Mdm2 inhibitors synergize with topoisomerase II inhibitors to induce p53-independent pancreatic cancer cell death
Open accessAbstract
Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer death in the western world, with a 5‐year survival rate below 5%. Murine double minute 2 (Mdm2) is an important negative regulator of the tumor suppressor p53. Reactivation of wild‐type p53 is a promising treatment strategy, and inhibitors of Mdm2 have already entered clinical trials. To investigate the effects of Mdm2 inhibitors in PDAC, we used a murine cell line platform with a genetically defined status of p53. Here, we describe that Mdm2 inhibitors can act on a subset of murine PDAC cell lines p53 independently. Furthermore, we observed that Mdm2 inhibitors increase the sensitivity of murine PDAC cell lines toward topoisomerase II inhibitors by inducing effector caspase‐independent cell death. The combination of Mdm2 inhibitors with topoisomerase II inhibitors acts independent of the survival factor NFκB/RelA. Mechanistically, Mdm2 inhibitors increase topoisomerase II inhibitor‐induced DNA double‐strand breaks. We show that Mdm2 binds to Nbs1 of the Mre11–Rad50–Nijmegen breakage syndrome (Nbs) 1 DNA repair complex. In addition, we provide evidence that Mdm2 inhibitors delay DNA repair. These findings may help to design novel therapeutic strategies to overcome therapeutic resistance of PDAC.
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- Findingown data
Multiple-dose combinations of Nutlin 3a and etoposide had a combination index below 0.85 in all five murine pancreatic ductal adenocarcinoma cell lines tested, indicating synergism.
“The calculation of the CIs of multiple-dose combinations of Nutlin 3a and etoposide (!1 lg/ml) revealed CI < 0.85 in all five cell lines investigated, demonstrating synergism.”
Quote in the paperSubstantial claim - Findingown data
In PPT-5436 cells, zVAD-FMK completely blocked caspase 3/7 activity induced by Nutlin 3a plus etoposide.
“Although zVAD-FMK completely blocks caspase 3/7 activity induced by the Nutlin 3a/etoposide combination (Fig. 3e)”
Quote in the paperSubstantial claim - Findingown data
The IC50 of PXN822 in p53 wild-type murine pancreatic ductal adenocarcinoma cells was comparable to that in p53-proficient HCT116 control cells.
“IC 50 values of p53 wild-type murine PDAC cells were comparable to p53-proficient HCT116 cells, which were used as a control (Fig. 1f).”
Quote in the paperSubstantial claim - Findingown data
Nutlin 3a decreased etoposide IC50 values in a dose-dependent manner in murine pancreatic ductal adenocarcinoma cells.
“In addition, Nutlin 3a also reduced IC 50 values for etoposide in a dosedependent manner (Fig. 2f).”
Quote in the paperSubstantial claim - Findingown data
One p53 R172H-mutant murine pancreatic ductal adenocarcinoma cell line and one p53-deficient cell line responded significantly to PXN822 treatment.
“one p53 R172H -mutant cell line and one p53-deficient cell line responded significantly to the PXN822 treatment”
Quote in the paperSubstantial claim
- Findingown data
In p53-deficient PPT-W22 cells, PXN822 plus etoposide increased caspase 3/7 activity significantly more than etoposide alone.
“Although the combination of PXN822 and etoposide significantly increased the activation of caspase 3/7 in p53-deficient PPT-W22 cells compared to the etoposide treatment (Fig. 3b)”
Quote in the paperSubstantial claim - Findingown data
Doxorubicin synergized with Nutlin 3a in murine pancreatic ductal adenocarcinoma cells.
“In addition to etoposide, doxorubicin synergizes with Nutlin 3a in murine PDAC cells (Supporting Information Fig. S1).”
Quote in the paperNot a substantial claim - Findingown data
In p53-deficient PPT-W22 murine pancreatic ductal adenocarcinoma cells, etoposide and etoposide plus PXN822 induced similar G2/M-phase arrest.
“Etoposide as well as the combination of etoposide and PXN822 induced a similar G2/M-phase arrest in the p53-deficient murine PDAC cell line PPT-W22 (Fig. 3a).”
Quote in the paperNot a substantial claim - Findingown data
In p53-mutant PPT-5436 cells, Nutlin 3a plus etoposide increased caspase 3/7 activity compared to etoposide alone.
“In addition, in the p53-mutant PDAC cell line PPT-5436, the combination of the Mdm2 inhibitor Nutlin 3a with etoposide also distinctly increased caspase 3/7 activity compared to etoposide alone (Fig. 3e).”
Quote in the paperSubstantial claim - Findingown data
In PPT-W22 cells, the pan-caspase inhibitor zVAD-FMK did not rescue viability loss induced by PXN822 plus etoposide.
“the loss of viability induced by the PXN822/etoposide combination was not rescued by adding the pan-caspase inhibitor zVAD-FMK (Fig. 3c)”
Quote in the paperSubstantial claim - Findingown data
Cotreatment with PXN822 decreased etoposide IC50 values in murine pancreatic ductal adenocarcinoma cells.
“Furthermore, the IC 50 values for etoposide were obviously decreased on cotreatment with PXN822 (Fig. 2f).”
Quote in the paperSubstantial claim - Findingown data
PXN822 increased sensitivity to etoposide in all murine pancreatic ductal adenocarcinoma cell lines tested, regardless of p53 status.
“we observed that the sensitivity toward etoposide was distinctly increased in all murine PDAC cell lines investigated on the inhibition of Mdm2 with PXN822, irrespective of the p53 status.”
- Also stated in: § Abstract · plain
“we observed that Mdm2 inhibitors increase the sensitivity of murine PDAC cell lines toward topoisomerase II inhibitors by inducing effector caspase-independent cell death.”
Quote in the paperSubstantial claim - Also stated in: § Abstract · plain
- Findingown data
In PPT-W22 cells, zVAD-FMK did not rescue the cellular response to Nutlin 3a plus etoposide.
“Again, the cellular response of the Nutlin 3a and etoposide combination was not rescued by the zVAD-FMK cotreatment in PPT-W22 cells (Fig. 3d).”
Quote in the paperSubstantial claim - Findingcitation
PXN822 did not stabilize the p53 R172H mutant in the murine pancreatic ductal adenocarcinoma cell lines PPT-3107 and PPT-5436.
“The p53mutant R172H was not stabilized by the PXN822 treatment in PPT-3107 and PPT-5436 cells”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [24] source
Not checked in the free run.
- Findingown data
In PPT-5436 cells, zVAD-FMK failed to rescue the loss of viability induced by Nutlin 3a plus etoposide.
“zVAD-FMK failed to rescue the therapeutic response of the Nutlin 3a/etoposide combination in PPT-5436 cells (Fig. 3f).”
Quote in the paperSubstantial claim - Findingown data
Apoptosis induced by the combination of etoposide and Nutlin 3a is not blocked by zVAD-FMK.
“apoptosis induced by the combination of etoposide and Nutlin 3a was not blocked by zVAD-FMK”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Findingown data
Increased activation of ATM is observed in response to the Nutlin 3a and etoposide combination.
“As we observed increased activation of ATM in response to the Nutlin 3a and etoposide combination”
Quote in the paperSubstantial claim - Findingown data
A subset of p53-mutant human PDAC cell lines responds significantly to Nutlin 3a.
“we observed that a subset of p53-mutant human PDAC cell lines responds significantly to Nutlin 3a (data not shown).”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Findingown data
After washout, H2AX was dephosphorylated to a similar extent in PPT-5436, PPT-6554, and PPT-W22 cells regardless of pretreatment with etoposide or etoposide plus Nutlin 3a.
“H2AX was dephosphorylated to a similar extent in PPT-5436, PPT-6554 and PPT-W22 cells, irrespectively of the pretreatment with etoposide or the combination of etoposide and Nutlin 3a (Figs.5f-5h).”
Quote in the paperNot a substantial claim- Rests on an assumption: Etoposide is a potent inducer of DNA double-strand breaks.
- Findingown data
A subset of murine p53-deficient or -mutant PDAC cells shows a significant cellular response to Mdm2 inhibitors.
“In our work, we describe a subset of murine p53-deficient or -mutant PDAC cells revealing a significant cellular response to Mdm2 inhibitors.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Findingown data
Nuclear Mdm2 directly binds to Nbs1.
“we show that nuclear Mdm2 directly binds to Nbs1”
Quote in the paperSubstantial claim - Findingown data
The cellular response to the combination of etoposide and Mdm2 inhibitor is not rescued by the pan-caspase inhibitor zVAD-FMK.
“the cellular response was not rescued by the pan-caspase inhibitor zVAD-FMK.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Findingown data
Mdm2 inhibitors increase topoisomerase II inhibitor-induced DNA double-strand breaks, as indicated by increased H2AX phosphorylation in murine pancreatic ductal adenocarcinoma cells.
“Mechanistically, Mdm2 inhibitors increase topoisomerase II inhibitor-induced DNA double-strand breaks.”
- Also stated in: § Mdm2 inhibitors interfere with repair of DNA DSBs · plain
“The treatment of the p53-mutant and -deficient murine PDACs cells PPT-5436, PPT-6554 and PPT-W22 with the combination of the Mdm2 inhibitor PXN822 and etoposide distinctly increased phosphorylation of H2AX (Fig. 5b).”
Quote in the paperSubstantial claim- Rests on an assumption: Etoposide is a potent inducer of DNA double-strand breaks.
- Also stated in: § Mdm2 inhibitors interfere with repair of DNA DSBs · plain
- Findingown data
Mdm2 inhibitors delay DNA double-strand break repair, as shown by persistent H2AX phosphorylation when Nutlin 3a was maintained in the medium.
“we provide evidence that Mdm2 inhibitors delay DNA repair.”
- Also stated in: § Mdm2 inhibitors interfere with repair of DNA DSBs · boosted
“However, when Nutlin 3a was included in the medium, H2AX remains significantly phosphorylated, arguing that DNA DSB repair is significantly delayed by the Mdm2 inhibitor (Figs.5f-5h).”
Quote in the paperSubstantial claim- Rests on an assumption: Etoposide is a potent inducer of DNA double-strand breaks.
- Also stated in: § Mdm2 inhibitors interfere with repair of DNA DSBs · boosted
- Findingown data
Sole treatment with Mdm2 inhibitors did not induce significant phosphorylation of H2AX in p53-deficient and -mutant murine PDAC cells.
“the sole treatment with Mdm2 inhibitors did not induce significant phosphorylation of H2AX in our p53-deficient and -mutant murine PDAC cells.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Findingown data
In murine PDAC cell lines with a genetically defined p53 status, Mdm2 inhibitors increase the efficacy of topoisomerase II inhibitors.
“In our study, we demonstrated murine PDAC cell lines with a genetically defined p53 status, in which Mdm2 inhibitors increase the efficacy of topoisomerase II inhibitors.”
- Also stated in: § Discussion · boosted
“In summary, we demonstrate that an Mdm2 inhibitor favorably combines with topoisomerase II inhibitors in a murine PDAC cell model.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Also stated in: § Discussion · boosted
- Findingown data
In RelA-reconstituted murine pancreatic ductal adenocarcinoma clones, gemcitabine-induced loss of viability was diminished.
“As expected, the gemcitabine-induced loss of viability was diminished in RelA-reconstituted clones (Fig. 4f).”
Quote in the paperNot a substantial claim - Findingown data
ATM phosphorylation, a measure of DNA double-strand break downstream signaling, was increased in cells cotreated with etoposide and Nutlin 3a.
“DSB-induced downstream signaling, as measured by phosphorylation of ATM, was augmented in etoposide/Nutlin 3a cotreated cells (Fig. 5e).”
Quote in the paperSubstantial claim- Rests on an assumption: Etoposide is a potent inducer of DNA double-strand breaks.
- Findingown data
Increased activation of effector caspases is detected on treatment with the combination of etoposide and the Mdm2 inhibitor compared to etoposide alone.
“we detected increased activation of effector caspases on treatment with the combination of etoposide with the Mdm2 inhibitor compared to the sole etoposide treatment”
Quote in the paperNot a substantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Findingown data
In PPT-3590 cells, Nutlin 3a plus etoposide significantly decreased viability compared to etoposide alone.
“Again, the combination of the Mdm2 inhibitor Nutlin 3a and etoposide significantly decreased viability compared to the sole treatment with etoposide (Figs.4c-4e).”
Quote in the paperSubstantial claim - Findingown data
Mdm2 inhibitors modulate DNA double-strand break repair without requiring functional p53 in murine PDAC cells.
“provide evidence that Mdm2 inhibitors modulate DNA DSB repair without the requirement for functional p53 in murine PDAC cells.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Findingown data
Nutlin 3a, etoposide, or their combination did not significantly change the binding of Nbs1 to Mdm2.
“However, treatment with Nutlin 3a, etoposide or the combination of Nutlin 3a/etoposide did not significantly alter the binding of Nbs1 to Mdm2 (Figs. 6a-6c).”
Quote in the paperNot a substantial claim - Findingown data
In PPT-5436 and PPT-W22 cells, neither etoposide nor etoposide plus Nutlin 3a significantly changed Mdm2 expression.
“Neither etoposide nor the combination of etoposide with Nutlin 3a induces a significant change of Mdm2 expression in PPT-5436 (p53 R172H ) and PPT-W22 (p53 null) cells (Fig. 5a).”
Quote in the paperNot a substantial claim - Findingown data
Mdm2 binds to Nbs1 of the MRN DNA repair complex, as observed by immunoprecipitation in murine pancreatic ductal adenocarcinoma cell lines.
“We show that Mdm2 binds to Nbs1 of the Mre11-Rad50-Nijmegen breakage syndrome (Nbs) 1 DNA repair complex.”
- Also stated in: § Mdm2 inhibitors interfere with repair of DNA DSBs · plain
“Consistently, using immunoprecipitations of nuclear extracts from the p53-mutant PPT-5436 and the p53-deficient PPT-6554 and PPT-W22 cell lines, we observed direct interaction of Nbs1 with Mdm2 (Figs. 6a-6c).”
Quote in the paperSubstantial claim - Also stated in: § Mdm2 inhibitors interfere with repair of DNA DSBs · plain
- Findingown data
The cellular response to Nutlin 3a plus etoposide was not modulated by RelA status in murine pancreatic ductal adenocarcinoma cells.
“However, the cellular response toward the combination of Nutlin 3a with etoposide was not modulated by RelA (Figs. 4c-4e).”
- Also stated in: § Abstract · plain
“The combination of Mdm2 inhibitors with topoisomerase II inhibitors acts independent of the survival factor NFjB/RelA.”
Quote in the paperSubstantial claim - Also stated in: § Abstract · plain
- Findingown data
Fluorescence microscopy showed increased H2AX phosphorylation with Nutlin 3a plus etoposide compared to etoposide alone.
“The increased phosphorylation of H2AX was also observed with fluorescence microscopy using the combination of the Mdm2 inhibitor Nutlin 3a and etoposide compared to the sole treatment with etoposide (Fig. 5d).”
Quote in the paperSubstantial claim- Rests on an assumption: Etoposide is a potent inducer of DNA double-strand breaks.
- Findingown data
Nutlin 3a plus etoposide significantly increased the fraction of apoptotic cells in PPT-5436 and PPT-W22 murine pancreatic ductal adenocarcinoma cells compared to etoposide alone.
“Compared to the etoposide treatment, the combination of Nutlin 3a with etoposide significantly increases the fraction of apoptotic cells in PPT-5436 (Fig. 3g) and PPT-W22 (Fig. 3h) PDAC cells.”
Quote in the paperSubstantial claim - Findingown data
No overt change in the interaction between Nbs1 and Mdm2 is observed upon treatment with the Mdm2 inhibitor, the topoisomerase II inhibitor, or their combination.
“However, we observed no overt change in the interaction of Nbs1 with Mdm2 on treatment of the cells with the Mdm2 inhibitor, the topoisomerase II inhibitor or the combination of both.”
Quote in the paperSubstantial claim - Findingreasoning
The Mdm2 inhibitor delays DSB repair in the investigated murine PDAC model.
“the Mdm2 inhibitor delays DSB repair in the investigated murine PDAC model”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Interpretationown data
Mdm2 inhibitors can reduce viability in a subset of murine pancreatic ductal adenocarcinoma cell lines independently of p53 status.
“Mdm2 inhibitors can act on a subset of murine PDAC cell lines p53 independently.”
- Also stated in: § Cancer Cell Biology 2250 · plain
“arguing for p53-independent functions of Mdm2 in the murine PDAC models investigated.”
Quote in the paperSubstantial claim - Also stated in: § Cancer Cell Biology 2250 · plain
- Premisecitation
Mdm2 inhibition sensitizes cancer cells to chemotherapeutic agents.
“As Mdm2 inhibition sensitizes cancer cells to chemotherapeutics, 7 we tested”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [7] source
Not checked in the free run.
- Premisecitation
In preclinical in vivo models, Mdm2 inhibition was tolerated by nonmalignant tissues without overt toxicity.
“Nonmalignant tissues tolerate inhibition of Mdm2, and no overt toxicity was observed in preclinical in vivo models.9,11”
Quote in the paperSubstantial claim0 of 2 sources checked - Premisecitation
A cisplatin plus MI-319 combination was efficacious in human pancreatic ductal adenocarcinoma models in vitro and in vivo independently of p53 status.
“In addition, a cisplatin/MI-319 combination demonstrated efficacy in human PDAC models in vitro and in vivo independent of the p53 status.15”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15] source
Not checked in the free run.
- Premisecitation
About half of human cancers retain wild-type p53.
“As p53 remains wild type in $50% of human cancers, 6 reactivation of p53 by small molecules is an attractive therapeutic approach.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [6] source
Not checked in the free run.
- Interpretationown data
Mdm2 inhibition increases sensitivity of murine pancreatic ductal adenocarcinoma cells to topoisomerase II inhibitors independently of p53.
“These findings argue that Mdm2 inhibition increases sensitivity toward topoisomerase II inhibitors in a p53-independent manner.”
Quote in the paperSubstantial claim - Premisecitation
MI-319 suppressed growth of a Capan-2-based xenograft model in vivo.
“and MI-319 suppressed growth of a Capan-2-based xenotransplant model in vivo.15”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15] source
Not checked in the free run.
- Premisecitation
Mdm2 is overexpressed in pancreatic ductal adenocarcinoma tissue specimens.
“Furthermore, Mdm2 was shown to be overexpressed in PDAC tissue specimens.13”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [13]
Not checked in the free run.
- Premisecitation
Human pancreatic ductal adenocarcinoma cell lines show higher Mdm2 protein expression than immortalized ductal cells.
“Consistently, high Mdm2 protein expression was observed in human PDAC cell lines compared to immortalized ductal cells.14”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] source
Not checked in the free run.
- Premisecitation
The Mdm2 inhibitors MI-319 and Nutlin 3a efficiently induce apoptosis in the human p53 wild-type pancreatic ductal adenocarcinoma cell line Capan-2 in vitro.
“The Mdm2 inhibitors MI-319 and Nutlin 3a efficiently induce apoptosis in the human p53 wild-type PDAC cell line Capan-2 in vitro 14,15”
Quote in the paperSubstantial claim0 of 2 sources checked - Premisecitation
Overexpression of Mdm2 delays DSB repair independently of p53 and of Mdm2's E3 ubiquitin ligase activity, but strictly requires Nbs1.
“Interestingly, overexpression of Mdm2 leads to a delay in DSB repair independent of p53 and the E3 ubiquitin ligase activity of Mdm2, but strictly dependent on Nbs1.30”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [30] source
Not checked in the free run.
- Interpretationown data
The apoptosis induced by the combination of etoposide and Nutlin 3a is caspase-independent.
“arguing for caspase-independent apoptosis.43”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [43] source
Not checked in the free run.
- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Interpretationown data
ATM activation is intact in the murine pancreatic ductal adenocarcinoma cells tested.
“Thus, activation of the DNA damage protein ATM is intact in the cells tested.”
Quote in the paperSubstantial claim - Premisecitation
Mdm2 directly interacts with Nbs1 of the Mre11-Rad50-Nbs1 DNA double-strand break repair complex.
“Mdm2 was demonstrated to directly interact with Nbs1 of the Mre11-Rad50-Nijmegen breakage syndrome (Nbs) 1 (MRN) DNA DSB repair complex.[28][29][30]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [28][29][30]
Not checked in the free run.
- Premisecitation
Regulation of MYC, c-Jun and cyclin D1 was only detected after RNAi targeting Mdm2, but not recapitulated using the Mdm2 inhibitor Nutlin 3a.
“the regulation of MYC, c-Jun and cyclin D1 was only detected after RNAi targeting Mdm2, but not recapitulated using the Mdm2 inhibitor Nutlin 3a. 14”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] source
Not checked in the free run.
- Interpretationown data
The signaling hub function of the MRN complex is not disturbed by the Mdm2 inhibitor.
“the signaling hub function of the MRN complex is not disturbed by the Mdm2 inhibitor.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Interpretationown data
The combination of an Mdm2 inhibitor and topoisomerase II inhibitors acts by modulating the repair of etoposide-induced DNA DSBs.
“Their combination acts by modulating the repair of etoposide-induced DNA DSBs”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- Interpretationreasoning
Mdm2 inhibitors may be therapeutically effective by delaying DNA double-strand break repair in a subset of pancreatic cancer cells.
“thus providing evidence that Mdm2 inhibitors may be therapeutically effective by delaying repair of DNA double strand breaks in a subset of pancreatic cancer cells.”
Quote in the paperSubstantial claim - Interpretationown data
The effect of RelA on the cellular response of pancreatic ductal adenocarcinoma cells to treatment is specific to the stress induced by a particular therapeutic agent or combination.
“Altogether, these data argue that the influence of RelA toward the cellular response of PDACs on a certain treatment regime is specific for the stress induced by a certain therapeutic or a combination thereof.”
Quote in the paperSubstantial claim - Interpretationreasoning
Mdm2 inhibitors block Mdm2 functions needed for sufficient DNA repair.
“argues that Mdm2 inhibitors block Mdm2 functions needed for sufficient DNA repair.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
- May combine two claims. 9
- Classified as probably not a substantial claim. 20
May combine two claims.
Findingown dataIn p53 wild-type murine pancreatic ductal adenocarcinoma cell lines, PXN822 treatment stabilized p53 and increased the p53 target p21 Cip1.
“Treatment with PXN822 induces stabilization of p53 and upregulation of the p53 target gene p21 Cip1 (Fig. 1b).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn murine pancreatic ductal adenocarcinoma cells, zVAD-FMK significantly blocked bortezomib-induced apoptosis but did not significantly change cell death induced by Nutlin 3a plus etoposide.
“Although apoptosis induced by the proteasome inhibitor bortezomib was significantly blocked by the pan-caspase inhibitor zVAD-FMK, cell death induced by the Nutlin 3a/etoposide combination was not significantly changed by zVAD-FMK (Figs.3g and 3h)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn the RelA-deficient murine pancreatic ductal adenocarcinoma cell line PPT-3590, p53 was highly expressed, was not stabilized by etoposide, and Bax was not induced by etoposide.
“In PPT-3590 cells, p53 was highly expressed, not stabilized by the treatment with etoposide, and the p53 target gene Bax was not induced by the etoposide treatment (Fig. 4b).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataH2AX phosphorylation was detectable from 30 minutes after etoposide treatment and was increased by the Mdm2 inhibitor.
“In time kinetics, phosphorylation of H2AX was detected from 30 min after etoposide treatment and was augmented by the Mdm2 inhibitor (Fig. 5c).”
Quote in the paperSubstantial claim- Rests on an assumption: Etoposide is a potent inducer of DNA double-strand breaks.
May combine two claims.
Findingown dataA p53-independent G2/M cell cycle arrest is observed upon treatment with the topoisomerase II inhibitor, and it is not significantly altered by addition of the Mdm2 inhibitor.
“As expected, we observed a p53-independent G2/M cell cycle arrest on the treatment of the cells with the topoisomerase II inhibitor, which was not significantly altered by the addition of the Mdm2 inhibitor.”
Quote in the paperNot a substantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
May combine two claims.
PremisecitationPXN822 binds the p53-binding pocket of Mdm2 and disrupts a preformed p53/Mdm2 complex.
“PXN822 binds to the p53-binding pocket of Mdm2 and disrupts a preformed p53/Mdm2 complex 7,8”
Quote in the paperNot a substantial claim0 of 2 sources checkedClassified as probably not a substantial claim.
PremisecitationBinding of Mdm2 to the N-terminus of p53 inhibits the transactivation domain of p53.
“Binding of Mdm2 to the N-terminus of p53 inhibits the transactivation domain of the tumor suppressor.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [1] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationPreclinical data indicate that Mdm2 inhibitors have good efficacy in p53 wild-type cancers in vivo and in vitro.
“preclinical data point to a good efficacy of Mdm2 inhibitors for p53 wildtype cancers in vivo and in vitro.[9][10][11]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [9][10][11]
Not checked in the free run.
May combine two claims.
Interpretationown dataCaspases are not critical for the cell death induced by Mdm2 inhibitor plus etoposide, which is a caspase-independent programmed cell death pathway.
“arguing that caspases are not critical for the PXN822/etoposideinduced cellular response.”
- Also stated in: § Cancer Cell Biology · plain
“arguing for a caspase-independent programmed cell death pathway.”
Quote in the paperSubstantial claim- Also stated in: § Cancer Cell Biology · plain
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationBetween 50% and 75% of pancreatic ductal adenocarcinomas carry p53 mutations.
“About 50-75% of PDACs harbor p53 mutations.12”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [12] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRestoring p53 in murine cancer models causes tumor regression.
“Restoration of p53 in murine cancer models induces tumor regression.[3][4][5]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [3][4][5]
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationActivation of p53 target genes drives cell cycle arrest, apoptosis, and senescence in tumor cells.
“Consequently, p53 target genes become activated and drive cellular responses of tumor cells, which include cell cycle arrest, apoptosis and senescence. 2”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [2] source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationAs an E3 ubiquitin ligase, Mdm2 mediates ubiquitination and 26S proteasomal degradation of p53.
“Furthermore, as E3 ubiquitin ligase, Mdm2 leads to the ubiquitination and degradation of p53 by the 26S proteasome. 1”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [1] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationInhibitors of Mdm2 have entered clinical trials.
“Beyond these strategies, inhibitors of Mdm2 have already entered clinical trials, 7,8”
Quote in the paperNot a substantial claim0 of 2 sources checkedClassified as probably not a substantial claim.
PremisecitationMdm2 acts as a critical negative regulator of the tumor suppressor p53.
“Murine double minute 2 [Mdm2; termed as human double minute 2 (HDM2) in Homo sapiens] is a critical negative regulator of the transcription factor and tumor suppressor p53. 1”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [1] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOn DNA DSBs, the MRN complex is involved in the activation of ATM, a kinase initiating DNA damage signaling pathways.
“On DNA DSBs, the MRN complex is involved in the activation of ATM, a kinase initiating DNA damage signaling pathways.[34][35][36]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [34][35][36]
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationPhosphorylation of the histone H2A variant H2AX marks DNA double-strand breaks.
“The phosphorylation of this histone H2A variant indicates DSBs.27”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [27] source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationTopoisomerase II inhibitors are potent inducers of DNA DSBs.
“Topoisomerase II inhibitors are potent inducers of DNA DSBs.32”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [32] source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable).
Interpretationown dataThese findings may help design novel therapeutic strategies to overcome therapeutic resistance in pancreatic ductal adenocarcinoma.
“These findings may help to design novel therapeutic strategies to overcome therapeutic resistance of PDAC.”
Quote in the paperNot a substantial claim- Rests on an assumption: Effective therapies for locally advanced or metastatic pancreatic ductal adenocarcinoma are lacking.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe MRN complex coordinates signaling to induce cellular responses such as cell cycle arrest and DNA repair.
“Moreover, it coordinates signaling to induce cellular responses like cell cycle arrest and repair of DNA.33”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [33] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe canonical Kras-Mek-Erk pathway drives the expression of Mdm2 in human PDAC cells.
“A recent report demonstrated that the canonical Kras-Mek-Erk pathway drives the expression of Mdm2 in human PDAC cells.14”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationNutlin 3a induces cH2AX foci in Mdm2-deficient mouse embryonic fibroblasts.
“Valentine et al. 39 recently described that Nutlin 3a induces cH2AX foci in Mdm2-deficient mouse embryonic fibroblasts.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [39] source
Not checked in the free run.
May combine two claims.
Interpretationown dataMdm2 possesses p53-independent functions in PDAC cells that can be targeted by Mdm2 inhibitors.
“Together, these data argue that Mdm2 possesses p53-independent functions in PDAC cells, which can be targeted by Mdm2 inhibitors.”
Quote in the paperSubstantial claim- Rests on an assumption: The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRelA promotes apoptosis induced by the Mdm2 inhibitor MI-219 plus oxaliplatin in the human pancreatic ductal adenocarcinoma cell line Capan-2.
“Recently, the NFjB subunit RelA was shown to promote apoptosis induced by a combination of the Mdm2 inhibitor MI-219 and oxaliplatin in the human PDAC cell line Capan-2.25”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [25] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIf DNA repair fails or the number of DNA damage lesions exceeds a threshold, DSBs are potent inducers of apoptosis to eliminate cells with inappropriate genomic information.
“If DNA repair fails or the number of DNA damage lesions exceeds a certain threshold, DSBs are potent inducer of apoptosis to eliminate cells with inappropriate genomic information.42”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [42]
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe MRN complex is a central hub in the detection of DNA damage.
“The MRN complex is a central hub in the detection of DNA damage.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [33] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMdm2 maintains the expression of tumor-relevant proteins such as MYC, c-Jun and cyclin D1 in a p53-independent manner.
“Mdm2 was shown to maintain the expression of tumor-relevant proteins, such as MYC, c-Jun and cyclin D1, in a p53-independent manner.14”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationNbs1 is important to localize the MRN complex into the nucleus and to act as a regulatory and protein recruitment adaptor in the MRN complex.
“Nbs1 is important to localize the MRN complex into the nucleus and to act as a regulatory and protein recruitment adaptor in the MRN complex.33”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [33] source
Not checked in the free run.
The paper relies on this without support.
Effective therapies for locally advanced or metastatic pancreatic ductal adenocarcinoma are lacking.
“Currently, there is a lack of effective therapies for locally advanced or metastatic tumors, pointing to the need to define novel therapeutic strategies.”
The paper relies on this without support.
Etoposide is a potent inducer of DNA double-strand breaks.
“The topoisomerase II inhibitor etoposide is a potent inducer of DNA DSBs.”
The paper relies on this without support.
The Mdm2 inhibitors PXN822 and Nutlin 3a act specifically on Mdm2, and their observed effects are not primarily due to off-target activities.
“the Mdm2 inhibitor delays DSB repair in the investigated murine PDAC model argues that Mdm2 inhibitors block Mdm2 functions needed for sufficient DNA repair.”
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?
In addition to activating p53, Nutlin stabilizes Mdm2, raising Mdm2 levels that dampen the p53 response to DNA damage and increase sensitivity to genotoxic drugs.
“Our data suggest that in addition to p53 activation, Nutlin is also stabilizing Mdm2 causing an increase in the levels of Mdm2, which dampens the p53 response to DNA damage, resulting in increased sensitivity to genotoxic drugs (22,23).”
From Pharmacologically increasing Mdm2 Inhibits DNA repair and cooperates with genotoxic agents to kill p53-inactivated ovarian cancer cells (2015) · cites this paper as [23]
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.
Figure 4 panel e is assigned two different treatments
MinorThe caption assigns panels c-e to the Nutlin 3a/etoposide experiment, then assigns panel e to the gemcitabine experiment. The results text refers to the gemcitabine experiment as Figure 4f, suggesting the second panel label is incorrect.
“(c-e) The indicated cell lines were treated with Nutlin 3a (5 lM), etoposide (5 lg/ml), the combination thereof or were left as vehicle-treated controls.”
“(e) The indicated cell lines were treated with gemcitabine (0.01 lM) or were left as vehicle-treated controls.”
The citation lists haven’t been fetched yet
References 44 · Cited by 30
Research works printed but not linked: 1
- Roos WP, DNA damage‐induced apoptosis: from specific DNA lesions to the DNA damage response and apoptosis, Cancer Lett
- Authors
- Conradt, L., Henrich, A., Wirth, M., Reichert, M., Lesina, M., Algül, H., Schmid, R. M., Krämer, O. H., Saur, D., & Schneider, G.
- Affiliations
- Technical University of Munich (DE)
- Abramson Cancer Center (US)
- Friedrich Schiller University Jena (DE)
- Corresponding author
- Schneider, Günter
- ORCID
- Venue
- International Journal of Cancer
- Publisher
- Wiley
- Published
- 2012-11-26
- Volume
- 132
- Issue
- 10
- Pages
- 2248-2257
- ISSN
- 0020-7136 (ISSN-L)
- 1097-0215
- OpenAlex
- W1969546668
- Semantic Scholar
- bf28077e195ab71b50e982d17b11d36478556986
- Cited by
- 30
- References
- 44
- Citations by year
- 2024: 1 · 2023: 2 · 2022: 3 · 2021: 2 · 2020: 2 · 2019: 4 · 2018: 3 · 2017: 1 · 2016: 6 · 2015: 2 · 2014: 4
- FWCI
- 0.95
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- International Journal of Cancer (publishedVersion)
- PubMed (publishedVersion)
- mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion)
- Funders
- Bayerische Forschungsstiftung
- Deutsche Krebshilfe
- Topics
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Health Sciences › Medicine › Oncology › Pancreatic and Hepatic Oncology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
- Keywords
- MDM2 inhibitors, topoisomerase II inhibitors, pancreatic ductal adenocarcinoma, p53-independent cell death, caspase-independent cell death, DNA double-strand breaks, Mre11-Rad50-Nbs1 complex, DNA repair, combination therapy, treatment resistance
- MeSH terms
- Gemcitabine, Amino Acid Chloromethyl Ketones, Animals, Antineoplastic Agents, Deoxycytidine, Disease Models, Animal, DNA Damage, Doxorubicin, Drug Synergism, Etoposide, Imidazoles, Immunohistochemistry, Indoles, Pancreatic Neoplasms, Piperazines, Pyrrolidinones, Blotting, Western, Apoptosis, Cell Line, Tumor, Immunoprecipitation, Mice, Proto-Oncogene Proteins c-mdm2, Topoisomerase II Inhibitors
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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 2026-10-08.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.
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