CDK4 inhibition diminishes p53 activation by MDM2 antagonists
Open accessAbstract
The genes encoding MDM2 and CDK4 are frequently co-amplified in sarcomas, and inhibitors to both targets are approved or clinically tested for therapy. However, we show that inhibitors of MDM2 and CDK4 antagonize each other in their cytotoxicity towards sarcoma cells. CDK4 inhibition attenuates the induction of p53-responsive genes upon MDM2 inhibition. Moreover, the p53 response was also attenuated when co-depleting MDM2 and CDK4 with siRNA, compared to MDM2 single knockdown. The complexes of p53 and MDM2, as well as CDK4 and Cyclin D1, physically associated with each other, suggesting direct regulation of p53 by CDK4. Interestingly, CDK4 inhibition did not reduce p53 binding or histone acetylation at promoters, but rather attenuated the subsequent recruitment of RNA Polymerase II. Taken together, our results suggest that caution must be used when considering combined CDK4 and MDM2 inhibition for patient treatment. Moreover, they uncover a hitherto unknown role for CDK4 and Cyclin D1 in sustaining p53 activity.
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Written from the paper’s full text.
TL;DR
CDK4 inhibition antagonizes MDM2 antagonists in sarcoma cells by attenuating p53 target gene expression and cytotoxicity rather than producing synergy. The paper links this to physical association of CDK4–Cyclin D1 with p53/MDM2 and reduced RNA Polymerase II recruitment at p53 target promoters.
Key findings
- Palbociclib did not enhance Nutlin-induced cytotoxicity in SJSA cells; instead, Nutlin-treated cells survived significantly more when co-treated with Palbociclib, and long-term survival was rescued by Palbociclib (Fig. 1).
- CDK4/6 inhibitors Palbociclib, Ribociclib, and Abemaciclib decreased Nutlin-induced p53 target products MDM2 and p21 at protein and mRNA levels; co-depletion of MDM2 and CDK4 by siRNA also induced p21/p53 less than MDM2 knockdown alone (Fig. 2).
- RNA-Seq in SJSA cells identified 2368 Nutlin-vs-DMSO differentially regulated genes (base mean >20, log2 fold >1 or <−1, adjusted p < 0.05); most p53-responsive genes were expressed less when cells were pretreated with CDK4/6 inhibitor, with p53-responsive genes among the most differentially expressed after cell cycle targets by GSEA (FDR < 25%) (Fig. 4).
- CDK4/6 inhibition did not reduce p53 promoter binding or H3K27ac at p53-responsive promoters; it increased p53 Lys382 acetylation and p53 occupancy/H3K27ac, but decreased RNA Polymerase II association at p21/CDKN1A, MDM2, PUMA/BBC3, and PIG3/TP53I3 TSSs (Fig. 6).
Methods
- Cell lines: SJSA osteosarcoma and CRL-3043, CRL-3044, GOT-3 liposarcoma cells, plus H1299 for overexpression Co-IP; treatments included Nutlin-3a, Palbociclib/PD0332991, Ribociclib/LEE011, Abemaciclib/LY2835219, neocarzinostatin, and MG-132.
- Assays: cell viability by CellTiter-Glo, proliferation by Celigo confluence, cell cycle by propidium iodide flow cytometry, immunoblot, qRT-PCR, RNA-Seq, endogenous and exogenous co-immunoprecipitation, and ChIP for p53, H3K27ac, and RNA Polymerase II.
- Statistics: unpaired t-test with Sidak–Bonferroni correction, significance at p ≤ 0.05; RNA-Seq analysis used TopHat/Bowtie 2, HTSeq, DESeq2, and GSEA.
Limitations
- The paper states that it cannot rule out that the drugs might cooperate more favorably under clinical conditions, and that each combination with CDK4 inhibitors remains to be tested individually.
- The paper notes that the antagonism was observed “at least under some circumstances” and that a prior synergy report may have used Idasanutlin concentrations with little single-agent effect.
- Evident from the text: findings are based on in vitro sarcoma cell lines and biochemical assays, with no in vivo tumor model or clinical trial data; some experiments report only two biological replicates (e.g., Fig. 1b, Fig. 6d), and the proposed co-factor mechanism is not shown by direct CDK4 phosphorylation of p53.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Cites
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Claims the paper makes
0 of 31 cited sources checked · 0 supported · 0 flagged
- Findingown data
Nutlin induced p53 accumulation and its target genes p21 and MDM2 in sarcoma cells.
“Nutlin induced p53 accumulation and its target genes p21 and MDM2 (Fig. S1b)”
- Also stated in: § CDK4 is required for p53-induced gene expression · plain
“Nutlin alone increased the levels of p53 and its target gene products MDM2 and CDKN1A/p21, as expected.”
Quote in the paperSubstantial claim - Also stated in: § CDK4 is required for p53-induced gene expression · plain
- Findingcitation
Nutlin profoundly decreased the viability of SJSA cells.
“Nutlin profoundly decreased the viability of SJSA cells, as reported previously26”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26] Tovar, C. et al. Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy. Proc. Natl Acad. Sci. USA 103, 1888–1893 (2006). source
Not checked in the free run.
- Findingcitation
PD0332991 abolished the phosphorylation of the CDK4 substrate pRb at Serine 807/811.
“PD0332991 abolished the phosphorylation of the CDK4 substrate pRb at Serine 807/81125 (Fig. S1c).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [25] Kato, J., Matsushime, H., Hiebert, S. W., Ewen, M. E. & Sherr, C. J. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev. 7, 331–342 (1993). source
Not checked in the free run.
- Findingown data
Combination of the drugs at different concentrations in various MDM2-amplified sarcoma cell lines revealed no synergism with regard to viability of cells.
“Combination of the drugs at different concentrations in various MDM2-amplified sarcoma cell lines also revealed no synergism with regard to viability of cells (Fig. S1d–g; note that here viability was assayed immediately after 72 h of treatment, without allowing the cells to further proliferate).”
Quote in the paperSubstantial claim - Findingown data
Palbociclib also reduced the viability of SJSA cells.
“Palbociclib also reduced their viability (Fig. 1a, b).”
Quote in the paperNot a substantial claim
- Findingown data
p53 interacts with Cyclin D1, confirmed by plasmid-based overexpression.
“The interaction of p53 with Cyclin D1 was further confirmed by plasmid-based overexpression of both components (Fig. 5d).”
Quote in the paperSubstantial claim - Findingown data
CDK4/6 inhibition mostly reduced pre-mRNA levels proximal to the promoters of p53-responsive genes in response to Nutlin.
“We observed that CDK4/6 inhibition mostly reduced pre-mRNA levels proximal to the promoters of p53-responsive genes in response to Nutlin (Fig. 6d), arguing that the initiation of transcription depends most strongly on cyclin-dependent kinases.”
Quote in the paperSubstantial claim - Findingown data
CDK4 inhibition did not reduce histone acetylation at promoters.
“CDK4 inhibition did not reduce p53 binding or histone acetylation at promoters”
Quote in the paperSubstantial claim - Findingown data
CDK4 inhibition did not reduce p53 binding at promoters.
“CDK4 inhibition did not reduce p53 binding or histone acetylation at promoters”
Quote in the paperSubstantial claim - Findingown data
The complexes of p53 and MDM2, as well as CDK4 and Cyclin D1, physically associate with each other.
“The complexes of p53 and MDM2, as well as CDK4 and Cyclin D1, physically associated with each other”
- Also stated in: § Introduction · plain
“In parallel, the complexes of MDM2 and p53, as well as CDK4 and Cyclin D1, physically associate with each other.”
Quote in the paperSubstantial claim - Also stated in: § Introduction · plain
- Findingown data
The HIPK2 inhibitor A64 did not cause any detectable change in p53 activity.
“we performed a parallel experiment replacing CDK4 inhibitors with the HIPK2 inhibitor A64 (PubChem Substance ID 329826044) but did not observe any detectable change in p53 activity (Fig. S2, f, g).”
Quote in the paperSubstantial claim - Findingown data
p53 target gene expression was decreased when Nutlin was combined with the CDK4/6 inhibitors Ribociclib and Abemaciclib at the protein and mRNA levels.
“Again, p53 target gene expression was decreased when Nutlin was combined with the CDK4/6 inhibitors at the protein (Fig. 2b) and mRNA (Fig. 2c) levels.”
Quote in the paperSubstantial claim - Findingown data
CDK4 inhibition attenuates the induction of p53-responsive genes upon MDM2 inhibition.
“CDK4 inhibition attenuates the induction of p53-responsive genes upon MDM2 inhibition.”
- Also stated in: § CDK4 is required for p53-induced gene expression · plain
“in combination with Nutlin, Palbociclib markedly decreased the protein levels of the p53 target gene products MDM2 and p21 (Fig. 2a).”
Quote in the paperSubstantial claim - Also stated in: § CDK4 is required for p53-induced gene expression · plain
- Findingown data
The p53 response was attenuated when co-depleting MDM2 and CDK4 with siRNA, compared to MDM2 single knockdown.
“Moreover, the p53 response was also attenuated when co-depleting MDM2 and CDK4 with siRNA, compared to MDM2 single knockdown.”
- Also stated in: § CDK4 is required for p53-induced gene expression · plain
“This revealed corresponding changes in p53-induced p21, i.e., induction by MDM2 knockdown alone, but far less induction by the simultaneous depletion of MDM2 and CDK4 (Fig. 2e).”
Quote in the paperSubstantial claim - Also stated in: § CDK4 is required for p53-induced gene expression · plain
- Findingown data
Association of RNA Polymerase II with p53-responsive genes was decreased in response to CDK4/6 inhibition.
“On the other hand, however, the association of RNA Polymerase II with p53-responsive genes was decreased in response to CDK4/6 inhibition (Fig. 6g), suggesting that CDK4/6 activity is required for p53-mediated recruitment of RNA Polymerase II.”
Quote in the paperSubstantial claim - Findingown data
Palbociclib arrested the majority of treated cells in G1.
“As expected, flow cytometric analyses verified that Palbociclib arrested the majority of treated cells in G1 (Fig. 3a, b).”
Quote in the paperNot a substantial claim - Findingown data
CDK4/6 inhibition did not decrease but further increased the extent of p53 occupancy on target gene promoters.
“Interestingly, however, CDK4/6 inhibition did not decrease but even further increased the extent of p53 occupancy on target gene promoters (Fig. 6e), despite the decreased expression of the respective target genes.”
Quote in the paperSubstantial claim - Findingown data
In SJSA cells, pretreatment with a CDK4/6 inhibitor reduced the expression of most p53-responsive genes induced by Nutlin.
“This approach revealed that most p53-responsive genes, as identified through their induction by Nutlin, were expressed to a lesser degree when cells were pretreated with the CDK4/6 inhibitor (Fig. 4b).”
Quote in the paperSubstantial claim - Findingown data
Palbociclib pretreatment strongly decreased the accumulation of p53 and its target gene product p21 in response to NCS.
“Strikingly, Palbociclib pretreatment strongly decreased the accumulation of p53 and its target gene product p21 in response to NCS (Fig. 2d).”
Quote in the paperSubstantial claim - Findingown data
p53 activity was markedly increased upon Palbociclib removal compared to cells treated with Nutlin in the continued presence of Palbociclib.
“However, p53 activity was still markedly increased upon Palbociclib removal, when compared to cells that were treated with Nutlin in the continued presence of Palbociclib, as determined by immunoblot analysis of p53 target gene products (Fig. 3c) and also by assessing the mRNA levels corresponding to such genes (Fig. 3d).”
Quote in the paperSubstantial claim - Findingown data
Acetylation of p53 at Lys382 was stronger when PD0332991 was combined with Nutlin than with Nutlin alone.
“Surprisingly, we observed that the acetylation of p53 was even stronger when PD0332991 was combined with Nutlin than Nutlin alone (Fig. 6a, b).”
Quote in the paperSubstantial claim - Findingown data
Co-immunoprecipitation in SJSA cell lysates revealed an interaction between the MDM2-p53 complex and the CDK4-Cyclin D complex.
“Indeed, co-immunoprecipitation analyses revealed the interaction of the MDM2–p53 and the CDK4–Cyclin D complexes (Fig. 5a).”
- Also stated in: § Discussion · boosted
“We show that the complexes of CDK4 and Cyclin D1 on the one hand, and MDM2 and p53 on the other hand, associate with each other.”
Quote in the paperSubstantial claim - Also stated in: § Discussion · boosted
- Findingown data
CDK4 inhibition attenuated the subsequent recruitment of RNA Polymerase II.
“but rather attenuated the subsequent recruitment of RNA Polymerase II.”
Quote in the paperSubstantial claim - Findingown data
Nutlin increased the amount of p53 associated with its cognate promoter elements.
“Nutlin increased the amount of p53 associated with its cognate promoter elements, as expected.”
Quote in the paperSubstantial claim - Findingown data
Brief removal of the CDK4 inhibitor was not enough to resume the cell cycle.
“This brief removal of the CDK4 inhibitor was not enough to resume the cell cycle, at least as far as can be judged by propidium iodide staining (Fig. 3b).”
Quote in the paperSubstantial claim - Findingown data
Expression of p53 target genes at the protein and mRNA levels was decreased when adding the CDK4/6 inhibitor to Nutlin.
“In contrast, we still observed decreased expression of p53 target genes, at the protein (Fig. 6b) and mRNA (Fig. 6c) level, when adding the CDK4/6 inhibitor to Nutlin.”
Quote in the paperSubstantial claim - Findingown data
Impaired p53 target gene expression upon CDK4 inhibition was also observed when using different time schedules, drug concentrations, or the alternate MDM2 antagonist RG-7388.
“Similarly, impaired p53 target gene expression upon CDK4 inhibition was also observed when using different time schedules, drug concentrations or the alternate MDM2 antagonist RG-7388 (Fig. S2, a–e).”
Quote in the paperSubstantial claim - Findingown data
p53 and CDK4 interact in Nutlin-treated cells.
“The interaction of p53 and CDK4 was also found in Nutlin-treated cells (Fig. 5b, c).”
Quote in the paperSubstantial claim - Findingown data
Among genes differentially expressed between Nutlin-treated cells and cells treated with Nutlin plus CDK4 inhibitor, p53-responsive genes were enriched to the greatest extent after cell cycle targets.
“Comparing Nutlin-treated cells with or without CDK4 inhibitor, it was still the p53-responsive genes that were differentially expressed to the greatest extent after the cell cycle targets, as determined by gene set enrichment analysis (GSEA) (Fig. 4c, d).”
Quote in the paperSubstantial claim - Findingown data
Combined inhibition of MDM2 and CDK4 decreases the association of Cyclin D1 and CDK4 compared to Nutlin alone.
“Combined inhibition of MDM2 and CDK4 led to decreased association of Cyclin D1 and CDK4 when compared to Nutlin alone (Fig. 5c with quantification in Fig. S3).”
Quote in the paperNot a substantial claim - Findingown data
p53 physically associates with CDK4 and Cyclin D1.
“In sum, p53 physically associates with CDK4 and Cyclin D1, suggesting a mechanism by which CDK4 might directly regulate the activity of p53 as a transcription factor.”
Quote in the paperSubstantial claim - Findingown data
We were unable to observe synergy between Palbociclib and Idasanutlin in killing liposarcoma cells.
“Despite testing numerous conditions, we were unable to observe a similar synergy.”
Quote in the paperSubstantial claim - Interpretationown data
Inhibitors of MDM2 and CDK4 antagonize each other in their cytotoxicity towards sarcoma cells.
“we show that inhibitors of MDM2 and CDK4 antagonize each other in their cytotoxicity towards sarcoma cells.”
- Also stated in: § Inhibitors of CDK4 and MDM2 lack synergistic cytotoxicity towards sarcoma cells · hedged
“Taken together, these results strongly suggest that, at least under some circumstances, Palbociclib is capable of antagonizing the cytotoxic activity of Nutlin.”
Quote in the paperSubstantial claim - Also stated in: § Inhibitors of CDK4 and MDM2 lack synergistic cytotoxicity towards sarcoma cells · hedged
- Interpretationreasoning
The failure of Nutlin to fully induce pro-apoptotic genes in the presence of CDK4 inhibitor may explain why CDK4 inhibition attenuates the cytotoxic effects of Nutlin.
“The failure of Nutlin to fully induce pro-apoptotic genes in the presence of CDK4 inhibitor may also explain why CDK4 inhibition attenuates the cytotoxic effects of Nutlin (Fig. 1).”
Quote in the paperSubstantial claim - Interpretationown data
CDK4 activity is required for maximal p53-induced gene expression, regardless of cell cycle progression.
“Thus, we conclude that CDK4 activity is required for maximal p53-induced gene expression, regardless of cell cycle progression.”
Quote in the paperSubstantial claim - Interpretationown data
CDK4 inhibition or depletion severely diminishes the transcriptional activity of p53 in response to MDM2 antagonists.
“Taken together, these results strongly suggest that CDK4 inhibition or depletion severely diminishes the transcriptional activity of p53 in response to MDM2 antagonists.”
Quote in the paperSubstantial claim - Interpretationown data
CDK4 inhibition can also interfere with the p53-inducing ability of DNA damaging drugs.
“Hence, CDK4 inhibition can also interfere with the p53-inducing ability of DNA damaging drugs”
Quote in the paperSubstantial claim - Interpretationown data
CDK4 might directly regulate the activity of p53 as a transcription factor via physical association.
“In sum, p53 physically associates with CDK4 and Cyclin D1, suggesting a mechanism by which CDK4 might directly regulate the activity of p53 as a transcription factor.”
Quote in the paperSubstantial claim - Interpretationown data
CDK4 activity has a direct and specific impact on p53-induced transcription.
“This suggests that CDK4 activity has a direct and specific impact on p53-induced transcription.”
Quote in the paperSubstantial claim - Interpretationown data
CDK4 inhibition generally reduces p53 activity.
“Thus, CDK4 inhibition generally reduces p53 activity.”
Quote in the paperSubstantial claim - Interpretationown data
When cells are subjected to MDM2 inhibition, a CDK4/6 inhibitor specifically attenuates the expression of p53-responsive genes more than any other distinguishable group of genes except the cell cycle regulators.
“We conclude that, when cells are subjected to MDM2 inhibition, a CDK4/6 inhibitor specifically attenuates the expression of p53-responsive genes more than any other distinguishable group of genes except the cell cycle regulators.”
Quote in the paperSubstantial claim - Interpretationown data
The physical association of p53-MDM2 and CDK4-Cyclin D1 complexes suggests direct regulation of p53 by CDK4.
“suggesting direct regulation of p53 by CDK4.”
Quote in the paperSubstantial claim - Interpretationown data
Initiation of transcription depends most strongly on cyclin-dependent kinases.
“We observed that CDK4/6 inhibition mostly reduced pre-mRNA levels proximal to the promoters of p53-responsive genes in response to Nutlin (Fig. 6d), arguing that the initiation of transcription depends most strongly on cyclin-dependent kinases.”
Quote in the paperSubstantial claim - Interpretationcitation
CDK4/Cyclin D1 might act as a co-factor for p53 in target gene activation.
“These observations further support the idea that CDK4/Cyclin D1 might act as a co-factor for p53 in target gene activation.”
Quote in the paperSubstantial claim - Interpretationnone given
The CDK4-Cyclin D1 complex activity is required for gene induction and RNA Polymerase II recruitment by p53.
“The CDK4–Cyclin D1 complex associates with p53, and its activity is required for gene induction and RNA Polymerase II recruitment by p53.”
Quote in the paperSubstantial claim - Premisecitation
Some conventional chemotherapeutic drugs depend on the activation of p53 for their efficacy.
“Some conventional chemotherapeutic drugs depend on the activation of p53 for their efficacy.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [44] Bunz, F. et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J. Clin. Invest. 104, 263–269 (1999). source
Not checked in the free run.
- Interpretationown data
CDK4/6 activity is required for p53-mediated recruitment of RNA Polymerase II.
“On the other hand, however, the association of RNA Polymerase II with p53-responsive genes was decreased in response to CDK4/6 inhibition (Fig. 6g), suggesting that CDK4/6 activity is required for p53-mediated recruitment of RNA Polymerase II.”
Quote in the paperSubstantial claim - Interpretationcitation
CDK4 inhibition can be expected to increase the accumulation of hypophosphorylated Rb.
“CDK4 inhibition can be expected to increase the accumulation of hypophosphorylated Rb, since CDK4 is a key driver of Rb phosphorylation25.”
Quote in the paperSubstantial claim - Premisecitation
CDK4 is a key driver of Rb phosphorylation.
“CDK4 inhibition can be expected to increase the accumulation of hypophosphorylated Rb, since CDK4 is a key driver of Rb phosphorylation25.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [25] Kato, J., Matsushime, H., Hiebert, S. W., Ewen, M. E. & Sherr, C. J. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev. 7, 331–342 (1993). source
Not checked in the free run.
- Interpretationreasoning
This would result in decreased apoptosis.
“This, perhaps in addition to the diminished p53 activity reported here, would then result in decreased apoptosis27.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [27] Du, W., Wu, J., Walsh, E. M., Zhang, Y., Chen, C. Y. & Xiao, Z. X. Nutlin-3 affects expression and function of retinoblastoma protein: role of retinoblastoma protein in cellular response to nutlin-3. J. Biol. Chem. 284, 26315–26321 (2009). source
Not checked in the free run.
- Premisecitation
In HCT116 cells, comparing p53-proficient and -deficient cells, a dependency on p53 was found in particular for 5-fluorouracil (5-FU).
“Comparing p53-proficient and -deficient HCT116 cells, such a dependency was found in particular for 5-fluorouracil (5-FU)44.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [44] Bunz, F. et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J. Clin. Invest. 104, 263–269 (1999). source
Not checked in the free run.
- Interpretationown data
Targeting both CDK4 and MDM2 simultaneously may be counterproductive for cancer therapy.
“Despite the co-amplification of genes encoding CDK4 and MDM2 in human malignancies, our results indicate that targeting both simultaneously may be counterproductive for cancer therapy.”
Quote in the paperSubstantial claim- Rests on an assumption: CDK4 and MDM2 are co-amplified in human malignancies.
- Premisecitation
In vitro kinase assays did not reveal direct phosphorylation of p53 by CDK4.
“However, in vitro kinase assays did not reveal any such direct phosphorylation39.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [39] Wang, Y. & Prives, C. Increased and altered DNA binding of human p53 by S and G2/M but not G1 cyclin-dependent kinases. Nature 376, 88–91 (1995). source
Not checked in the free run.
- Interpretationreasoning
The dependence of p53 activity on CDK4 appears as a mechanism to channel tumor suppression on cancer-prone, proliferating cells, while sparing differentiated cells despite DNA damage.
“Thus, the dependence of p53 activity on CDK4 appears as a mechanism to channel tumor suppression on cancer-prone, proliferating cells, while sparing differentiated cells despite DNA damage.”
Quote in the paperSubstantial claim - Interpretationreasoning
The positive impact of the CDK4-Cyclin D1 complex on p53 activity may constitute a greater sensitivity of cycling cells towards p53 activation, as compared to resting cells.
“The positive impact of the CDK4–Cyclin D1 complex on p53 activity may thus constitute a greater sensitivity of cycling cells towards p53 activation, as compared to resting cells.”
Quote in the paperSubstantial claim - Interpretationreasoning
Combining CDK4 inhibitors with DNA damaging chemotherapy might protect non-cancerous tissue in a patient.
“Thus, combining CDK4 inhibitors with DNA damaging chemotherapy might turn out to protect non-cancerous tissue in a patient, giving rise to a potential strategy for avoiding undesired general toxicities.”
Quote in the paperSubstantial claim
- May combine two claims. 14
- Classified as probably not a substantial claim. 23
May combine two claims.
Findingown dataPalbociclib completely failed to enhance the cytotoxic effects elicited by Nutlin; instead, Nutlin-treated cells survived to a significantly greater extent when co-treated with Palbociclib.
“Palbociclib completely failed to enhance the cytotoxic effects elicited by Nutlin. Instead, Nutlin-treated cells even survived to a significantly greater extent when co-treated with Palbociclib.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe long-term survival of SJSA cells was strongly decreased by Nutlin alone, but was rescued by co-treatment with Palbociclib.
“Similarly, the long-term survival of SJSA cells was strongly decreased by Nutlin alone, but was rescued by co-treatment with Palbociclib (Fig. 1c, d).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPalbociclib alone had little effect on p53 target gene products, while it reduced the amount of phosphorylated pRb.
“Palbociclib alone had little effect on them, while it did reduce the amount of phosphorylated pRb.”
Quote in the paperSubstantial claimMay combine two claims.
FindingreasoningCell proliferation is diminished in response to Nutlin alone but partially re-established by co-treatment with Nutlin and a CDK4 inhibitor.
“Moreover, cell proliferation is diminished in response to Nutlin alone but partially re-established by co-treatment with Nutlin and a CDK4 inhibitor.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataCDK4 and Cyclin D1 play a role in sustaining p53 activity.
“Moreover, they uncover a hitherto unknown role for CDK4 and Cyclin D1 in sustaining p53 activity.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationAcetylation of p53 on Lys382 is an activating modification of p53.
“we performed immunoblot analysis to detect the acetylation of p53 on Lys 382, an activating modification of p5335,36.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [35] Barlev, N. A. et al. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Mol. Cell 8, 1243–1254 (2001). source
Not checked in the free run.
- cites [36] Sakaguchi, K. et al. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev. 12, 2831–2841 (1998). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMDMX is phosphorylated by CDK4.
“These findings are in line with a previous report that MDMX, another close binding partner of MDM2 and p53, is phosphorylated by CDK437.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [37] Gerarduzzi, C., de Polo, A., Liu, X. S., El Kharbili, M., Little, J. B. & Yuan, Z. M. Human epidermal growth factor receptor 4 (Her4) suppresses p53 protein via targeting the MDMX-MDM2 protein complex: implication of a novel MDMX SER-314 phosphosite. J. Biol. Chem. 291, 25937–25949 (2016). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationCyclin D1 and the CDK4-related kinase CDK6 can associate with target genes and function as transcriptional regulators.
“Previous studies revealed that Cyclin D140,41 as well as the CDK4-related kinase CDK642 can associate with target genes and function as transcriptional regulators.”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [40] Pestell, R. G. New roles of cyclin D1. Am. J. Pathol. 183, 3–9 (2013). source
Not checked in the free run.
- cites [41] Bienvenu, F. et al. Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen. Nature 463, 374–378 (2010). source
Not checked in the free run.
- cites [42] Tigan, A. S., Bellutti, F., Kollmann, K., Tebb, G. & Sexl, V. CDK6-a review of the past and a glimpse into the future: from cell-cycle control to transcriptional regulation. Oncogene 35, 3083–3091 (2016). source
Not checked in the free run.
Probably not a substantial claim: may not pass S1 (asserted).
PremisecitationThe cytotoxic effects of Nutlin on p53-proficient cells might depend on the ability of MDM2 to degrade the retinoblastoma protein Rb.
“Another previous report argued that the cytotoxic effects of Nutlin on p53-proficient cells might depend on the ability of MDM2 to degrade the retinoblastoma protein Rb27.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [27] Du, W., Wu, J., Walsh, E. M., Zhang, Y., Chen, C. Y. & Xiao, Z. X. Nutlin-3 affects expression and function of retinoblastoma protein: role of retinoblastoma protein in cellular response to nutlin-3. J. Biol. Chem. 284, 26315–26321 (2009). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPPM1D/Wip1 might represent a suitable target of drugs that synergize with MDM2 antagonists.
“Our previous work suggests that the phosphatase PPM1D/Wip1 might represent a suitable target of drugs that synergize with MDM2 antagonists19,45.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [19] Sriraman, A., Radovanovic, M., Wienken, M., Najafova, Z., Li, Y. & Dobbelstein, M. Cooperation of Nutlin-3a and a Wip1 inhibitor to induce p53 activity. Oncotarget 7, 31623–31638 (2016). source
Not checked in the free run.
- cites [45] Sriraman, A., Li, Y. & Dobbelstein, M. Fortifying p53 - beyond Mdm2 inhibitors. Aging 8, 1836–1837 (2016). source
Not checked in the free run.
Classified as probably not a substantial claim.
Premisecitationp53-deficient cells displayed a higher sensitivity towards topoisomerase inhibitors such as Adriamycin.
“On the other hand, the same report showed that p53-deficient cells displayed a higher sensitivity towards topoisomerase inhibitors such as Adriamycin.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [44] Bunz, F. et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J. Clin. Invest. 104, 263–269 (1999). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCyclin D1 physically associates with MDM2.
“This notion is further strengthened by a recent report on a physical association between Cyclin D1 and MDM238.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [38] Yang, P. et al. Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3. Oncotarget 7, 32652–32663 (2016). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCDK4-mediated phosphorylation of MDMX stabilizes its interaction with MDM2 to antagonize p53.
“According to this report, CDK4-mediated phosphorylation of MDMX stabilizes its interaction with MDM2 to antagonize p53.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [37] Gerarduzzi, C., de Polo, A., Liu, X. S., El Kharbili, M., Little, J. B. & Yuan, Z. M. Human epidermal growth factor receptor 4 (Her4) suppresses p53 protein via targeting the MDMX-MDM2 protein complex: implication of a novel MDMX SER-314 phosphosite. J. Biol. Chem. 291, 25937–25949 (2016). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCyclin D1 binds with some preference to sites that also associate with p53.
“Interestingly, one of the genome-wide ChIP analyses revealed that Cyclin D1 binds with some preference to sites that also associate with p53, among other transcription factors43.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [43] Casimiro, M. C. et al. ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice. J. Clin. Invest. 122, 833–843 (2012). source
Not checked in the free run.
May combine two claims.
Interpretationown dataCDK4/6 inhibition interferes with the recruitment of RNA Polymerase II by p53, thereby diminishing the initiation of transcription at p53 target genes.
“In conclusion, CDK4/6 inhibition interferes with the recruitment of RNA Polymerase II by p53, thereby diminishing the initiation of transcription at p53 target genes.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataCDK4 inhibition attenuates the p53 response to MDM2-targeted drugs, resulting in decreased cytotoxicity.
“CDK4 inhibition attenuates the p53 response to MDM2-targeted drugs, resulting in decreased cytotoxicity.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationIn cells where accumulated MDM2 leads to degradation of hypophosphorylated Rb, apoptosis can be induced, but when Rb remains, the cells merely arrest.
“In cells where accumulated MDM2 leads to the degradation of hypophosphorylated Rb, apoptosis can be induced, but when Rb remains, the cells merely arrest.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [27] Du, W., Wu, J., Walsh, E. M., Zhang, Y., Chen, C. Y. & Xiao, Z. X. Nutlin-3 affects expression and function of retinoblastoma protein: role of retinoblastoma protein in cellular response to nutlin-3. J. Biol. Chem. 284, 26315–26321 (2009). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationcitationThe combination of CDK4 inhibitors and the resulting p53 attenuation would be counterproductive for 5-FU but might be beneficial for Adriamycin.
“In analogy, the combination of CDK4 inhibitors and the resulting p53 attenuation would be counterproductive for 5-FU but might be beneficial for Adriamycin.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationMDM2 antagonists cooperated efficiently with MEK or PI3K inhibitors, BH3 mimetics, BCR-ABL antagonists, and histone deacetylase inhibitors in preclinical investigations and cell culture.
“In preclinical investigations and cell culture, MDM2 antagonists also cooperated efficiently with mitogen-activated protein kinase kinase (MEK) or phosphatidylinositol-3 kinase (PI3K) inhibitors, BH3 mimetics, BCR-ABL antagonists, and histone deacetylase inhibitors47.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [47] Saiki, A. Y. et al. MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways. Oncotarget 5, 2030–2043 (2014). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisereasoningInterfering with this dephosphorylation enhances activating p53 modifications.
“Interfering with this dephosphorylation enhances activating p53 modifications.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based).
Premisenone givenWip1 dephosphorylates p53, thereby compromising its activity as a transcription factor.
“Wip1 dephosphorylates p53, thereby compromising its activity as a transcription factor.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFor neuroblastoma cells, MDM2 inhibitors cooperate with anaplastic lymphoma kinase (ALK) inhibition.
“For neuroblastoma cells, MDM2 inhibitors cooperate with anaplastic lymphoma kinase (ALK) inhibition48.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [48] Wang, H. Q. et al. Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models. Elife 6, pii: e17137 (2017). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe DNA damaging drug Trabectedin was reported to synergize with the MDM2 inhibitor RG7112.
“The DNA damaging drug Trabectedin, currently used in second line for treating soft tissue sarcoma, was also reported to synergize with the MDM2 inhibitor RG711246, perhaps as a result of p53 accumulation (through MDM2 inhibition) and activating p53 modifications (through DNA damage response).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [46] Obrador-Hevia, A. et al. RG7112, a small-molecule inhibitor of MDM2, enhances trabectedin response in soft tissue sarcomas. Cancer Invest. 33, 440–450 (2015). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
PremisecitationWhen combined with MDM2 inhibitors, both stability and activity of p53 are increased, leading to pronounced cell death.
“When this is combined with MDM2 inhibitors, both stability and activity of p53 are increased, leading to pronounced cell death.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [19] Sriraman, A., Radovanovic, M., Wienken, M., Najafova, Z., Li, Y. & Dobbelstein, M. Cooperation of Nutlin-3a and a Wip1 inhibitor to induce p53 activity. Oncotarget 7, 31623–31638 (2016). source
Not checked in the free run.
- cites [45] Sriraman, A., Li, Y. & Dobbelstein, M. Fortifying p53 - beyond Mdm2 inhibitors. Aging 8, 1836–1837 (2016). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCDK4 inhibitors were found to cooperate with inhibitors of signaling kinases, such as MEK and PI3K.
“On the other hand, CDK4 inhibitors were found to cooperate with inhibitors of signaling kinases, such as MEK and PI3K49,50.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [49] Franco, J., Witkiewicz, A. K. & Knudsen, E. S. CDK4/6 inhibitors have potent activity in combination with pathway selective therapeutic agents in models of pancreatic cancer. Oncotarget 5, 6512–6525 (2014). source
Not checked in the free run.
- cites [50] Laroche, A., Chaire, V., Algeo, M. P., Karanian, M., Fourneaux, B. & Italiano, A. MDM2 antagonists synergize with PI3K/mTOR inhibition in well-differentiated/dedifferentiated liposarcomas. Oncotarget 8, 53968–53977 (2017). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationIn breast cancer treatment, the CDK4/6 inhibitor Palbociclib is often combined with Letrozole, an inhibitor of aromatase in estrogen production, but this only makes sense when treating estrogen-dependent tumors.
“In breast cancer treatment, the CDK4/6 inhibitor Palbociclib is often combined with Letrozole, an inhibitor of aromatase in estrogen production, but this only makes sense when treating estrogen-dependent tumors1.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [1] O’Leary, B., Finn, R. S. & Turner, N. C. Treating cancer with selective CDK4/6 inhibitors. Nat. Rev. Clin. Oncol. 13, 417–430 (2016). source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCDK4 inhibition was reported to antagonize drugs that require entry into mitosis for their efficacy, such as taxanes.
“CDK4 inhibition was reported to antagonize drugs that require entry into mitosis for their efficacy, such as taxanes49, probably due to inhibited cell cycle progression.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [49] Franco, J., Witkiewicz, A. K. & Knudsen, E. S. CDK4/6 inhibitors have potent activity in combination with pathway selective therapeutic agents in models of pancreatic cancer. Oncotarget 5, 6512–6525 (2014). source
Not checked in the free run.
May combine two claims.
PremisecitationUnder physiological conditions, the CDK4-Cyclin D1 complex is active in cycling cells and stem cells, whereas it is inactive in post-mitotic and terminally differentiated cells.
“Under physiological conditions, the CDK4–Cyclin D1 complex is active in cycling cells and stem cells, whereas it is inactive in post-mitotic and terminally differentiated cells51.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [51] Latella, L. et al. Reconstitution of cyclin D1-associated kinase activity drives terminally differentiated cells into the cell cycle. Mol. Cell Biol. 21, 5631–5643 (2001). source
Not checked in the free run.
Probably not a substantial claim: may not pass S4 (based).
Premisenone givenIn cases where p53 is mutated in a tumor, p53 activation by chemotherapeutics can be considered irrelevant.
“In cases where p53 is mutated in a tumor, p53 activation by chemotherapeutics can be considered irrelevant.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningIn such a scenario, it may be beneficial for normal cells if p53-induced cell death is attenuated.
“In such a scenario, it may be beneficial for normal cells if p53-induced cell death is attenuated.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningThe drug antagonism reported here may be used in a beneficial way.
“On the other hand, the drug antagonism reported here may be used in a beneficial way.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The co-amplification of MDM2 and CDK4 in sarcomas constitutes tumor cell addiction to the simultaneous activity of both gene products.
“The co-amplification of both genes might constitute tumor cell addiction to the simultaneous activity of both gene products.”
The paper relies on this without support.
The effects observed in sarcoma cell lines predict clinical outcomes in patients.
“Taken together, our results suggest that caution must be used when considering combined CDK4 and MDM2 inhibition for patient treatment.”
The paper relies on this without support.
CDK4 and MDM2 are co-amplified in human malignancies.
“Despite the co-amplification of genes encoding CDK4 and MDM2 in human malignancies, our results indicate that targeting both simultaneously may be counterproductive for cancer therapy.”
The paper relies on this without support.
The p53-responsive genes induced by Nutlin are representative of p53 transcriptional targets.
“most p53-responsive genes, as identified through their induction by Nutlin”
The paper relies on this without support.
PD0332991 (Palbociclib) specifically inhibits CDK4/6 at the doses used.
“pretreated with the CDK4/6 inhibitor”
The paper relies on this without support.
Co-immunoprecipitation of proteins indicates a physical association.
“co-immunoprecipitation analyses revealed the interaction”
The paper relies on this without support.
The SJSA cell line is a valid model for CDK4/MDM2 co-amplified human tumors.
“SJSA cells”
The paper relies on this without support.
The effects observed in cell culture are relevant to clinical cancer therapy.
“our results do give rise to caution when treating patients”
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?
Simple combination therapy of MDM2 and CDK4 inhibitors in preclinical experiments demonstrated paradoxical results in sarcoma.
“Although the simple combination therapy of MDM2 and CDK4 inhibitors in preclinical experiments were reported recently, the two independent reports demonstrated the paradoxical results in sarcoma51,52.”
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.
Two different identifiers are given for Abemaciclib
MinorThe Introduction identifies Abemaciclib as LY2835319, while the Methods identify it as LY2835219.
“Abemaciclib (LY2835319)1.”
“Abemaciclib (LY2835219, Selleckchem S7158)”
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MDM2 inhibition: an important step forward in cancer therapy
Konopleva, M. Y., Martinelli, G., Daver, N. G., Papayannidis, C., Wei, A. H., Higgins, B., Ott, M., Mascarenhas, J. O., & Andreeff, M. (2020) · Leukemia · doi:10.1038/s41375-020-0949-z · Cited by 376 · see citing works
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- Authors
- Sriraman, A., Dickmanns, A., Najafova, Z., Johnsen, S. A., & Dobbelstein, M.
- Affiliations
- Universitätsmedizin Göttingen (DE)
- Corresponding author
- Dobbelstein, Matthias
- Venue
- Cell Death & Disease
- Published
- 2018-09-11
- Volume
- 9
- Issue
- 9
- Pages
- 918-918
- ISSN
- 2041-4889 (ISSN-L)
- PMC
- PMC6133967
- OpenAlex
- W2890791082
- Semantic Scholar
- a0331b8fe00e3fdaee1a1478ac1f18ef60f6a614
- Cited by
- 40
- References
- 52
- Citations by year
- 2026: 5 · 2025: 3 · 2023: 5 · 2022: 7 · 2021: 5 · 2020: 8 · 2019: 6 · 2018: 1
- FWCI
- 1.88
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Cell Death and Disease (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, cc-by)
- Funders
- Wilhelm Sander-Stiftung
- Universitätsmedizin Göttingen
- Deutsche Forschungsgemeinschaft
- Else Kröner-Fresenius-Stiftung
- Göteborgs Universitet
- Deutsche Krebshilfe
- Topics
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
- Keywords
- CDK4 inhibition, MDM2 inhibitors, p53 activation, sarcoma, cyclin D1, p53-MDM2 complex, drug synergy, RNA polymerase II recruitment, histone acetylation, combination therapy
- MeSH terms
- Aminopyridines, Antineoplastic Agents, Benzimidazoles, Humans, Piperazines, Purines, Pyridines, Sarcoma, Tumor Suppressor Protein p53, Cyclin D1, RNA Interference, RNA, Small Interfering, Cell Line, Tumor, Cell Proliferation, Cyclin-Dependent Kinase 4, Proto-Oncogene Proteins c-mdm2
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