Coculture of AML with adipocytes activates β-oxidation in AML cells. (A) Primary AML blasts or nonmalignant CD34⁺ cells were cultured on adipocytes for 3 days and then starved for 4 hours before measuring OCR (pMoles/min) using the Seahorse XFp Analyzer, at baseline and then after injection of palmitate (18 minutes) and ETX (36 minutes). Circles represent ETX (40 μM) treatment, and squares represent no ETX treatment. (B) Primary AML blasts or nonmalignant CD34⁺ cells were cultured alone for 3 days, and AML blasts cultured on adipocytes for 3 days and then starved for 4 hours. AML blasts were then treated with ETX (40 μM), and OCR was measured as above (n = 4). Data are represented as mean ± standard deviation. (C) Primary AML blasts cultured alone for 3 days and AML blasts cultured on adipocytes or BMSC for 3 days and then starved for 4 hours. Data are represented as mean ± standard deviation. (D) AML were infected with FABP4-targeted shRNA or control shRNA lentivirus, and after 72 hours, incubated with adipocytes or BMSC for 24 hours, and OCR was measured in the AML (n = 4). Data are represented as mean ± standard deviation. (E) Primary AML blasts were in monoculture or cocultured on adipocytes or BMSC with and without treatment with ETX for 72 hours. AML blasts were counted using flow cytometry and Trypan blue exclusion (n = 4). Data are represented as mean ± standard deviation. (F) AML blasts were infected with CPT1A shRNA or control shRNA lentivirus, and after 72 hours, analyzed for CPT1A mRNA and protein expression using RT-PCR and western blotting. Blots were reprobed for β-actin to show equal sample loading. Data are represented as mean ± standard deviation. (G) AML were infected with CPT1A shRNA or control shRNA lentivirus and then cocultured on BMSC or adipocytes. AML blasts were counted using flow cytometry and Trypan blue exclusion (n = 4). Data are represented as mean ± standard deviation. (H) Two primary AML samples were infected with CPT1A shRNA or control shRNA lentivirus, and after 96 hours, were then grown on adipocytes for 48 hours; subsequently, 2 × 10⁶ primary AML cells (n = 4) were IV injected into NSG mice. Survival of the NSG mice is represented by a Kaplan-Meier plot. P = .025 for mice injected with AML CPT1-KD compared with AML control-KD mice. *P < .05.