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 4

OE of ONECUT3 leads to dysregulation of the CPC and mitotic defects.

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OE of ONECUT3 leads to dysregulation of the CPC and mitotic defects. (A)...
(A) Time course of Western blot analysis of CPC components. Left: Control (–Dox) and ONECUT3 OE (+Dox) Tp53-KO MEFs were treated with nocodazole (Noc) (75 ng/mL) for 15 hours and released at the indicated time points. Cell lysates were blotted with antibodies against Incenp, Borealin, Survivin, and Aurora B. Protein levels were comparatively analyzed at the indicated time points, with gray values determined using ImageJ software (NIH). (B) Representative confocal images of the coimmunostaining against DAPI (blue), tubulin (yellow), and Aurora B (green) in each mitotic phase. (C) 3D model generated by Huygens, based on photos from Z-stack scanning. White dashes indicate the putative cleavage furrow. (D) Cells were immunolabeled at the indicated phases with α-tubulin (red) and Incenp (green) antibodies. White arrows indicate the differential localization of Incenp in Onecut3-overexpressing cells compared with the control. (E) Flow cytometry was performed to determine the fraction of mitotic cells, using H3S10ph and propidium iodide costaining following nocodazole release, normalized to the initial point (t = 0) for each time point. (F) Following Dox treatment and subsequent nocodazole treatment and washout, DAPI (blue) and H3S10ph (green) coimmunostaining was performed. Shown is a representative confocal image 1 hour after nocodazole release. Graph shows the mitotic index, evaluated 1 hour after nocodazole release. Randomly counted the number of H3S10ph+ cells from 62–120 cells/slide; n = 3. (G) Costaining with α-tubulin (red), H3S10ph (green), and DAPI (blue). White dashes outline control cells (outlined areas 1 and 2) and Onecut3-overexpressing cells (outlined areas 3 and 4). (H) Percentages of cells showing chromosome lagging, chromosome bridging, and multipolarity (n = 3). Scale bars: 11.6 μm (B and D), 10 μm (C and G), 100 μm (F). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA and 2-tailed, paired Student’s t test (E, F, and H).