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Cited by 95Protein Degradation and InhibitorsColorectal Cancer Treatments and StudiesUbiquitin and proteasome pathways

Degradation of CCNK/CDK12 is a druggable vulnerability of colorectal cancer

Open access
Dieter, Sebastian M., Siegl, Christine, Codó, Paula L., Huerta, Mario, Ostermann-Parucha, Anna L., Schulz, Erik, Zowada, Martina K., Martin, Sylvia +24 more·2021·Cell Reports
DOI 10.1016/j.celrep.2021.109394
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Abstract

Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.

AI snapshot

Written from the paper’s full text.

TL;DR

NCT02 was identified from a phenotypic screen of ~80,000 small molecules against patient-derived colorectal cancer tumor spheroid cultures and acts as a molecular glue degrader of CCNK/CDK12. Targeted CCNK/CDK12 degradation is a vulnerability in a subset of colorectal cancers, particularly TP53-deficient and CMS4 tumors.

Key findings

  • In a screen of ~80,000 non-characterized small molecules against TSC03 cells, 346 compounds showed >95% inhibition; after triage, 14 compounds (NCT01–NCT14) had IC50 <20 µM, and NCT02 showed the most differential response across TSCs while lacking activity against normal primary fibroblasts.
  • Across 24 patient-derived TSCs, 13 were sensitive (IC50 <4 µM), 7 resistant (IC50 >10 µM), and 4 intermediate; NCT02 decreased self-renewing spheroid-forming cell frequency, induced apoptosis, downregulated DNA damage response genes, and increased γH2AX.
  • NCT02 induced ubiquitination and proteasomal degradation of CCNK and CDK12; degradation was rescued by MG132 or MLN4924, depended on DDB1, and involved molecular glue activity between CDK12/CCNK and DDB1.
  • TP53-deficient TSCs were significantly more sensitive to NCT02, and CMS4 TSCs were significantly more sensitive than CMS2/CMS3; CDK12 knockout reduced viability in 9/31 CRC cell lines, and SR-4835 halted tumor growth in TSC03-derived PDXs but not TSC60-derived PDXs.

Methods

  • A biobank of 24 primary CRC TSCs from primary and metastatic samples was characterized by whole-exome/whole-genome sequencing and transcriptome analysis, including CMS assignment.
  • High-throughput screening used ~80,000 compounds at 10 µM for 96 h with ATPlite viability readout, followed by dose-response validation and testing across TSCs and non-malignant fibroblasts.
  • Target deconvolution combined transcriptome analysis after 12 h NCT02 treatment, thermal proteome profiling of >5,000 proteins, chemical proteomics pull-downs, ubiquitin coIP, CRISPR/Cas9 knockouts, xenograft/PDX experiments, and Bliss synergy testing with oxaliplatin or irinotecan.

Limitations

  • Stated: the biobank’s limited size of 24 TSCs was not expected to provide robust statistics, and its heterogeneity required selecting individual TSCs for secondary assays.
  • Stated: NCT02 was not metabolically stable enough for in vivo experiments, so its potency was not tested in vivo; SR-4835 was used as a surrogate for targeted CCNK/CDK12 degradation in PDXs.
  • Stated: combination synergy with oxaliplatin or irinotecan was tested only in vitro, so the authors cannot formally rule out less synergy in vivo. Evident: direct biochemical binding of NCT02 to CDK12 was not detected, consistent with low/undetectable affinity typical of molecular glues.

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

Integrity signals

Registry

Crossref · 10.1016/j.celrep.2021.109394

Cites

59 of 59 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 41 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Serine 5 phosphorylation of POLR2 was not changed after NCT02 treatment.

    “Moreover, serine 5 phosphorylation of POLR2, which is the major target of CDK9, was not changed after NCT02 treatment (Figure S3A).”
    Quote in the paperSubstantial claim
  • Findingown data

    The biobank of TSCs captured key aspects of the genomic and transcriptomic diversity of CRCs.

    “In summary, our biobank of TSCs captured key aspects of the genomic and transcriptomic diversity of CRCs.”
    • Also stated in: § A collection of primary TSCs reflects CRC heterogeneity · boosted
      “The most commonly mutated genes in patients with CRC as identified by The Cancer Genome Atlas Network (Cancer Genome Atlas, 2012) were well represented in our biobank”
    • Also stated in: § A collection of primary TSCs reflects CRC heterogeneity · plain
      “all CMSs could be detected in our biobank”
    Quote in the paperSubstantial claim
    • Rests on an assumption: These tumor cell models recapitulate the original patient tumor after transplantation into immunodeficient mice.
  • Findingown data

    NCT02 was largely inert in biochemical interaction screens with 68 targets causing critical side-effects and a collection of 468 kinases.

    “In biochemical interaction screens with 68 targets causing critical side-effects in patients and a collection of 468 kinases, NCT02 was largely inert, indicating that our selection strategy did not identify a promiscuous or non-specifically toxic compound (Table S3).”
    Quote in the paperSubstantial claim
  • Findingown data

    NCT02 caused a concentration-dependent increase in cleaved PARP-positive cells in four out of five sensitive TSCs, indicating that apoptosis contributed to inhibition.

    “we detected a concentration-dependent increase in the percentage of cleaved PARP (cPARP)-positive cells in four out of five sensitive TSCs after treatment with NCT02, indicating that induction of apoptosis contributed to inhibition of sensitive TSCs by NCT02 (Figure 2D).”
    Quote in the paperNot a substantial claim
  • Findingown data

    CDK13 was found with a significant reduction of protein intensity, although at low levels.

    “CDK13, an additional binding partner of CCNK and not detected in previous TPP experiments, was found with a significant reduction of protein intensity (although at low levels).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 37
  • The quote only partly states this claim. 3
  • Classified as probably not a substantial claim. 32

  • May combine two claims.

    Findingown data

    Decrease of CDK12 was also detected in two resistant TSCs after 2 h, but CDK12 protein levels increased back to baseline after 6 h.

    “Interestingly, decrease of CDK12 was also detected in two resistant TSCs after 2 h, but CDK12 protein levels increased back to baseline after 6 h, indicating that reduction of CDK12 protein levels was compensated in at least some resistant TSCs over time.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Combined overexpression of CCNK/CDK12 decreased sensitivity toward NCT02, confirming the functional relevance of CCNK/CDK12 for the inhibitory effect of NCT02.

    “Combined overexpression of CCNK/CDK12 decreased sensitivity toward NCT02, confirming the functional relevance of CCNK/CDK12 for the inhibitory effect of NCT02 (Figures 4F and 4G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fourteen compounds (NCT01-NCT14) showed IC50 below 20 mM and appropriate dose-response curves with Hill slope between 0.5 and 2.5.

    “Of the 120 tested compounds, 14 (NCT01-NCT14) (Figure 1D; Table S2) were chosen that showed an IC 50 below 20 mM and appropriate doseresponse curves (Hill slope between 0.5 and 2.5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Thermal proteome profiling identified 43 proteins with significantly differing melting curves, of which 15 showed increased stability and 28 showed decreased stability.

    “We identified 43 proteins with significantly differing melting curves, of which 15 proteins showed a shift toward increased stability, and 28 proteins showed a shift toward decreased stability (Figure 4B; Table S5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NCT02 showed the most pronounced differential response profile and lacked activity against normal primary fibroblasts.

    “The compound that showed the most pronounced differential response profile and lacked activity against normal primary fibroblasts was NCT02 (Figure 1E).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    A high-throughput screen of approximately 80,000 small molecules against TSC03 identified 346 active compounds using a 95% inhibition cutoff.

    “Using a cut-off of 95% inhibition, 346 active compounds were identified (Figure 1C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TP53 deficiency and CMS4 predict sensitivity to NCT02.

    “TP53 deficiency and consensus molecular subtype 4 predict sensitivity to NCT02”
    • Also stated in: § TP53-deficient TSCs assigned to CMS4 are particularly sensitive to NCT02 · plain
      “TP53-deficient TSCs assigned to CMS4 are particularly sensitive to NCT02”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CDK9 and its target gene MCL1 levels were not affected by NCT02.

    “Levels of CDK9, which can also phosphorylate serine 2 of POLR2, and its target gene MCL1 were not affected by NCT02 (Figure 5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ingenuity upstream regulator analysis identified CCNK as potentially inhibited by NCT02, with the lowest p value (p = 9.22 x 10^-8) and the highest Z score for inhibition (-3.65).

    “Ingenuity upstream regulator analysis identified several transcriptional regulators potentially inhibited by NCT02, of which cyclin K (CCNK) showed both the lowest p value (p = 9.22 3 10 À8 ) and the highest Z score for inhibition (À3.65) (Figure 3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Combination treatment with MLN4924 substantially rescued NCT02-induced CCNK degradation, demonstrating that NCT02-induced CCNK degradation is dependent on Cullin-RING E3 ubiquitin ligases.

    “Combination treatment of TSC03 cells with NCT02 and MLN4924 revealed a substantial rescue of NCT02-induced CCNK degradation, demonstrating that NCT02-induced CCNK degradation is dependent on CRE3s (Figure 5H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SR-4835 highly selectively binds CDK12 and CDK13, induces degradation of CCNK similar to NCT02, and shows a differential sensitivity pattern across different TSCs comparable to NCT02.

    “we treated PDXs with SR-4835 that highly selectively binds CDK12 and CDK13, induces degradation of CCNK similar to NCT02, and shows a differential sensitivity pattern across different TSCs comparable to NCT02 (Figure S4B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Two CDK12 mutations were found in sensitive TSCs: one nonsense mutation and one missense mutation of unknown significance outside the kinase domain.

    “Two CDK12 mutations were found in sensitive TSCs, one of them being a nonsense mutation, the other one being a missense mutation of unknown significance outside the kinase domain.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Ubiquitin coimmunoprecipitation revealed additional higher molecular weight bands for CCNK, but not CDK12, after combination treatment, indicating ubiquitination of CCNK induced by NCT02.

    “which revealed additional bands of higher molecular weight than the unmodified protein for CCNK, but not CDK12, after combination treatment (Figure 5G), indicating ubiquitination of CCNK induced by NCT02”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Combination of NCT02 and MG132 partially rescued CCNK and CDK12 protein levels, apoptosis, and the impact of NCT02 on cell viability, indicating that the inhibitory effect of NCT02 was dependent on protein degradation.

    “Indeed, protein levels of CCNK and CDK12, induction of apoptosis and the impact of NCT02 on cell viability could be partially rescued upon combination treatment (Figures 5D-5F), clearly indicating that the inhibitory effect of NCT02 was dependent on protein degradation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SR-4835 halted tumor growth in PDXs derived from TSC03, whereas PDXs derived from TSC60 cells continued growing despite treatment.

    “SR-4835 halted tumor growth in PDXs derived from TSC03, whereas PDXs derived from TSC60 cells continued growing despite treatment (Figure 6D)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: NCT02 was metabolically not stable enough for in vivo experiments.

  • May combine two claims.

    Findingown data

    Transcriptomic and proteomic approaches characterized NCT02 as a molecular glue degrader that depletes CCNK and induces degradation of its binding partners CDK12 and CDK13.

    “Transcriptomic and proteomic approaches characterized NCT02 as a molecular glue degrader that depletes CCNK and induces degradation of its binding partners CDK12 and CDK13.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    NCT02 lacks detectable CDK12 kinase inhibitory activity, distinguishing it from CR8 and SR-4835, which both act as degrader and kinase inhibitors.

    “Notably, the lack of detectable CDK12 kinase inhibitory activity distinguishes NCT02 from CR8 and SR-4835 that both act as degrader and kinase inhibitors.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    Fourteen inhibitory compounds from the primary screen were validated against different TSCs and non-malignant primary human fibroblasts.

    “Validation of 14 inhibitory compounds from the primary screen against different TSCs and non-malignant primary human fibroblasts (HD-F; x = no data available; color scale for sensitivity [logIC 50 mM] identical to B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only small variations like replacement of the 5-methyl substituent with ethyl or trifluoromethyl were tolerated in NCT02, while other heterocycles or substituent patterns led to inactive compounds.

    “Only small variations like replacement of the 5-methyl substituent with ethyl or trifluoromethyl were tolerated, whereby other heterocycles or substituent patterns led to inactive compounds (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    CCNK-eGFP degradation is induced by NCT02 alone or in combination with increasing doses of competitive, non-degrading CDK12 inhibitors.

    “CCNK-eGFP degradation induced by 2 h treatment with NCT02 alone or in combination with increasing doses of competitive, non-degrading CDK12 inhibitors (presented are mean + SD).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    During SAR exploration of NCT02, the northern benzofuran derivative part tolerated high structural variability while the southern triazole part showed a very steep SAR.

    “During a first structure-activity relationship (SAR) exploration of the lead molecule NCT02 a very high structural variability at the northern bezofuranderivative part of the molecule was encountered, while the southern triazole part showed a very steep SAR.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    NCT02 induced downregulation of genes involved in DNA damage repair, which was associated with an increase in unrepaired DSBs and genomic instability.

    “Thus, NCT02 induced the downregulation of genes involved in DNA damage repair, which was associated with an increase in unrepaired DSBs and genomic instability.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Molecular glue degraders deplete CCNK by promoting an interaction between CCNK and the CUL4 adaptor protein DDB1 via CDK12, thereby presenting CCNK for ubiquitination and degradation.

    “Recently, molecular glue degraders that deplete CCNK were identified (Lv et al., 2020;Mayor-Ruiz et al., 2020;S1abicki et al., 2020).These compounds act by promoting an interaction between CCNK and the CUL4 adaptor protein DDB1 via CDK12, thereby presenting CCNK for ubiquitination and degradation.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Lv et al., 2020

      Not checked in the free run.

    • cites Mayor-Ruiz et al., 2020 source

      Not checked in the free run.

    • cites S1abicki et al., 2020

      Not checked in the free run.

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

    Premisecitation

    The interaction of CDK12 with CCNK occurs via the kinase domain.

    “which occurs via the kinase domain (Lv et al., 2020)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lv et al., 2020

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    TICs that have the capacity to self-renew and maintain long-term tumor growth are usually rare within the tumor mass but highly enriched within spheroid cultures.

    “Moreover, TICs that have the capacity to self-renew and maintain long-term tumor growth are usually rare within the tumor mass but highly enriched within spheroid cultures (Dieter et al., 2011;Ishiguro et al., 2017).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Dieter et al., 2011 source

      Not checked in the free run.

    • cites Ishiguro et al., 2017 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Complex formation of CCNK and CDK12 contributes to stability of both complex partners.

    “complex formation of CCNK and CDK12, which contributes to stability of both complex partners (Barette et al., 2001)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Barette et al., 2001 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Loss of CCNK and CDK12 decreased viability of CRC cells, suggesting that degradation of these proteins by NCT02 at least contributed to its inhibitory effect.

    “Thus, loss of CCNK and CDK12 decreased viability of CRC cells, suggesting that degradation of these proteins by NCT02 at least contributed to its inhibitory effect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CCNK degradation can be triggered by structurally diverse compounds, and selective CDK12 inhibitors display a variable degree of kinase inhibition and degradation induction.

    “indicating that CCNK degradation can be triggered by structurally diverse compounds, and selective CDK12 inhibitors display a variable degree of kinase inhibition and degradation induction”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    NCT02D active kept its activity in cellular assays with a highly correlating differential response profile across individual TSCs, indicating that relevant target(s) of NCT02 were preserved.

    “NCT02D was selected, because it kept its activity in cellular assays with a highly correlating differential response profile across individual TSCs (Figures S2C and S2D), indicating that relevant target(s) of NCT02 were preserved.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The heterogeneous response profile of TSCs toward targeted CDK12/CCNK degradation was recapitulated in vivo and confirms its therapeutic value for a subset of CRC.

    “indicating that the heterogeneous response profile of TSCs from our biobank toward targeted CDK12/CCNK degradation was recapitulated in vivo and confirming its therapeutic value for a subset of CRC”
    Quote in the paperSubstantial claim
    • Rests on an assumption: NCT02 was metabolically not stable enough for in vivo experiments.

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

    Premisecitation

    Binding of a compound to a protein can result in thermal stabilization of the protein and a detectable shift of its melting curve.

    “This assay is based on the concept that binding of a compound to a protein can result in thermal stabilization of the protein and-consequently-in a detectable shift of its melting curve.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Savitski et al., 2014 source

      Not checked in the free run.

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

    Premisecitation

    Identification of the molecular target and selection of a promising candidate from a list of toxic compounds remains a challenge for phenotypic screens.

    “Still, identification of the molecular target and selection of a promising candidate from a list of toxic compounds remains a challenge for phenotypic screens (Eder et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Eder et al., 2014 source

      Not checked in the free run.

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

    Premisecitation

    3D patient-derived tumor models of CRC allow maintenance and expansion of functionally relevant cell subfractions and harbor the patients' individual genetic lesions.

    “3D patient-derived tumor models of CRC (i.e., organoid and spheroid cultures) allow maintenance and expansion of functionally relevant cell subfractions and harbor the patients' individual genetic lesions (Ishiguro et al., 2017;van de Wetering et al., 2015).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Ishiguro et al., 2017 source

      Not checked in the free run.

    • cites van de Wetering et al., 2015 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Phenotypic screens can identify compounds that modulate targets that are not considered druggable.

    “Notably, phenotypic screens can identify compounds that modulate targets that are not considered druggable-a prominent example being Lenalidomide that induces degradation of transcription factors that are critical drivers of multiple myeloma (Kro ¨nke et al., 2014).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kro ¨nke et al., 2014

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    These molecular glue degraders induce complex formation between CDK12/CCNK and the CUL4 adaptor protein DDB1, presenting CCNK for ubiquitination and degradation via DDB1-CUL4-RBX1 ubiquitin ligases.

    “These molecules induce complex formation between CDK12/CCNK and the CUL4 adaptor protein DDB1, presenting CCNK for ubiquitination and degradation via DDB1-CUL4-RBX1 ubiquitin ligases.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Lv et al., 2020

      Not checked in the free run.

    • cites Mayor-Ruiz et al., 2020 source

      Not checked in the free run.

    • cites S1abicki et al., 2020

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    WRN has been identified as a synthetically lethal target of MSI-H cancers.

    “recent reports have identified WRN as a synthetically lethal target of MSI-H cancers”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Chan et al., 2019 source

      Not checked in the free run.

    • cites Dubbury et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    TP53 deficiency might be important, but not sufficient, to induce sensitivity to NCT02.

    “indicating that TP53 deficiency might be important, but not sufficient to induce sensitivity.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Fitness Scores calculated from large-scale genome-wide CRISPR/Cas9 screens across 31 independent CRC cell lines represent a quantitative measure of the reduction of cell viability elicited by gene inactivation via CRISPR/Cas9.

    “''Fitness Scores'' that were calculated from large-scale genome-wide CRISPR/Cas9 screens across 31 independent CRC cell lines and represent a quantitative measure of the reduction of cell viability elicited by a gene inactivation via CRISPR/Cas9 (https://score.depmap.sanger.ac.uk) (Behan et al., 2019;van der Meer et al., 2019).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Behan et al., 2019 source

      Not checked in the free run.

    • cites van der Meer et al., 2019 source

      Not checked in the free run.

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

    Premisecitation

    Molecular glues frequently bind to their target proteins with low or undetectable affinities.

    “consistent with the observation that molecular glues frequently bind to their target proteins with low or undetectable affinities (Lv et al., 2020).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lv et al., 2020

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    DDR deficiencies may sensitize to ATM/ATR inhibitors due to increased reliance on DDR checkpoints.

    “It has been suggested that DNA damage response (DDR) deficiencies may sensitize to ATM/ATR inhibitors due to increased reliance on DDR checkpoints (Weber and Ryan, 2015).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Weber and Ryan, 2015

      Not checked in the free run.

  • The quote only partly states this claim.

    Interpretationown data

    WRN mRNA downregulation potentially explains the sensitivity of MSI-H TSCs to NCT02.

    “potentially explaining the sensitivity of MSI-H TSCs to NCT02.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Interpretationown data

    The particular sensitivity of CMS4 TSCs (all TP53 deficient) to NCT02 is similar to the lower CDK12 Fitness Scores in CMS4 TP53 deficient cell lines.

    “similar to the particular sensitivity of CMS4 TSCs (that were all TP53 deficient ) to NCT02.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Synergy between CDK12 kinase inhibitors and drugs causing DNA damage or interfering with DNA damage repair pathways has recently been reported in other tumor entities.

    “Synergy between CDK12 kinase inhibitors and drugs causing DNA damage or interfering with DNA damage repair pathways has recently been reported in other tumor entities (Quereda et al., 2019).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Quereda et al., 2019 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    CMS1 marks highly microsatellite-instable (MSI-H) and hypermutated TSCs.

    “CMS1 marks highly microsatelliteinstable (MSI-H) and hypermutated TSCs (Guinney et al., 2015).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Guinney et al., 2015 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    WRN has been proposed to be a CDK12 target.

    “WRN has been proposed to be a CDK12 target (Chan et al., 2019;Dubbury et al., 2018).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Chan et al., 2019 source

      Not checked in the free run.

    • cites Dubbury et al., 2018 source

      Not checked in the free run.

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

    Premisecitation

    CMS4 is associated with particularly poor prognosis.

    “CMS4 is associated with particularly poor prognosis (Guinney et al., 2015)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Guinney et al., 2015 source

      Not checked in the free run.

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

    Premisecitation

    An association of TP53-deficiency and sensitivity to ATR inhibitors has been described as profound synthetic lethality.

    “an association of TP53-deficiency and sensitivity to ATR inhibitors has been described as profound synthetic lethality (Reaper et al., 2011;Toledo et al., 2011)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Reaper et al., 2011 source

      Not checked in the free run.

    • cites Toledo et al., 2011 source

      Not checked in the free run.

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

    Study design and conductreasoning

    The heterogeneous nature of the biobank of CRC TSCs required selection of individual TSCs suitable for individual secondary assays.

    “A limitation of our study is that the heterogeneous nature of our biobank of CRC TSCs with differences in e.g., tolerance of dissociation methods and cell sorting, efficiency of the CRISPR/ Cas9 system, and resistance toward selection antibiotics, required selection of individual TSCs suitable for individual secondary assays.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Surface-exposed moieties can confer gain-of-function glue properties to compounds.

    “which further supports the hypothesis that surfaceexposed moieties can confer gain-of-function glue properties to compounds (S1abicki et al., 2020).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites S1abicki et al., 2020

      Not checked in the free run.

  • May combine two claims.

    Study design and conductnone given

    NCT02 could not be applied in animal experiments and its potency was not tested in vivo.

    “Moreover, NCT02 could not be applied in animal experiments and its potency thus not be tested in vivo.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Oxaliplatin and irinotecan are both part of chemotherapy regimen for metastatic CRC.

    “that are both part of chemotherapy regimen for metastatic CRC (Grothey et al., 2004).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Grothey et al., 2004 source

      Not checked in the free run.

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

    Study design and conductnone given

    Positive and negative controls in the library were defined based on results across previously screened cell lines.

    “Positive and negative controls in the library were defined based on results across previously screened cell lines.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    NCT02 adds a different small molecule to the spectrum of drug-induced proteolysis and might open new therapeutic strategies for many tumor types including CRC by expanding the druggable proteome.

    “In summary, NCT02 adds a different small molecule to the spectrum of drug-induced proteolysis, which has just recently begun to be exploited by the class of proteolysis targeting chimeras (PROTACs) and molecular glue degraders and might open new therapeutic strategies for many tumor types including CRC by expanding the druggable proteome.”
    Quote in the paperSubstantial claim

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

    Study design and conductreasoning

    The combination therapies might act less synergistically in vivo.

    “we cannot formally rule out that these combination therapies might act less synergistically in vivo.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The limited size of 24 TSCs was not expected to provide robust statistics.

    “although the limited size of 24 TSCs was not expected to provide robust statistics.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductown data

    The high-throughput compound screen assay was validated against a pilot library of 4,080 compounds at 10 μM in two independent experiments.

    “The assay was validated against a pilot library, a representative sample of 4,080 compounds, at a final concentration of 10 mM in two independent experiments.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    CDK12 has recently emerged as a potential vulnerability for different cancer types including Ewing sarcoma, triple-negative breast cancer, and hepatocellular carcinoma.

    “Although CDK12 has only recently emerged as a potential vulnerability for different cancer types including Ewing sarcoma, triple-negative breast cancer, and hepatocellular carcinoma (Iniguez et al., 2018;Quereda et al., 2019;Wang et al., 2020)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Iniguez et al., 2018 source

      Not checked in the free run.

    • cites Quereda et al., 2019 source

      Not checked in the free run.

    • cites Wang et al., 2020 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CMS4 is associated with a TIC-like and/or epithelial-mesenchymal transition (EMT) signature.

    “and a TIC-like and/or epithelial-mesenchymal transition (EMT) signature (Le Corre et al., 2019)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Le Corre et al., 2019 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 given

    NSG mice were housed at a specific-pathogen-free animal facility according to all applicable laws and regulations subsequent to approval by the institution's animal care and ethical committee.

    “Female eight-to ten-week-old NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice were used in this study, obtained from the Jackson Laboratory and housed at a specific-pathogen-free animal facility according to all applicable laws and regulations subsequent to approval by the institution's animal care and ethical committee.”
    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 conductown data

    CCNK/CDK12 co-overexpression in transduced TSC03 cells was confirmed at protein level and monitored based on mCherry/eGFP signal.

    “Overexpression was confirmed at protein level and monitored based on mCherry/eGFP-signal.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Human CRC samples were obtained from Heidelberg University Hospital in accordance with the Declaration of Helsinki.

    “Human CRC samples were obtained from Heidelberg University Hospital in accordance with the Declaration of Helsinki.”
    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 conductnone given

    TSCs and cell lines were authenticated using Multiplex Cell Authentication, and purity of TSCs was validated using the Multiplex cell contamination test (Multiplexion).

    “TSCs and cell lines were authenticated using Multiplex Cell Authentication and purity of TSCs was validated using the Multiplex cell contamination test (Multiplexion).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    No Mycoplasma, SMRV or interspecies contaminations were detected in the cultures.

    “No Mycoplasma, SMRV or interspecies contaminations were detected.”
    Quote in the paperNot a substantial claim

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

    Study design and conductcitation

    Purity of established cultures was monitored to assure pure epithelial cell content and exclude contaminations with hematopoietic or stroma cells.

    “Purity of established cultures was monitored to assure pure epithelial cell content and exclude contaminations with hematopoietic or stroma cells as described before (Dieter et al., 2017a).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Dieter et al., 2017a

      Not checked in the free run.

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

    Study design and conductnone given

    Informed consent on tissue collection was received from each patient, as approved by the University Ethics Review Board.

    “Informed consent on tissue collection was received from each patient, as approved by the University Ethics Review Board.”
    Quote in the paperNot a substantial claim
65 of 65

  • The paper relies on this without support.

    These tumor cell models recapitulate the original patient tumor after transplantation into immunodeficient mice.

    “These tumor cell models recapitulate the original patient tumor after transplantation into immunodeficient mice.”

  • The paper relies on this without support.

    NCT02 was metabolically not stable enough for in vivo experiments.

    “Because NCT02 was metabolically not stable enough for in vivo experiments, we treated PDXs with SR-4835”

  • The paper relies on this without support.

    Activating mutations in KRAS or BRAF characterize CRC subgroups associated with poor prognosis.

    “two subgroups of CRCs that are characterized by activating mutations in KRAS (KRAS mut ) or BRAF (BRAF mut ) and associated with poor prognosis.”

  • The paper relies on this without support.

    CMS2 and CMS3 identify epithelial CRCs, and CMS4 defines a mesenchymal CRC subtype associated with unfavorable prognosis.

    “In microsatellite-stable (MSS) CRCs, CMS2 and CMS3 identify epithelial CRCs, and CMS4 defines a mesenchymal CRC subtype associated with unfavorable prognosis.”
4 of 4

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?

  • Numerous CycK degraders have been discovered in both serendipitous screens and through more targeted approaches.

    “and since then numerous cycK degraders have been discovered in both serendipitous screens and through more targeted approaches (Dieter et al. 2021;Houles et al. 2023;Kozicka et al. 2024).”

    From From serendipity to strategy: molecular glue degraders in cancer therapeutics (2025) · cites this paper as Dieter et al. 2021

    The Introduction and Discussion describe identifying the cyclin K degrader NCT02 through a phenotypic compound screen, and the Discussion reports additional cyclin K degraders. The paper does not establish that numerous degraders were discovered through both serendipitous screens and targeted approaches; it only cites a separate study titled “Rational discovery of molecular glue degraders” in its References. Page numbers are unavailable in our copy. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • NCT02 induces the degradation of CycK and CDK12.

    “Characterization of the top hit, NCT02, revealed that it induces the degradation of CycK and CDK12.”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [135]

  • Four separate classes of compounds were found to have molecular glue-like properties in potentiating the interaction of DDB1 and CDK12, resulting in CCNK degradation.

    “In fact, not only CR8, but four separate classes of compounds, each discovered by a different screening method, were found to have molecular glue-like properties in potentiating the interaction of DDB1 and CDK12, resulting in CCNK degradation84–87.”

    From PROTAC targeted protein degraders: the past is prologue (2022) · cites this paper as [87]

Self-check

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

  • 280 mg acid is assigned too few millimoles

    Minor

    The preceding mass spectrum reports m/z 397 (M+H)+, implying a molar mass of approximately 396 g/mol. Thus 280 mg is about 0.71 mmol rather than 0.64 mmol.

    Reported 0.64 · recomputed 0.70529

  • 460 mg intermediate is assigned too few millimoles

    Minor

    The preceding mass spectrum reports m/z 411 (M+H)+, implying a molar mass of approximately 410 g/mol. Thus 460 mg is about 1.12 mmol rather than 0.9 mmol.

    Reported 0.9 · recomputed 1.119221

  • 550 mg intermediate is assigned too few millimoles

    Minor

    The preceding mass spectrum reports m/z 397 (M+H)+, implying a molar mass of approximately 396 g/mol. At that mass, 550 mg is about 1.39 mmol, not 1.2 mmol.

    Reported 1.2 · recomputed 1.38539

3 of 3

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Related

Authors
Dieter, S. M., Siegl, C., Codó, P. L., Huerta, M., Ostermann-Parucha, A. L., Schulz, E., Zowada, M. K., Martin, S., Laaber, K., Nowrouzi, A., Blatter, M., Kreth, S., Westermann, F., Benner, A., Uhrig, U., Putzker, K., Lewis, J., Haegebarth, A., Mumberg, D., Holton, S. J., Weiske, J., Toepper, L.-M., Scheib, U., Siemeister, G., Ball, C. R., Kuster, B., Stoehr, G., Hahne, H., Johannes, S., Lange, M., Herbst, F., & Glimm, H.
Affiliations
  • German Cancer Research Center (DE)
  • Merck KGaA, Darmstadt (Germany) (DE)
  • University Hospital Heidelberg (DE)
  • Heidelberg University (DE)
  • European Molecular Biology Laboratory (DE)
  • Bayer (Germany) (DE)
  • Nuvisan (Germany) (DE)
Corresponding author
  • Dieter, Sebastian M.
  • Glimm, Hanno
ORCID
Venue
Cell Reports
Publisher
Elsevier BV
Published
2021-07
Volume
36
Issue
3
Pages
109394
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2021.109394
OpenAlex
W3183737123
Semantic Scholar
ec96e50f6d898943d9068feca71c12ac9aa4791a
Cited by
95
References
59
Citations by year
2026: 16 · 2025: 25 · 2024: 18 · 2023: 12 · 2022: 20 · 2021: 4
FWCI
4.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion)
  • mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion, other-oa)
Funders
  • Spark Therapeutics
  • Bayer (NCT3.0_2015.54 DysregPT)
  • European Commission
  • Deutsches Krebsforschungszentrum
  • Universität Heidelberg
  • Merck KGaA
  • Deutsche Krebshilfe
  • Universitätsklinikum Heidelberg
  • European Molecular Biology Laboratory
  • Horizon 2020 Framework Programme
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Health Sciences › Medicine › Oncology › Colorectal Cancer Treatments and Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
Keywords
colorectal cancer, CDK12, molecular glues, ubiquitination, proteasomal degradation, TP53 deficiency, patient-derived spheroids, patient-derived xenografts, small molecule screening, metastatic colorectal cancer
MeSH terms
Cell Self Renewal, Antineoplastic Agents, DNA Damage, Female, Humans, Colorectal Neoplasms, Tumor Suppressor Protein p53, Cyclins, Apoptosis, Cyclin-Dependent Kinases, Spheroids, Cellular, Proteomics, Ubiquitin-Protein Ligases, Cell Line, Tumor, Proteasome Endopeptidase Complex, Cell Proliferation, Ubiquitination, High-Throughput Screening Assays, Proteolysis

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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