Page 293 - 2021_02-Haematologica-web

Letters to the Editor
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Figure 2. Characterization of CHMFL-FLT3-362 in cell and in vivo in preclinical acute myeloid leukemia (AML) models with FLT3-ITD mutants. (A) The phospho- rylation levels of FLT3 (Tyr 589/591), STAT5 (Tyr 694), ERK1/2 (Tyr 202/204), and AKT (Ser 308) were detected by western blot in MOLM13, MOLM14 and MV4- 11 cell lines. These cells were incubated with the indicated concentrations of CHMFL-FLT3-362 for 4 hours (h) before lysis. (B) Western blot analysis for the expression of PARP, cleaved PARP, caspase-3, and cleaved caspase-3 in MOLM-13, MOLM-14, and MV4-11 cells treated with CHMFL-FLT3-362 for 24 h. (C) Growth inhibition effects of CHMFL-FLT3-362 against FLT3-ITD+ AML patient primary cells, FLT3-wild-type (wt) AML patient primary cells, and peripheral blood mononuclear cells using cell Titer-Glo assay after 72 h drug treatment (maximum concentration 10 mM, mean±SEM, n=3). (D) Effects of CHMFL-FLT3-362 on zebrafish normal myelopoiesis and the FLT3/ITD-induced myeloid cells expansion. WT granulopoiesis were measured by the number of myeloperoxidase positive cells using in situ hybridization assay with mpx specific RNA probe. Inject-the injection of FLT3-ITD gene mRNA during the fertilized egg period, uninject-the FLT3- ITD gene mRNA was not injected during the fertilized egg period; DMSO uninject vs. CHMFL-FLT3-362 uninject, P=0.4165; DMSO uninject vs. DMSO inject, P=0.0067; CHMFL-FLT3-362 uninject vs. CHMFL-FLT3-362 inject, P=0.4385; DMSO inject vs. CHFML-FLT3-362 inject, P=0.0039, **P<0.01, ns: not significant. (E) Bone marrow (BM) smear of mouse 14-day sub-drug test. (F) Anti-leukemia effects of CHMFL-FLT3-362 with once daily (QD) dosing at 50, 100, and 150 mg/kg. Total study length was 28 days. Each treatment group=5 animals. (Left) Representative graphs of relative tumor size are shown. (Right) Representative graphs of tumor weight are shown of different groups. Error bars, mean±SEM, n=5. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. (G) Anti-tumor efficacy of CHMFL-FLT3-362 in a BM engrafted mouse model. Kaplan-Meier plots of survival. (Left) The disseminated NOD/SCID mice were intravenously inoculated with MV4-11 cells. Mice received daily oral administration of CHMFL-FLT3-362 dosing at 50, 100, and 150 mg/kg. Total study length was 174 days. Each treatment group = 5 animals. (Right) Disseminated NOD/SCID mice were intravenously inoculated with MOLM-13 cells. Mice received daily oral administration of CHMFL- FLT3-362 dosing at 50, 100, and 150 mg/kg. Total study length was 32 days. Each treatment group=5 animals.
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