FIGURE 5 from Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors

<p>Activation of stress pathways is induced by pitavastatin and the ISR inhibitor, ISRIB, partially rescues from statin-mediated sensitization of BH3 mimetics. <b>A,</b> ATF4 and stress activated factors were assessed across our panel of cell lines by Western blot analysis, including five statin sensitive, and two statin insensitive MMCL (RPMI-8226 and U266). <b>B,</b> Puromycin incorporation was measured by flow cytometry as a marker for active translation, and was assessed after 15 minutes of puromycin exposure. MMCLs were treated with 20 or 40 hours of pitavastatin or pitavastatin/ISRIB. Fifteen minutes prior to puromycin exposure, control wells were treated with sodium arsenite (NaArs), NaArs/ISRIB, or cycloheximide. MFI of cycloheximide-treated cells served as background fluorescence and was subtracted from the MFI of all other samples. Data are presented relative to vehicle treated cells. Significance of ISRIB rescue was determined by one-way ANOVA and multiple comparisons were corrected by Holm-Sidak multiple comparisons test, *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001. <b>C,</b> AnnexinV-PI viability assays of L363 and OPM2. ISRIB rescues from the apoptosis induced by venetoclax and pitavastatin in L363 and OPM2, <i>n</i> = 3. <b>D,</b> Western blot analysis of ATF4 upregulation induced by pitavastatin after 16 or 40 hours for the purpose of assessing the effect of AMG-PERK44 (PERKi) in L363, OPM2, and NCI-H929. Upregulation of ATF4 is delayed in L363 relative to the t(4;14) MMCLs, OPM2 and NCI-H929, consistent with puromycin incorporation results. PERK inhibition blocks ATF4 upregulation in L363, but not OPM2 and NCI-H929.</p>