Multimodal single-cell sequencing reveals a severely altered leukemic BM. (A) Schematic illustration of multiomic profiling of BCP-ALL (n = 23) and NBM (n = 9) samples. Illustration created with BioRender.com. (B) UMAP visualization of scRNA-seq gene expression data representing 188 546 cells from the BCP-ALL and NBM samples. Each cell is colored according to the cell type. (C) UMAP visualization of scATAC-seq data representing 41 892 nuclei from the BCP-ALL and NBM samples. (D) Cell composition based on scRNA-seq data in DUX4-r, BCR::ABL1⁺, ETV6::RUNX1⁺, and HeH ALL samples, as well as mononuclear cells (MNC), CD34⁺-enriched cells, and CD19⁺-enriched cells from NBM samples. ALL blast cells, regardless of subtype, are marked in gray. (E) Protein expression by scADT-seq of CD10, CD19, CD22, CD34, CD20, and CD45 on BCP-ALL blast cells and nonleukemic cells from BCP-ALL and NBM samples, visualized using ridge plots. (F) TF footprinting of the DUX4-binding motif shows that DUX4 is exclusively active in DUX4-r blast cells (marked in purple). The annotation “all other cell types” denotes all other detected cell types using the color legend from C. (G) UMAP visualization of T cells extracted from the larger scRNA-seq data set colored by sample type (upper) and predicted cell type (lower) showing a specific cell cluster of an accumulation of CD4⁺ T cells exclusively from DUX4-r ALL. (H) Gene set enrichment scores using defined gene sets from dysfunctional CD4⁺ T cells in cancer by Li et al (left) and Zheng et al (right).^31,32 The gene set enrichment scores are based on differentially expressed genes between memory CD4⁺ T cells in DUX4-r ALL and NBM-MNC. GMP, granulocyte-macrophage progenitors; LMPP, lympho-myeloid primed progenitor; NK, natural killer; scBCR-seq, single-cell BCR sequencing; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.