(A) Schematic illustration of paired RNA-Seq to find the potential transcription regulator for a CK. (i) RNA-Seq was performed on RNA samples from 5 paired patients with MDS with a CK or a NK. Each sample was matched for sex, age, and BM blasts (Supplemental Table 1). (ii) Volcano map showed the differential expression genes. Blue dots represent downregulated genes in the CK versus the NK (P < 0.05); orange dots represent upregulated genes (P < 0.05, log₂ fold change [FC] <4.7 in CK); and red dots indicate the top 100 upregulated genes (P < 0.05, log₂ FC >4.7) in Supplemental Table 2. (iii) Functional enrichment analysis of the top 100 upregulated genes by Metascape (metascape.org). (iv) Heatmap visualization of the mRNA abundance (normalized to fragments per kilobase per million mapped reads [FPKM]) for 6 TFs among the top 100 genes. (v) Only ONECUT3 was expressed at relatively low levels in the BM samples (see Supplemental Figure 1, A and B). (B–D) Quantitative real-time PCR was performed to measure mRNA levels of ONECUT3 in 165 MDS patients harboring WT TP53 with different cytogenetic aberrations and 31 volunteers. The mean value ± 1 SD for the healthy donor was used as a cutoff. (B) Kaplan-Meier survival curves showing the OS of patients with low or high expression of ONECUT3. (C) The forest plot was generated based on the multivariable analysis of factors associated with OS. (D) Each data point represents ONECUT3 expression for an individual patient. RQ, relative quantification. (E) Western blot analysis was applied to detect endogenous ONECUT3 expression using a homemade antibody (Supplemental Figure 1, C and D). A positive control (Ctrl) was achieved by transfecting HEK293T cells with pcDNA3.1-hONECUT3 for 48 hours. The lysate was from the BM mononuclear cells of MDS patients with a CK (P1 and P2), a NK (P3 and P4), and 2 volunteer donors of HSPCs (D1 and D2). (F) ONECUT3 MFI detected by flow cytometry (see gating strategy in Supplemental Figure 1E) in CD34⁺CD38^– (left panel) and CD34⁺CD38⁺ (right panel) of BM mononuclear cells from volunteer donors of HSPCs (n = 14), MDS patients with a NK (n = 8), and MDS patients with an aberrant karyotype (1–2 cytogenetic aberrations, n = 8; CK, n = 6). Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple-comparison test (D and F). *P < 0.05, **P < 0.01, and ***P < 0.001, and ****P < 0.0001.