TKI treatment leads to metabolic reprogramming and selection of CML progenitor subpopulations. (A) Overview of the SCENITH assay performed using the BM of CML mice treated with vehicle or nilotinib (TKI) for 2 days and 2 weeks. The SCENITH assay uses flow cytometry to measure changes in the level of translation in response to inhibitors as a measurement for cellular metabolism. BM cells were divided and separately treated with 2-deoxyglucose (DG), oligomycin (O), and DG + O, together with controls (Cos) incubated without inhibitors, and labeled with puromycin (Puro). After cell surface labeling and intracellular labeling for Puro, different subpopulations were analyzed via flow cytometry for Puro-MFI response to the various inhibitors. Calculations of metabolic dependencies and capacities based on Puro-MFI are shown. (B-C) GMP, common myeloid progenitor (CMP), MEP, and LSK cell frequency within CML c-Kit⁺ cells (n = 5-6), as assessed via flow cytometry, after treatment with TKI for 2 days (B) or 2 weeks (C). (D-E) Protein synthesis (Puro-MFI) within committed progenitors (GMPs, CMPs, and MEPs) after 2 days (D) and 14 days (E) of TKI treatment. (F-I) Mitochondrial dependence and glycolytic capacity within GMPs, CMPs, and MEPs after 2 days (F,H) and 14 days (G,I) of TKI treatment. Significance values: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. Results represent mean ± SEM of multiple replicates. AAO, amino acid oxidation; MFI, mean fluorescence intensity.