Fig. 3

ONC213 and venetoclax target AML progenitor and stem cells. (A) Primary AML patient samples were cultured with ONC213 (ONC) and venetoclax (VEN), alone or in combination, for 48 h and then plated in methylcellulose and incubated for 2 weeks. The number of surviving AML cells capable of generating leukemia colonies (AML-CFUs) were enumerated. Technical triplicates were performed. Data are presented as mean ± SEM. ** p < 0.01 and *** p < 0.001 compared to vehicle control; ## p < 0.01 and ### p < 0.001 compared to single drug treatments. (B) Human CD34 + cord blood cells were treated with ONC213 and VEN, alone or in combination, for 24 h and then plated in methylcellulose and incubated for 2 weeks. The number of surviving normal hematopoietic cells capable of generating colonies were counted and colonies are presented as mean ± SEM. The number of BFU-E, CFU-E, CFU-G, CFU-M, CFU-GM, and CFU-GEMM is presented as mean ± SEM (left panel). Total erythroid and myeloid colonies are presented as mean ± SEM (right panel). (C) AML cells were isolated from spleens of NSGS mice injected with J000106565 patient-derived AML cells and treated with VEN + AZA. CD34 MicroBeads were used to enrich human CD34 + cells. These cells were treated with VEN, ONC, or the combination for 48 h, and then stained with anti-human CD45 and annexin V/PI and analyzed via flow cytometry. *** p < 0.001 compared to control and single drug treatments. (D-F) NSGS mice were injected with J000106565 patient-derived AML cells. Engraftment was confirmed on day 18. On day 19, mice were randomized into treatment groups (n = 5) and treated as shown in panel D. On days 32 (part way through treatment) and 42 (at the end of treatment) peripheral blood was collected, and cells were stained with anti-human CD45, -CD34, -CD38, and -CD123 antibodies and analyzed via flow cytometry. Bulk AML cells were determined by gating for CD45 + cells (panel E). AML stem cells were determined by gating for CD45dim+/CD34+/CD38-/CD123 + cells (panel F). * p < 0.05 and ** p < 0.01 compared to control. # p < 0.05 compared to single drug treatments (left panel). ## p < 0.01 and ### p < 0.001 compared to VEN treatment (right panel)