Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN

In the paper

“Previous work has implicated the second C2H2 ZF domain in IKZF1 and IKZF3 (5, 7), and ZF4 of ZFP91 as the drug-inducible degrons (5, 7, 16).”

What the sources say

• ?Not checked — no full text of the sourcecites [5] source
• Partly supported

  The “CRBN functions as a DCAF for the CRL4CRBN ligase” section (page unavailable) reports drug-induced degradation of IKZF1 and IKZF3. It does not identify their second C2H2 zinc finger as a degron, and the searched passages do not address ZFP91 or its ZF4 domain. (LLM, conf=0.83)

  cites [7] sourceExperimental — accuracy not yet measured
• Supported

  The Discussion identifies a common structural motif in ZFP91 ZnF4 and IKZF1/3 ZnF2 that mediates interaction with the CRBN–IMiD complex. The ZFP91 substrate section also reports that disrupting ZnF4 largely disrupts drug-induced binding, supporting the cautious wording “implicated” as drug-inducible degrons. (LLM, conf=0.92)

  cites [16] source

In the paper

“proteome-wide mass-spectrometry demonstrated selective degradation of endogenous IKZF1/3, and ZFP91 in multiple cell lines (3, 4, 16).”

What the sources say

• Partly supported

  The copy’s Abstract (page not provided) reports SILAC-based mass spectrometry showing reduced IKZF1 and IKZF3 abundance in the MM1S cell line. It separately reports decreased endogenous IKZF1 and IKZF3 in cell lines, but does not report proteome-wide mass spectrometry across multiple cell lines or degradation of ZFP91. (LLM, conf=0.88)

  cites [3] sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section reports global mass-spectrometry profiling in KG-1 cells, and Extended Data Table 1 shows reduced IKZF1 and ZFP91 protein abundance after lenalidomide treatment. Extended Data Figure 8 also describes proteomic measurement of IKZF1 in MDS-L cells. The copy does not show mass-spectrometric degradation of endogenous IKZF3 or establish degradation of all three proteins across multiple cell lines; its IKZF3 assay used a luciferase reporter. (LLM, conf=0.87)

  cites [4] sourceExperimental — accuracy not yet measured
• Partly supported

  The ‘Identification of novel CRL4 CRBN targets through pSILAC MS’ section reports mass-spectrometry detection of altered ZFP91 turnover in HEK293T and Hct116 cells, and the ‘ZFP91 is a lenalidomide-dependent CRL4 CRBN substrate’ section confirms its degradation in additional cell lines. The paper describes IKZF1/3 degradation as an established finding, but says its own proteomics experiments used non-hematopoietic cells to look beyond IKZF1/3; it does not show proteome-wide mass-spectrometry detection of their degradation across multiple cell lines. (LLM, conf=0.85)

  cites [16] sourceExperimental — accuracy not yet measured

In the paper

“Thalidomide and its derivatives, lenalidomide and pomalidomide, are effective therapies for the hematologic malignancies multiple myeloma, del(5q) myelodysplastic syndrome, and mantle cell lymphoma (1).”

What the sources say

• Partly supported

  The Introduction states that lenalidomide is highly effective for multiple myeloma, mantle cell lymphoma, and del(5q) myelodysplastic syndrome. It identifies thalidomide and pomalidomide as related drugs in widespread clinical use, but does not establish that each is an effective treatment for all three diseases. (LLM, conf=0.87)

  cites [1] sourceExperimental — accuracy not yet measured

In the paper

“The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase.”

What the sources say

• Partly supported

  The indexed passages describing Figs. 1 and 3 (section labeled Abstract; page unavailable) report lenalidomide-dependent binding of IKZF1 and IKZF3 to cereblon, IKZF1 ubiquitylation, and proteasome-dependent loss of both proteins; the Fig. 1 passage also reports similar down-regulation with pomalidomide. The copy does not establish that thalidomide causes their degradation, or specifically identify the recruited region as a C2H2 zinc finger domain. It describes cereblon as a substrate-recognition component of a cullin-dependent ligase but does not give the CRL4CRBN designation. (LLM, conf=0.88)

  cites [2] sourceExperimental — accuracy not yet measured
• Partly supported

  The indexed text (section labelled Abstract; page unavailable) shows that lenalidomide recruits IKZF1 and IKZF3 to CRBN through a region within zinc finger domain 2, promoting their ubiquitination and degradation. It also reports that thalidomide and pomalidomide decrease both proteins’ abundance, but does not establish that those two drugs use the same zinc-finger recruitment and ubiquitination mechanism. (LLM, conf=0.87)

  cites [3] sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion and Differential substrate specificity sections (pages not provided) state that all three drugs induce CRL4CRBN-mediated ubiquitination and degradation of IKZF1 and IKZF3. The copy mentions an IKZF1/3 degron but does not identify it as a C2H2 zinc finger domain or explicitly describe the degradation of these proteins as proteasomal. (LLM, conf=0.86)

  cites [4] sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [5] source
• ?Not checked — no full text of the sourcecites [6] source
• Partly supported

  The 'CRBN functions as a DCAF' section identifies CRBN as a substrate receptor and reports IMiD-dependent degradation of IKZF1/3, noting that thalidomide is less efficient than lenalidomide and pomalidomide. The 'Thalidomide is an agonist and antagonist' section proposes recruitment of Ikaros to IMiD-bound CRBN but says the detailed binding mode remains to be established. Our copy does not identify a C2H2 zinc finger domain as the recruited portion, so that specific mechanism is unsupported. (LLM, conf=0.88)

  cites [7] sourceExperimental — accuracy not yet measured

In the paper

“Given the CRL4CRBN protein architecture and its ~100 Å ubiquitination radius (6, 7, 22)”

What the sources say

• ?Not checked — no full text of the sourcecites [6] source
• Partly supported

  In “CRBN functions as a DCAF for the CRL4CRBN ligase” (page unavailable), the paper describes a ubiquitination zone created by rotation of the ligase arm. It gives the zone’s dimensions as up to 340 × 110 × 30 Å, not a ubiquitination radius of approximately 100 Å. (LLM, conf=0.85)

  cites [7] sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [22] source

In the paper

“Thalidomide, lenalidomide and pomalidomide mediate CRL4CRBN-dependent degradation of a larger number of proteins than previously appreciated through a C2H2 ZF degron (3, 4, 16).”

What the sources say

• Partly supported

  The paper reports CRL4–CRBN-dependent degradation of IKZF1 and IKZF3 by lenalidomide, and says thalidomide and pomalidomide also decreased their abundance (Abstract; page unavailable). It identifies a degradation-determining sequence within an IKZF3 zinc-finger domain, but does not establish the claimed broader set of proteins degraded by all three drugs through a C2H2 zinc-finger degron. (LLM, conf=0.84)

  cites [3] sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion and Differential substrate specificity sections report CRL4CRBN-mediated degradation of IKZF1 and IKZF3 by all three drugs and identify CK1α as an additional substrate strongly affected by lenalidomide, but not thalidomide. The CK1α is a substrate of CRL4CRBN section says its responsive region has no evident homology to the previously identified IKZF1/3 degron. The paper therefore does not support the claim that the expanded set of proteins is degraded through a C2H2 zinc-finger degron. (LLM, conf=0.88)

  cites [4] sourceExperimental — accuracy not yet measured
• Partly supported

  The ‘ZFP91 is a lenalidomide-dependent CRL4 CRBN substrate’ section (page not provided) reports degradation of the additional substrate ZFP91 with lenalidomide and pomalidomide, but says thalidomide’s effect in cells was minimal. The paper proposes a common zinc-finger motif for IMiD-dependent binding; it does not establish degradation of a larger set of proteins by all three drugs through a C2H2 degron. (LLM, conf=0.88)

  cites [16] sourceExperimental — accuracy not yet measured

In the paper

“Examination of published proteome-wide mass spectrometry data confirmed degradation of endogenous, full-length WIZ in WSU-DLCL2 (human diffuse large B cell lymphoma) and TMD8 (human diffuse large B cell lymphoma) cells in the presence of lenalidomide and the thalidomide analog CC-122 (fig. S7C) (20).”

What the sources say

• Supported

  The ‘Differential activities of CC-122 and lenalidomide in DLBCL’ section describes mass-spectrometry proteomics of TMD8 and WSU-DLCL2 cells treated with both drugs. The citation identifies the published data that the citing authors examined; the WIZ finding is attributed to their examination, not to a finding reported in the source paper (page unavailable in our copy). (LLM, conf=0.88)

  cites [20] source

In the paper

“and could contribute to developmental abnormalities in fetuses exposed to thalidomide (25–30).”

What the sources say

• Partly supported

  The Introduction (page not provided) links HIC1 haploinsufficiency to Miller–Dieker syndrome, supporting a possible connection between HIC1 and developmental abnormalities. It does not address thalidomide exposure or the proposed roles of HIC2, GZF1, OSR1, OSR2, and SALL4 in exposed fetuses. (LLM, conf=0.82)

  cites [25] sourceExperimental — accuracy not yet measured
• Partly supported

  The Hic2 Has a Haploinsufficient Cardiovascular Phenotype section (page unavailable) reports developmental delay and cardiac defects in Hic2-mutant mouse embryos, supporting a developmental role for HIC2. The paper does not address thalidomide exposure or establish that HIC2 contributes to abnormalities in exposed fetuses; it also does not establish the proposed roles of GZF1, OSR1, OSR2, or SALL4. (LLM, conf=0.86)

  cites [26] sourceExperimental — accuracy not yet measured
• Partly supported

  The section “Osr1 regulates embryonic myogenesis” (page not provided) reports limb muscle patterning defects in Osr1-deficient mouse embryos, supporting a developmental role for OSR1. It does not link those defects to thalidomide exposure or address the proposed contribution of HIC1, HIC2, GZF1, OSR2, and SALL4; searches for thalidomide and the gene list found no such passage. (LLM, conf=0.82)

  cites [27] sourceExperimental — accuracy not yet measured
• Partly supported

  The Results and in situ hybridization passages (page unavailable) show Osr2 expression in developing mouse limbs and craniofacial tissues, providing developmental context for one gene in the claim. They do not link Osr2 to thalidomide exposure or fetal abnormalities, and this paper does not address HIC1, HIC2, GZF1, or SALL4. (LLM, conf=0.78)

  cites [28] sourceExperimental — accuracy not yet measured
• Partly supported

  The section on GZF1 and the HOXA10 regulatory region proposes a possible role for GZF1 in normal kidney development. It does not address thalidomide exposure or fetal abnormalities, and it does not implicate the other five named genes in those abnormalities. (LLM, conf=0.82)

  cites [29] sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page unavailable) supports SALL4 as a cause of developmental abnormalities in a family previously thought to have thalidomide embryopathy. It does not address HIC1, HIC2, GZF1, OSR1, or OSR2. It also argues that the family's phenotype was likely misdiagnosed as thalidomide embryopathy, rather than showing that these genes contribute to abnormalities caused by thalidomide exposure. (LLM, conf=0.86)

  cites [30] sourceExperimental — accuracy not yet measured

In the paper

“Several of the ZFs identified as degraded in functional experiments, or in vitro binders that could be targeted with further drug development, have been implicated in human disease.”

What the sources say

• ?Not checked — no full text of the sourcecites [23] source
• ?Not checked — no full text of the sourcecites [24] source
• Supported

  The Introduction identifies HIC1 as a zinc-finger transcription factor and links it to human tumors and Miller–Dieker syndrome. This supports the paper's use as an example of a disease-implicated zinc-finger protein; the degradation experiments and potential drug development are the citing authors' framing, not findings attributed to this source. (LLM, conf=0.87)

  cites [25] source
• Supported

  The Results section reports that loss of HIC2 is associated with congenital cardiac disease in distal 22q11 deletion syndrome. The Discussion identifies HIC2 as a contributor to the syndrome’s phenotype, supporting its use as an example of a disease-implicated protein. (LLM, conf=0.85)

  cites [26] source
• ?Could not verify

  The References section identifies Osr1 and Osr2 as zinc-finger transcription factors, while the experimental sections concern Osr1’s role in embryonic limb development. Searches for degraded zinc-finger proteins, in vitro binders, drug development, and their implication in human disease found no passage addressing the attributed claim. Page numbers were not available in our copy. (LLM, conf=0.75)

  cites [27] sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Results section identifies mouse OSR2 as a zinc-finger protein, and §2.1 identifies a putative human OSR2 gene. Neither passage links any zinc-finger protein to human disease; searches for disease implications, experimental degradation, in vitro binding, and drug development found no such discussion in our copy. Page numbers were not available. (LLM, conf=0.76)

  cites [28] sourceExperimental — accuracy not yet measured
• ?Could not verify

  The analysis of GZF1 zinc finger motifs reports DNA-binding experiments, but does not link GZF1 or several zinc finger proteins to human disease. A search for “human disease” found that phrase only in a reference about GDNF/RET signaling, not in a finding about the zinc finger proteins described in the claim. The copy also does not address their degradation or potential drug targeting. (LLM, conf=0.76)

  cites [29] sourceExperimental — accuracy not yet measured
• Supported

  The Figure 3 caption identifies zinc-finger motifs in SALL4, and the Discussion states that SALL4 mutations cause a range of clinical phenotypes. This supports SALL4 as an example of a zinc-finger protein implicated in human disease; the citing paper’s degradation and drug-development framing is not a finding attributed to this source. (LLM, conf=0.90)

  cites [30] source

In the paper

“Replacing ZF3 with ZF1 in the context of the ZF2-ZF3 construct (ZF2-ZF1) decreased the binding affinity by a factor of 6 (Ki 1027±302 nM).”

In the paper

“Four of these full-length proteins were previously unknown thalidomide analog targets (fig. S2D).”

In the paper

“We confirmed degradation of endogenous ZNF692, ZFP91, ZNF276, ZNF653 and ZNF827 in the presence of pomalidomide using western blotting (Fig. 2, C–E).”

In the paper

“At a false discovery rate (FDR) of <0.01, pomalidomide depleted 11 C2H2 ZFs, each from different proteins, including IKZF1/IKZF3 ZF2 (Fig. 1E).”

In the paper

“Lenalidomide and thalidomide targeted a variable subset of these 11 ZFs.”

In the paper

“Insertion of a glycine residue between the β-hairpin residues N148 and Q149 of the IKZF1 ZF2-ZF3 construct (IKZF3 N149 and Q150; fig. S1D) compromised binding in vitro (fig. S3A).”

In the paper

“with 4,661 out of 6,572 carrying a glycine residue at an equivalent position”

In the paper

“The screen highlighted 9 loci in IKZF3 ZF2 whose amino acid identities were critical for degradation (Fig. 3, B and C, and fig. S3, C and D).”

In the paper

“Higher affinity binding to CRBN-pomalidomide in vitro was observed with an IKZF1 construct spanning ZF2 and ZF3 (aa 143–243(Δ197–238); Ki 165±37 nM).”

In the paper

“These loci again included residues that maintain the tertiary ZF fold (IKZF3 C148, C151, F155, L161, H164, H168) and the β-hairpin glycine residue (IKZF3 G152).”

In the paper

“This model positions the C-terminus of ZF2 adjacent to the N-terminus of ZF3 consistent with the 5 amino acid linker between the two ZFs.”

In the paper

“When cloned into the degradation reporter vector and tested in isolation, the 11 ZFs exhibited degradation in the presence of all three compounds (Fig. 2A and fig. S2A).”

In the paper

“Additionally, we demonstrated binding of hemagglutinin (HA)-tagged, full-length ZFP91 and ZNF692 to endogenous CRBN in the presence of all three compounds by immunoprecipitation (fig. S2C).”

In the paper

“Besides the inter-cysteine spacing, residues that stabilize the ternary ZF fold, or residues that frequently appear in C2H2 ZF domains, no discernable sequence consensus could be detected among the 11 ZFs (Fig. 3A).”

In the paper

“In contrast, the more than 28 C2H2 ZFs accommodated by the drug-CRBN interface show surprisingly little sequence conservation, apart from residues that stabilize the ternary ZF fold.”

In the paper

“all three thalidomide analogs exhibited distinct ZF degradation patterns across the ZF library (Fig. 6C).”

In the paper

“Using the ZNF692 template structure, we accurately predicted all 11 ZFs identified in the library screen (Fig. 6A)”

In the paper

“Small differences in affinity between the ZF and the drug-CRBN complex in vitro translate into large differences in degradation in cells”

In the paper

“Thus, of the 33 ZFs individually tested in this study, 28 are recruited to pomalidomide-engaged CRBN.”

In the paper

“The 11 previously identified ZF hits again scored in the screen with pomalidomide (fig. S8B).”

In the paper

“16 ZFs bound CRBN-pomalidomide in vitro (BCL6, BCL6B, EGR1, EGR4, GZF1, HIC1, HIC2, SALL1, SALL3, SALL4, OSR1, OSR2, WIZ, ZBTB7A, ZBTB7B), with similar (WIZ ZF7) or lower binding affinities (~5–20 fold) than the respective IKZF1 ZF2-ZF3 or ZF2 constructs (Fig. 6B and fig. S6, C, D, and E).”

In the paper

“Strikingly, 7 of the 11 ZF degrons carry an equivalent glutamine residue at this position (Fig. 3A).”

In the paper

“IKZF2 ZF2 differs from IKZF3 ZF2 by a single amino acid substitution at the drug-ZF interface, Q147H (Fig. 5A and fig. S1D).”

In the paper

“Crystal structures of CRBN-pomalidomide bound to IKZF1 ZF2 and ZNF692 ZF4 reveal the detailed side chain interactions between CRBN, pomalidomide and the ZFs.”

In the paper

“Replacing the IKZF3 ZF2 β-hairpin with the ZFP91 ZF4 β-hairpin resulted in a construct that was degraded more efficiently than either of the parent molecules”

In the paper

“however, replacing the IKZF3 α-helix with the ZFP91 α-helix resulted in a ZF domain that was resistant to degradation (fig. S5F).”

In the paper

“The model highlights three consecutive arginine residues of ZF3 (IKZF1 R183-R185) at the CRBN-ZF2 interface that are not present in the ZF2-ZF1 construct (Fig. 1A and fig. S4C).”

In the paper

“The crystal structures suggest that ZF side chains, particularly those proximal to the drug at IKZF1 position 146, 148 and 153 (IKZF3 147, 149 and 154), interact with thalidomide analogs”

In the paper

“found 40 ZFs with lower interface energy scores than the ZNF692 ZF4 template”

In the paper

“We identified 15 individual ZFs and 7 full-length ZF-containing proteins that are degraded by thalidomide derivatives in functional or computational screens.”

In the paper

“and identified 108 ZFs scoring better than the lowest ranking ZF found degraded in the library screen.”

In the paper

“Notably, IKZF2 ZF2 was selectively degraded by CC-220 (Fig. 6C).”

In the paper

“Crystal structures, in vitro binding, and cellular degradation assays illustrate that 28 ZFs (including IKZF2) with diverse amino acid sequences bind the same drug-CRBN interface (fig. S6E).”

In the paper

“IKZF1 residues A152 and L166 (IKZF3 A153 and L167) face CRBN residues V388, I371 and A395 located on the opposite side of the groove (Fig. 4, C and D).”

In the paper

“Overall, different docking trials revealed an overlapping set of ~50–150 ZFs with interface scores similar to, or better than those of ZFs found degraded in the library screen (Fig. 6A and fig. S6, A and B)”

In the paper

“Supporting this notion, 28 of the ~50–150 additional C2H2 ZFs nominated by in silico docking were shown to bind the drug-CRBN complex”

In the paper

“In validation experiments, the IKZF2 ZF2 reporter was degraded by more than 40% in the presence of CC-220 (fig. S8, C and D).”

In the paper

“Equivalent mutations in IKZF3 ZF2 (Q147I/A/H) stabilized the degradation reporter in cells (Fig. 5D, fig. S5D)”

In the paper

“We observed that thalidomide analogs with chemical modifications at the drug-ZF interface are capable of converting ZFs with weak affinity for the CRBN-pomalidomide complex into degraded targets.”

In the paper

“This suggests that the ZF ‘degrome’ is restricted to an inter-cysteine spacing of two residues.”

In the paper

“Unexpectedly, the known thalidomide analog targets, IKZF1/3, ZFP91, CK1a and GSPT1 do not share obvious primary sequence similarity with the exception of a glycine residue located in a β-hairpin, which diverges from the canonical destruction motif paradigm.”

In the paper

“Degradation of these targets in part explains the therapeutic effects of thalidomide analogs.”

In the paper

“IKZF1 binding to CRBN-pomalidomide is thus driven by ZF2 in vitro, with minor but specific contributions from the C-terminal ZF3.”

In the paper

“Of the 6,572 ZFs, 5,611 had sufficient representation in the sequencing data to be assayed (>200 read counts).”

In the paper

“These data show that in addition to the residues maintaining the ZF fold, non-structural amino acids (IKZF3 Q147, G152 and A153) contribute to degron specificity.”

In the paper

“The identified ZF degrons thus revealed structural features common to C2H2 ZF domains that alone cannot explain selective degradation of only 11 ZFs, while 4,661 structurally similar ZFs were present in the library.”

In the paper

“Despite the low degree of sequence similarity across the 11 ZF degrons (Fig. 3A), only distinct amino acid combinations at the drug-CRBN interface result in degradation.”

In the paper

“In addition to the complex ZF-CRBN side chain interaction, direct contacts between thalidomide analogs and the ZF provide additional layers of specificity (fig. S5D).”

In the paper

“and that chemical alterations can modulate ZF specificity (fig. S5D).”

In the paper

“which are likely due to competition for CRBN occupancy by multiple ZFs as well as other substrates that utilize the same binding surface”

In the paper

“demonstrating that the CRBN surface is more permissive than suggested by the number of targets identified in the ZF library screens.”

In the paper

“Thus, the two structures demonstrate how CRBN accommodates ZF degrons with varying side chain properties at the ZF-CRBN interface (Fig. 3A; IKZF3 N148: E, N, K, R, V, P; Q149: I, V, R, L, A; A153: Y, F, L, R and L167: V, I, K, N).”

In the paper

“Our in silico ZF docking approach is thus capable of predicting ZFs that interact with the drug-CRBN complex with over 80% accuracy (fig. S6F).”

In the paper

“ZF degrons bind a complementary groove on the CRBN surface that fits the overall shape of the C2H2 ZF domain, bringing different amino acids of different ZF degrons in contact with the same drug-CRBN interface.”

In the paper

“suggesting that an unexpectedly large number of ZFs are capable of binding the drug-CRBN interface.”

In the paper

“Our results suggest that degradation of C2H2 ZF-containing proteins through novel thalidomide derivatives may be a generalizable paradigm for targeting “undruggable” transcription factors”