FLT3 ITD kinase-inhibition rescues DEP-1 activity in cell lines and AML patient blasts. The AML cell lines MOLM-13 (A), or EOL-1 (B), harboring FLT3 ITD, or WT FLT3, respectively, were treated with the FLT3 inhibitor AG1295 (20μM, 3 hours), or DMSO solvent, and subsequently ROS formation was measured in a plate reader using carboxy-H2DFFDA (left panels). Endogenous DEP-1 was immunoprecipitated and assayed using a phosphopeptide substrate and malachite green detection (means of normalized values for 3 separate experiments ± SD, significance tested by t test). (C) AML patient blasts were freshly isolated from blood of patients taken before start of therapy. AG1295 treatment and measurement of ROS production and DEP-1 activity were performed as in (A-B). Values are based on duplicate (DEP-1 activity) and triplicate (ROS generation) determinations for each patient and means for all patients are presented (± SD, significance tested by t test). The data are normalized to values in vehicle (DMSO)–treated cells (100%). Data for ROS production and DEP-1 activity and the effects of AG1295 for individual patients are shown in supplemental Figure 4.
