American Society of Hematology

Figure 3.

$Identification and functional validation of LSC transcriptional programs. (A) HSCs were subclustered in a BCR::ABL1 GE feature space, as described in supplemental Figure 6A and in the supplemental Methods. (A, left) UMAP feature plots of HSCs colored by the presence or absence of the BCR::ABL1 GE signature. The dotted line demarcates the area that was identified as positive for the BCR::ABL1 GE signature. These cells are referred to as the BCR::ABL1+ stem cells or LSCs. (A, right) UMAP feature plots of HSCs colored by group label. The zoomed-in inset shows stem cells from CML samples clustering with normal stem cells from controls. These cells are referred to as BCR::ABL1− stem cells. (B) BCR::ABL1 fusion reads were amplified from 10× barcoded full-length complementary DNA using the universal primer from the 10× Genomics 5′ GE kit as the forward primer (FP) and a primer spanning exon 3 of ABL1 gene as the reverse primer (RP) from 4 CML and 2 NBM CD34+ HSPC fractions, and products were subjected to long-read sequencing (supplemental Methods). GE signature-based assignment of BCR::ABL1 status was correlated with the direct detection of BCR::ABL1 junction-spanning reads. Fisher exact test, 7.5e−11. (C) HSCs from controls and LSCs from patients with CML were analyzed using SCENIC. Scores for regulons that showed significant differential activity (P value < .05) between any pair of comparisons are shown. Up to 2 regulons are identified for each transcription factor (TF). (D) Patient-specific pseudobulk regulon scores for selected TFs are plotted. Each dot represents 1 patient (E) GSEA comparisons between LSCs from groups A and C for the selected gene sets. (For a complete analysis, refer to supplemental Tables 19 and 20; supplemental Figure 7.) (F) scRNA-seq UMAP, colored by fold enrichment of group A cells among 300 neighboring cells in transcriptome space. Enriched areas demarcated by the dotted line correspond to ERPs in the CML single-cell atlas. (G) scRNA-seq; ERP abundance is plotted as a proportion of total HSPCs in each patient. Each dot represents 1 patient. (H) CFU-E/CFU-GM ratio: CD34+ HSPCs were purified from all 3 patient groups and subjected to colony-forming assay (CFA). Colonies were scored after 14 days as erythroid (CFU-E) or myeloid (CFU-GM). E/GM ratio is plotted for each patient (n = 6 group A; n = 4 group B; and n = 4 group C). (I) CD34+ cells were purified from all groups, and subjected to CFAs in the presence or absence of imatinib. The surviving fraction with respect to dimethyl sulfoxide controls is plotted. (J) Purified CD34+ cells were plated in methylcellulose-based media in the presence/absence of imatinib/IFN-γ. Colony numbers for CFU-E were plotted relative to vehicle (dimethyl sulfoxide) control. The data for total colony numbers and for CFU-GM are shown in supplemental Figure 7E. Statistical tests: Figure 3D,F-I; 2-tailed t test. Supplemental Figures 6 and 7 and supplemental Tables 17-20 are linked with data shown in Figure 3. CFU-E, colony forming unit-erythroid; CFU-GM, colony forming unit-granulocyte-macrophage.$

Identification and functional validation of LSC transcriptional programs. (A) HSCs were subclustered in a BCR::ABL1 GE feature space, as described in supplemental Figure 6A and in the supplemental Methods. (A, left) UMAP feature plots of HSCs colored by the presence or absence of the BCR::ABL1 GE signature. The dotted line demarcates the area that was identified as positive for the BCR::ABL1 GE signature. These cells are referred to as the BCR::ABL1⁺ stem cells or LSCs. (A, right) UMAP feature plots of HSCs colored by group label. The zoomed-in inset shows stem cells from CML samples clustering with normal stem cells from controls. These cells are referred to as BCR::ABL1⁻ stem cells. (B) BCR::ABL1 fusion reads were amplified from 10× barcoded full-length complementary DNA using the universal primer from the 10× Genomics 5′ GE kit as the forward primer (FP) and a primer spanning exon 3 of ABL1 gene as the reverse primer (RP) from 4 CML and 2 NBM CD34⁺ HSPC fractions, and products were subjected to long-read sequencing (supplemental Methods). GE signature-based assignment of BCR::ABL1 status was correlated with the direct detection of BCR::ABL1 junction-spanning reads. Fisher exact test, 7.5e−11. (C) HSCs from controls and LSCs from patients with CML were analyzed using SCENIC. Scores for regulons that showed significant differential activity (P value < .05) between any pair of comparisons are shown. Up to 2 regulons are identified for each transcription factor (TF). (D) Patient-specific pseudobulk regulon scores for selected TFs are plotted. Each dot represents 1 patient (E) GSEA comparisons between LSCs from groups A and C for the selected gene sets. (For a complete analysis, refer to supplemental Tables 19 and 20; supplemental Figure 7.) (F) scRNA-seq UMAP, colored by fold enrichment of group A cells among 300 neighboring cells in transcriptome space. Enriched areas demarcated by the dotted line correspond to ERPs in the CML single-cell atlas. (G) scRNA-seq; ERP abundance is plotted as a proportion of total HSPCs in each patient. Each dot represents 1 patient. (H) CFU-E/CFU-GM ratio: CD34⁺ HSPCs were purified from all 3 patient groups and subjected to colony-forming assay (CFA). Colonies were scored after 14 days as erythroid (CFU-E) or myeloid (CFU-GM). E/GM ratio is plotted for each patient (n = 6 group A; n = 4 group B; and n = 4 group C). (I) CD34⁺ cells were purified from all groups, and subjected to CFAs in the presence or absence of imatinib. The surviving fraction with respect to dimethyl sulfoxide controls is plotted. (J) Purified CD34⁺ cells were plated in methylcellulose-based media in the presence/absence of imatinib/IFN-γ. Colony numbers for CFU-E were plotted relative to vehicle (dimethyl sulfoxide) control. The data for total colony numbers and for CFU-GM are shown in supplemental Figure 7E. Statistical tests: Figure 3D,F-I; 2-tailed t test. Supplemental Figures 6 and 7 and supplemental Tables 17-20 are linked with data shown in Figure 3. CFU-E, colony forming unit-erythroid; CFU-GM, colony forming unit-granulocyte-macrophage.

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