GLS1 controls AML survival by modulating the TCA cycle. (A) ShGLS1#5 was induced with doxycycline for 4 days in OCI-AML2, HL-60, MV4-11, MOLM-14, and OCI-AML3 leukemic cells, and apoptosis was evaluated based on Annexin-V binding. (B) CD34⁺ HPCs from 3 healthy donors were transfected with a lentiviral vector that expressed a noninducible GLS1 shRNA (#5), and transfected cells were selected with puromycin for 2 days; 3 days later, apoptosis was evaluated based on Annexin-V staining, and protein extracts were immunobloted with anti-GAC and anti-ACTIN antibodies. (C) MOLM-14 shGLS1 cells, MOLM-14 shCTR cells (9 mice each), and OCI-AML2 shGLS1 cells or OCI-AML2 shCTR cells (9 mice each) were intravenously injected into NSG mice (2 × 10⁶ cells per mouse). Mice were treated with doxycycline by oral gavage. The Kaplan-Meier survival curves of mice treated with doxycycline are shown for both cell lines. (D) The spleens of the mice injected with the OCI-AML2 shGLS1 and OCI-AML2 shCTR cell lines were measured. (E) A total of 11 cell lines were cultured for 4 days with or without CB-839 (1 µM), and apoptosis was evaluated based on Annexin-V staining. (F) OCI-AML2 and HL-60 cells were cultured for 4 days with or without CB-839 (1 µM), and dmαKG (5 mM) and apoptosis was evaluated based on Annexin-V staining. (G) OCI-AML2 cells that were stably infected with GAC^WT or GAC^K320A were cultured for 2 days with or without doxycycline or CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. (H) AML samples from 10 patients and CD34⁺ HPCs from 6 healthy donors were cultured for 4 days with or without CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. *P < .05, **P < .01, ***P < .001.