Combination treatment with SLC-391 and venetoclax synergistically inhibits proliferation and long-term clonogenic activities of cells from patients with AML in vitro. (A) CD34⁺ cells from AML patients were treated with DMSO (0.05%), SLC-391 (SLC; 0.25 µM), or venetoclax (VEN; 0.2 µM), alone or in combination for 72 hours, and absolute viable cells were counted (n = 18). Each point represents an individual patient sample with indicated symbols. (B) Quantification of Annexin V⁺ (apoptotic) cells after performing Annexin V/propidium iodide (PI) staining and flow cytometry on cells from panel A (n = 16). (C) Total CFCs counted 2 weeks after plating 1200 CD34⁺ AML cells (n = 9) in semisolid media containing the indicated drugs. (D) Colonies harvested from panel C were replated in semisolid media for another 2 weeks and counted (n = 4). (E) CFC outputs from CD34⁺ AML cells (n = 3) maintained in 6-week LTC-IC assays to which the indicated drugs were added for the first 2 weeks. (F) Cells from patients with AML were treated with VEN, alone or in combination with SLC (0.25 or 0.35 µM) for 72 hours, and viable cells were counted. IC₅₀ values were calculated based on drug concentrations that result in 50% cell viability compared with DMSO-treated control cells. The boxplot shows IC₅₀ relative to VEN alone (which equates to 100%) per sample. The corresponding raw IC₅₀ values are shown in supplemental Figure 17. Patients with AML with monocytic features or FAB-M5 subtype are denoted with an apostrophe (‘). (G) Colonies obtained from CD34⁺ NBM cells (n = 4) 2 weeks after plating in semisolid media containing the indicated drugs. (H) Colonies harvested from panel G were replated in semisolid media for another 2 weeks and counted (n = 3). (I) Distributions of colony types, including GEMM (granulocytes, erythrocytes, monocytes, and megakaryocytes), GM (granulocyte and macrophage), and BFU-E (burst-forming unit-erythroid) under each treatment condition. (J) Western blot analysis of AXL protein levels in AML cells transduced with LentiCRISPRv2/GFP or AXL-knockdown (KD) constructs and sorted for GFP⁺ cells 7 days posttransduction. (K) Cells from patients with AML were transfected with AXL-KD construct (KD1 and KD2), sorted for GFP⁺ cells, and treated with SLC (0.25 µM) and VEN (0.2 µM), alone or combination, for 72 hours; viable cells were counted (n = 4, patients #2, #3, #6, and #14). (L) CFC outputs from CD34⁺GFP⁺ AXL-KD cells from panel K compared with untreated cells and cells transfected with control construct and treated with the indicated drugs. (M) Western blotting analysis of AXL in THP-1 cells transfected with CRISPR control or with 2 AXL-KD constructs (KD1 and KD2). (N) THP-1 cells in panel M were treated with the indicated drugs for 72 hours, and viable cells were counted (n = 2). (O) The same treated cells as in panel N were subjected to Annexin V/PI staining and flow cytometry analysis. Annexin V⁺ cells were graphed as apoptotic cells (n = 2). All data are presented as mean ± SD of measurements for the indicated number of patients or samples. The P values were calculated using a 2-tailed, paired Student t test (A-H,K-L), or a 2-tailed, unpaired Student t test (N-O).