EPHB4 in AML can be targeted with a monoclonal antibody (MAb131). (A) EPHB4⁺ leukemia cells (K562 and Molm14) and EPHB4^– (KG1) cells treated with 1 dose of MAb131 at time 0 and cultured under normal conditions. Live cells were counted by trypan blue exclusion at specified time points. MAb131 treatment reduced cell growth in EPHB4⁺ cells only. The average counts from 3 separate experiments are displayed, and error bars represent standard error of the mean. P values were calculated at 72 hours by pairwise comparison between control IgG-treated and MAb131-treated cells. *P < .01. (B) MAb131 induces apoptosis in EPHB4⁺ leukemia cells. K562 cells were treated with MAb131 (1, 10, and 100 μg/mL) and assayed for apoptosis by Annexin V/PI staining. The percentage of cells live (double negative), early apoptotic (Annexin V⁺/PI⁺), and late apoptotic/dead (Annexin V⁺/PI⁺) are noted. (C) K562 cells were treated with MAb131 and harvested for protein analysis by immunoblot (top) or by flow cytometry (bottom). MAb131 treatment causes loss of cell surface EPHB4 and degradation of total EPHB4 in cells. (D) K562 cells were treated with Alexa 488–labeled MAb131 and incubated at 37°C and 4°C for 1 hour, followed by imaging (40× objective). Incubation at 4°C (top) prevents the internalization and degradation of EPHB4, as evidenced by the bright cell surface staining, whereas incubation at 37°C (bottom) leads to internalization of labeled antibody and decreased overall staining. (E) EPHB4⁺ primary AML blasts (n = 3) were treated with MAb131. Live cells were counted on a hemacytometer by trypan blue exclusion at 24 and 48 hours and normalized to untreated controls. Treatment with MAb131 lead to increased cell death in primary AML samples. (F) Primary AML samples were treated with MAb131 and show downregulation of cell surface EPHB4 as measured by flow cytometry. (G) K562 cells were treated with increasing concentrations of soluble EPHB4, which blocks the cytotoxic effect of MAb131, confirming that MAb131 effects are due to EPHB4 binding and not off-target effects. Cells were treated and live cells counted by trypan blue exclusion at 72 hours. The average counts from 3 separate experiments are displayed, and error bars represent the standard error of the mean. P values for selected pairwise comparison are noted on the chart.