Figure 2. MRX-2843 inhibits MERTK activation and downstream signaling and has functional antitumor effects in MERTK⁺ FLT3-WT cell culture and animal models.

(A–D) Kasumi-1 and/or NOMO-1 AML cells were treated with the indicated concentrations of MRX-2843, vehicle (DMSO), or a nontargeting control TKI (Ctrl TKI). (A) After 1 hour, Kasumi-1 cells were treated with pervanadate phosphatase inhibitor for 10 minutes to stabilize p-MERTK, and cell lysates were prepared. MERTK protein was immunoprecipitated, and p-MERTK and total MERTK were detected by immunoblot analysis. (B) After 2 hours, lysates were prepared from Kasumi-1 cells without pervanadate treatment, and phosphorylated and total STAT6, AKT, and ERK1/2 proteins were detected by immunoblotting. 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 the number of viable cells. (D) After 72 hours, cells were stained with Yo-Pro-1 iodide and propidium iodide, and apoptotic and dead cells were detected by flow cytometry. ***P < 0.001, by 1-way ANOVA. (E) Kasumi-1 or NOMO-1 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 NSGS mice with orthotopic NOMO-1 xenografts that were treated with MRX-2843 or vehicle (saline) beginning on day 21 after transplantation. Median survival was 51 days for MRX-2843–treated mice and 37 days for vehicle-treated control mice (n ≥10 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; NSGS, NOD-SCID-γ mice expressing Tg human cytokines; p, phosphorylated.