CoChat
Sign Up
Cited by 36Protein Degradation and InhibitorsUbiquitin and proteasome pathwaysMultiple Myeloma Research and Treatments

Genome-wide screening reveals a role for subcellular localization of CRBN in the anti-myeloma activity of pomalidomide

Open access
Tateno, Shumpei, Iida, Midori, Fujii, Satoshi, Suwa, Tetsufumi, Katayama, Miki, Tokuyama, Haruka, Yamamoto, Junichi, Ito, Takumi +3 more·2020·Scientific Reports
DOI 10.1038/s41598-020-61027-wPMC PMC7055313
Open this paper in CoChat — free accountOpen PDF

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 CRL4 CRBN. 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 CRL4 CRBN 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.

AI snapshot

Written from the paper’s full text.

TL;DR

A genome-wide shRNA screen in OPM-2 multiple myeloma cells identified 445 genes affecting pomalidomide sensitivity, including CRL4CRBN and ubiquitin-proteasome components. The study further shows that KPNB1 mediates nuclear import of CRBN, and that CRBN’s subcellular localization determines whether pomalidomide- or CC-885-dependent substrates are degraded.

Key findings

  • The screen used 82,500 shRNAs targeting 15,377 human genes and identified 445 pomalidomide-affecting genes: 141 with |log2(maxFC)| > log2(3), 360 with FDR < 0.1, and 56 meeting both criteria.
  • CRL4CRBN subunits and ubiquitin-proteasome pathway components were enriched, and KPNB1 was identified as a novel pomalidomide-affecting gene. KPNB1 knockdown decreased nuclear FH-CRBN but did not affect nuclear localization of Aiolos or IRF4, and prevented pomalidomide-induced Aiolos degradation.
  • Subcellular localization of CRBN was critical: NLS-fused CRBN supported pomalidomide-induced Aiolos degradation but not CC-885-induced GSPT1 degradation, whereas NES-fused CRBN supported GSPT1 degradation but not Aiolos degradation. Cells expressing NLS-fused CRBN were resistant to CC-885 growth inhibition.
  • GST pull-down assays showed KPNB1 interacts with CRBN and, to a lesser extent, CUL4A and ROC1.

Methods

  • Pooled lentiviral shRNA libraries (Cellecta Human Modules 1–3) were used to infect OPM-2 cells at MOI 0.3; cells were treated with 1 µM pomalidomide or vehicle for 5 days, and barcodes were quantified by high-throughput sequencing on a HiSeq 2000 with approximately 300 million reads per sample. Genes were called using maxFC and a modified rank product method for FDR.
  • Validation used individual shRNAs in OPM-2, MM1.S, and H929 cells, with competition assays, quantitative RT-PCR, immunoblotting, immunofluorescence, CRISPR/Cas9 CRBN-knockout OPM-2 and 293T cells, GST pull-down assays, and cell viability assays.
  • GO enrichment analysis of the 445 genes was performed with DAVID v. 6.8.

Limitations

  • The paper states that how individual genes identified by the screen affect pomalidomide sensitivity remains largely unknown and is the subject of future study; the precise role of IPO11 is also unclear. It also notes that it cannot exclude the possibility that KPNB1 imports CRBN indirectly through another CRL4CRBN component, and that CRBN subcellular distribution varies among cell lines and may be influenced by pathological or physiological conditions.
  • Evident from the text: the primary screen was performed only in OPM-2 cells using shRNA knockdown rather than CRISPR knockout, although validation was done in additional myeloma cell lines; several mechanistic experiments used 293T overexpression systems, and no patient-derived samples were analyzed.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41598-020-61027-w

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 63 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Knockdown of a subset of shRNAs conferred resistance to pomalidomide in MM1.S and H929 multiple myeloma cell lines.

    “we repeated the experiments using a subset of the shRNAs in two other multiple myeloma cell lines, namely MM1.S and H929. Since components of CRL4CRBN and the COP9 signalosome were independently identified in similar genetic screens using other cell lines17,18, they were removed from further analysis. Consequently, similar results were obtained (Fig. 2c,d)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The phenotypic effects of the shRNAs used are due to knockdown of their intended target genes and not to off-target effects.
  • Findingown data

    Nuclear localization of Aiolos was not affected by either KPNB1 or IPO11 knockdown.

    “On the other hand, nuclear localization of Aiolos was not affected by either knockdown.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The phenotypic effects of the shRNAs used are due to knockdown of their intended target genes and not to off-target effects.
  • Findingown data

    KPNB1 was identified as a novel pomalidomide-affecting gene.

    “Karyopherin beta 1 (KPNB1) was identified as a novel pomalidomide-affecting gene.”
    • Also stated in: § KPNB1 is important for nuclear import of CRBN and pomalidomide activity · plain
      “members of the importin-β family19, such as importin 11 (IPO11) and karyopherin beta 1 (KPNB1), were also enriched (GO:0006610).”
    Quote in the paperSubstantial claim
  • Findingown data

    KPNB1 is required for CRBN-directed, pomalidomide-dependent degradation of Aiolos.

    “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.”
    • Also stated in: § KPNB1 is important for nuclear import of CRBN and pomalidomide activity · plain
      “Although Aiolos was substantially degraded following pomalidomide treatment in control cells, the Aiolos level was not significantly decreased by pomalidomide in KPNB1 knockdown cells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The phenotypic effects of the shRNAs used are due to knockdown of their intended target genes and not to off-target effects.
  • Findingown data

    GSPT1 was degraded following treatment with CC-885 only when CRBN was present in the cytoplasm.

    “the cytoplasmic translation factor GSPT1 was degraded following treatment with the thalidomide derivative CC-885 only when CRBN was present in the cytoplasm”
    • Also stated in: § Subcellular localization of CRBN affects the efficacy of CRBN modulators · plain
      “When these cells were treated with CC-885, GSPT1, a CC-885–dependent CRBN substrate localized in the cytoplasm, was degraded in cells expressing wild-type or NES-fused CRBN, but not in cells expressing NLS-fused CRBN (Fig. 4d)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The NLS and NES tags do not alter CRBN's function other than its subcellular localization.
  • May combine two claims. 16
  • Classified as probably not a substantial claim. 18

  • May combine two claims.

    Findingown data

    A genome-wide shRNA library screen identified 445 genes affecting pomalidomide sensitivity, with 141 genes having |log2(maxFC)| > log2(3), 360 genes having FDR < 0.1, and 56 genes meeting both criteria.

    “we performed a genome-wide shRNA library screen and identified 445 genes as those affecting pomalidomide sensitivity.”
    • Also stated in: § Identification of pomalidomide-affecting genes by shRNA library screening · plain
      “Based on FDR and maxFC, 445 genes were identified by at least one criterion: 141 genes had |log2(maxFC)| > log2(3) in pomalidomide-treated vs. untreated samples, and 360 genes had FDR < 0.1; 56 genes met both criteria (Fig. 1c and Supplementary Table 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Under control conditions, FH-CRBN was present in both the nucleus and the cytoplasm, whereas Aiolos was localized in the nucleus; CRBN was partially colocalized with Aiolos in the nucleus.

    “Immunofluorescence microscopy revealed that under control conditions, FH-CRBN was present in both the nucleus and the cytoplasm, whereas Aiolos was localized in the nucleus, i.e., CRBN was partially colocalized with Aiolos in the nucleus (Fig. 3a).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The subcellular localization of FH-CRBN reflects that of endogenous CRBN.

  • May combine two claims.

    Findingown data

    A fraction of both wild-type and NLS-fused CRBN was imported into the nucleus and overlapped with Aiolos.

    “Immunofluorescence microscopy analysis revealed that a fraction of both wild-type and NLS-fused CRBN was imported into the nucleus and overlapped with Aiolos (Fig. 4b).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The NLS and NES tags do not alter CRBN's function other than its subcellular localization.

  • May combine two claims.

    Findingown data

    KPNB1 interacts with CRBN and, to a lesser extent, CUL4A and ROC1.

    “Pull-down assays using GST-tagged KPNB1 and CRL4CRBN subunits individually overproduced using the baculovirus expression system showed that KPNB1 interacts with CRBN and, to a lesser extent, CUL4A and ROC1 (Fig. 3e)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    KPNB1 is critical for the nuclear import of CRBN, but not of Aiolos and IRF4.

    “We showed that KPNB1 is critical for the nuclear import of CRBN, but not of Aiolos and IRF4 (Fig. 3a–c), suggesting that CRL4CRBN is a primary cargo of KPNB1 in the context of regulating pomalidomide activity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    CRBN was predicted to be nuclear using both PSORT II and PredictNLS.

    “Using PSORT II, a program for predicting protein subcellular localization sites22, no putative NLS was identified in CRBN; however, CRBN was predicated to be nuclear using both PSORT II and PredictNLS.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Nakai, K. & Horton, P. PSORT: a program for detecting the sorting signals of proteins and predicting their subcellular localization. Trends Biochem. Sci. 24, 34–35 (1999). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The shRNA screen identified 445 pomalidomide-affecting genes, including CRBN and other components of the ubiquitin-proteasome pathway such as ROC1, DDB1, NEDD8, COP9 signalosome subunits, and 26S proteasome subunits.

    “Our screen identified 445 pomalidomide-affecting genes, including CRBN and other components of the ubiquitin-proteasome pathway, such as ROC1, DDB1, NEDD8, subunits of the COP9 signalosome, and subunits of the 26S proteasome.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    KPNB1 was identified only in our screen.

    “On the other hand, KPNB1 was identified only in our screen.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    NES-fused CRBN was found only in the cytoplasm and did not overlap with Aiolos.

    “By contrast, NES-fused CRBN was found only in the cytoplasm and did not overlap with Aiolos.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The NLS and NES tags do not alter CRBN's function other than its subcellular localization.

  • May combine two claims.

    Findingown data

    In OPM-2 and 293T cells, CRBN is detected in both the cytoplasm and the nucleus, with slight accumulation in the nucleus.

    “In OPM-2 and 293T cells, we detected CRBN in both the cytoplasm and the nucleus, with slight accumulation in the nucleus (Fig. 3a,b).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lenalidomide induces degradation of CK1α, a clinical target of 5q- syndrome.

    “Lenalidomide also induces degradation of CK1α, a clinical target of 5q– syndrome11,12.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523 (2015). source

      Not checked in the free run.

    • cites [12] Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CC-885 induces degradation of translation factor GSPT1, thereby eliciting broad-spectrum growth inhibition against a variety of cancer cell lines.

    “CC-885 induces degradation of the translation factor GSPT1, thereby eliciting broad-spectrum growth inhibition against a variety of cancer cell lines3.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252–257 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CC-885 is a newly developed compound with potent antitumor activity against hematological and epithelial cancers.

    “CC-885 is a newly developed compound with potent antitumor activity against not only hematological cancers but also epithelial cancers3.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252–257 (2016). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    CRBN is a primary target of thalidomide and is responsible for the various pharmacological activities of the drug and related compounds.

    “CRBN is a primary target of thalidomide and is responsible for the various pharmacological activities of the drug and related compounds1,4,5.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [1] Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–50 (2010). source

      Not checked in the free run.

    • cites [4] Zhu, Y. X. et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood 118, 4771–4780 (2011). source

      Not checked in the free run.

    • cites [5] Lopez-Girona, A. et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia 26, 2326–2335 (2012). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    CRBN enables its various modulators to induce their pharmacological effects by recruiting specific substrates to CRL4CRBN.

    “CRBN enables its various modulators to induce their pharmacological effects by recruiting specific substrates to CRL4CRBN.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cullin-based ubiquitin ligase activity is stimulated by neddylation and downregulated by deneddylation mediated by the COP9 signalosome.

    “Cullin-based ubiquitin ligase activity is stimulated by neddylation (i.e., NEDD8 conjugation) to cullin and downregulated by deneddylation mediated by the COP9 signalosome6,7.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [6] Soucy, T. A., Dick, L. R., Smith, P. G., Milhollen, M. A. & Brownell, J. E. The NEDD8 Conjugation Pathway and Its Relevance in Cancer Biology and Therapy. Genes Cancer 1, 708–16 (2010). source

      Not checked in the free run.

    • cites [7] Cope, G. A. & Deshaies, R. J. COP9 signalosome: A multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell 114, 663–671 (2003). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Each CRBN modulator acts as a molecular glue that mediates the interaction between CRBN and specific substrates.

    “each CRBN modulator acts as a molecular “glue” that mediates the interaction between CRBN and specific substrates16.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Ito, T. & Handa, H. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs. Int. J. Hematol. 104, 293–299 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Thalidomide possesses immunomodulatory effects and therapeutic potential against leprosy and cancer.

    “subsequent studies revealed that thalidomide possesses immunomodulatory effects and has therapeutic potential against leprosy and cancer1,2.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [1] Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–50 (2010). source

      Not checked in the free run.

    • cites [2] Melchert, M. & List, A. The thalidomide saga. Int. J. Biochem. Cell Biol. 39, 1489–1499 (2007). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Lenalidomide and pomalidomide induce CRBN-dependent ubiquitination and degradation of Ikaros and Aiolos, transcription factors required for development of lymphoids and survival of multiple myeloma.

    “lenalidomide and pomalidomide induce CRBN-dependent ubiquitination and degradation of Ikaros and Aiolos, transcription factors required for the development of lymphoids and survival of multiple myeloma8–10.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [8] Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–5 (2014). source

      Not checked in the free run.

    • cites [9] Lu, G. et al. The Myeloma Drug Lenalidomide Promotes the Cereblon-Dependent Destruction of Ikaros Proteins. Science 343, 305–309 (2013). source

      Not checked in the free run.

    • cites [10] Gandhi, A. K. et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4 CRBN. Br. J. Haematol. 164, 811–821 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The transcription factors Ikaros, Aiolos, and MEIS2 are localized to the nucleus.

    “The subcellular localizations of these factors differ: the transcription factors Ikaros, Aiolos, and MEIS2 are localized to the nucleus, whereas GSPT1, CK1α, and glutamine synthetase reside in the cytoplasm.”
    Quote in the paperSubstantial claim0 of 10 sources checked
    • cites [3] Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252–257 (2016). source

      Not checked in the free run.

    • cites [11] Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523 (2015). source

      Not checked in the free run.

    • cites [12] Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016). source

      Not checked in the free run.

    • cites [13] Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. Elife 7, e38430 (2018). source

      Not checked in the free run.

    • cites [14] Matyskiela, M. E. et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 14, 981–987 (2018). source

      Not checked in the free run.

    • cites [15] Asatsuma-Okumura, T. et al. p63 is a cereblon substrate involved in thalidomide teratogenicity. Nat. Chem. Biol, https://doi.org/10.1038/s41589-019-0366-7. source

      Not checked in the free run.

    • cites [16] Ito, T. & Handa, H. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs. Int. J. Hematol. 104, 293–299 (2016). source

      Not checked in the free run.

    • cites [24] Nguyen, T. V. et al. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon. Mol. Cell 61, 809–820 (2016). source

      Not checked in the free run.

    • cites [25] Fischer, E. S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014). source

      Not checked in the free run.

    • cites [26] Liu, J. et al. CRL4ACRBN E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis. Nat. Commun. 5, 1–9 (2014).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Over a dozen proteins, including Ikaros, Aiolos, GSPT1, CK1α, glutamine synthetase, MEIS2, KCNMA1/SLO1, CLC1, and AMPKα, have been identified as substrates for CRBN.

    “To date, over a dozen proteins including Ikaros, Aiolos, GSPT1, CK1α, glutamine synthetase, MEIS2, KCNMA1/SLO1, CLC1, and AMPKα have been identified as substrates for CRBN3,11–16,24–28.”
    Quote in the paperNot a substantial claim0 of 10 sources checked
    • cites [3] Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252–257 (2016). source

      Not checked in the free run.

    • cites [11] Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523 (2015). source

      Not checked in the free run.

    • cites [12] Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016). source

      Not checked in the free run.

    • cites [13] Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. Elife 7, e38430 (2018). source

      Not checked in the free run.

    • cites [14] Matyskiela, M. E. et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 14, 981–987 (2018). source

      Not checked in the free run.

    • cites [15] Asatsuma-Okumura, T. et al. p63 is a cereblon substrate involved in thalidomide teratogenicity. Nat. Chem. Biol, https://doi.org/10.1038/s41589-019-0366-7. source

      Not checked in the free run.

    • cites [16] Ito, T. & Handa, H. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs. Int. J. Hematol. 104, 293–299 (2016). source

      Not checked in the free run.

    • cites [24] Nguyen, T. V. et al. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon. Mol. Cell 61, 809–820 (2016). source

      Not checked in the free run.

    • cites [25] Fischer, E. S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014). source

      Not checked in the free run.

    • cites [26] Liu, J. et al. CRL4ACRBN E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis. Nat. Commun. 5, 1–9 (2014).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Each importin-β family member is thought to recognize and import a partially overlapping but distinct set of cargo proteins.

    “The importin-β family includes at least 22 members in humans, and each member is thought to recognize and import a partially overlapping but distinct set of cargo proteins19.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Ström, A. C. & Weis, K. Importin-beta-like nuclear transport receptors. Genome Biol. 2, reviews 3008.1–3008.9 (2001). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    GSPT1, CK1α, and glutamine synthetase reside in the cytoplasm.

    “The subcellular localizations of these factors differ: the transcription factors Ikaros, Aiolos, and MEIS2 are localized to the nucleus, whereas GSPT1, CK1α, and glutamine synthetase reside in the cytoplasm.”
    Quote in the paperSubstantial claim0 of 10 sources checked
    • cites [3] Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase. Nature 535, 252–257 (2016). source

      Not checked in the free run.

    • cites [11] Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523 (2015). source

      Not checked in the free run.

    • cites [12] Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016). source

      Not checked in the free run.

    • cites [13] Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. Elife 7, e38430 (2018). source

      Not checked in the free run.

    • cites [14] Matyskiela, M. E. et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 14, 981–987 (2018). source

      Not checked in the free run.

    • cites [15] Asatsuma-Okumura, T. et al. p63 is a cereblon substrate involved in thalidomide teratogenicity. Nat. Chem. Biol, https://doi.org/10.1038/s41589-019-0366-7. source

      Not checked in the free run.

    • cites [16] Ito, T. & Handa, H. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs. Int. J. Hematol. 104, 293–299 (2016). source

      Not checked in the free run.

    • cites [24] Nguyen, T. V. et al. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon. Mol. Cell 61, 809–820 (2016). source

      Not checked in the free run.

    • cites [25] Fischer, E. S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014). source

      Not checked in the free run.

    • cites [26] Liu, J. et al. CRL4ACRBN E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis. Nat. Commun. 5, 1–9 (2014).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The importin-β family includes at least 22 members in humans.

    “The importin-β family includes at least 22 members in humans, and each member is thought to recognize and import a partially overlapping but distinct set of cargo proteins19.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Ström, A. C. & Weis, K. Importin-beta-like nuclear transport receptors. Genome Biol. 2, reviews 3008.1–3008.9 (2001). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    KPNB1 is critical for the nuclear import of CRBN, but not of Aiolos and IRF4, and CRBN does not spatially overlap with its substrate Aiolos in the absence of KPNB1.

    “Thus, KPNB1 is critical for the nuclear import of CRBN, but not of Aiolos and IRF4, and CRBN does not spatially overlap with its substrate Aiolos in the absence of KPNB1.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The phenotypic effects of the shRNAs used are due to knockdown of their intended target genes and not to off-target effects.
    • Rests on an assumption: The subcellular localization of FH-CRBN reflects that of endogenous CRBN.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The two studies identified UBE2D3 and UBE2G1 as E2 ubiquitin-conjugating enzymes specific to CRL4CRBN.

    “The two studies also identified UBE2D3 and UBE2G1 as E2 ubiquitin-conjugating enzymes specific to CRL4CRBN.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [17] Sievers, Q. L., Gasser, J. A., Cowley, G. S., Fischer, E. S. & Ebert, B. L. Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity. Blood 132, 1293–1303 (2018). source

      Not checked in the free run.

    • cites [18] Patil, A., Manzano, M. & Gottwein, E. Genome-wide CRISPR screens reveal genetic mediators of cereblon modulator toxicity in primary effusion lymphoma. Blood Adv. 3, 2105–2117 (2019). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Two recent studies independently conducted genome-wide CRISPR knockout screens to identify factors affecting the efficacy of lenalidomide.

    “Two recent studies independently conducted genome-wide CRISPR knockout screens to identify factors affecting the efficacy of lenalidomide17,18.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [17] Sievers, Q. L., Gasser, J. A., Cowley, G. S., Fischer, E. S. & Ebert, B. L. Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity. Blood 132, 1293–1303 (2018). source

      Not checked in the free run.

    • cites [18] Patil, A., Manzano, M. & Gottwein, E. Genome-wide CRISPR screens reveal genetic mediators of cereblon modulator toxicity in primary effusion lymphoma. Blood Adv. 3, 2105–2117 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    For CRL4DDB2, DDB2 plays a key role in its nuclear translocation and accumulation to sites of DNA damage in response to UV irradiation.

    “For CRL4DDB2, the CRL4-based E3 ligase complex into which DDB2 is incorporated as a substrate receptor, DDB2 plays a key role in its nuclear translocation and accumulation to the sites of DNA damage in response to UV irradiation23.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Alekseev, S. et al. Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage. Mol. Cell. Biol. 28, 7402–7413 (2008). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    KCNMA1/SLO1 and CLC1 are transmembrane proteins whose CRBN-mediated degradation is thought to occur during membrane trafficking.

    “The potassium channel KCNMA1/SLO1 and the chloride channel CLC1 are transmembrane proteins whose CRBN-mediated degradation is thought to occur during membrane trafficking26,27.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [26] Liu, J. et al. CRL4ACRBN E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis. Nat. Commun. 5, 1–9 (2014).

      Not checked in the free run.

    • cites [27] Chen, Y.-A. et al. The Cullin 4A/B-DDB1-Cereblon E3 ubiquitin ligase complex mediates the degradation of CLC-1 chloride channels. Sci. Rep. 5, 10667 (2015). source

      Not checked in the free run.

29 of 29

  • The paper relies on this without support.

    The phenotypic effects of the shRNAs used are due to knockdown of their intended target genes and not to off-target effects.

    “Knockdown efficiency of the individual shRNAs employed was evaluated by quantitative RT-PCR following transduction of lentiviral vectors expressing shRNAs into OPM-2 cells (Supplementary Fig. 1).”

  • The paper relies on this without support.

    The subcellular localization of FH-CRBN reflects that of endogenous CRBN.

    “We made CRBN-knockout OPM-2 cells using the CRISPR/Cas9 system and transduced them with a lentiviral vector expressing FLAG- and HA-tagged CRBN (FH-CRBN) for imaging.”

  • The paper relies on this without support.

    The NLS and NES tags do not alter CRBN's function other than its subcellular localization.

    “Coimmunoprecipitation study showed that ectopically expressed CRBN proteins were incorporated into CUL4-based E3 ligase complexes (Fig. 4a).”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Several functional genomics campaigns have helped map how the repertoire of CRLs and their regulators shape degrader efficacy, outlining putative resistance mechanisms.

    “Several functional genomics campaigns in recent years have helped map how the repertoire of CLRs and their regulators shape degrader efficacy, thus also outlining putative resistance mechanisms.141-151”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [147]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • OPM-2 validation incubation is reported as both 6 and 4 days

    Major

    The validation experiment described here uses OPM-2 cells and says they were incubated for 6 days. The Figure 2 caption specifies 4 days for OPM-2 cells and 6 days only for MM1.S and H929 cells.

  • RNA-splicing GO accession differs from Table 1

    Minor

    The text identifies RNA splicing with accession GO:008380, whereas Table 1 assigns it GO:0008380.

2 of 2

The citation lists haven’t been fetched yet

References 35 · Cited by 36

Authors
Tateno, S., Iida, M., Fujii, S., Suwa, T., Katayama, M., Tokuyama, H., Yamamoto, J., Ito, T., Sakamoto, S., Handa, H., & Yamaguchi, Y.
Affiliations
  • Tokyo Institute of Technology (JP)
  • Kyushu Institute of Technology (JP)
  • Tokyo Medical University (JP)
Corresponding author
Yamaguchi, Yuki
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-03-04
Volume
10
Issue
1
Pages
4012-4012
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-020-61027-w
PMC
PMC7055313
OpenAlex
W3010312076
Semantic Scholar
a25819e1bc06ef9ea2ee93aaf9b9a90a1bb119aa
Cited by
36
References
35
Citations by year
2026: 2 · 2025: 6 · 2024: 5 · 2023: 4 · 2022: 8 · 2021: 10 · 2020: 1
FWCI
1.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • MEXT | Japan Society for the Promotion of Science (KAKENHI 24118008, KAKENHI 17K14996, KAKENHI 17H04213, KAKENHI 18H05502, KAKENHI 17H06112, KAKENHI 24118002)
  • MEXT | JST | Precursory Research for Embryonic Science and Technology (JPMJPR1531)
  • Ministry of Education, Culture, Sports, Science and Technology (Strategic Research Foundation at Private Universities S1411011)
  • Takeda Science Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Health Sciences › Medicine › Hematology › Multiple Myeloma Research and Treatments
Keywords
pomalidomide, cereblon, multiple myeloma, subcellular localization, KPNB1, nuclear import, GSPT1, thalidomide derivatives, ubiquitin-proteasome system, COP9 signalosome, proteasome, drug sensitivity
MeSH terms
Humans, Multiple Myeloma, Neoplasm Proteins, Phenylurea Compounds, Thalidomide, Ubiquitin-Protein Ligases, Cell Line, Tumor, Adaptor Proteins, Signal Transducing, Genome-Wide Association Study

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review