Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders
Open accessPublished version of a preprint
Abstract
Abstract Targeted protein degradation is a new pharmacologic paradigm established by drugs that recruit target proteins to E3 ubiquitin ligases via a ternary ligase-degrader-target complex. Based on the structure of the degrader and the neosubstrate, different E3 ligase interfaces are critically involved in this process, thus forming defined “functional hotspots”. Understanding disruptive mutations in functional hotspots informs on the architecture of the underlying assembly, and highlights residues prone to cause drug resistance. Until now, their identification was driven by structural methods with limited scalability. Here, we employ haploid genetics to show that hotspot mutations cluster in the substrate receptors of the hijacked ligases and find that type and frequency of mutations are shaped by the essentiality of the harnessed ligase. Intersection with deep mutational scanning data revealed hotspots that are either conserved, or specific for chemically distinct degraders or recruited neosubstrates. Biophysical and structural validation suggest that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we identified and validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and experimentally widely accessible methodology that empowers the characterization of small-molecule degraders and informs on associated resistance mechanisms.
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Written from the paper’s full text.
TL;DR
This study uses haploid genetics and deep mutational scanning to map functional hotspots in the E3 substrate receptors CRBN and VHL that determine sensitivity or resistance to small-molecule degraders. It shows that hotspot mutations often alter ternary complex assembly, can be degrader- or neo-substrate-specific, and overlap with CRBN mutations found in multiple myeloma patients relapsing from IMiD treatment.
Key findings
- In KBM7 cells, resistance clones arose ten-fold more frequently after treatment with the CRBN-based PROTAC dBET6 than with the VHL-based PROTAC ARV-771; dBET6-resistant cells mostly had disruptive CRBN alterations, with 55% frameshifts/stop codons in CRBN, whereas ARV-771-resistant cells had fewer direct VHL defects and 60% of VHL alterations were missense, with additional mutations in CRL2VHL components such as CUL2 and ELOB.
- Deep mutational scanning libraries covering residues within 10 Å of the degrader-binding pocket identified 1,442 VHL variants and 1,738 CRBN variants; conserved hotspots localized to the ligand-binding pockets, while neo-substrate-specific VHL hotspots included N67, R69, and H110 for ACBI1 and P71 for MZ1/macroPROTAC-1.
- VHLN67Q decreased ACBI1 ternary complex affinity/cooperativity by ~7-fold without substantially affecting BRD4BD2 binding, and VHLP71I affected MZ1 cooperativity by 6–7 fold; VHLH110L caused ACBI1 resistance but sensitized cells to MZ1 (5-fold) and ARV-771 (6-fold).
- CRBN hotspots included glue-selective V388, CRBNN351D causing CC-90009 resistance but 15-fold sensitization to THAL-SNS-032, and CRBNH397 mutations; patient relapse mutations CRBNP352S, CRBNF381S, and CRBNH57D overlapped with DMS hotspots.
Methods
- Resistance mechanisms were studied in near-haploid KBM7 cells by outgrowth after dBET6 or ARV-771 treatment, followed by hybrid-capture targeted sequencing of CRL complex members and recruited targets.
- DMS was performed in VHL-/- or CRBN-/- RKO cells reconstituted with site-saturation variant libraries, selected with VHL- or CRBN-based degraders, and analyzed by next-generation sequencing to calculate variant enrichment over vehicle.
- Mechanistic validation used cell viability, clonogenic assays, western blotting, fluorescence polarization with recombinant VCB and bromodomain proteins, and crystallography of the BRD4BD2:AT7:VCB ternary complex at 3.0 Å (PDB: 7ZNT).
Limitations
- The paper states that DMS focused on residues proximal to the degrader-binding site (<10 Å) to keep libraries manageable (~1,500 variants each), which prevented identification of hotspots outside the dimerization interface.
- The paper states that its sequencing strategy is limited in detecting copy number loss or splicing defects and does not cover the full spectrum of possible causative mutations; it also notes that DMS reconstitution mimics homozygous mutations, whereas patient mutations may be heterozygous.
- Evident from the text: the study is restricted to CRBN and VHL, and the authors note that for TPD-compatible E3 ligases lacking structural data, variant library design and mechanistic interpretation will be more challenging.
Model-written summary. Check it against the paper before relying on it.
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Claims the paper makes
0 of 29 cited sources checked · 0 supported · 0 flagged
- Findingown data
In VHL, 60% of alterations were missense point mutations.
“In contrast, the majority (60%) of alterations in VHL were missense point mutations (Fig. 1D and E, Supplementary Dataset).”
Quote in the paperSubstantial claim - Findingown data
The occurrence of resistant clones was ten-fold increased after exposure to dBET6 compared to ARV-771.
“Despite their matched cellular efficacy, occurrence of resistant clones was ten-fold increased after exposure to dBET6 compared to ARV-771 (Fig. 1B).”
Quote in the paperSubstantial claim - Findingcitation
VHL is essential in 935 of the profiled cell lines.
“Despite its well-established role as a tumor suppressor in renal carcinoma25, VHL proved essential in 935 of the profiled cell lines.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [24] Meyers, R. M. et al. Computational correction of copy number effect improves specificity of CRISPR–Cas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779–1784 (2017). source
Not checked in the free run.
- Findingown data
In CRBN, 55% of alterations were frameshifts and gained stop-codons.
“We found a higher fraction (55 %) of frameshifts and gained stop-codons in CRBN.”
Quote in the paperSubstantial claim - Findingcitation
CRBN is a non-essential gene across 1070 cell lines profiled by genome-scale CRISPR/Cas9 knockout screens.
“Mining publicly available data from the DepMap Consortium, CRBN presents as a non-essential gene across 1070 cell lines that were profiled via genome-scale CRISPR/Cas9 knockout screens (Fig. 1A)24.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [24] Meyers, R. M. et al. Computational correction of copy number effect improves specificity of CRISPR–Cas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779–1784 (2017). source
Not checked in the free run.
- Findingown data
BRD3/4 destabilization by the assayed BET degraders was unaffected by VHLN67 mutation.
“In contrast, BRD3/4 destabilization by the assayed BET degraders was unaffected by VHLN67 mutation (Fig. 3C and Extended Data Fig. 4E).”
Quote in the paperSubstantial claim - Findingown data
Averaging log2 fold-enrichment for each mutation across all 5 degraders generated a map of consensus VHL hotspots.
“Averaging log2 fold-enrichment for each mutation across all 5 degraders generated a map of consensus VHL hotspots (Fig. 2B).”
Quote in the paperSubstantial claim - Findingown data
Mutations in CRBNH397 also prompted resistance to molecular glue degraders.
“Intriguingly, mutations in this position also prompted resistance to molecular glue degraders (Fig. 5A and B and Extended Data Fig. 6G).”
Quote in the paperSubstantial claim - Findingown data
In cells expressing a VHLN67 mutant, ACBI1 failed to induce SMARCA2/4 degradation at conditions where profound degradation is observed in isogenic VHLWT cells.
“In cells expressing a VHLN67 mutant, ACBI1 failed to induce SMARCA2/4 degradation at conditions where profound degradation is observed in isogenic VHLWT cells.”
Quote in the paperSubstantial claim - Findingown data
CRBNH397 mutation to only negatively charged amino acids aspartate or glutamate abrogated the cellular and degradation efficacy of dBET57.
“Interestingly, our DMS data suggested that mutation to only the negatively charged amino acids aspartate or glutamate abrogated the cellular and degradation efficacy of the BET PROTAC dBET57 (Extended Data Fig. 6E).”
Quote in the paperSubstantial claim - Findingown data
VHLP71 is selectively critical for the efficacy of MZ1 and macroPROTAC-1.
“Comparative analysis of DMS enrichments revealed that VHLP71 is selectively critical for the efficacy of MZ1 and macroPROTAC-1(Fig. 4A and Extended Data Fig. 5A).”
Quote in the paperSubstantial claim - Findingown data
Mutations in VHLN67 (VHLN67Q) decrease the ternary complex affinity and cooperativity of ACBI1 binding to SMARCA4BD by ~7-fold.
“This led us to identify that mutations in VHLN67 (here VHLN67Q) decrease the ternary complex affinity and cooperativity of ACBI1 binding to SMARCA4BD by ~7-fold (Fig. 3D).”
Quote in the paperSubstantial claim - Findingown data
Some identified and validated functional hotspots could not sufficiently be rationalized based on existing structural models.
“Intriguingly, some of the identified and validated functional hotspots could not sufficiently be rationalized based on existing structural models.”
Quote in the paperSubstantial claim - Findingown data
The cooperativity of ARV-771-induced ternary complex formation is not affected by VHLP71I.
“In contrast, the cooperativity of ARV-771-induced ternary complex formation is not affected (Fig. 4E), suggesting that the ARV-771-induced ternary complex features a unique architecture that is likely distinct from the architecture observed for MZ1.”
Quote in the paperSubstantial claim - Findingown data
The CRBNH397 mutational effect is not observed for the closely related dBET6.
“We validated that this mutational effect is not observed for the closely related dBET6 (Fig. 5B, E and F and Extended Data Fig. 6F).”
Quote in the paperSubstantial claim - Findingnone given
ARV-771-induced ternary complex assemblies have thus far proven unsuccessful to crystallization efforts.
“In support of these predictions are the observations that (i) ARV-771-induced ternary complex assemblies have thus far proven to be unsuccessful to crystallization efforts;”
Quote in the paperSubstantial claim - Findingown data
CRBNN351D led to a marked sensitization (15-fold shift in EC50) to THAL-SNS-032.
“Simultaneously, it led to a marked sensitization (15-fold shift in EC50) to the CDK9-targeting PROTAC THAL-SNS-03240 (Extended Data Fig. 6B and C).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [40] Olson, C. M. et al. Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation. Nat. Chem. Biol. 14, 163–170 (2017). source
Not checked in the free run.
- Findingown data
The binding cooperativity between MZ1, BRD4BD2 and VCB is significantly (6-7 fold) affected upon introducing the VHLP71I mutation.
“Fluorescence polarization assays indicated that the binding cooperativity between MZ1, BRD4BD2 and VCB is significantly (6-7 fold) affected upon introducing the VHLP71I mutation (Fig. 4E).”
Quote in the paperSubstantial claim - Findingown data
VHLH110L simultaneously sensitized cells to treatment with certain BET PROTACs, such as MZ1 (5-fold) or ARV-771 (6-fold).
“Intriguingly, this mutation simultaneously sensitized cells to treatment with certain BET PROTACs, such as MZ1 (5-fold) or ARV-771 (6-fold, Fig. 3B and Extended Data Fig. 4G and H).”
Quote in the paperSubstantial claim - Findingown data
CRBN V388 is a key determinant of cellular efficacy of CC-90009.
“Among the enriched, glue-selective hotspots, we identified V388 as a key determinant of cellular efficacy of CC-90009.”
Quote in the paperSubstantial claim - Findingown data
Functional CRBN hotspots enriched across all tested degraders localized to the glutarimide (ligand-) binding pocket.
“As observed for VHL, functional CRBN hotspots that were enriched across all tested degraders localized to the glutarimide (ligand-) binding pocket. (Extended Data Fig. 3D).”
Quote in the paperSubstantial claim - Findingown data
Residues of shared relevance primarily localized to the binding pocket of the closely related VHL ligands of the various assayed PROTACs.
“As expected, residues of shared relevance primarily localized to the binding pocket of the closely related VHL ligands of the various assayed PROTACs (Fig. 2C).”
Quote in the paperSubstantial claim - Findingown data
The affinity and cooperativity of the VHL:MZ1 binary complex to BRD4BD2 was largely unaffected by mutations in VHLN67 (within 2-fold those of wild-type).
“In contrast, the affinity and cooperativity of the VHL:MZ1 binary complex to BRD4BD2 was largely unaffected by mutations in VHLN67 (within 2-fold those of wild-type, Fig. 3D).”
Quote in the paperSubstantial claim - Findingcitation
Several mutations in relapsed patients, such as CRBNP352S, CRBNF381S and CRBNH57D, overlapped with CRBN hotspots identified by DMS.
“Upon closer inspection, several mutations in relapsed patients, such as CRBNP352S, CRBNF381S and CRBNH57D overlapped with CRBN hotspots identified by DMS (Fig. 2D, 5G and H and Extended Data Fig. 6G and H)42.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [42] Gooding, S. et al. Multiple cereblon genetic changes are associated with acquired resistance to lenalidomide or pomalidomide in multiple myeloma. Blood 137, 232–237 (2021). source
Not checked in the free run.
- Interpretationown data
The SR is the most frequently mutated CRL component in degrader-resistant clones.
“Together, these data implicate the SR as the most frequently mutated CRL component in degrader-resistant clones.”
Quote in the paperSubstantial claim - Study design and conductown data
The DMS experimental setup was robust between biological replicates (R = 0.92).
“We initially validated the robustness of this experimental setup between biological replicates (R = 0.92, Extended Data Fig. 3A).”
Quote in the paperSubstantial claim - Premisecitation
dBET6 and ARV-771 have matched cellular potency, including in KBM7 cells.
“we focused on two BET Bromodomain targeting PROTACs: dBET6 (CRBN-based) and ARV-771 (VHL-based) that have matched cellular potency, including in the near-haploid human leukemia cell line KBM7 (Extended Data Fig. 1A)26,27.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [26] Raina, K. et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl Acad. Sci. USA 113, 7124–7129 (2016). source
Not checked in the free run.
- cites [27] Winter, G. E. et al. BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol. Cell 67, 5–18 (2017). source
Not checked in the free run.
- Interpretationreasoning
KBM7 cells are a valid model to capture the overall essentiality profile of both ligases.
“KBM7 cells, which are a frequently used tool to study mechanisms of drug resistance are thus a valid model to capture the overall essentiality profile of both ligases. 28–30.”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [28] Forment, J. V. et al. Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells. Nat. Chem. Biol. 13, 12–14 (2017). source
Not checked in the free run.
- cites [29] Volz, J. C., Schuller, N. & Elling, U. Using functional genetics in haploid cells for drug target identification. Methods Mol. Biol. 1953, 3–21 (2019). source
Not checked in the free run.
- cites [30] Winter, G. E. et al. The solute carrier SLC35F2 enables YM155-mediated DNA damage toxicity. Nat. Chem. Biol. 10, 768–773 (2014). source
Not checked in the free run.
- Premisecitation
CRBN loss is inconsequential for KBM7 proliferation.
“Previous studies have shown that CRBN loss is inconsequential for KBM7 proliferation15.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15] Mayor-Ruiz, C. et al. Plasticity of the Cullin-RING ligase repertoire shapes sensitivity to ligand-induced protein degradation. Mol. Cell 75, 849–858 (2019). source
Not checked in the free run.
- Study design and conductown data
An even distribution of possible substitutions was present for almost all residues in the DMS libraries.
“Furthermore, an even distribution of possible substitutions was present for almost all residues (Extended Data Fig. 2B, see also Methods section).”
Quote in the paperNot a substantial claim - Premisecitation
Loss of CUL2 has previously been reported as an acquired resistance mechanism to VHL-based PROTACs in OVCAR8 cells.
“Supporting these results, loss of CUL2 has previously been reported as an acquired resistance mechanism to VHL-based PROTACs in OVCAR8 cells16.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [16] Zhang, L., Riley-Gillis, B., Vijay, P. & Shen, Y. Acquired resistance to BET-PROTACs (proteolysis-targeting chimeras) caused by genomic alterations in core components of E3 ligase complexes. Mol. Cancer Ther. 18, 1302–1311 (2019). source
Not checked in the free run.
- Interpretationreasoning
Both the frequency and the type of alterations appear to be influenced by the essentiality of the co-opted SR.
“However, both the frequency and the type of alterations appear to be influenced by the essentiality of the co-opted SR.”
Quote in the paperSubstantial claim - Premisecitation
Up to one-third of patients refractory to pomalidomide treatment present with various types of CRBN alterations.
“Accumulating data has shown that up to one-third of patients refractory to pomalidomide treatment present with various types of CRBN alterations41,42,49.”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [41] Barrio, S. et al. IKZF1/3 and CRL4 CRBN E3 ubiquitin ligase mutations and resistance to immunomodulatory drugs in multiple myeloma. Haematologica 105, E237–E241 (2020). source
Not checked in the free run.
- cites [42] Gooding, S. et al. Multiple cereblon genetic changes are associated with acquired resistance to lenalidomide or pomalidomide in multiple myeloma. Blood 137, 232–237 (2021). source
Not checked in the free run.
- cites [49] Kortum, K. M. et al. Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes. Blood 128, 1226–1233 (2016). source
Not checked in the free run.
- Interpretationown data
Nuanced, differentiated mutational profiles and sensitivities can arise even with degraders which share otherwise highly similar chemical structures, mechanisms, and cellular activities.
“Hence, this and related observations emerging from this study underscore that nuanced, differentiated mutational profiles and sensitivities can arise even with degraders which share otherwise highly similar chemical structures, mechanisms, and cellular activities.”
Quote in the paperSubstantial claim - Premisecitation
A mutation in CRBNH397 was identified in a multiple myeloma patient who presented refractory to IMiD treatment.
“Furthermore, a mutation in CRBNH397 was also identified in a multiple myeloma (MM) patient who presented refractory to IMiD treatment 41.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [41] Barrio, S. et al. IKZF1/3 and CRL4 CRBN E3 ubiquitin ligase mutations and resistance to immunomodulatory drugs in multiple myeloma. Haematologica 105, E237–E241 (2020). source
Not checked in the free run.
- Study design and conductown data
VHL hotspots were highly robust and conserved over a wide concentration range.
“Hotspots were highly robust and conserved over a wide concentration range (Extended Data Fig. 3B).”
Quote in the paperSubstantial claim - Interpretationown data
The DMS approach enabled robust and reproducible identification of functional hotspots of general relevance over different degrader modalities, ligases and neo-substrates.
“In sum, the presented deep mutational scanning approach empowered the robust and reproducible identification of functional hotspots of general relevance over different degrader modalities, ligases and neo-substrates.”
Quote in the paperSubstantial claim - Premisecitation
ARV-771 and MZ1 displayed distinct intra-BET bromodomain cooperativity profiles in FP ternary complex assays.
“ARV-771 and MZ1 displayed distinct intra-BET bromodomain cooperativity profiles in FP ternary complex assays46.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [46] Klein, V. G., Bond, A. G., Craigon, C., Lokey, R. S. & Ciulli, A. Amide-to-ester substitution as a strategy for optimizing PROTAC permeability and cellular activity. J. Med. Chem. 64, 18082–18101 (2021). source
Not checked in the free run.
- Interpretationown data
The essentiality of the co-opted SR appears to correlate with the frequency, type and topology of the identified alterations.
“Intriguingly, our data highlight that the essentiality of the co-opted SR appears to correlate with the frequency, type and topology of the identified alterations, even though we can’t exclude the contribution of additional factors.”
Quote in the paperNot a substantial claim - Premisecitation
Position V388 corresponds to position 391 in mouse Crbn, which features the critical isoleucine variant responsible for the lack of IMiD activity in mouse cells, masking thalidomide teratogenicity.
“Intriguingly, this site corresponds to position 391 in mouse Crbn, which features the critical isoleucine variant that is responsible for the lack of IMiD activity in mouse cells, hence masking the teratogenicity of thalidomide39.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [39] Fink, E. C. et al. Crbn I391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice. Blood 132, 1535–1544 (2018). source
Not checked in the free run.
- Premisecitation
Published co-crystal structures and TR-FRET data previously validated the importance of VHLR69 in SMARCA2BD recognition within the ternary complex.
“In support of this, published co-crystal structures and TR-FRET data previously validated the importance of VHLR69 in SMARCA2BD recognition within the ternary complex35.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [35] Farnaby, W. et al. BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat. Chem. Biol. 15, 672–680 (2019). source
Not checked in the free run.
- Premisecitation
Mutations can also arise on the neo-substrate, as reported for CDK12-targeting PROTACs.
“While it appears reasonable to conclude that resistance-causing mutations will be enriched in the ligase, mutations can also arise on the neo-substrate, as for instance reported for CDK12-targeting PROTACs47.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [47] Jiang, B. et al. Discovery and resistance mechanism of a selective CDK12 degrader. Nat. Chem. Biol. 17, 675–683 (2021). source
Not checked in the free run.
- Interpretationcitation
Most emerging mutations occur directly in the SR of the involved E3 ligase.
“In line with previous studies that employed CRISPR/Cas9 screens13–15, we show that most emerging mutations occur directly in the SR of the involved E3 ligase.”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [13] Shirasaki, R. et al. Functional genomics identify distinct and overlapping genes mediating resistance to different classes of heterobifunctional degraders of oncoproteins. Cell Rep. 34, 108532 (2021). source
Not checked in the free run.
- cites [14] 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 CRL4(CRBN) activity. Blood 132, 1293–1303 (2018). source
Not checked in the free run.
- cites [15] Mayor-Ruiz, C. et al. Plasticity of the Cullin-RING ligase repertoire shapes sensitivity to ligand-induced protein degradation. Mol. Cell 75, 849–858 (2019). source
Not checked in the free run.
- Study design and conductreasoning
The focus on residues proximal to the degrader binding site prevented identification of hotspots outside the dimerization interface.
“This focus was chosen to obtain a relatively manageable library size of around 1500 variants each, yet prevented the identification of hotspots outside the dimerization interface.”
Quote in the paperSubstantial claim - Interpretationown data
The ARV-771-induced ternary complex features a unique architecture that is likely distinct from the architecture observed for MZ1.
“In contrast, the cooperativity of ARV-771-induced ternary complex formation is not affected (Fig. 4E), suggesting that the ARV-771-induced ternary complex features a unique architecture that is likely distinct from the architecture observed for MZ1.”
Quote in the paperSubstantial claim - Premisecitation
Structurally diverse degraders can prompt ternary assemblies of different architectures.
“Enabled by the plasticity of a given protein-protein interface, structurally diverse degraders can prompt ternary assemblies of different architectures2,9.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [2] Gerry, C. J. & Schreiber, S. L. Unifying principles of bifunctional, proximity-inducing small molecules. Nat. Chem. Biol. 16, 369–378 (2020). source
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- cites [9] Maniaci, C. & Ciulli, A. Bifunctional chemical probes inducing protein-protein interactions. Curr. Opin. Chem. Biol. 52, 145–156 (2019). source
Not checked in the free run.
- Interpretationown data
DMS highlighted functional hotspots that are disrupted by mutations in patients relapsing from IMiD treatment.
“Importantly, DMS also highlighted functional hotspots that are disrupted by mutations in patients relapsing from IMiD treatment.”
Quote in the paperSubstantial claim - Premisecitation
An essential step in targeted protein degradation is the drug-induced formation of a ternary complex.
“An essential step in targeted protein degradation is the drug-induced formation of a ternary complex10,43.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [10] Gadd, M. S. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521 (2017). source
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- cites [43] Roy, M. J. et al. SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate. ACS Chem. Biol. 14, 361–368 (2019). source
Not checked in the free run.
- May combine two claims. 18
- Classified as probably not a substantial claim. 22
- The quote only partly states this claim. 1
May combine two claims.
Findingown dataAll assayed degraders blocked the proliferation of RKO cells in a VHL-dependent manner, enabling sufficient selective pressure.
“All degraders blocked the proliferation of RKO cells in a VHL dependent manner, enabling sufficient selective pressure (Extended Data Fig. 2E and H).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataVHL is essential in KBM7 cells, validated by CRISPR/Cas9-mediated disruption in competitive growth assays.
“First, we validated the essentiality of VHL in KBM7 cells by CRISPR/Cas9-mediated disruption of VHL in competitive growth assays (Extended Data Fig. 1B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn ARV-771-resistant cells, there was a lower proportion of genetic defects directly in VHL and an equal number of alterations in various other components of the CRL2VHL complex, such as CUL2 and ELOB.
“In contrast, cells resistant to ARV-771 featured a lower proportion of genetic defects directly in VHL and an equal number of alterations in various other components of the CRL2VHL complex, such as CUL2 and ELOB.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn dBET6-resistant cells, the majority of disruptive alterations were directly in CRBN, while other members of the CRL4CRBN ligase complex were not affected.
“In dBET6 resistant cells, we identified the majority of disruptive alterations directly in CRBN, while other members of the CRL4CRBN ligase complex were not affected (Fig. 1C, Supplementary Dataset).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataCRBNN351D prompted resistance to CC-90009 and was inconsequential for dBET6.
“While cellular expression of CRBNN351D prompted resistance to CC-90009, it was inconsequential for dBET6 (Fig. 5A and B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataVHLY112C elicited selective resistance to BET degraders while having nearly no effect on ACBI1 potency.
“Intriguingly, the mutant VHLY112C elicited selective resistance to BET degraders while having nearly no effect on ACBI1 potency (Extended Data Fig. 4L).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataMutating VHLN67 rescued the efficacy of ACBI1 without modulating the efficacy of BET PROTACs.
“Indeed, mutating VHLN67 rescued the efficacy of ACBI1 without modulating the efficacy of BET PROTACs.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataVHLN67, VHLR69 and VHLH110 are functional hotspots specifically required to sustain ACBI1 activity, while they appear inconsequential for the tested BET PROTACs.
“This allowed identification of the functional hotspots VHLN67, VHLR69 and VHLH110, which appear to be specifically required to sustain the activity of ACBI1, while they seem inconsequential for the tested BET PROTACs.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.
Findingown dataARV-771 has proven intractable to structural exploration via crystallography.
“This is best exemplified by complexes induced by the BET protein degrader ARV-771, which has, intriguingly, at least in our hands so far proven intractable to structural exploration via crystallography.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataVHLH110L causes resistance to ACBI1, validated via single point mutant reconstitutions.
“Accordingly, DMS also identified VHLH110L as a mutation that causes resistance to ACBI1, which we could validate via single point mutant reconstitutions (Fig. 3A and B).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataThe VHL library had a coverage of 135x and the CRBN library had a coverage of 54x.
“Dilutions of the bacterial suspension were plated and counted to determine a library coverage of 135x and 54x for VHL and CRBN libraries respectively.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataThe DMS screens yielded a calculated library representation of 1664 cells per VHL variant and 1380 cells per CRBN variant.
“Eight million RKO CRBN-/- or VHL-/- were transduced at a MOI of 0.3 yielding a calculated library representation of 1664 and 1380 cells per variant for VHL and CRBN respectively.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
FindingreasoningGiven our sequencing strategy, 98.89 % of reads constituted wild type sequences and were therefore filtered out during this step.
“Given our sequencing strategy, 98.89 % of reads constituted wild type sequences and were therefore filtered out during this step.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown data1442 of 1500 possible VHL variants and 1738 of 1740 CRBN substitutions were recovered in the libraries.
“1442 of 1500 possible VHL variants and 1738 of 1740 CRBN substitutions were recovered in the libraries.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataResidues marked in red were identified in hybrid capture analysis.
“Residues marked in red were identified in hybrid capture analysis.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataThe crystals belonged to space group P32, with two copies of the ternary complex in the asymmetric unit.
“The crystals belonged to space group P32, with two copies of the ternary complex in the asymmetric unit.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationDMS strategies have previously been successfully employed to investigate functional relationships between small molecules and target proteins.
“Noteworthy, DMS strategies have previously been successfully employed to investigate functional relationships between small molecules and target proteins31,32.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [31] Suiter, C. C. et al. Massively parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity. Proc. Natl Acad. Sci. USA 117, 5394–5401 (2020). source
Not checked in the free run.
- cites [32] Awad, M. M. et al. Acquired resistance to KRAS G12C inhibition in cancer. N. Engl. J. Med. 384, 2382–2393 (2021). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe prepared DMS libraries mostly contained expected missense variants.
“To initially ensure quality control, we sequenced the prepared libraries and mostly identified expected missense variants (Extended Data Fig. 2A).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenEach treatment was performed in biological triplicates.
“Each treatment was performed in biological triplicates.”
- Also stated in: § Extended Data · plain
“Mean ± s.e.m.; n = 3 independent treatments.”
Quote in the paperNot a substantial claim- Also stated in: § Extended Data · plain
Classified as probably not a substantial claim.
Study design and conductnone givenIndependent mutational scanning resistance screens were performed in replicates by treating 2.5 million cells, splitting and retreating after 4 days and harvesting 2.5 million cell pellets after a total of 7 day treatment with the indicated drug and dose.
“Independent mutational scanning resistance screens were performed in replicates by treating 2.5 million cells, splitting and retreating after 4 days and harvesting 2.5 million cell pellets after a total of 7 day treatment with the indicated drug and dose.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationA complementary CRISPR-suppressor scanning strategy identifies resistance-causing mutations localized in neo-substrates of known molecular glue degraders.
“Moreover, an elegant recent study described a complementary approach, which is based on a CRISPR-suppressor scanning strategy, to identify resistance-causing mutations that are localized in neo-substrates of known molecular glue degraders48.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [48] Gosavi, P. M. et al. Profiling the landscape of drug resistance mutations in neosubstrates to molecular glue degraders. ACS Cent. Sci. 8, 417–429 (2022). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenThe DMS reconstitution mimics the scenario of homozygous mutations, while mutations in patients might also be heterozygous.
“Of note, our DMS reconstitution mimics the scenario of homozygous mutations, while mutations in patients might also be heterozygous.”
Quote in the paperNot a substantial claimMay combine two claims.
Study design and conductnone givenThe sequencing strategy is limited in detecting copy number loss or splicing defects and does not cover the full spectrum of possible causative mutations.
“Of note, our sequencing strategy is limited in detecting copy number loss or splicing defects, and hence doesn’t cover the full spectrum of possible causative mutations.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenQuality control of the library distribution was performed via next-generation sequencing of the plasmid preparation.
“Quality control of the library distribution was performed via next-generation sequencing of the plasmid preparation as outlined for the screens below, except that the mentioned PCR was performed for 5 cycles.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S1 (asserted).
InterpretationreasoningARV-771 induces a ternary complex of a different geometry than the ones previously resolved for MZ110 or macroPROTAC-134.
“Based on the presented DMS data, for instance exemplified by VHLP71I and VHLH110L, it is conceivable that ARV-771 induces a ternary complex of a different geometry than the ones previously resolved for MZ110 or macroPROTAC-134.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataSome identified hotspots appeared specific for molecular glues, such as CRBNP352, while others were similarly required for PROTAC potency, for example CRBNF381.
“Some of the identified hotspots appeared to be specific for molecular glues, such as CRBNP352, while others were similarly required for PROTAC potency, for example CRBNF381.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenAssays were run in triplicate using 384-well plates.
“Assays were run in triplicate using 384-well plates (Corning, 3544)”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe cycle number for specific amplification of the 700 base-pair (VHL) and 1.4 kilo-base-pair (CRBN) targets was confirmed by agarose gel electrophoresis.
“The cycle number for specific amplification of the 700 base-pair (VHL) and 1.4 kilo-base-pair (CRBN) targets was confirmed by agarose gel electrophoresis.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenData represents mean ± s.d. of n = 3 biological replicates.
“Data represents mean ± s.d. of n = 3 biological replicates.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataMass spectrometry analysis and agarose gel electrophoresis was carried out to ensure purity of the recombinant proteins.
“Mass spectrometry analysis and agarose gel electrophoresis was carried out to ensure purity of the recombinant proteins (see Supplementary Figure 3).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe purity and integrity of the PCR products were analysed on an Agilent 2100 Bioanalyzer.
“The purity and integrity of the PCR products were analysed on an Agilent 2100 Bioanalyzer following manufacturer recommendations for high sensitivity DNA chips (Agilent, 5067-4626).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationModel geometry and steric clashes were validated using the MOLPROBITY server.
“Model geometry and steric clashes were validated using the MOLPROBITY server.60”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe structure has been deposited in the protein data bank (PDB: 7ZNT).
“The structure has been deposited in the protein data bank (PDB: 7ZNT); data collection and refinement statistics are presented in Supplementary Table 3.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The abundant VHL F119I mutant in the library has no functional consequence.
“The VHL library included an abundant mutant (F119I) caused by library synthesis, which had no functional inconsequence.”
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?
Both deep mutational scanning (DMS) and CRISPR tiling have recently highlighted hotspots in ligase and POI that are functionally relevant for degrader activity.
“For instance, both DMS and CRISPR tiling have recently highlighted hotspots in ligase and POI that are functionally relevant for degrader activity.237,238”
From Advancing Targeted Protein Degradation via Multiomics Profiling and Artificial Intelligence (2023) · cites this paper as [237]
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.
Recombinant VHL mutant is assigned two different residue positions
MinorThe recombinant-protein methods describe a glutamine substitution at VHL residue R67, but the results identify the mutant used for the binding experiments as VHLN67Q. These specify different starting amino acids at position 67.
“The VCB mutants, in which R67 and P71I of VHL (54-213) were mutated to glutamine and isoleucine respectively”
“mutations in VHLN67 (here VHLN67Q)”
The citation lists haven’t been fetched yet
References 76 · Cited by 133
- Authors
- Hanzl, A., Casement, R., Imrichova, H., Hughes, S. J., Barone, E., Testa, A., Bauer, S., Wright, J., Brand, M., Ciulli, A., & Winter, G. E.
- Affiliations
- Austrian Academy of Sciences (AT)
- University of Dundee (GB)
- Corresponding author
- Ciulli, Alessio
- Winter, Georg E.
- ORCID
- Venue
- Nature Chemical Biology
- Published
- 2022-11-03
- Volume
- 19
- Issue
- 3
- Pages
- 323-333
- ISSN
- 1552-4450 (ISSN-L)
- 1552-4469
- PMC
- PMC7614256
- OpenAlex
- W4308099893
- Cited by
- 133
- References
- 76
- Citations by year
- 2026: 40 · 2025: 30 · 2024: 28 · 2023: 31 · 2022: 4
- FWCI
- 7.64
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Nature Chemical Biology (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion, cc-by)
- Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
- Funders
- Pfizer
- Wellcome Trust (FP7/2007–2013, 094090/Z/10/, 100476, 094090/Z/10/Z, 094090, 100476/Z/12/Z)
- Diamond Light Source (MX14980)
- European Commission (2007–2013, FP7 2007-2013, HORIZON2020, FP7/2007, ERC-2012-StG-311460, 851478)
- Österreichischen Akademie der Wissenschaften (P7909, P32125)
- Austrian Science Fund (P31690, P 32125, P7909)
- Directorate for Biological Sciences
- Biotechnology and Biological Sciences Research Council (BB/M010996/1, BB/M010996/, M010996, M010996/1)
- 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
- E3 ubiquitin ligase, targeted protein degradation, PROTACs, molecular glues, drug resistance, mutational hotspots, substrate receptor, gene essentiality, deep mutational scanning, relapse
- MeSH terms
- Carrier Proteins, Ubiquitin-Protein Ligases, Proteolysis
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