mcl 1 specific bh3  (Mimetics)

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: MMCLs are sensitized to BH3 mimetics by simvastatin. We assessed a panel of seven myeloma cell lines including: OPM2, L363, KMS-12-PE, NCI-H929, MOLP8, RPMI-8226, and U266. A, Representative plots of statin-sensitive (black) MMCLs are shown alongside statin insensitive (red). The initial screen used PI exclusion to determine viability by flow cytometry. Rescue with 1 mmol/L mevalonate was demonstrated at the highest concentration of simvastatin to demonstrate on-target activity. Of note, statin-mediated killing of RPMI-8226 is not fully rescued by mevalonate, perhaps due to inefficient cellular uptake. n = 3, error bars indicate SD. B, IC 50 of venetoclax or C, S63845 at concentrations of statins necessary to significantly shift the IC 50 . Significance ( P value <0.05) was determined by extra sum-of-squares F test for differences in log IC 50 . Achieving significance was the classification determinant for statin-sensitive and statin-insensitive cell lines. *, P < 0.05; **, P < 0.01; *** , P < 0.001; ****, P < 0.0001 of Bonferroni-adjusted P values.

Article Snippet: MCL-1–specific BH3 mimetics, which have entered clinical trials to treat MCL-1–dependent blood cancers like multiple myeloma , may also benefit from combination agents to broaden their therapeutic utility.

Techniques: Flow Cytometry, Concentration Assay, Activity 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: Simvastatin induces p53-independent PUMA upregulation. A, A BCL2 family model figure depicting protein interactions and how BH3 mimetics can cooperate with apoptotic sensitizers. B, A representative Western blot analysis of statin-insensitive (red) and statin-sensitive (black) MMCLs treated for 16 hours with 10 µmol/L simvastatin. n = 3. C, Quantification by densitometry of PUMA upregulation. Mean ± SD, n = 3, significance (α<0.05) determined by one-sample t test of log-transformed fold changes against the value of 0. *, P < 0.05; **, P < 0.01. D, Representative ( n = 3) Western blots showing that PUMA upregulation by 10 µmol/L simvastatin is not dependent on the transcriptional activity of p53. Expression of the p53 dominant negative, GSE56, was accomplished in three MMCLs, L363, NCI-H929, and OPM2 wherein the basal p53 status is mutated [*L363 cells have a point mutation in exon7/intron 7 splicing junction (base C782G) that impairs splicing of intron 7], wild-type, and mutated (R175H), respectively. GSE56 expression was confirmed with a rodent-specific p53 antibody (Cell Signaling Technology# 32532). Activation of p53 using 10 µmol/L of the topoisomerase inhibitor, etoposide, was used as a positive control to confirm blockade of p53 activation of PUMA upregulation.

Article Snippet: MCL-1–specific BH3 mimetics, which have entered clinical trials to treat MCL-1–dependent blood cancers like multiple myeloma , may also benefit from combination agents to broaden their therapeutic utility.

Techniques: Western Blot, Transformation Assay, Activity Assay, Expressing, Dominant Negative Mutation, Mutagenesis, Activation Assay, Positive Control

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 is three times more potent than simvastatin in cell-based assays of apoptosis and biomarker responses, and increases apoptosis in combination with venetoclax in a subset of patient samples treated ex vivo . A, Cell Titer Glo was used to assess viability in statin/BH3 mimetic combinations after 48 hours; % viable cells was normalized to vehicle. Mean ± SD, n = 3. B, Pitavastatin increases PUMA and blocks prenylation of RAP1A at concentrations lower than simvastatin. C, Results of a glucocorticoid resistance cell line models MM.1S (sensitive) and MM.1R (resistant) after 48 hours treatment. Mean ± SD; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA with Tukey post-test correction for multiple comparisons. D, Buffy coats from multiple myeloma patient bone marrow aspirates were treated in vitro with 1 µmol/L pitavastatin and 10 nmol/L venetoclax for 24 hours before staining with CD45, CD38, and Annexin V to assess viability.

Article Snippet: MCL-1–specific BH3 mimetics, which have entered clinical trials to treat MCL-1–dependent blood cancers like multiple myeloma , may also benefit from combination agents to broaden their therapeutic utility.

Techniques: Biomarker Discovery, Ex Vivo, In Vitro, Staining

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: MCL-1–specific BH3 mimetics, which have entered clinical trials to treat MCL-1–dependent blood cancers like multiple myeloma , may also benefit from combination agents to broaden their therapeutic utility.

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: 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: MCL-1–specific BH3 mimetics, which have entered clinical trials to treat MCL-1–dependent blood cancers like multiple myeloma , may also benefit from combination agents to broaden their therapeutic utility.

Techniques: Binding Assay, Activation Assay, Activity Assay

Journal: eLife

Article Title: The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes

doi: 10.7554/eLife.88329

Figure Lengend Snippet: ( A ) Transient transfection of HEK293, HCT116 and BMK cell lines to express exogenous BH3-only protein: BIM, PUMA, BIMdCTS, PUMAdCTS as well as PUMA-4E in which the 4 key hydrophobic residues in the BH3 motif of PUMA were changed to glutamic acid to disrupt the pro-apoptotic function of PUMA (I137E, L141E, M144E, and L148E). Cell death was scored using Annexin V staining to quantify cells that were both Venus and Annexin V positive out of the total Venus-positive cell population. ( B ) Two-way titration of two BH3-mimetics S63845 (MCL-1 inhibitor) and ABT-263 (BCL-X L , BCL-2, and BCL-W inhibitor) confirmed that HEK293 cells cannot be killed by inhibiting anti-apoptotic proteins (are unprimed) whereas BMK and HCT116 cells are primed for apoptosis and were killed by both BH3-mimetics. Annexin V positivity was scored as in ( A ) for cells that were incubated overnight with the indicated concentrations of S63845 and ABT-263. Results are displayed in a heatmap colored as indicated in the scale at the left, the values within each square indicate the average % Annexin V positive (dead cells) from three independent biological replicates.

Article Snippet: Similar to the results obtained for binding to C BCL-X L , the addition of MCL-1 specific BH3-mimetics; S63845 and S64315, resulted in higher dissociation constants for binding to MCL-1 by both mutant proteins compared to V PUMA ( ).

Techniques: Transfection, Staining, Titration, Incubation