Figure 3. MRX-2843 inhibits FLT3 activation and downstream signaling and has functional antitumor effects in MERTK^negFLT3-ITD cell lines.

(A–D) MOLM-14 (MERTK^negFLT3-ITD) and MV4-11 (MERTK^dimFLT3-ITD) AML cells were treated with the indicated doses of MRX-2843, vehicle (DMSO), or a nontargeting control TKI. (A) After 1 hour, MOLM-14 cells were treated with pervanadate phosphatase inhibitor for 3 minutes and lysates prepared. FLT3 protein was immunoprecipitated, and phosphorylated and total FLT3 were detected by immunoblot analysis. (B) Lysates were prepared from MOLM-14 cells without pervanadate treatment, and phosphorylated and total STAT5, AKT, and ERK1/2 proteins were detected by immunoblot analysis. Actin was used as a loading control. (C) After 48 hours, MTS reagent was added to cultures for an additional 2 hours ,and absorbance was determined as an indicator of viable cell numbers. (D) After 72 hours of drug treatment, cells were stained with YoPro-1 iodide and propidium iodide dyes, and apoptotic and dead cells were detected by flow cytometry. **P < 0.01 and ***P < 0.001, by 1-way ANOVA. (E) MOLM-14 or MV4-11 cells were cultured in soft agar overlaid with media containing the indicated concentrations of MRX-2843, vehicle (DMSO), or control TKI. Colonies were stained and counted after 14 to 21 days. Results are shown relative to vehicle-treated controls. Mean values and standard errors were derived from 3 independent experiments. **P < 0.01 and ***P < 0.001, by 1-way ANOVA. (F) Kaplan-Meier curve showing survival of NSG mice with orthotopic MOLM-14 xenografts that were treated with MRX-2843 or vehicle (saline) beginning on day 21 after transplantation. Median survival for MRX-2843-treated mice was 126 days compared with 38 days for vehicle-treated controls (n = 8 per group). P < 0.001, by log-rank test. AML, acute myeloid leukemia; Ctrl TKI, control tyrosine kinase inhibitor; FLT3, FMS-like tyrosine kinase 3; ITD, internal tandem duplication; NSG, NOD-SCID-γ; p, phosphorylated.