Gene and protein expression changes upon menin-MLL inhibition in NPM1^mut and MLL-r AML. (A) Human (left) and murine (right) AML cells were treated for 11 days with MI-503. Viable (4′,6-diamidino-2-phenylindole [DAPI]–negative) cells were assessed by flow cytometry, and IC₅₀ values were calculated with GraphPad Prism software. (B) Summary of IC₅₀ values (MI-503), MLL-rearrangement, and NPM1 and FLT3 mutation status in the AML cells assessed. (C) Venn diagram showing downregulated genes identified by RNA-seq (more than twofold decrease; adjusted P < .05), in NPM1^mut OCI-AML3, MLL-r MOLM13, and MV411 cells after MI-503 treatment (2.5 µM) compared with the DMSO control. (D) Volcano plots of RNA-seq data obtained from OCI-AML3, MOLM13, and MV411 cells treated with MI-503 (2.5 µM). FLT3 and selected MLL-fusion targets are labeled. (E) FLT3 and MEIS1 mRNA expression in human and murine leukemia cells after 4 days of MI-503 treatment (2.5 µM), as assessed by qRT-PCR. ChIP was performed with antibodies against menin (F) or MLL1 (G) and IgG as the negative control, followed by qPCR to detect a sequence within the MEIS1 gene body or SOX2 as negative control. Cells were treated with MI-503 (2.5 µM) or vehicle control for 4 days. (H) FLT3 protein (cell surface) expression assessed by flow cytometry in human and murine NPM1^mut and MLL-rearranged AML cells after MI-503 treatment (2.5 µM for 4 or 7 days, as indicated). Representative histograms of 3 independent experiments are shown. Bar graphs in panels A and E-G represent the mean of 3 independent experiments, each performed in technical triplicate. Bar graph in panel H showing Npm1^CA/+Flt3^ITD/+ cells represents 2 independent experiments performed in technical triplicate. Error bars represent standard deviation.