ANP32A Regulates Histone 3 Acetylation and Promotes Leukemogenesis in AML
Authors: Yang Xuejing , Sun Xueqin , Lu Bin , Wang Qianfei , Huang Zan 
Published: October 2019

Leukemia initiation and progression not only depend on genetic alterations but also on abnormal epigenetic changes that cause disorder of gene expression. Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) binds directly to histone tails, preferentially to unmodified histone H3 tails (Schneider et al., 2004; Seo et al., 2002). Although ANP32A inhibits histone acetyltransferase activity in vitro (Seo et al., 2002), loss of ANP32A leads to decreased histone 3 acetylation (acetyl-H3) levels on interferon-stimulated genes in human cervical carcinoma HeLa S3 cells (Kadota et al., 2011). Aberrant ANP32A expression has been observed in many solid tumors, which involves in diverse processes depending on the cell context (Brody et al., 2004; Hoffarth et al., 2008), yet mechanisms are still unknown. Furthermore, there has as yet been no specific investigation into the role of ANP32A in acute myeloid leukemia (AML).

We found that ANP32A expression was upregulated at both mRNA and protein levels in patients with AML. In AML primary cells from patients, ANP32A KD decreased cell proliferation and colony formation ability. In addition, ANP32A KD induced apoptosis and cell cycle arrest at G1 phase accompanied with the downregulated protein expression of survival or cell cycle progress genes BCL2, CDK4, CCND1 and the upregulated protein expression of pro-apoptosis genes BAK, BAD, cleaved caspase 3. These findings were also verified in various subtypes of AML cell lines and Mll-Af9 pre-leukemic cells. Thus, ANP32A is essential for cell growth via the promotion of cell-cycle progression and inhibition of apoptosis in AML.

Given that ANP32A associates with histone acetylation modification, we analyzed the effect of ANP32A depletion on acetyl-H3 and histone 4 acetylation (acetyl-H4). Western blot analysis showed that ANP32A KD caused global decrease of acetyl-H3, but no effect on the global level of acetyl-H4. To investigate the mechanism of ANP32A in AML, ChIP-seq and gene expression microarray experiments were performed to determine acetyl-H3 enrichment and transcriptome profiles, respectively, in ANP32A KD and control K562 cells. We found a positive correlation between changes in gene expression and changes in acetyl-H3 (r=0.16, p<10-4), especially for differential expression genes (DEGs; r=0.40, p<10-4). Moreover, we also found genes with decreased acetyl-H3 enrichment displayed lower expression levels compared with genes with increased acetyl-H3 enrichment (p<10-3). Thus, ANP32A alters acetyl-H3 modification in association with changes in gene expression.

Gene set enrichment analysis (GSEA) revealed that ANP32A KD significantly decreased the acetyl-H3 enrichment levels at genes of lipid metabolism signatures (e.g. lipid metabolic process, cholesterol homeostasis). Transcriptome profile analysis using GSEA showed that gene expression levels of lipid metabolic process signature were also reduced after ANP32A KD. We further identified 4 genes (APOC1, CNKSR3, H19, PCSK9) which had significantly lower levels of mRNA expression (p<10-2, FDR<10-2, FC>1.5) and decreased levels of acetyl-H3 enrichment (p<10-2, FDR<10-2, FC>1.5) in ANP32A KD cells. Apolipoprotein CI(APOC1) was the most significantly downregulated gene among these 4 genes, with decreased acetyl-H3 on its promoter. To explore whether lipid metabolism as a downstream effector of ANP32A contributes to ANP32A function in AML, re-introduction of APOC1 was performed in ANP32A KD THP1 or K562 cells. Re-introduction of APOC1 significantly rescued the inhibition of cell growth in ANP32A KD AML cells, also upregulated protein expression of BCL2, CDK4 and CCND1, showing that ANP32A functions in AML partially depending on APOC1. Importantly, APOC1 depletion decreased cell proliferation of AML cells in vitro, consistent with the point of view that complex lipid alterations contribute to endoplasmic reticulum stress-induced apoptosis in cancer cells (Beloribi-Djefaflia et al., 2016; Iurlaro et al., 2016).

Overall, our data suggest ANP32A as a novel regulator of leukemogenesis in AML. ANP32A may involve in the regulation of genome-wide histone 3 acetylation that alters the expression of multiple genes, including lipid metabolism related gene APOC1. Thus, ANP32A is a promising drug target for AML therapy.

Disclosures

No relevant conflicts of interest to declare.