STAT3 promotes glutaminolysis in LSCs via MYC’s regulation of the transporter SLC1A5. (A) Cartoon depiction of experimental design. LSCs are isolated from primary human AML cells and then treated with 5 μM of STAT3i vs vehicle control for 3 hours. Stable-isotope labeled ¹³C₅,¹⁵N₂-L-Glutamine is then added to the media, and cells are collected at 1, 8, and 16 hours and analyzed by mass spectrometry. (B) Tracing experiments showing changes in intracellular glutamine, glutamate, and glutathione in LSCs after 4 hours of treatment with 5 μM of STAT3i compared with vehicle control (n = 4). (C) Cartoon depiction of hypothesized pathway by which STAT3 regulates glutaminolysis. (D) Data from BloodSpot showing SLC1A5 gene expression in AML cells compared with HSCs. CBF, core-binding factor; Complex, complex cytogenetics. (E) Western blot showing protein levels of SLC1A5 in LSCs after a 4-hour incubation period with 5 μM of STAT3i compared with vehicle control. (F) Western blot showing protein levels of SLC1A5 after knocking down MYC in a human primary AML patient sample. (G) Western blot showing SLC1A5 protein levels after 48 hours of siRNA knockdown compared with scramble control. (H) Heat map of steady-state metabolomics in primary AML cells treated with siSCR versus siSLC1A5. (I) Seahorse assay showing OCR after 40 minutes of 5 μM of STAT3i-treated LSCs in the presence or absence of media supplemented with 10× glutamine. These conditions were then recapitulated in the presence of a plasma membrane permeabilizer (n = 4). Statistical analyses were performed using the Student t-test. P values are represented as follows: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.