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. 2021 Mar;35(3):887-892.
doi: 10.1038/s41375-020-0989-4. Epub 2020 Jul 20.

Bortezomib resistance mutations in PSMB5 determine response to second-generation proteasome inhibitors in multiple myeloma

Kira Allmeroth  1 , Moritz Horn  2 , Virginia Kroef  1 , Stephan Miethe  1 , Roman-Ulrich Müller  3   4   5 , Martin S Denzel  6   7   8
Affiliations

Bortezomib resistance mutations in PSMB5 determine response to second-generation proteasome inhibitors in multiple myeloma

Kira Allmeroth et al. Leukemia. 2021 Mar.
No abstract available

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Conflict of interest statement

MH and MSD are shareholders in ACUS Laboratories GmbH, which uses genetic screens for target deconvolution. The other authors declare no potential conflict of interests.

Figures

Fig. 1
Fig. 1. Unbiased identification and characterization of clinically relevant bortezomib resistance mutations in Psmb5 using haploid cells.
a Schematic representation of diploid cells, in which the presence of a wild-type allele masks the phenotype caused by a mutant allele. In haploid cells, there is a direct genotype–phenotype correlation, enabling functional analysis. b Schematic representation of experimental workflow for bortezomib resistance screen using ENU mutagenesis. c Schematic representation of PSMB5. Amino acid substitutions identified in the screen are highlighted in red. Positions with reported resistance are marked with an asterisk. For more information, see Supplementary Table 1. d Cell viability assay (XTT) of wild-type (WT) control cells and isolated clones treated with 10 nM bortezomib. Statistical significance was calculated by one-way ANOVA Dunnett’s post-hoc test. ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant. Mean + SEM (n = 4). e Crystal structure of human PSMB5 (gray) in complex with bortezomib (green). Identified substitutions are highlighted in red. Hydrogen bonds between bortezomib and the amino acids in the binding pocket are shown (black dashed lines). PDB: 5LF3. f Chymotrypsin-like proteasome activity of wild-type and CRISPR/Cas9-engineered AN3-12 cells with the indicated PSMB5 substitutions using suc-LLVY-AMC as a substrate. Mean + SEM (n = 3). g Correlation of mean chymotrypsin-like activity (Fig. 1f) with mean proliferation on day 3 (Supplementary Fig. 2e) of wild-type and CRISPR/Cas9-engineered AN3-12 cells with the indicated PSMB5 substitutions. R2 was calculated by linear regression fit using GraphPad Prism.
Fig. 2
Fig. 2. Bortezomib-resistant Psmb5 mutant cells display differential response to second-generation proteasome inhibitors.
a Cell viability of wild-type and CRISPR/Cas9-engineered AN3-12 cells with the indicated PSMB5 substitutions treated with 50 nM ixazomib. Mean + SEM (n = 5; n = 3 for V31L). b Cell viability of wild-type and CRISPR/Cas9-engineered AN3-12 cells with the indicated PSMB5 substitutions treated with 15 nM carfilzomib. Mean + SEM (n = 5; n = 3 for V31L). c Cell viability of wild-type CRISPR/Cas9-engineered AN3-12 cells with the indicated PSMB5 substitutions treated with 80 nM oprozomib. Mean + SEM (n = 3). a–c T21A is highlighted in pink and A49V is highlighted in orange. Statistical significance was calculated by one-way ANOVA Dunnett’s post-hoc test. ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant. d Cell viability of wild-type (black) and PSMB5 T21A KMS-18 cells (pink) treated with the indicated PIs. e Cell viability of wild-type (black) and PSMB5 A49V KMS-27 cells (orange) treated with the indicated PIs. d, e Data are presented as mean ± SEM (n = 3).
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References

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