MP-A08 induces ATF4 dependent Noxa transcription. (A) MV411 cells were treated with MP-A08 (20 μM), daunorubicin (DNR) (1 μM), or cytarabine (Ara-C; 1 μM) for 6 hours; lysed; and subjected to immunoblot analysis with the indicated antibodies. (B) The UPR. (C) MV411 cells were treated with MP-A08 (20 μM) over a 6-hour period, lysed, and subjected to immunoblot analysis with the indicated antibodies. (D) MV411 cells were stably transduced with 2 different CRISPR guide sequences targeting SPHK1 (g1 and g2), lysed, and subjected to immunoblot analysis with the indicated antibodies. The efficiency of the SPHK1 knockout was confirmed via SPHK1 activity assays of lysates from those cells in assay conditions largely selective for SPHK1 over SPHK2 (supplemental Figure 5C). (E) MV411 cells were treated with MP-A08 (20 μM), alone or in combination with the eIF2b agonist, ISRIB (200 nM) over 6 hours for quantitative polymerase chain reaction analysis of Noxa messenger RNA levels and immunoblot analysis with the indicated antibodies. Statistical significance was assessed by Student t test. *P < .05 (n = 3). (F) MV411 cells were stably transduced with a doxycycline-inducible shRNA targeting ATF4. Cells were treated with 1 μg/mL doxycycline for 48 hours and MP-A08 (20 μM) for 6 hours before cell lysis for immunoblot analysis. ns, a nonspecific band. (G) Chromatin immunoprecipitation analysis of the Noxa promoter in response to MP-A08 treatment (20 μM) of MV411 cells for 6 hours. Statistical significance was assessed by Student t test. *P < .05 (n = 4). (H) Primary AML samples were treated with increasing concentrations of MP-A08 and subjected to immunoblot analysis with the indicated antibodies. All qualitative data are representative of at least 3 independent experiments, and all quantitative data represent the mean ± standard error of the mean of at least 3 independent experiments.