CerS1-C₁₈-ceramide–mediated lethal mitophagy is required for FLT3-targeted therapy in the AML mouse model and human AML blasts. (A-B) FACS analysis to measure the percentage of injected AML cells (hCD45⁺ cells) in the bone marrow. Values indicate mean ± SD of n = 6 mice. P values were generated using ANOVA. (C) qPCR to measure CerS1 mRNA in sorted hCD45⁺ cells. (D) Western blot to measure CerS1, ACO2, and LC3B in sorted hCD45⁺ cells. (E) HPLC-MS-MS measuring C₁₈-ceramide in sorted hCD45⁺ cells. Values indicate mean ± SD of n = 3 mice. P values were generated using the Student t test. (F) EM visualization of sorted hCD45⁺ cells from bone marrow of NSG mice with AML xenografts. (G) CD34⁺ bone marrow blasts obtained from FLT3-ITD⁺ patients were treated with 5 μM crenolanib for 24 hours, followed by western blot to detect p-Drp1 S637, CerS1, ACO2, and LC3B. (H) EM visualization of crenolanib-treated FLT3⁺ AML patient blasts labeled with ceramide antibody. White arrows indicate ceramide gold label in mitochondria and black arrows indicate autophagosomes. (I) Percentage of viability of AML patient blasts pretreated with either FB-1, bafilomycin, or mdivi1, followed by 5 μM crenolanib treatment of 24 hours. *P value of <.05 using the 2-sided Student t test.