Fig. 6: Targeting the WDR5-NANOG axis suppresses the self-renewal of AML1-ETO9a (AE9a) leukemia stem cells.

a mRNA expression levels of Nanog and Wdr5 in WT and AE9a hematopoietic stem and progenitor cells (HSPCs). Data are mean ± SD from three independent replicates. *P < 0.05, ****P < 0.0001 by unpaired two-sided t-test. b Representative images and quantification of colony-forming units (CFUs) generated by WT and AE9a c-Kit⁺ cells cultured in methylcellulose following treatment with DMSO (Mock), C16 (2 μM), or Revumenib (2 μM). Data are mean ± SD from three independent replicates. ns: P > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001 by unpaired two-sided t-test. Scale bars, 2 mm. c Flow cytometry analysis (FACS) of stem cell populations in AE9a cells treated with DMSO (Mock), C16 (2 μM), or Revumenib (2 μM). Data are mean ± SD from three independent replicates. ns: P > 0.05, **P < 0.01, ***P < 0.001 by unpaired two-sided t-test. d Volcano plots of differentially expressed genes (DEGs) in C16-treated AE9a and WT c-Kit⁺ cells compared to DMSO controls, analyzed with DESeq2. P values were calculated using the two-sided Wald test and adjusted by Benjamini-Hochberg method (FDR). Dashed lines indicate the thresholds |log₂[fold change]| ≥1 and FDR < 0.05. RNA-seq was performed using three biological replicates. e Venn diagram showing the overlap of downregulated genes between C16-treated AE9a and WT c-Kit⁺ cells. f GO enrichment analysis of downregulated and upregulated genes in C16-treated AE9a cells relative to DMSO-treated controls, analyzed with DAVID. Downregulated and upregulated gene sets were analyzed separately using a one-sided hypergeometric test, and P values were adjusted with the Benjamini-Hochberg method (FDR). Dot size indicates gene number and color indicates -log₁₀ (adjusted P value). g Schematic of the C16 in vivo inhibition assay. h FACS analysis of stem cell populations in AE9a recipient mice following 21-day treatment with C16 or Revumenib. Data represent mean ± SEM (n = 7 mice). ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired two-sided t-test. Source data are provided as a Source Data file. Figure 6b, g: Created in BioRender. Shi, Y. (2026) https://BioRender.com/h76z570.