GLS1 inhibition induces mitochondrial apoptosis and sensitizes leukemic cells to priming with ABT-199. (A) OCI-AML2 WT cells or cells transfected with shGLS1#5 were cultured with or without CB-839 (1 µM) or doxycycline as indicated. Apoptosis was evaluated by western blotting using anti-CASPASE-8, cleaved CASPASE-3, anti-PARP, and anti-ACTIN antibodies. (B) OCI-AML2 cells were cultured with or without CB-839 (1 µM) and with or without a CASPASE-8 inhibitor (IETD) or a pan-caspase inhibitor (QVD). Protein extracts were immunoblotted using anti-CASPASE-8, anti-cleaved CASPASE-3, and anti-ACTIN antibodies. Mitochondrial depolarization was evaluated using TMRE staining and apoptosis was quantified based on Annexin V staining. (C) OCI-AML2 cells were cultured for 1 day with or without the indicated doses of CB-839 and ABT-199; apoptosis was evaluated based on Annexin V staining. (D) MOLM-14 cells were cultured for 1 day with or without the indicated doses of CB-839 and ABT-199; apoptosis was evaluated based on Annexin-V staining. Histograms show data that are representative of 3 independent experiments. The response of the combination was compared with its single agents against the widely used Loewe model for drug-with-itself dose additivity using Chalice software²⁶  and presented as an isobologram. *P < .05, **P < .01, ***P < .001.