Fig 2: Inhibitory efficacy of LT-171-861 against patient blasts. (A) AML blasts harboring FLT3-ITD (patient 1 and 5) and FLT3-wt (patient 2-4 and 6) were treated with indicated concentrations of LT-171-861 for 36 h. Cells then stained with propidium iodide (PI)/annexin V and then evaluated by flow cytometry. (B) AML patient blasts expressing FLT3-ITD (patient 7, 9 and 10) were incubated with indicated concentrations of LT-171-861, sorafenib and AC220 for 72 h and cell viabilities were evaluated with MTT assay. (C) Blasts from AML patients harboring FLT3-ITD (patient 7 and 8) were incubated with LT-171-861 for 2 h, and p-FLT3, p-STAT5 and p-ERK1/2 were tested by western blot.

Fig 3: Potent pro-apoptotic ability of LT-171-861 in leukemia cells harboring FLT3-ITD mutation. (A) MOLM13 cells and MV4-11 cells were respectively incubated with indicated concentrations of LT-171-861, sorafenib and AC220 for 2 h. Cell lysates were used to perform western blot assay. (B) MV4-11 cells and MOLM13 cells were treated with LT-171-861 for 36 h. Cells then stained with propidium iodide (PI) /annexin V-FITC and then evaluated by flow cytometry. Annexin V+/PI- and annexin V+/PI+ cells were considered dead cells and shown in the bottom chart (n=3). (C) MOLM13 cells and MV4-11 cells were treated with indicated concentrations of LT-171-861 for 36 h. PARP, caspase 3, caspase 8, Mcl-1 were evaluated in the cell lysate by western blot. (D) MV4-11 cells were treated with LT-171-861 (200 nM), AC220 (200nM) or sorafenib (200 nM) for indicated time periods (0, 4, 8, 12, 24, and 36 h) respectively. Then Cells were stained with propidium iodide/annexin V-FITC and evaluated by flow cytometry. (E) MOLM13 cells and MV4-11 cells were treated with cytarabine, LT-171-861 or a combination of cytarabine and LT-171-861 (ratio of 10:1), and cell viability was measured by MTT assay. Error bars show standard deviation (SD), and the combination index (CI) value was calculated by CompuSyn 1.0 software.

Fig 4: Inhibition of FLT3 in BaF3 cell models and in stroma-protecting setting. (A) Indicated FLT3 mutations were expressed in BaF3 cells respectively. Transformed BaF3 cells were incubated for 2 h with indicated concentrations of LT-171-861, and (B-E) for direct comparison with 3 inhibitors. The autophosphorylation level of FLT3 was visualized and compared by western blot analysis. (E) MOLM13 cells were treated with indicated concentrations of LT-171-861, sorafenib and AC220 for 72 h in condition medium (CM) and normal culture medium (NM). The CM was prepared from HS-5 cell culture for 4 days under routine culture conditions, clarified by centrifugation, and used immediately. The CM was added to complete medium at a final concentration of 40%. Cell viabilities were measured by MTT assay. Data were shown as mean±SD. (F) MOLM13 cells were incubated for 8 h in CM with indicated concentrations of LT-171-861 and AC220. The FLT3 phosphorylation levels of FLT3, STAT5 and ERK 1/2 were determined by Western blot.

Fig 5: Anti-leukemia activity of LT-171-861 in xenograft and engrafted mouse model. (A) Mice (n=6) received LT-171-861, sorafenib and vehicle every two days for around 24 days. The group of mice treated with LT-171-861 (10 mg/kg) was subsequently monitored for an additional 20 days to examine tumor regrowth (ANOVA with Dunnett's posttest was performed, ***P< 0.001, **P< 0.01, *P< 0.05). Data were shown as mean±SD. (B) Representative photographs of tumors in each group after LT-171-861 (10 mg/kg), sorafenib (20 mg/kg) and the vehicle treatment. Comparison of the final tumor weights in each group after the treatment period (ANOVA with Dunnett's posttest was performed, ***P< 0.001, **P< 0.01, *P< 0.05, * represents control vs LT-171-861 and sorafenib, # represents LT-171-861 vs sorafenib). Data were shown as mean±SD. (C) An engrafted tumor model was used to evaluate in vivo antitumor activity. The NOD/SCID mice were intravenously inoculated with MV4-11 cells. From 12 days after inoculation, mice (n=6) received LT-171-861 (10 mg/kg) every other day. Log-rank testing was used to compare survival curves among treatment groups. LT-171-861 treatment resulted in significant survival advantage (***P< 0.001, **P< 0.01, *P< 0.05; * represents control vs LT-171-861 and sorafenib, # represents LT-171-861 vs sorafenib). (D) Apoptotic assays in engraft model were evaluated by TUNEL assay and cell proliferative activity was measured by Ki67 assay. Images were acquired at ×40 magnification. (E) BALB/c nude mice were subcutaneously inoculated with MV4-11 cells (5×106) and bodyweight of each mouse was recorded every other day. (F) Representative photo of tumors from BALB/c nude mice which were inoculated with MV4-11 cells. (G) BALB/c nude mice were subcutaneously inoculated with BaF3 cells bearing FLT3-ITD-N676D (n=6) and FLT3-ITD-F691L (n=6) respectively. We administered 10 mg/kg LT-171-861 or vehicle once every other day. (H) BALB/c mice were intravenously inoculated with cells used in (G). Mice were treated with LT-171-861 (10 mg/kg) or vehicle once every other day. Log-rank testing was used to compare survival curves among treatment groups. LT-171-861 treatment resulted in significant survival advantage (***P< 0.001, **P< 0.01, *P< 0.05; * represents control vs LT-171-861). (I) Representative photos of spleens dissected from mice in (H) inoculated with BaF3 cells bearing FLT-3-ITD-F691L.