Projection of KMT2A-r leukemia cells to normal hematopoietic trajectory reveals larger intratumor heterogeneity in younger patients. (A) UMAPs based on scRNA-Seq (top panel) and scATAC-Seq data from healthy pediatric donors (bottom panel). scATAC-Seq includes the dataset generated in this study and previously published datasets.⁴⁹ Cell type annotation for scATAC-Seq data were transferred from scRNA-Seq data using Seurat. Total numbers of sequenced cells are indicated on top. (B) Violin plots of marker gene expression used for defining the hematopoietic cell populations. (C) Projection of patient cells onto normal hematopoietic trajectories. Left panel, representative projection of patient 1154 scRNA-Seq data. Right panel, representative projection of patient 1154 scATAC-Seq data. Gray dots, cells from healthy donors; colored dots, patient cells. (D) Composition of CD19⁺ leukemic blasts in different hematopoietic lineages based on projected scRNA-Seq data (left bar) and scATAC-Seq data (right bar). M-lineage, myeloid lineage. (E) Frequency of B-lineage blasts from 18 infant KMT2A-r patients stratified along the normal hematopoietic trajectory (pseudotime). B-lineage pseudotime from HSC/MPPs to mature B cells is ordered into 20 bins. Upper heatmap shows the frequency of each bin from all 18 infant KMT2A-r patients. Lower line plot shows the frequency of each developmental stage along the pseudotime trajectory using healthy donor data. (F) UMAP shows coembedded snmC-Seq2 and scATAC-Seq data. Total numbers of sequenced cells of each modality are indicated. (G) Blasts from younger patients (<6 months old) show higher developmental heterogeneity based on scRNA-Seq, scATAC-Seq, and snmC-Seq2 data, respectively. Heterogeneity was quantified using Shannon’s entropy. P values are based on Student t test. (H) Fractions of cells aberrantly coexpressing B (VPREB1, IGLL1, CD79A, and CD19) and myeloid (MPO, CEBPA, and CD33) lineage antigens in younger and older infant patients. P value was computed using a one-sided binomial test.