ATRA plus sorafenib targets leukemia stem cells in a genetically engineered mouse model of AML. (A) CFU counts at day 7 of 5 × 10³ NHD13;FLT3/ITD leukemic mouse BM cells treated with sorafenib (20 nM) and/or ATRA (100 nM). Data indicate average colony number ± SD, and are representative of 3 independent experiments (**P < .01, ***P < .001). (B-D) MPP cells were isolated from a CD45.2⁺ NHD13;FLT3/ITD leukemic donor mouse and treated with DMSO or ATRA (10 nM) ex vivo for 48 hours. On day 2, 1 × 10⁴ viable donor cells along with 5 × 10⁵ CD45.1⁺ helper cells were transplanted into sublethally irradiated CD45.1⁺ recipients (n = 20) and randomly divided into 4 cohorts: DMSO/Veh, DMSO/Sor, ATRA/Veh, ATRA/Sor. Starting on day 9, mice received vehicle (Veh) or sorafenib (Sor) for 2 weeks. (B) Mouse xenograft schematic. (C) PB engraftment on day 58 posttransplant, represented by percentage CD45.2⁺ cells of a total of CD45.1⁺ and CD45.2⁺ cells. Data represent average of n = 5 mice per cohort ± SD (*P < .05, **P < .01, ***P < .001). (D) Kaplan-Meier survival of mouse cohorts (n = 5 each), indicating median survival of DMSO/Veh (69 days), DMSO/Sor (109 days), ATRA/Veh (80 days), and ATRA/Sor (127) days (**P < .01, relative to vehicle). Treatment period is indicated by black arrows. (E) Mice received 5 × 10⁵ whole BM cells from a NHD13;FLT3/ITD leukemic donor along with 5 × 10⁵ CD45.1⁺ helper cells and were treated in vivo with sorafenib (5 mg/kg daily), ATRA (5 mg/kg daily), sorafenib + ATRA (Sor+ATRA), or vehicle, all via oral gavage, for 2 weeks. Kaplan-Meier survival of mouse cohorts (n = 6 each), indicating median survival of vehicle (26 days), ATRA (25 days), sorafenib (34 days), and Sor+ATRA (55.5) days (**P < .01, ***P < .001, relative to vehicle). Treatment period is indicated by black arrows. (F) Mouse secondary transplant schematic. Whole BM cells were isolated from a CD45.2⁺ NHD13;FLT3/ITD leukemic donor mouse and 5 × 10⁵ donor cells, along with 5 × 10⁵ CD45.1⁺ helper cells, were transplanted into sublethally irradiated CD45.1⁺ recipients. Starting on day 7, mice received vehicle, sorafenib, ATRA, or Sor+ATRA (n = 3 per cohort) for 2 weeks. PB and BM were harvested on day 21 posttransplant, and pooled BM was transplanted into healthy secondary recipients and mice were monitored for PB engraftment and survival. (G) PB engraftment on day 28 and (H) day 84 postsecondary transplant, represented by the percentage of CD45.2⁺ cells of a total of CD45.1⁺ and CD45.2⁺ cells. Data represent average of n = 3 mice per cohort ± SD (**P < .01, ***P < .001). (I) Kaplan-Meier survival of mouse cohorts (n = 5 each), indicating median survival of vehicle (43 days), ATRA (96 days), sorafenib (>120 days), and Sor+ATRA (>120 days) treated mouse BM recipient mice (**P < .01 for vehicle to ATRA, vehicle to sorafenib, and vehicle to Sor+ATRA comparisons, *P < .05 for ATRA to Sor+ATRA comparison). (J) Limiting dilution transplantation analysis of pooled whole BM from mice treated as in panels E and F. Detection of >1% hCD45⁺ of a total of CD45.1⁺ and CD45.2⁺ cells in the PB was used as a marker for successful engraftment of leukemia. TP, transplant; WBM, whole bone marrow.