Evi1^high cells show distinct features in murine AML models. (A) Uniform manifold approximation and projection (UMAP) plot of single-cell RNA-seq (scRNA-seq) data of AML cells from patient AML328 with inv(3)(q21.3q26.2), showing 9 clusters. (B) Violin plot of MECOM expression in the 9 clusters. (C) The scheme of the experimental model of EVI1-GFP KMT2A-MLLT1 AML mice. (D) Frequency of GFP⁺ cells in the whole live KuO⁺ AML cells and L-GMPs from the BM of EVI1-GFP KMT2A-MLLT1 AML mice. (E) qPCR showing the relative expression of Evi1 in GFP^high and GFP^low AML cells compared with normal LSKs. (F) Colony-forming units of GFP^high and GFP^low L-GMPs from 3 independent AML mice. (G-I) A Kaplan-Meier survival curve for secondary recipient mice that underwent transplantation with an indicated number of GFP^high or GFP^low L-GMPs, after exposure to 6.5 Gy total body irradiation (TBI). Significance between the same number of cells was examined by a log-rank test. (J) Frequency of GFP⁺ cells in L-GMPs in secondary recipient mice intravenously treated with vehicle (phosphate-buffered saline ) or cytarabine (AraC; 100 mg/kg) for 5 days through days 15 and 19 after transplant. Mice were analyzed on day 19. (K) Top-ranked differentially expressed genes between GFP^high and GFP^low L-GMPs. (L) Top-ranked pathways enriched in GFP^high L-GMPs. (M-N) Gene set enrichment analysis (GSEA) showing that downregulated genes in leukemia stem cells (M) and multiple drug-resistant–related genes (N) are upregulated in GFP^high L-GMPs. (O) Colony-forming units of 200 KMT2A-MLLT1 AML cells with or without exogenous EVI1 expression from 3 independent primary recipients (supplemental Figure 1K). Mean ± standard deviation (SD). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. FDR, false discovery rate (q value); NES, normalized enrichment score.