Enrichment of primitive CML stem cell subpopulations after TKI treatment. LSK cells were isolated from the BM of CML mice treated with vehicle or nilotinib (Nil) for 2 days or 2 weeks, and scRNA sequencing was performed using the 10x Genomics platform. Cluster identification was based on gene profiles. Clusters are color coded. (A) Uniform manifold approximation and projection (uMAP) display of scRNA sequencing from CML LSK cells treated with vehicle (n = 42 238 single cells, 4 samples), nilotinib for 2 days (n = 15 911 single cells; 2 samples), or nilotinib for 2 weeks (n = 11 449 single cells; 2 samples). (B) The percent of indicated clusters within CML LSK after treatment with vehicle, NIL for 2 days, or NIL for 2 weeks. (C) Gene set enrichment analysis subpopulations of gene sets (FDR < 0.05) comparing TKI-persistent with TKI-depleted clusters. Net enrichment score (NES), and statistical significance (FDR) are represented by color and size, respectively. (D-G) The SCENITH assay was performed on BM cells from CML mice treated with vehicle or NIL (TKI) for 2 days and 14 days (n = 5-6). (D) LTHSC, short-term HSC (STHSC), MPP-GM, and MPP-MKE frequency within CML LSK cells, assessed via flow cytometry, after treatment with TKI for 2 days (left) and 14 days (right). (E) Protein synthesis (Puro-MFI) within LSK subpopulations after 2 days (top) and 14 days (bottom) of TKI treatment. (F-G) Glucose dependence (F) and FAO/AAO capacity (G) within LSK subpopulations after 2 days (top) and 14 days (bottom) of TKI treatment. Significance values: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. Results represent mean ± SEM of multiple replicates. FDR, false discovery rate; MK, megakaryocytic progenitors; TNF, tumor necrosis factor.