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. 2020 Mar 4;10(1):4012.
doi: 10.1038/s41598-020-61027-w.

Genome-wide Screening Reveals a Role for Subcellular Localization of CRBN in the Anti-Myeloma Activity of Pomalidomide

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Free PMC article

Genome-wide Screening Reveals a Role for Subcellular Localization of CRBN in the Anti-Myeloma Activity of Pomalidomide

Shumpei Tateno et al. Sci Rep. .
Free PMC article

Abstract

Pomalidomide, a derivative of thalidomide, is an effective treatment for multiple myeloma. The drug exerts its effects through CRBN, a component of the E3 ubiquitin ligase complex CRL4CRBN. To search for novel factors involved in the anti-cancer activity of pomalidomide, we performed a genome-wide shRNA library screen and identified 445 genes as those affecting pomalidomide sensitivity. Genes encoding components of the ubiquitin-proteasome pathway, such as subunits of the CRL4CRBN complex, the COP9 signalosome, and the 26S proteasome, were among the pomalidomide-affecting genes. Karyopherin beta 1 (KPNB1) was identified as a novel pomalidomide-affecting gene. KPNB1 was required for the nuclear import of CRBN and for the CRBN-directed, pomalidomide-dependent degradation of a clinically relevant substrate, the transcription factor Aiolos. By contrast, the cytoplasmic translation factor GSPT1 was degraded following treatment with the thalidomide derivative CC-885 only when CRBN was present in the cytoplasm, indicating that subcellular distribution of CRBN is critical for the efficacy of thalidomide-based medications.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
shRNA library screen for discovery of pomalidomide-affecting genes. (a) Outline of shRNA library screen. OPM-2 cells were infected with lentiviral shRNA libraries, split, and cultured with or without 1 μM pomalidomide for 5 days. Genomic DNA was extracted, and the abundance of each barcode, representing the number of cells expressing each shRNA, was quantified by high-throughput sequencing. (b) Scatter plot showing the results of shRNA library screening. Blue dots represent pomalidomide-affecting genes. Red dots represent CRL4CRBN subunits and importin-β family members identified by the screen. (c) Venn diagram showing the number of pomalidomide-affecting genes identified by two statistical criteria. (d) Illustration depicting the CRBN-dependent ubiquitin-proteasome pathway and its regulatory factors. Genes identified by the screen are colored in red.
Figure 2
Figure 2
Validation experiments for Pomalidomide-affecting genes. (a) Calculation of the resistance index. An equal number of shRNA-transduced RFP-positive cells and control GFP-positive cells were mixed and incubated for 4 (OPM-2) or 6 (MM1.S and H929) days in the presence or absence of 1 µM pomalidomide (Pom). Fluorescent live cells were counted using an image-based cytometer, and the resistance index was calculated as indicated. (b–d) Bar graphs indicating the resistance indices of shRNAs identified by the screen. Error bars represent standard deviations (S.D.) (n = 3). *P < 0.05, **P < 0.01 (Student’s t-test).
Figure 3
Figure 3
KPNB1, but not IPO11, is important for nuclear import of CRBN. (a,c) Immunofluorescence microscopy analysis of CRBN-knockout OPM-2 cells stably expressing FLAG-HA (FH)-CRBN. Following lentiviral transduction of shRNA targeting KPNB1 or IPO11, or treatment with 1 µM pomalidomide for 48 h, the cells were stained with anti-HA, anti-Aiolos, or anti-IRF4 antibody (red) and counterstained with DAPI (blue). Scale bar, 10 µm. (b) Immunofluorescence microscopy analysis was performed using CRBN-knockout 293 T cells stably expressing FH-CRBN. Following transient transfection of Aiolos- and shRNA-expression vectors, the resultant cells were subjected to immunofluorescence staining. (d) Immunoblot analysis of 293 T cells transiently expressing Aiolos and the indicated shRNAs. The cells were treated with the indicated concentrations of pomalidomide for 12 h before analysis. e, Control GST or GST-KNPB1 coupled to glutathione beads was incubated with lysates of High Five insect cells overproducing the indicated proteins. Input (10%) and bound proteins were subjected to immunoblot analysis.
Figure 4
Figure 4
Spatial overlaps of CRBN and its substrates are important for the efficacy of CRBN modulators. (a) CRL4CRBN was immunoprecipitated (IP) using anti-FLAG beads from CRBN-knockout 293 T cells expressing FH-CRBN, FH-NLS-CRBN, or FH-NES-CRBN, and their subunit compositions were examined by immunoblot analysis. Asterisks denote nonspecific signals. (b) Immunofluorescence microscopy analysis of CRBN-knockout 293 T cells transiently expressing Aiolos and FH-CRBN. Cells were stained with anti-HA (red), anti-Aiolos (green), and DAPI (blue). Scale bar, 10 µm. (c,d) CRBN-knockout 293 T cells expressing Aiolos and FH-CRBN, FH-NLS-CRBN, or FH-NES-CRBN were treated with the indicated concentrations of pomalidomide for 12 h (c) or CC-885 for 24 h (d), and then subjected to immunoblot analysis using the indicated antibodies. (e) CRBN-knockout 293 T cells and the knockout cells re-expressing wild-type (WT), NLS-fused, or NES-fused CRBN were treated with the indicated concentrations of CC-885 for 48 h. Cell viability was assayed, normalized against the number of untreated cells, and displayed as the mean ± S.D. of three independent experiments.

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