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

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.

A. The data is based on HPA RNA-seq tissue data from the Human Protein Atlas version 22.0.nTPM (normalized protein-coding transcripts per million), corresponds to the mean values of the different individual samples from each tissue.B. The expression of ONECUT3 was generated from the dataset of GSE13159, GSE15434, GSE61804, GSE14468, and The Cancer Genome Atlas (TCGA).Data has been batch corrected.AML with CK contains: AML with Complex, AML with Complex +other and AML with Complex+5q.HSC/ HSPC includes Hematopoietic stem cell (HSC), Multipotential progenitors (MPP), Common myeloid progenitor cell (CMP), Granulocyte monocyte progenitors (GMP), Megakaryocyte-erythroid progenitor cell (MEP).C-D.The validation of newly generated anti-ONECUT3 antibody.The cell lysate of the bone marrow (BM) mononuclear cells from one patient of complex karyotype was used for immunoblotting.1 μg homemade antibody (rabbit anti-human ONECUT3 antibody) was not blocked [(-) blocking peptide] and another 1 μg antibody that pre-incubated with the peptide [(+) 1 μg blocking peptide (aa 469-482)] for overnight (C).HEK293 cells were transiently transfected with pLKO-TET-ONECUT3.In addition, the cell lysate from ShRNA-Ctrl (-Dox) or shRNA-ONECUT3 (+Dox) for the immunoblotting was also performed to confirm the specificity of the rabbit anti-ONECUT3 polyclonal antibody (D).Loading control is ACTIN.E. Gating strategy for Lin -D34 + CD38 -stem cells and Lin -CD34 + CD38 + progenitor cells from volunteers and patients with MDS in Figure 1 F. A. Schematic overview of the experiment: in the setting of Tp53-WT or Tp53-KO MEF cells that were stably transduced with Retro-ONECUT3, the reduction of Incenp expression was achieved using small interfering RNA (siRNA), and this reduction was coordinated with the regulation of Onecut3 expression, both in the absence and presence of doxycycline (DOX).After a period of 48 hours, the cells were collected for quantitative polymerase chain reaction (qPCR) or Western blotting (WB).Additionally, to ensure synchronization of the cell cycle, nocodazole was introduced for an additional 15 hours, followed by washout for immuno-staining and confocal A. Virtual Flow for Virtual Screening.Alpha-Fold2 was used to obtain the three-dimensional structure of the human ONECUT3 protein, and the hydrophobic core cavity near D358, K364, R468, N471, and R472 was served as a possible binding site.As the target region, hydrogen bonds, salt bridges, etc., were added, which were used as structural templates for subsequent virtual screening.Subsequently, we used the 500,000 compounds from Hit2Lead compound library (ChemBridge) for 3D optimization.Blasticidin S HCl.These drugs were removed before the transfection.The above cells were cultured in a 37°C, 5% CO2 incubator.

Plasmids of ONECUT3-OE, transfection, and transduction
ORF clone of human ONECUT3 (NM_001080488.2) was originally synthesized in the pcDNA3.1 vector.pRetroX-TetOne system was gifted by Prof. Kosei Ito (Nagasaki University, Nagasaki, Japan).The full-length, truncated mutants and point mutants of ONECUT3 were amplified with PCR using the listed primers (Supplemental Table 11) and subcloned into Retro-X-TET-ON system using the BamHI and EcoRI restriction enzyme.Retroviruses were generated by calcium phosphate transient transfection of the retroviral plasmids into PlatE cells.The supernatant was harvested at 48 hours and 72 hours and filtrated with a 0.45 um filter.MEF cells, MOLM13 and HL-60 were plated in a 6-well plate one day before the transduction.On the day of transduction, a fresh medium and retroviral supernatant was added.Polybrene was used at the final concentration of 4 µg/ml.Puromycin (0.5-1 µg/ml) was used for selection to generate stable cell lines and ONECUT3 expression was induced by the doxycycline (100 ng/ml).
Nucleotide sequence of the PGK-EFP-ONECUT3-pA with CCR5 homologous arms was annealed as ssODN by GeneScript Inc.Sequences and maps of the relevant parts were available in Supplemental Figure 2A.
acquired data were subjected to fitting using the BIAcore T200 analysis software, employing a 1:1 Langmuir binding model to ascertain kinetic constants such as the association rate constant, dissociation rate constant, and binding and dissociation equilibrium constants.

Drug Affinity Responsive Target Stability (DARTS) for target identification
The target identification of the small-molecule C5484617 were conducted using Drug Affinity Responsive Target Stability by Target Pharmaceutical (Shanghai) co., Ltd.The protocol is as reference (2) .293T cells were lysed in M-PER buffer with the addition of both protease inhibitors and phosphatase inhibitors.After chilled TNC buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM CaCl2) was added to the protein lysate, the protein concentration of the lysate was measured by the BCA Protein Assay kit (Pierce, 23227).The protein lysate was then incubated for 1 hours at room temperature with either vehicle control (DMSO) or 100 μM compound C5484617; materials were shaken at 600 rpm in an Eppendorf Thermomixer.Pronase digestions (1:200), which were performed for 30 min at room temperature, were stopped by adding SDS loading buffer and immediately heating at 70 °C for 10 min.Samples were both subjected to SDS-PAGE.
For the decolorization of the gel strip, the reduction and alkylation process involved the use of DTT and Iodoacetimide.Subsequently, a trypsin enzyme solution was introduced, and the mixture was kept at a temperature of 4°C for a duration of 60 minutes, followed by overnight digestion at 37°C.To extract the digested peptides, a blend of formic acid and acetonitrile was prepared and subjected to ultrasonic treatment at 37°C.The resulting mixture of peptides was then dried and reconstituted in a 0.1% formic acid (FA) solution.After vacuum drying, the sample was dissolved in FA once again, and an equal volume of the sample was extracted for mass spectrometry analysis using the Q-Exactive HF system (Thermo Scientific, USA).The sample was subjected to separation using the EASY-nLC 1200 system (Thermo Scientific, USA),

Total RNA extraction, reverse transcription, and qPCR assay
Total RNA was extracted using Trizol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen).
Then 1 μg of total RNA was reverse transcribed into cDNA using the SuperScript IV First-Strand Systhesis System (Invitrogen).Primers for qPCR are listed in Supplemental Table 14.Real-time PCR was conducted using the TB Green Premix Ex Taq (Takara-Bio) on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems).The relative mRNA expression level of each gene was calculated as 2-△△Ct to the internal reference (ACTIN).

Supplemental Figure 2 (
Figure 3A).C. Distribution map of Onecut3 binding loci in ChIP-seq.D. BETA suit found Onecut3 in Tp53-KO MEF had both activating and repressing functions, while it dominated to activate other genes.E. Top binding motif in Onecut3-specific peaks.F. The integrative assays of RNAseq and ChIP-seq were conducted by Binding and Expression Target Analysis (BETA).The top 10 predicted target genes are listed in the table.G.The enrichment of Incnep and Cdca8 was found upon Onecut3-OE via quantitative PCR analysis in Tp53-WT MEF.H.The correlation of the IHC intensity level (H-Score) of ONECUT3 and Aurora B, INCENP or Borealin/CDCA5 in MDS biopsy tissue.The black line represents the regression curve derived from Pearson correlation analysis.The shaded area in the plot indicates the confidence interval (95%).Statistical analysis was performed using a two-tailed paired Student t-test (G.) or Pearson correlation tests (H.); n. s. not significant, *P <0.05, **P <0.01.
analysis.B-C.The cell lysates were harvested after the treatment with or without 48h Doxycycline (100 ng ml−1) in control or Incenp-silenced Tp53-WT and Tp53-KO MEF and were then blotted against anti-Incenp antibodies (B.) or quantitated of mRNA level of Incenp and Onecut3 (C.).Loading control is Actin.D. Left: The representative co-immunostaining image against H3S10ph (green) and Tubulin (red) of control or Incenp-silenced Tp53-WT MEF with (red box) or without Onecut3-OE (green box); Right: The frequencies of unaligned chromosomes and multi-polar cells were quantified in control (Si-NC) or si-Incenp cells; n=3.E. Corresponding Tp53-KO part as D. Scale bar, 12.3 μm (D-E.);Statistical analysis was performed using a twotailed paired Student t-test (B and E); n. s. not significant, *P <0.05, **P <0.01, *** P <0.001, **** P <0.0001.Supplemental Figure 7 (Related to Figure 5) ONECUT3-overexpressing cells present multiple drug resistance, which could be mitigated by targeting to ONECUT3-CPC axis.A-B.Overexpressed Onecut3 in Tp53-KO MEFs were induced for 7-10 days, and the control cells (-Dox) were cultured simultaneously.The cells were later treated with the chemo-drugs for 48 hours, and the cell viability was tested by CellTiter-Luminescence.The drug sensitivity of the topoisomerase II inhibitor, Doxorubicin (left), the tubulin inhibitor Vincristine (middle), and the DNA methyltransferase inhibitor Decitabine (right) was indicated as IC50.B. Overexpressed Onecut3 in Tp53-WT MEFs was also induced in the same condition as above and treated with Doxorubicin (left), Vincristine (middle), and Decitabine (right).Supplemental Figure 8 (Related to Figure 5) ONECUT3-overexpressing cells present multiple drug resistance, which could be mitigated by targeting to ONECUT3-CPC axis. A. Schematic representation of ONECUT3 protein.The full length of ONECUT3 protein includes two domains: CUT (green) and HOX (Homeodomain, orange).The plasmids of truncated ONECUT3 with 3x Flag-tag were constructed.B-C.Truncated variants of FLAG-tagged ONECUT3 plasmids were transiently transfected in HEK293T cells.ChIP-qPCR assays were conducted using IgG and FLAG antibodies after 48 hours of Doxycycline induction.The graph shows the comparative analysis o of INCENP (B) and CDCA8 (C) enrichment in control (-Dox) and truncated-ONECUT3-OE (+Dox); n=3.D-E.qPCR and Western-blot were also conducted to assess the mRNA (D) and protein levels of CPC components (E).Supplemental Figure 9 (Related to Figure 5) ONECUT3-overexpressing cells present multiple drug resistance, which could be mitigated by targeting to ONECUT3-CPC axis.
B. The three-dimensional structure of the human ONECUT3 protein was modeled using the Alpha-Fold v2.0.C. Western blot analysis for recombinant ONECUT3 protein purified the inclusion body proteins using Ni-Smart affinity chromatography.Lane 1: inclusion body; lane 2: flow-through; lane 3-4: eluted sample; D. The target hits were obtained by LC-MS/MS data after Drug Affinity Responsive Target Stability (DARTS); n =2.E. Western blot analysis of ONECUT3 in ONECUT3-OE HEK293T cells treated with C5484617 for 48 hours; F. HEK293T cells were transiently transfected with control (MSCVvector), ONECUT3-OE (MSCV-ONECUT3-WT) and ONECUT3 point mutation (ONECUT3-D358A, ONECUT3-K364A, ONECUT3-K370A, and ONECUT3-R472A) constructs for a duration of 48 hours.Following this, ChIP-qPCR assays were performed using IgG and FLAG antibodies.after an additional 36-hour treatment with either Vehicle (DMSO) or 2.5 μM C5484617.The graph illustrates the comparative analysis of the enrichment of INCENP promoter in different treatment groups; N=3.Supplemental Figure 10 (Related to Figure 5) ONECUT3-overexpressing cells present multiple drug resistance, which could be mitigated by targeting to ONECUT3-CPC axis. A. The Overexpressed Onecut3 in Tp53-KO MEFs was induced for 7 days, and the cells were later treated with the above drugs for 48 h.Dose-response surface for Azacitidine plus C5484617 (lead compound targeting ONECUT3) and Azacitidine plus Barasertib was calculated by SynergyFinder 2.0.ZIP synergy score of Azacitidine plus C5484617: 11.925, ZIP synergy score of Azacitidine plus Barasertib: 15.276.B. The IC50 of Azacitidine in 'Azacitidine alone' group, in 'Azacitidine with 1μM C5484617' group; the green line indicates that the concentration of Azacitidine decreased after the combination with C5484617'.Supplemental Figure 11 (Related to Figure 5) ONECUT3-overexpressing cells present multiple drug resistance, which could be mitigated by targeting to ONECUT3-CPC axis. A. Left: The mRNA level heatmap of ONECUT3 was normalized in 15 newly diagnosed MDS patients before and after treatment with C5484617 and Azacitidine with C5484617.Right: Each data point represents the individual expression of ONECUT3 before and after treatment with C5484617 and Azacitidine with C5484617; the green line indicates that the level of ONECUT3 decreased after treatment.B. The representative data from the BM mononuclear cells of MDS patient following 48 hours of drug treatments (Azacitidine, C5484617, Azacitidine with C5484617): the quantification of clone numbers (upper), and the assessment of ONECUT3/ INCENP/CDCA8/AURKB/BIRC5 mRNA levels (lower).C. The representative images (left) and proportions (upper right) of morphological changes in five patients diagnosed with MDS following treatment with Azacitidine, C5484617, Azacitidine with C5484617 for 48 h; a schematic diagram illustrating the morphological changes associated with types A-D (lower right).Scale bar: 100 μm.
employing a C18 analytical column (1.9μm particle size, 75 μm×15 cm) at a flow rate of 300 nL/min.Subsequently, tandem mass spectrometry detection was performed in Data Dependent Acquisition (DDA) mode.on a BD FACS-Canto II Flow Cytometer (BD) and analyzed with FlowJo 10.3 (TreeStar).Wright-Giemsa stainingMEF cells were washed with PBS and harvested by trypsin.Place 0.3 M cells (in 100 µl medium per slide) in Cytospin 4 Centrifuge (Thermo Scientific, USA), centrifuge them at 500x rpm for 3 minutes, and dry them at room temperature for 20-30 minutes.The slides were then stained by Camco Stain Pak (Camco, USA) according to the manual.Briefly, fix them in Fixative Solution for 10 seconds; then dye in Solution I and II for 30 seconds and 20 seconds, respectively.Cell morphology was photographed in a 100x oil len by an ECLIPSE E400 microscope (Nikon, Japan).
was conducted by the Genomics, Epigenomics and Sequencing Core of the University of Cincinnati(Cincinnati, Ohio).NEBNext Ultra II Directional RNA Library Prep Kit was used for library preparation, and adapter trimmed reads were generated in fastq format.: AGATCGGAAGAGCGTCGTGT. RNA-seq was performed using the NextSeq 550 platform (Illumina, San Diego, CA).General bioinformatic analysis was performed via SEQUENCE HUB app RNA-Seq Alignment v2.0.2 followed by RNA-Seq Differential Expression version 1.0.1.The analysis used STAR for alignment and Salmon for quantification (Transcripts Per Million, TPM), followed by DESeq2 to identify differentially expressed genes.Afterward, the significantly regulated biological processes were identified by GO term analysis and Gene-set enrichment analysis (GSEA).Chromatin Immunoprecipitation sequencing (ChIP-seq) and ChIP-qPCR AssayMEF cells were crosslinked with 1% formaldehyde at room temperature for 10 minutes.1.5 M Glycine solution was used to terminate the fixation reaction.After washing with the iced PBS and homogenizing, cells were then lysed with SDS cell-lysis buffer (50 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 0.1% wt/vol SDS, and 1 mM PMSF) for 10 min on ice, and the chromatin was sonicated to 100-500bp in a Bioruptor (Diagenode).The size of the sheared chromatin was verified by agarose gel electrophoresis after reverse crosslinking.For immunoprecipitation, Dynabeads Protein G (Thermo, 10003D) was pre-washed was 0.5% BSA and conjugated to 5 μg of ChIP antibody or normal IgG at 4 °C for 4-6h.After washing with the RIPA/150mM NaCl washing buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% wt/vol sodium deoxycholate, 1% vol/vol Triton X-100, 0.1% wt/vol SDS and 1 mM PMSF), the conjugated beads were added to the chromatin slurry and incubated at 4 °C for 12 hours.Afterward, beads were washed twice with RIPA/150mM NaCl washing buffer, and twice with RIPA/500mM NaCl washing buffer (50 mM Tris pH 8.0, 500 mM NaCl, 1 mM EDTA pH 8.0, 1% wt/vol sodium deoxycholate, 1% vol/vol Triton X-100, 0.1% wt/vol SDS and 1 mM PMSF), four times LiCl washing buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA pH 8.0, 0.5% vol/vol NP-40 and 0.5% vol/vol Sodium Deoxycholate) and twice with TE buffer (10 mM Tris pH 8.0, 300 mM NaCl and 0.5 mM EDTA pH 8.0).The precipitated beads were diluted with ChIP elution buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA pH 8.0, 0.5% vol/vol NP-40, and 0.5% wt/vol SDS).One-fourth of the solution with beads was added with SDS protein lysis buffer for WB to verify the specificity of ChIP.The rest of the ChIP'd beads and the input were reverse crosslinking with 5 M NaCl solution at 65 °C overnight.The procedure for DNA recovery from immunoprecipitation is as follows.First, the above reversed crosslinking solution was mixed with 2x SDS lysis buffer (200 mM Tris pH 8.0, 1 M NaCl, 100mM EDTA pH 8.0, and 0.5% wt/vol SDS) with Proteinase K at 200x rpm for 15 minutes and