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Cited by 270Protein Degradation and InhibitorsUbiquitin and proteasome pathwaysChemical Synthesis and Analysis

Small-molecule-induced polymerization triggers degradation of BCL6

Open access
Słabicki, Mikołaj, Yoon, Hojong, Koeppel, Jonas, Nitsch, Lena, Roy Burman, Shourya S., Di Genua, Cristina, Donovan, Katherine A., Sperling, Adam S. +13 more·2020·Nature
DOI 10.1038/s41586-020-2925-1PMC PMC7816212
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Abstract

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets 1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches 4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL6 6. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

AI snapshot

Written from the paper’s full text.

TL;DR

BI-3802 binds the BCL6 BTB domain and induces reversible polymerization into filaments and cellular foci, leading to SIAH1-dependent ubiquitination and proteasomal degradation of BCL6. This drug-induced polymerization mechanism yields superior BCL6 degradation and anti-proliferative activity compared with non-degrading BCL6 inhibitors or BCL6 PROTACs.

Key findings

  • In SuDHL4 cells, quantitative mass spectrometry after 4 h treatment showed BCL6 was the only protein with significantly decreased abundance upon BI-3802 treatment; the structurally related inhibitor BI-3812 did not alter protein abundance.
  • Cryo-EM of BI-3802-induced BCL6 filaments gave a nominal 3.7 Å reconstruction (EMD-22265, PDB-6XMX), with BI-3802 at the dimer-dimer interface contacting Y58 and C84, plus an R28–E41 interaction; mutations Y58A, R28A, E41A, and C84A impaired foci formation, and E41A did not impair drug binding.
  • SIAH1 was the only gene scoring significantly in both genome-scale CRISPR-Cas9 screens; BCL6 residues 249–251 contain a VxP motif required for SIAH1 interaction, and BI-3802 enhanced BCL6–SIAH1 interaction (EC50 = 64 nM) and polymerized BCL6 affinity (KDapp = 0.2 μM), accelerating ubiquitination.
  • BI-3802-induced degradation required the 26S proteasome and UBA1 but not neddylation; the first 275 amino acids of BCL6 were sufficient for degradation, whereas BCL61–250 formed drug-induced foci but was not degraded.

Methods

  • Cell-based assays used HEK293T, SuDHL4, Raji, and DEL cells with eGFP-BCL6/mCherry reporters, flow cytometry, live-cell imaging, immunofluorescence, immunoblots, qPCR, and whole-proteome tandem mass tag mass spectrometry.
  • Structural and biochemical work used recombinant BCL6 and SIAH1 proteins, negative-stain EM, cryo-EM single-particle analysis, Rosetta docking, ITC, TR-FRET, BRET, and in vitro ubiquitination assays.
  • Genetic screens included genome-scale CRISPR-Cas9 Brunello screens in HEK293T and SuDHL4 cells, a targeted BISON ubiquitin-ligase library, and an alanine scan of BCL6 BTB residues 32–99.

Limitations

  • The paper states that cryo-EM 2D class averages showed flexibility and variable helical pitch, making the data unsuitable for helical processing; single-particle analysis had significant preferred orientations and anisotropic resolution, and the final map was nominally 3.7 Å but model resolution at FSC = 0.5 was 8.1 Å, with side-chain placement approximated from crystal structure 5MW2.
  • Evident from the text: several supporting experiments are reported with n = 1 or n = 2, including some ITC measurements, the E2 enzyme ubiquitination screen, and the BISON CRISPR screen, so those specific mechanistic supports are less statistically robust.
  • Evident from the text: the structural conclusions depend on fitting the BCL6–BI-3802 crystal structure into an anisotropic cryo-EM map, which limits independent validation of side-chain-level details in the filament.

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

Integrity signals

Registry

Crossref · 10.1038/s41586-020-2925-1

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 37 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    BI-3802 results in proteasomal degradation of BCL6.

    “BI-3802 is a small molecule that binds the BTB domain of the oncogenic transcription factor BCL6 and results in proteasomal degradation6.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Kerres, N. et al. Chemically induced degradation of the oncogenic transcription factor BCL6. Cell Rep. 20, 2860–2875 (2017). source

      Not checked in the free run.

  • Findingown data

    The solvent-exposed moiety of a BCL6 inhibitor contributes to a composite ligand/protein surface that engages BCL6 homodimers to form a supramolecular structure.

    “We used cryo-EM to reveal how the solvent-exposed moiety of a BCL6 inhibitor contributes to a composite ligand/protein surface that engages BCL6 homodimers to form a supramolecular structure.”
    Quote in the paperSubstantial claim
  • Findingown data

    Mutations R28A or E41A prevented foci formation upon BI-3802 treatment.

    “mutations R28A or E41A prevented foci formation upon BI-3802 treatment”
    Quote in the paperSubstantial claim
  • Findingown data

    At the interface between BCL6 dimers, density representing BI-3802 was observed, overlapping with the location of the compound in the crystal structure.

    “At the interface between BCL6 dimers, we observed density representing BI-3802, overlapping with the location of the compound in the crystal structure”
    Quote in the paperSubstantial claim
  • Findingown data

    BI-3812 did not alter the abundance of any protein.

    “Treatment with the structurally similar BCL6 inhibitor BI-3812 (Fig. 1a) did not alter the abundance of any protein (Extended Data Fig. 1c).”
    Quote in the paperSubstantial claim
  • May combine two claims. 40
  • Classified as probably not a substantial claim. 14
  • The quote only partly states this claim. 2

  • May combine two claims.

    Findingown data

    BI-3802 binds a groove between BCL6 dimers, directly in contact with Y58 of BTB α, and facilitates higher order assembly through hydrophobic interactions of the compound with C84 on an adjacent BCL6 dimer.

    “BI-3802 binds a groove between BCL6 dimers, directly in contact with Y58 of BTB α, and facilitates higher order assembly through hydrophobic interactions of the compound with C84 on an adjacent BCL6 dimer”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the absence of BI-3802, BCL6 is present as monodisperse particles; upon incubation with BI-3802, regular structures with a sinusoidal shape form.

    “In the absence of BI-3802, BCL6 is present as monodisperse particles. However, upon incubation of BCL6 with BI-3802, we observed the formation of regular structures with a sinusoidal shape”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BI-3802-induced degradation of eGFPBCL6FL was attenuated by chemical inhibition of the 26S proteasome with MG132 or inhibition of UBA1 by MLN7243, but not by inhibition of the neddylation pathway with MLN4924.

    “BI-3802-induced degradation of eGFPBCL6FL, was attenuated by chemical inhibition of the 26S proteasome with MG132 or inhibition of the ubiquitin activating enzyme UBA1 by MLN7243, but not by inhibition of the neddylation pathway with MLN4924”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BI-3802 induced degradation of the full-length BCL6 reporter, while BI-3812 did not alter stability of the reporter.

    “BI-3802 induced degradation of the full-length BCL6 reporter, while the inhibitor, BI-3812, did not alter stability of the reporter.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The eGFP signal and foci subsequently disappeared, consistent with BCL6 degradation.

    “The eGFP signal and foci subsequently disappeared, consistent with BCL6 degradation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutation of Y58 to alanine in BCL6 prevents BI-3802 binding in vitro and consequently BCL6 foci formation in cells.

    “Mutation of Y58 to alanine in BCL6 prevents BI-3802 binding in vitro (Extended Data Fig. 4f) and consequently BCL6 foci formation in cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Presence of BI-3802, but not BI-3812, led to higher molecular weight species of BCL6 during purification.

    “the presence of BI-3802, but not BI-3812, led to higher molecular weight species of BCL6”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overexpression of E41A, G55A, Y58A, and C84A variants in BCL6-dependent SuDHL4 and Raji cell lines conferred resistance to BI-3802 but had no effect in the BCL6-independent DEL cell line.

    “Overexpression of these variants in the BCL6-dependent SuDHL4 and Raji cell lines conferred resistance to BI-3802 but had no effect in the BCL6-independent DEL cell line”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BI-3802 induces polymerization of the BCL6-BTB domain and foci formation in cells while BI-3812 does not.

    “BI-3802 induces polymerization of the BCL6-BTB domain and foci formation in cells while BI-3812 does not.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    C84 forms a hydrophobic interaction with the methyl group of the neighboring BI-3802 molecule, and a C84A mutation significantly reduced foci formation in cells.

    “C84 forms a hydrophobic interaction with the methyl group of the neighboring BI-3802 molecule (Fig. 2e), and a C84A mutation significantly reduced foci formation in cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The residues identified in the unbiased mutagenesis experiment were located either close to the drug-binding site (G55A, Y58A) or on the polymerization interface (E41A, C84A).

    “the residues identified in this unbiased mutagenesis experiment were located either close to the drug-binding site (G55A, Y58A), likely preventing drug-binding, or on the polymerization interface (E41A, C84A)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In vitro ubiquitination assays with recombinant proteins demonstrated that BCL6 is a substrate for SIAH1, and that the rate and magnitude of ubiquitination is accelerated by BI-3802.

    “In vitro ubiquitination assays with recombinant proteins demonstrated that BCL6 is a substrate for SIAH1 (Extended Data Fig. 8d), and that the rate and magnitude of ubiquitination is accelerated by BI-3802”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Targeting SIAH1 with multiple independent sgRNAs attenuated eGFPBCL6FL degradation upon BI-3802 treatment and induced resistance to BI-3802 treatment.

    “Each sgRNA attenuated eGFPBCL6FL degradation upon BI-3802 treatment and induced resistance to BI-3802 treatment”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with an excess of BI-3812 shortly before protein harvest reverts polymerization, solubilizes BCL6, and allows reliable quantification.

    “treatment with an excess of BI-3812 shortly before protein harvest reverts polymerization, solubilized BCL6, and allowed for reliable quantification”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overexpression of wild-type SIAH1 enhanced BI-3802-dependent BCL6 degradation and reduced BCL6 abundance in the absence of drug.

    “Overexpression of wild-type SIAH1 not only enhanced BI-3802-dependent BCL6 degradation, but also reduced BCL6 abundance in the absence of drug”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BCL6 and SIAH1 co-immunoprecipitate in cells and in vitro using recombinant proteins, and mutation or deletion of the VxP motif prevented the co-immunoprecipitation.

    “We found that BCL6 and SIAH1 co-immunoprecipitate in cells (Fig. 3c) and in vitro using recombinant proteins (Extended Data Fig. 8a), and that mutation or deletion of the VxP motif prevented the co-immunoprecipitation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    Multiple subsequent rounds of 3D classification, refinement, and polishing improved map resolution to a final overall resolution of 3.7 Å.

    “Multiple subsequent rounds of 3D classification, refinement, and polishing improved map resolution to a final overall resolution of 3.7 Å.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BI-3802 increased the interaction between BCL6 and SIAH1 (EC50 = 64 nM) both in vitro and in cells, while BI-3812 did not influence the BCL6-SIAH1 interaction, despite comparable affinity of both BI-3802 and BI-3812 to BCL6.

    “We found that BI-3802 increased the interaction between BCL6 and SIAH1 (EC50 = 64 nM) both in vitro (Extended Data Fig. 8f) and in cells (Extended Data Fig. 8g), while BI-3812 did not influence the BCL6-SIAH1 interaction, despite comparable affinity of both BI-3802 and BI-3812 to BCL6”
    Quote in the paperSubstantial claim

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

    Findingown data

    BCL6 mRNA expression (TPM) and BCL6 dependency (CERES score) are correlated in a set of 559 cancer cell lines from the Dependency Map Project.

    “Correlation of BCL6 mRNA expression (TPM) and BCL6 dependency (CERES score) in a set of 559 cancer cell lines from the Dependency Map Project.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Strongly preferred orientation causes a dip in Model vs Map FSC, leading to low model resolution (8.1 Å) estimation at FSC=0.5.

    “Strongly preferred orientation causes dip in Model vs Map FSC, leading to low model resolution (8.1 Å) estimation at FSC=0.5.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    BI-3802 and key interacting residues R28, E41, Y58, and C84 are involved in BCL6 polymerization.

    “Density of BI-3802 and key interacting residues (R28, E41, Y58, C84) for BCL6 polymerization.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    BCL6 is a promising drug target for non-Hodgkin lymphomas including diffuse large B cell lymphoma and follicular lymphoma.

    “BCL6 is a promising drug target for non-Hodgkin lymphomas including diffuse large B cell lymphoma (DLBCL)13,14 and follicular lymphoma15.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [13] Cerchietti, L. C. et al. A small-molecule inhibitor of BCL6 kills DLBCL cells in vitro and in vivo. Cancer Cell 17, 400–411 (2010). source

      Not checked in the free run.

    • cites [14] Cardenas, M. G. et al. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma. J. Clin. Invest. 126, 3351–3362 (2016). source

      Not checked in the free run.

    • cites [15] Bosga-Bouwer, A. G. et al. BCL6 alternative translocation breakpoint cluster region associated with follicular lymphoma grade 3B. Genes Chromosom. Cancer 44, 301–304 (2005). source

      Not checked in the free run.

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

    Premisecitation

    BCL6 represses a broad range of genes involved in the DNA damage response, cell cycle checkpoints, and differentiation.

    “BCL6 represses a broad range of genes involved in the DNA damage response18, cell cycle checkpoints19, and differentiation20.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [18] Ranuncolo, S. M., Polo, J. M. & Melnick, A. BCL6 represses CHEK1 and suppresses DNA damage pathways in normal and malignant B-cells. Blood Cells Mol. Dis. 41, 95–99 (2008). source

      Not checked in the free run.

    • cites [19] Tunyaplin, C. et al. Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation. J. Immunol. 173, 1158–1165 (2004). source

      Not checked in the free run.

    • cites [20] Phan, R. T., Saito, M., Basso, K., Niu, H. & Dalla-Favera, R. BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells. Nat. Immunol. 6, 1054–1060 (2005). source

      Not checked in the free run.

  • The quote only partly states this claim.

    Premisecitation

    Screens for novel BCL6 inhibitors led to the identification of small molecules that unexpectedly induce BCL6 degradation, including BI-3802.

    “Screens for novel BCL6 inhibitors led to the identification of small molecules that unexpectedly induce BCL6 degradation, including BI-38026.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Kerres, N. et al. Chemically induced degradation of the oncogenic transcription factor BCL6. Cell Rep. 20, 2860–2875 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    A small molecule can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to superior pharmacological activity.

    “Our findings demonstrate that a small molecule can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to superior pharmacological activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    A small molecule can induce highly specific, reversible polymerization, sequestration into cellular foci, and subsequent degradation of a target protein.

    “Here we report an alternative mechanism, whereby a small molecule induces highly specific, reversible polymerization, sequestration into cellular foci, and subsequent degradation of a target protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    BCL6 is recalcitrant to hetero-bifunctional degraders, which have shown insufficient target modulation to induce growth inhibition.

    “One such example is the B cell lymphoma 6 (BCL6) protein, for which hetero-bifunctional degraders have shown insufficient target modulation to induce growth inhibition5.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] McCoull, W. et al. Development of a novel B-cell lymphoma 6 (BCL6) PROTAC to provide insight into small molecule targeting of BCL6. ACS Chem. Biol. 13, 3131–3141 (2018). source

      Not checked in the free run.

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

    Premisecitation

    Overexpression of BCL6 is sufficient to drive lymphoma development in genetically engineered mice.

    “In genetically engineered mice, overexpression of BCL6 is sufficient to drive lymphoma development17.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Cattoretti, G. et al. Deregulated BCL6 expression recapitulates the pathogenesis of human diffuse large B cell lymphomas in mice. Cancer Cell 7, 445–455 (2005). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Pathologically increased BCL6 expression, as a result of somatic BCL6 translocation, exonic mutation, promoter mutation, or mutations in regulatory pathways, is a common driver of B cell malignancies.

    “Pathologically increased BCL6 expression, as a result of somatic BCL6 translocation, exonic mutation, promoter mutation, or mutations in regulatory pathways, is a common driver of B cell malignancies16.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Hatzi, K. & Melnick, A. Breaking bad in the germinal center: how deregulation of BCL6 contributes to lymphomagenesis. Trends Mol. Med. 20, 343–352 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    BI-3802 induces rapid ubiquitination and degradation of BCL6.

    “BI-3802 induces rapid ubiquitination and degradation of BCL6, resulting in profound de-repression of BCL6 target genes and anti-proliferative effects in DLBCL cell lines, comparable to a genetic knock-out21, and superior to non-degrading BCL6 inhibitors such as BI-3812 or heterobifunctional BCL6 degraders5,6.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] McCoull, W. et al. Development of a novel B-cell lymphoma 6 (BCL6) PROTAC to provide insight into small molecule targeting of BCL6. ACS Chem. Biol. 13, 3131–3141 (2018). source

      Not checked in the free run.

    • cites [6] Kerres, N. et al. Chemically induced degradation of the oncogenic transcription factor BCL6. Cell Rep. 20, 2860–2875 (2017). source

      Not checked in the free run.

    • cites [21] Schlager, S. et al. Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis. Oncotarget 11, 875–890 (2020). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    BCL6 acts as a master transcriptional repressor enabling rapid expansion of germinal center B cells and tolerance to genomic instability caused by hypermutation of immunoglobulin genes and class switch recombination.

    “BCL6 acts as a master transcriptional repressor enabling rapid expansion of germinal center (GC) B cells and tolerance to genomic instability caused by hypermutation of the immunoglobulin genes and class switch recombination16.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Hatzi, K. & Melnick, A. Breaking bad in the germinal center: how deregulation of BCL6 contributes to lymphomagenesis. Trends Mol. Med. 20, 343–352 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Several peptide and small molecule inhibitors targeting BCL6 have shown efficacy in vivo, but only at high concentrations, which has limited their translation into clinical therapeutic agents.

    “Several peptide and small molecule inhibitors targeting BCL6 have shown efficacy in vivo, but only at high concentrations, which has limited their translation into clinical therapeutic agents13,14.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Cerchietti, L. C. et al. A small-molecule inhibitor of BCL6 kills DLBCL cells in vitro and in vivo. Cancer Cell 17, 400–411 (2010). source

      Not checked in the free run.

    • cites [14] Cardenas, M. G. et al. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma. J. Clin. Invest. 126, 3351–3362 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    These molecules bind the BTB domain, which mediates BCL6 homodimerization and its interactions with co-repressor proteins.

    “These molecules bind the Broad complex/Tramtrack/Bric-a-brac (BTB) domain, which mediates BCL6 homodimerization and its interactions with co-repressor proteins22.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [22] Ghetu, A. F. et al. Structure of a BCOR corepressor peptide in complex with the BCL6 BTB domain dimer. Mol. Cell 29, 384–391 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The effects of BI-3802 are comparable to a genetic knock-out and superior to non-degrading BCL6 inhibitors such as BI-3812 or heterobifunctional BCL6 degraders.

    “BI-3802 induces rapid ubiquitination and degradation of BCL6, resulting in profound de-repression of BCL6 target genes and anti-proliferative effects in DLBCL cell lines, comparable to a genetic knock-out21, and superior to non-degrading BCL6 inhibitors such as BI-3812 or heterobifunctional BCL6 degraders5,6.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] McCoull, W. et al. Development of a novel B-cell lymphoma 6 (BCL6) PROTAC to provide insight into small molecule targeting of BCL6. ACS Chem. Biol. 13, 3131–3141 (2018). source

      Not checked in the free run.

    • cites [6] Kerres, N. et al. Chemically induced degradation of the oncogenic transcription factor BCL6. Cell Rep. 20, 2860–2875 (2017). source

      Not checked in the free run.

    • cites [21] Schlager, S. et al. Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis. Oncotarget 11, 875–890 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    BI-3802 results in profound de-repression of BCL6 target genes and anti-proliferative effects in DLBCL cell lines.

    “BI-3802 induces rapid ubiquitination and degradation of BCL6, resulting in profound de-repression of BCL6 target genes and anti-proliferative effects in DLBCL cell lines, comparable to a genetic knock-out21, and superior to non-degrading BCL6 inhibitors such as BI-3812 or heterobifunctional BCL6 degraders5,6.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] McCoull, W. et al. Development of a novel B-cell lymphoma 6 (BCL6) PROTAC to provide insight into small molecule targeting of BCL6. ACS Chem. Biol. 13, 3131–3141 (2018). source

      Not checked in the free run.

    • cites [6] Kerres, N. et al. Chemically induced degradation of the oncogenic transcription factor BCL6. Cell Rep. 20, 2860–2875 (2017). source

      Not checked in the free run.

    • cites [21] Schlager, S. et al. Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis. Oncotarget 11, 875–890 (2020). source

      Not checked in the free run.

  • May combine two claims. The quote only partly states this claim.

    Interpretationown data

    Modeling BI-3812 onto the cryo-EM structure reveals a steric clash with the extended carboxamide group and explains the lack of BCL6 polymerization and consequent lack of degradation.

    “Modeling the structurally similar BCL6 inhibitor, BI-3812, onto the cryo-EM structure reveals a steric clash with the extended carboxamide group and explains the lack of BCL6 polymerization”
    Quote in the paperSubstantial claim

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

    Study design and conductreasoning

    Due to the limited resolution of the map, the placement of side-chains is approximated from the crystal structure (5MW2).

    “Due to the limited resolution of the map, the placement of side-chains is approximated from the crystal structure (5MW2).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A previous study has shown that introduction of amino acid mutations on the surface of symmetric proteins can trigger supramolecular self-assembly.

    “A previous study has shown that introduction of amino acid mutations on the surface of symmetric proteins can trigger supramolecular self-assembly27.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Garcia-Seisdedos, H., Empereur-Mot, C., Elad, N. & Levy, E. D. Proteins evolve on the edge of supramolecular self-assembly. Nature 548, 244–247 (2017). source

      Not checked in the free run.

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

    Study design and conductnone given

    All degradation assays were done in at least triplicates.

    “All degradation assays were done in at least triplicates.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    SIAH1 has a role in both drug-dependent and endogenous BCL6 degradation.

    “implicating a role of SIAH1 in both drug-dependent and endogenous BCL6 degradation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    BI-3802, as well as structurally related bioavailable analogs, have markedly increased activity against lymphoma cells compared to BI-3812, likely due to the combined effects of inhibiting co-activator binding, sequestering BCL6 into foci, and degrading BCL6.

    “BI-3802, as well as structurally related bioavailable analogs29, have markedly increased activity against lymphoma cells compared to BI-38126, which is likely due to the combined effects of inhibiting co-activator binding, sequestering BCL6 into foci, and degrading BCL6.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [29] Bellenie, B. R. et al. Achieving in vivo target depletion through the discovery and optimization of benzimidazolone BCL6 degraders. J. Med. Chem. 63, 4047–4068 (2020). source

      Not checked in the free run.

    • cites [6] Kerres, N. et al. Chemically induced degradation of the oncogenic transcription factor BCL6. Cell Rep. 20, 2860–2875 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    A subtle derivatization in solvent-exposed moiety distinguishes BI-3802 from BCL6 inhibitors that do not induce degradation, providing a potential path towards the rational design of molecules that induce polymerization.

    “A subtle derivatization in solvent-exposed moiety distinguishes BI-3802 from BCL6 inhibitors that do not induce degradation, providing a potential path towards the rational design of molecules that induce polymerization.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Polymerization promotes ubiquitination of BCL6 by SIAH1, an E3 ligase that recognizes a VxP motif distal to the drug binding site, and proteasomal degradation.

    “Polymerization promotes ubiquitination of BCL6 by SIAH1, an E3 ligase that recognizes a VxP motif distal to the drug binding site, and proteasomal degradation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    BI-3802-induced polymerization enhances the interaction between BCL6 and SIAH1, leading to accelerated ubiquitination and degradation of BCL6.

    “Together, our in vitro and cellular assays indicate that BI-3802-induced polymerization enhances the interaction between BCL6 and SIAH1, leading to accelerated ubiquitination and degradation of BCL6.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Drug-induced polymerization expands the repertoire of pharmacologic modalities that mediate targeted protein degradation, with likely applications to other transcription factors and proteins with internal symmetry that have traditionally been difficult to drug.

    “Drug-induced polymerization expands the repertoire of pharmacologic modalities that mediate targeted protein degradation as shown here for BCL6, with likely applications to other transcription factors and proteins with internal symmetry that have traditionally been difficult to drug.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    In the case of symmetric proteins, small molecules have the potential to induce polymerization which can then lead to degradation with extraordinary specificity.

    “In the case of symmetric proteins, small molecules have the potential to induce polymerization which can then lead to degradation with extraordinary specificity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    BI-3802 induces polymerization of BCL6, and blocking BCL6 polymerization impairs foci formation, BCL6 degradation, and BI-3802 cellular toxicity in lymphoma cells.

    “Together, the cryo-EM and mutagenesis studies established that BI-3802 induces polymerization of BCL6, and that blocking BCL6 polymerization impairs foci formation, BCL6 degradation, and BI-3802 cellular toxicity in lymphoma cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Inhibition of BCL6 or degradation by PROTACs results in insufficient inhibition of downstream targets and consequently only minor anti-proliferative effects.

    “It has been previously shown that inhibition of BCL6 or degradation by PROTACs results in insufficient inhibition of downstream targets and consequently only minor anti-proliferative effects5.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] McCoull, W. et al. Development of a novel B-cell lymphoma 6 (BCL6) PROTAC to provide insight into small molecule targeting of BCL6. ACS Chem. Biol. 13, 3131–3141 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The unique mechanism of action of BI-3802 overcomes these limitations and helps to explain its improved efficacy.

    “The unique mechanism of action of BI-3802 overcomes these limitations and helps to explain its improved efficacy.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The cryo-EM reconstruction suffers from an anisotropic resolution distribution because of a highly preferred orientation of the ribbon-like filament on the EM grid.

    “Because of a highly preferred orientation of the ribbon-like filament on the EM grid, the reconstruction suffers from an anisotropic resolution distribution.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    The model resolution at FSC=0.5 appears low (8.1 Å), which is due to a dip in the model-vs-map FSC resulting from the preferred orientation.

    “The model resolution at FSC=0.5 appears low (8.1 Å), which is due to a dip in the model-vs-map FSC resulting from the preferred orientation.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The BISON CRISPR library targets 713 E1, E2, and E3 ubiquitin ligases, deubiquitinases, and control genes and contains 2,852 guide RNAs.

    “The BISON CRISPR library targets 713 E1, E2, and E3 ubiquitin ligases, deubiquitinases, and control genes and contains 2,852 guide RNAs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Enkhbayar, P., Damdinsuren, S., Osaki, M. & Matsushima, N. HELFIT: helix fitting by a total least squares method. Comput. Biol. Chem. 32, 307–310 (2008). source

      Not checked in the free run.

52 of 52

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?

  • BI-3802 facilitates the self-assembly of a symmetric BCL6 dimer into polymers.

    “The authors showed that BI-3802 facilitates the self-assembly of a symmetric dimer into polymers.”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [133]

  • BCL6 polymerization leads to exacerbated ubiquitination by SIAH1.

    “These filaments led to an exacerbated ubiquitination by SIAH1, an E3 ligase of the RING family.”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [133]

  • BI-3802 does not directly glue BCL6 to the involved E3 but induces indirect dimerization between the polymerized protein and the E3.

    “BI-3802 does not directly glue BCL6 to the involved E3 but rather induces indirect ''dimerization'' between the polymerized protein and the E3.”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [133]

  • BI-3802 induces BCL6 destabilization via compound-induced polymerization.

    “The authors revealed that BCL6 destabilization was elicited via compound-induced polymerization.”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [133]

Self-check

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

  • Final cryo-EM class is not 95% of the particles

    Minor

    The stated class contains 112,048 of the 128,526 particles entering the additional classification, or about 87.2%, not 95%.

    Reported 95 · recomputed 87.179248

1 of 1

The citation lists haven’t been fetched yet

References 47 · Cited by 270

Authors
Słabicki, M., Yoon, H., Koeppel, J., Nitsch, L., Roy Burman, S. S., Di Genua, C., Donovan, K. A., Sperling, A. S., Hunkeler, M., Tsai, J. M., Sharma, R., Guirguis, A., Zou, C., Chudasama, P., Gasser, J. A., Miller, P. G., Scholl, C., Fröhling, S., Nowak, R. P., Fischer, E. S., & Ebert, B. L.
Affiliations
  • Harvard University (US)
  • Broad Institute (US)
  • Dana-Farber Cancer Institute (US)
  • German Cancer Research Center (DE)
Corresponding author
  • Fischer, Eric S.
  • Ebert, Benjamin L.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2020-11-18
Volume
588
Issue
7836
Pages
164-168
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-020-2925-1
PMC
PMC7816212
OpenAlex
W3099442067
Cited by
270
References
47
Citations by year
2026: 36 · 2025: 58 · 2024: 46 · 2023: 44 · 2022: 53 · 2021: 30 · 2020: 2
FWCI
10.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Zenodo (CERN European Organization for Nuclear Research) (submittedVersion, cc-by)
Funders
  • Howard Hughes Medical Institute
  • European Hematology Association (TRTH160)
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (174331)
  • National Heart, Lung, and Blood Institute (R01HL082945)
  • National Cancer Institute (F99CA253754, R35CA253125, P01CA108631, P50CA206963, R01CA214608, P01CA066996, R01CA218278)
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
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
Keywords
BCL6, protein polymerization, targeted protein degradation, BTB domain, SIAH1, ubiquitination, proteasomal degradation, oncogenic transcription factor, thalidomide analogues
MeSH terms
Seven in Absentia Proteins, Humans, Ligands, Models, Molecular, Nuclear Proteins, Solvents, Cryoelectron Microscopy, Ubiquitin-Protein Ligases, Proteasome Endopeptidase Complex, Proto-Oncogene Proteins c-bcl-6, Ubiquitination, Polymerization, Synthetic Biology, Proteolysis, In Vitro Techniques

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.

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