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. 2023 Apr 11;7(7):1103-1107.
doi: 10.1182/bloodadvances.2022008442.

Wnt inhibitors reduce the unfolded protein response and enhance bortezomib-induced cell death in multiple myeloma

Ingrid Spaan  1 , Niels van Nieuwenhuijzen  1   2 , Thomas Kimman  1 , Dedeke Rockx-Brouwer  1 , Ralph G Tieland  1 , Madelon M Maurice  3   4 , Monique C Minnema  2 , Reinier A Raymakers  2 , Victor Peperzak  1
Affiliations

Wnt inhibitors reduce the unfolded protein response and enhance bortezomib-induced cell death in multiple myeloma

Ingrid Spaan et al. Blood Adv. .
No abstract available

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Conflict of interest statement

Conflict-of-interest disclosure: V.P. received royalty payments related to venetoclax. M.C.M. received research funding from Celgene and honoraria from Celgene, Alnylam, Jansen Cilag, and Gilead. The remaining authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Inhibition of tankyrase and porcupine downregulates Wnt signaling in MM1.S and promotes cell death of primary MM cells. (A) Representative western blots showing β-catenin protein expression in MM cell line MM1.S after 24 hours exposure to indicated concentrations of tankyrase inhibitor (TNKSi) XAV939 or porcupine inhibitor (PORCi) C59, in the presence of 50% Wnt3a-conditioned medium (WCM) or 50% L-cell control medium (LCM). Alpha-tubulin was used as a loading control. (B) Transcriptional Wnt reporter activity as determined by TopFlash after 24 hours of exposure to the combination of 20 μM TNSKi and 5 μM PORCi in MM1.S. Data is normalized to internal Renilla and FOP negative control, and shown as fold change to untreated control. Bars show the mean of 3 individual experiments, error bars represent the standard error of the mean and statistical significance was determined by unpaired t-test. (C) Representative flow cytometry analysis plots of viability in CD38+ cells of a primary MM sample after 72 hours of exposure to 20 μM TNKSi, 5 μM PORCi, and the combination thereof, or untreated control cells, in the presence of IL-6 and APRIL. Gates represent viable (DiOC6+/TOPRO3−) cells. (D) Absolute number of viable CD38+ primary MM cells, represented as fold change relative to untreated control per time point, after 24 hours or 7 days of exposure to 20 μM TNKSi or 5 μM PORCi, in the presence of IL-6 and APRIL. Viable cells were identified as DiOC6+/TOPRO3- and cell counts were determined using flow cytometry beads. Bars indicate the mean of 14 included MM samples (MM1-MM14); dots represent individual MM samples. The dashed line represents the untreated control per time point. Statistical significance was determined by one-way analysis of variance (ANOVA) using Dunnett correction for multiple testing. (E) Absolute viable CD38+ primary MM cells, represented as fold change relative to untreated control per time point, after 24 hours, 72 hours and 7 days of exposure to the combination of 20 μM TNKSi and 5 μM PORCi, in the presence of IL-6 and APRIL. Viable cells were identified as DiOC6+/TOPRO3− and cell counts were determined using flow cytometry beads. Bars indicate the mean of the 7 included MM samples (MM7, MM9-MM14); dots represent individual MM samples. The dashed line represents the untreated control per time point. Statistical significance was determined by one-way ANOVA using Dunnett correction for multiple testing.
Figure 2.
Figure 2.
Targeting Wnt signaling downregulates expression of genes involved in the UPR and enhances BTZ-induced cell death in primary MM cells. Transcriptome analysis by single-cell RNA sequencing of viable CD38+CD138+ primary MM cells of samples MM9-MM13 after 72 hours of exposure to a combination of 20 μM tankyrase inhibitor XAV939 and 5 μM porcupine inhibitor C59, or untreated control cells, in the presence of IL-6 and APRIL. (A) Dimensionality reduction and clustering of transcriptome analysis by tSNE, and cluster distribution of the individual MM samples. The 6 clusters are denoted by digits 0 to 5 and corresponding colors. (B) Volcano plots of genes differentially expressed between MM cells exposed to the inhibitor combination vs control. Dark gray data points indicate significant differentially expressed (DE) genes with an adjusted P value of <.01; light gray data points indicate nonsignificant DE genes with an adjusted P value of >.01. The top 15 DE genes have been annotated with their corresponding gene name, whereas the genes within the top 15 that are functionally related to the UPR pathway have been colored red. (C) Violin plots showing expression of several important mediators of the UPR pathway: XBP1, MZB1, SEC61G, and HSP90B1, in MM cells exposed to control of the inhibitor combination. Individual data points represent the sequenced single cells, and the adjusted P value is annotated as P-adj, for which <.01 is considered statistically significant. (D) Representative example of intracellular protein expression of XBP1s measured by flow cytometry. Samples were treated with TNKSi and PORCi for 72 hours (red) or untreated as control (green). The dotted line shows a-specific background staining. (E) Comparison of intracellular XBP1s expression in 6 primary MM samples (MM13, MM18-22) treated with TNKSi and PORCi for 72 hours or untreated as control. XBP1s expression was denoted as mean fluorescence intensity (MFI), as measured by flow cytometry as indicated in (D). Statistical significance was determined with a paired t-test. (F) Plot showing specific apoptosis to BTZ at concentrations of 2, 4, and 8 nM in a cohort of 28 newly diagnosed MM samples. Viable cells were identified as DiOC6+/TOPRO3−. Bars indicate the mean of all included MM samples; dots represent individual MM samples. Statistical significance was determined by one-way ANOVA using Dunnett correction for multiple testing. (G) Absolute number of viable CD38+CD138+ cells, represented as fold change relative to untreated control, after 72 hours of exposure to 20 μM TNKSi and 5 μM PORCi, 24 hours exposure to 2 nM BTZ, or the combination thereof, in the presence of IL-6 and APRIL. Viable cells were identified as DiOC6+/TOPRO3- and cell counts were determined using flow cytometry beads. Bars indicate the mean of 6 included MM samples (MM4-5, MM12-13, MM16-17); dots represent individual MM samples. The dashed line represents the untreated control per time point. Statistical significance was determined by one-way ANOVA using Dunnett correction for multiple testing. (H) Plot comparing observed specific apoptosis to expected specific apoptosis. The 6 connected datapoints represent the 6 individual MM samples. Statistical significance was determined with a paired t-test. Specific apoptosis was calculated by comparing the number of surviving cells in treated conditions vs untreated control. OBS, observed; EXP, expected.
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References

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