endogenous KAT7 KO by lentiviral gRNA expression. C-D) Proliferation (C) and CD11b staining (D) of MOLM-13 expressing exogenous KAT7 WT or E508Q mutant with endogenous KAT7 disrupted by gKAT7. Data are shown as mean ± SD (n= 3).

Figure 3. Transcriptomic profiling of KAT7 KO in MOLM-13 (MLL-AF9), OCI-AML3 (MLL WT).

A) Numbers of differentially expressed (DE) genes in MOLM-13 and OCI-AML3 on day 3 and day 5 after gRNA-mediated KAT7 KO. B) GSEA on day 3 (top) and day 5 (bottom) transcriptomes in MOLM-13. Gene sets consisting of genes upregulated upon myeloid differentiation (left), MLL-AF9 target genes (centre), or MLL-AF9 spreading genes (right; Table S4) were used. C) MA plot of day 3 MOLM-13 comparing KAT7 WT and KO. Black, DE genes (adj.P<0.05); yellow, MLL-AF9 spreading genes. Key MLL-AF9 spreading genes, namely MEIS1, PBX3, JMJD1C, SENP6 and MEF2C were highlighted.

Figure 4. Transcriptomic profiling of the acute phase of KAT7 depletion in MOLM-13 using the degron system.

A) Time-course western blot analysis of KAT7 and H3K14ac in IAA-treated MOLM-13 KAT7-AID cells. B) Cell proliferation of IAA-treated KAT7-AID and parental MOLM-13. Data are shown as mean ± S.D. (n= 3). Two-tailed t-test was performed (***, P ≤ 0.001). C) GSEA on transcriptome of MOLM-13 KAT7-AID cells treated with IAA for 2- (top) or 4 h (bottom). The same genesets as Fig. 3B were used.

Figure 5. KAT7 binds to and is required for expression of a subset of MLL-AF9 targets.

A-B) Average occupancy of KAT7 per gene per base at the TSS of highly active genes (>10 FPKM), active genes (>0, ≤10 FPKM) and inactive genes (FPKM=0) in MOLM-13 (A) and OCI-AML3 (B). C-D) Comparison between KAT7 promoter occupancy and gene expression changes 3 days after KAT7 KO in MOLM-13 (C) and OCI-AML3 (D) for MLL-AF9 spreading genes. DE genes are highlighted in blue (up) or red (down).

Figure 6. KAT7-dependent recruitment of BRD4 and SEC complex to a subset of MLL-AF9 spreading genes.

A-C) ChIP-qPCR analysis of BRD4 (A), AFF1 (B) and pS5 Pol II (C) after 24 hours of IAA treatment at the promoter region of the key MLL-AF9 spreading genes. Data are shown as mean ± S.D. (n= 3). Two-tailed t-test was performed (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).