Journal: Cancer Research Communications

Article Title: Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors

doi: 10.1158/2767-9764.CRC-23-0350

Figure Lengend Snippet: Activation of stress pathways is induced by pitavastatin and the ISR inhibitor, ISRIB, partially rescues from statin-mediated sensitization of BH3 mimetics. A, 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, 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, *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, AnnexinV-PI viability assays of L363 and OPM2. ISRIB rescues from the apoptosis induced by venetoclax and pitavastatin in L363 and OPM2, n = 3. D, 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.

Article Snippet: Pitavastatin (AvaChem #2266), venetoclax (Active Biochem A-1231/Chemgood C-1008), S63845 (Selleck S8383/Chemgood C-1370), ISRIB (Selleck #50-136-4740), and AMG-PERK44 (MedChemExpress # HY-12661A) were dissolved in DMSO.

Techniques: Activation Assay, Western Blot, Flow Cytometry, Marker, Control, Fluorescence, Inhibition

Journal: Cancer Research Communications

Article Title: Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors

doi: 10.1158/2767-9764.CRC-23-0350

Figure Lengend Snippet: Pitavastatin-mediated activation of the ISR induces NOXA to sensitize to venetoclax separately from PUMA. Heat map summary of BCL2 family Western blot screen across all statin-sensitive cells (NCI-H929, MM.1S, L363, OPM2, MOLP8, and KMS12PE) for 16 ( A ) and 40 hours ( B ) treatments with 1 µmol/L pitavastatin. log 2 transformed mean fold change of three replicates of each MMCL are plotted on a double gradient scale. C, Western blot analysis of the t(4;14) OPM2 and non-t(4;14) MM1S treated with 1 µmol/L pitavastatin or pitavastatin/ISRIB. NOXA upregulation is partially blocked by ISRIB, but PUMA is mostly ISR independent. Representative of n = 2 for tested cell lines. D, The apoptosis sensitizing effects of pitavastatin are blocked in BBC3 (PUMA) knockout L363 cell line treated with ISRIB in a 40-hour Annexin V-PI viability assay, n = 4.

Article Snippet: Pitavastatin (AvaChem #2266), venetoclax (Active Biochem A-1231/Chemgood C-1008), S63845 (Selleck S8383/Chemgood C-1370), ISRIB (Selleck #50-136-4740), and AMG-PERK44 (MedChemExpress # HY-12661A) were dissolved in DMSO.

Techniques: Activation Assay, Western Blot, Transformation Assay, Knock-Out, Viability Assay

Journal: Cancer Research Communications

Article Title: Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors

doi: 10.1158/2767-9764.CRC-23-0350

Figure Lengend Snippet: Statins induce two independent responses in multiple myeloma. As observed in AML, DLBCL, and now multiple myeloma, statins upregulate PUMA in a p53-independent manner. PUMA can contribute to BH3 mimetic killing by promiscuously binding prosurvival BCL2 family members. Activation of the integrated stress response is a multiple myeloma–specific statin response that influences apoptosis. Through a currently undefined mechanism, statins reduced general translation and activate the translation of ATF4. ATF4 is a master mediator of the ISR. One such output of ATF4 is upregulation of NOXA. NOXA can be partially reduced with ISRIB which results in the partial rescue from statin-mediated apoptotic sensitization to venetoclax. The MCL-1 selective activity of NOXA promotes sensitivity to venetoclax. However, this property also makes it a direct competitor with S63845. As such, ISRIB did not rescue from S63845 and statin-mediated killing.

Article Snippet: Pitavastatin (AvaChem #2266), venetoclax (Active Biochem A-1231/Chemgood C-1008), S63845 (Selleck S8383/Chemgood C-1370), ISRIB (Selleck #50-136-4740), and AMG-PERK44 (MedChemExpress # HY-12661A) were dissolved in DMSO.

Techniques: Binding Assay, Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: Novel Bead-Based Epitope Assay is a sensitive and reliable tool for profiling epitope-specific antibody repertoire in food allergy

doi: 10.1038/s41598-019-54868-7

Figure Lengend Snippet: Relationship between epitope-specific IgE and IgG4 antibodies. ( A ) Boxplots of the average of 7 milk epitopes show dose-depended relationship between IgE and the OFC dose at month 32 of 44 patients; while the relationship is less clear for either IgG4 or IgG4-IgE ratio. ( B ) Scatterplot with the loess smoothed line of the average IgE and IgG4 to 7 milk epitopes; each point represents an individual patient, colored by the amount of protein they were able to consume during the OFC at month 32. ( C ) A classification tree determining best relationship between IgE and IgG4 and the binary outcome of the OFC, shows that having very high or low levels of IgE is enough to classify the OFC outcome, while at the intermediate IgE levels (0.81–2.4), high IgG4 (>7.81) can further improve classification. ( D ) Heatmaps of 73 peanut allergic (red) and 83 non-allergic (grey) patients and their IgE, IgG4, and IgG4-IgE binding to epitopes from 3 peanut proteins. The epitopes with FCH≥1.5 and FDR <0.05 are presented. All IgG4-IgE epitopes are also present among the IgE ones, indicating that IgE binding alone is most informative in identifying differences among allergic and non-allergic individuals.

Article Snippet: Slides were then washed with PBS-T and incubated for 1 hour at room temperature with a mixture of 3 monoclonal mouse anti-human IgE and IgG4 antibodies (working concentration, 0.4 μg/mL; mouse anti-human IgE from BD Biosciences Cat 555858, clone G726 and Invitrogen Cat 05-4740, and biotinylated mouse anti-human IgG4 from Southern Biotech Cat 9200-02) .

Techniques: Binding Assay