Ectopic expression of the transcription factor ONECUT3 drives a complex karyotype in myelodysplastic syndromes
Yingwan Luo, Xiaomin Feng, Wei Lang, Weihong Xu, Wei Wang, Chen Mei, Li Ye, Shuanghong Zhu, Lu Wang, Xinping Zhou, Huimin Zeng, Liya Ma, Yanling Ren, Jie Jin, Rongzhen Xu, Gang Huang, Hongyan Tong
Yingwan Luo, Xiaomin Feng, Wei Lang, Weihong Xu, Wei Wang, Chen Mei, Li Ye, Shuanghong Zhu, Lu Wang, Xinping Zhou, Huimin Zeng, Liya Ma, Yanling Ren, Jie Jin, Rongzhen Xu, Gang Huang, Hongyan Tong
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Research Article Hematology

Ectopic expression of the transcription factor ONECUT3 drives a complex karyotype in myelodysplastic syndromes

Abstract

Chromosomal instability is a prominent biological feature of myelodysplastic syndromes (MDS), with over 50% of patients with MDS harboring chromosomal abnormalities or a complex karyotype (CK). Despite this observation, the mechanisms underlying mitotic and chromosomal defects in MDS remain elusive. In this study, we identified ectopic expression of the transcription factor ONECUT3, which is associated with CKs and poorer survival outcomes in MDS. ONECUT3-overexpressing cell models exhibited enrichment of several notable pathways, including signatures of sister chromosome exchange separation and mitotic nuclear division with the upregulation of INCENP and CDCA8 genes. Notably, dysregulation of chromosome passenger complex (CPC) accumulation, besides the cell equator and midbody, during mitotic phases consequently caused cytokinesis failure and defective chromosome segregation. Mechanistically, the homeobox (HOX) domain of ONECUT3, serving as the DNA binding domain, occupied the unique genomic regions of INCENP and CDCA8 and transcriptionally activated these 2 genes. We identified a lead compound, C5484617, that functionally targeted the HOX domain of ONECUT3, inhibiting its transcriptional activity on downstream genes, and synergistically resensitized MDS cells to hypomethylating agents. This study revealed that ONECUT3 promoted chromosomal instability by transcriptional activation of INCENP and CDCA8, suggesting potential prognostic and therapeutic roles for targeting high-risk MDS patients with a CK.

Authors

Yingwan Luo, Xiaomin Feng, Wei Lang, Weihong Xu, Wei Wang, Chen Mei, Li Ye, Shuanghong Zhu, Lu Wang, Xinping Zhou, Huimin Zeng, Liya Ma, Yanling Ren, Jie Jin, Rongzhen Xu, Gang Huang, Hongyan Tong

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Figure 3

ONECUT3 activates the CPC components INCENP and CDCA8 through direct DNA binding.

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ONECUT3 activates the CPC components INCENP and CDCA8 through direct DNA...
(A) RNA-Seq was conducted on the sample collected after 48 hours of Dox (100 ng/mL) treatment (to induce ONECUT3 OE) compared with no Dox treatment (control) in Tp53-KO MEFs. Gene sets, which were significantly enriched (FDR ≤ 0.05) in the Onecut3-overexpressing cells, are listed according to the normalized enrichment score (NES), using the GO Biological Process program. The top 10 biological processes are shown. Colored bars represent the enrichment ratio. (B) Integrative Genomics Viewer (IGV) tracks by ChIP-Seq in Tp53-KO MEF depicts the normalized density profile on Incenp and Cdca8 gene loci. Onecut3 binds at 3 loci (the promoter, intron, and distal regions) of the Incenp gene, as well as at 2 loci of the Cdca8 gene. (C) Enrichment of Incenp and Cdca8 was found upon Onecut3 OE via ChIP-qPCR analysis in WT Tp53 MEFs. TSS, transcription start site. (D) Correlation between mRNA levels of ONECUT3 and INCENP, CDCA8, AURKB, and BIRC5 in 165 patients with MDS harboring WT TP53. (E) Representative images of immunohistochemical staining (ONECUT3, Aurora B, INCENP, and Borealin/CDCA8) of biopsy tissue from a volunteer (healthy donor) and a patient with MDS (the right black box was the magnified view of the indicated region in the left box). Single-cell color outlines indicate QuPath-analyzed expression (0, blue; 1+, yellow, 2+, orange, 3+, red). Scale bars: 625 μm (left) and ×2 magnification (right). (F) Heatmap shows the H-Score with a combination of the staining intensity and cell frequency in each sample. (G) Correlation of the abundance of ONECUT3 with Aurora B expression, as determined by IHC. The color and size of the dots indicate the correlation coefficient (R) and P values. RNA-Seq replicates = 3, ChIP-Seq replicates of OE-Input/IgG = 1, replicates of Ctrl/FLAG = 1, replicates of OE-FLAG = 3. The correlation coefficient (R) and P values from Pearson’s correlation tests are shown (D and G). *P < 0.05 and **P < 0.01, by 1-way ANOVA (C).