JNJ-75276617 inhibits proliferation and induces differentiation and apoptosis of AML and B-ALL cells with KMT2A alteration and NPM1 mutations and morphologic differentiation of KMT2A-AF9–transduced mouse bone marrow cells. (A) The antiproliferative activity of JNJ-75276617 was determined in a panel that included 8 AML cell lines (KMT2A-r [MOLM-14, MOLM-13, MV4-11, and THP-1], KMT2A–partial tandem duplication [PTD; EOL-1], NPM1c [OCI-AML3], KMT2A/NPM1-WT [KO-52 and HL-60]), 1 leukemia B-ALL cell line with KMT2A-r (RS4:11), and a KMT2A/NPM1-WT chronic myeloid leukemia (CML) cell line (K562). MOLM-14, MOLM-13, MV4-11, and THP-1 were originally derived from pediatric patients. Cells were treated with JNJ-75276617 for 8 days, and spheroid-like growth was measured in real time by live-cell imaging. Results are shown from a representative experiment in which all cell lines were evaluated in parallel (A). Absolute IC₅₀ and the mean ± standard deviation (SD) values were calculated as percentage change in confluence to DMSO-treated cells. The experiment was performed at least 3 times for each cell line (B). (C,D) Flow cytometry analysis of differentiation and apoptosis in KMT2A-r and NPM1c AML cells following treatment with various concentrations of JNJ-75276617 for 7 days (C). Expression of differentiation markers CD14 and CD11b was evaluated in the viable cell population only, and apoptosis was evaluated in the total cell population (D). Duplicate samples were tested for each condition, and bars represent the mean ± SD. The experiment was performed 3 times. (E) Effect of JNJ-75276617 on morphologic differentiation of KMT2A-AF9–transformed mouse BM cells was examined by May-Grünwald Giemsa staining. KMT2A-AF9–transformed mouse BM cells were treated with DMSO or 200 nM JNJ-75276617 for 10 days. Condensed nuclei, a readout of neutrophil-like morphologic differentiation, were counted after 10-day JNJ-75276617 treatment from 3 independently captured images. Representative images are shown. The experiment was performed once.