Functional characterization of recurrent somatic alterations in NUP98r leukemia. (A) Experimental schema of induction of cooperating alterations (RB1, WT1) in cbCD34/Cas9 models. (B) Cell growth assays of cbCD34/Cas9 NUP98::KDM5A with gRNAs targeting the AAVS, RB1, or WT1 loci (left), and cytospin of cells on day 35 (right; scale bars, 50 μm). (C) Rates of indel (insertions and deletions) at days 4 and 39 in each condition. Bars represent fractions of indel rates in all target sequence reads, and dots represent the out-of-frame indel ratio among total indels. (D) Flow gating (left), CD34+ CD41a+ positivity (middle), and CD34+ CD41a– (right) among mCherry+ GFP+ mAmetrine+ live cells. (E) Principal component analysis of RNAseq of gRNA-transduced NUP98::KDM5A cbCD34 models at day 35. (F) Differentially expressed genes analysis between AAVS controls and RB1-gRNA conditions (left), and gene ontology term analysis of differentially expressed genes (right). Colors indicate differentially expressed genes and gene ontology terms (red: high in RB1-gRNA conditions, blue: low in RB1-gRNA conditions). (G) Differentially expressed genes analysis between AAVS controls and WT1-gRNA conditions as shown in panel F. (H) UMAP plots of scRNAseq data from gRNA-transduced NUP98::KDM5A cbCD34 models at day 35, showing marker gene expression (left), annotated clusters (middle), and cell distributions among conditions (right). Colors in plots indicate relative expression levels, clusters, and cell density, respectively. (I) Enrichment of cells with each cluster indicated by colors and sizes. (J) Pseudotime along myeloid (HSC → GMP → monocytes) and platelet (HSC → MEP → MK → platelet) trajectories. Colors represent pseudotime scores of each single cell inferred by Slingshot. (K) RB1 (top) and CDKN2A (bottom) expression along the pseudotime axis in each condition, with red curves showing average expressions. (L) Differentially expressed genes analysis between the platelet-like and MK-like clusters in the AAVS-control condition (left) and gene ontology term analysis (right) of genes high in the platelet-like cluster (red) and the MK-like cluster (blue). (M) Schematics illustrating platelet differentiation in normal hematopoiesis and NUP98::KDM5A models (created in BioRender. Umeda, M. (2025) https://BioRender.com/kreq8kl). Assay data were obtained in technical triplicates from an established NUP98::KDM5A/Cas9 line and independent experiments. One data point in panel C was not obtained due to technical errors. RNAseq data were obtained from 6 independent experiments. In panels B-D, statistical tests were performed by linear mixed effect model (B) or 2-sided Student t test by comparing day 4 and day 39 (C) or gRNA conditions and AAVS controls (D), and limma (F-G), followed by the Benjamini-Hochberg adjustment when applicable. Differentially expressed genes in scRNAseq (L) were identified using the FindMarker function in the Seurat package with default settings, which calculates adjusted P values with limma implementation of the Wilcoxon rank-sum test followed by Bonferroni correction. Asterisks indicating P values or adjusted P values <.05. Error bars indicate mean ± standard error of the mean. HSC, hematopoietic stem cell; UMAP, uniform manifold approximation and projection.

Functional characterization of recurrent somatic alterations in NUP98r leukemia. (A) Experimental schema of induction of cooperating alterations (RB1, WT1) in cbCD34/Cas9 models. (B) Cell growth assays of cbCD34/Cas9 NUP98::KDM5A with gRNAs targeting the AAVS, RB1, or WT1 loci (left), and cytospin of cells on day 35 (right; scale bars, 50 μm). (C) Rates of indel (insertions and deletions) at days 4 and 39 in each condition. Bars represent fractions of indel rates in all target sequence reads, and dots represent the out-of-frame indel ratio among total indels. (D) Flow gating (left), CD34+ CD41a+ positivity (middle), and CD34+ CD41a– (right) among mCherry+ GFP+ mAmetrine+ live cells. (E) Principal component analysis of RNAseq of gRNA-transduced NUP98::KDM5A cbCD34 models at day 35. (F) Differentially expressed genes analysis between AAVS controls and RB1-gRNA conditions (left), and gene ontology term analysis of differentially expressed genes (right). Colors indicate differentially expressed genes and gene ontology terms (red: high in RB1-gRNA conditions, blue: low in RB1-gRNA conditions). (G) Differentially expressed genes analysis between AAVS controls and WT1-gRNA conditions as shown in panel F. (H) UMAP plots of scRNAseq data from gRNA-transduced NUP98::KDM5A cbCD34 models at day 35, showing marker gene expression (left), annotated clusters (middle), and cell distributions among conditions (right). Colors in plots indicate relative expression levels, clusters, and cell density, respectively. (I) Enrichment of cells with each cluster indicated by colors and sizes. (J) Pseudotime along myeloid (HSC → GMP → monocytes) and platelet (HSC → MEP → MK → platelet) trajectories. Colors represent pseudotime scores of each single cell inferred by Slingshot. (K) RB1 (top) and CDKN2A (bottom) expression along the pseudotime axis in each condition, with red curves showing average expressions. (L) Differentially expressed genes analysis between the platelet-like and MK-like clusters in the AAVS-control condition (left) and gene ontology term analysis (right) of genes high in the platelet-like cluster (red) and the MK-like cluster (blue). (M) Schematics illustrating platelet differentiation in normal hematopoiesis and NUP98::KDM5A models (created in BioRender. Umeda, M. (2025) https://BioRender.com/kreq8kl). Assay data were obtained in technical triplicates from an established NUP98::KDM5A/Cas9 line and independent experiments. One data point in panel C was not obtained due to technical errors. RNAseq data were obtained from 6 independent experiments. In panels B-D, statistical tests were performed by linear mixed effect model (B) or 2-sided Student t test by comparing day 4 and day 39 (C) or gRNA conditions and AAVS controls (D), and limma (F-G), followed by the Benjamini-Hochberg adjustment when applicable. Differentially expressed genes in scRNAseq (L) were identified using the FindMarker function in the Seurat package with default settings, which calculates adjusted P values with limma implementation of the Wilcoxon rank-sum test followed by Bonferroni correction. Asterisks indicating P values or adjusted P values <.05. Error bars indicate mean ± standard error of the mean. HSC, hematopoietic stem cell; UMAP, uniform manifold approximation and projection.

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