Drug-Induced p53 Activation Limits Pancreatic Cancer Initiation

  1. Jennifer J Twardowski
  2. Thomas I Heist
  3. Zamira Guerra Soares
  4. Emily S Berry
  5. Luis I Ruffolo
  6. Christoph Pröschel
  7. Stephano S Mello

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, initiated predominantly by mutations in Kras , which induce acinar-to-ductal metaplasia (ADM) and subsequent formation of precursor lesions, such as pancreatic intraepithelial neoplasia (PanIN). Progression to PDAC is frequently associated with mutations in the tumor suppressor TP53 , presumably via disrupting p53-mediated cellular senescence of PanINs. Whether TP53 also has tumor-suppressive activity in earlier phases of PDAC initiation has been less clear. In this study, we investigate the impact of pharmacological stabilization of the wild-type p53 protein on the formation of ADM in a Kras G12D -driven mouse model of PDAC. Our findings demonstrate that p53 stabilization via Nutlin-3a significantly reduces both ADM and PanIN formation by promoting the differentiation of ADM into acinar cells. This differentiation coincides with p53-dependent upregulation of the transcription factor Mist1 ( Bhlha15 ), a critical inducer of acinar cell identity. Our results reveal a role for p53 in tissue repair and maintenance of homeostasis in tumor suppression and suggest pharmacological engagement of p53 as an intervention strategy to prevent PDAC initiation.

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    Reply to the reviewers

    Reply to the Reviewers

    We thank the reviewers for their very thoughtful and insightful reviews. We have performed several new experiments and addressed their points in a revised manuscript, which has significantly improved the manuscript. Our detailed responses follow.

    Responses to Reviewer 1

    Major comments

    __ In the supplementary figures when looking at the KC vs KPC mice and the trichrome staining (S2) or looking at the muc5a and mucin levels in figure 2, the KPC mice appear to have a larger amount of PANIN formation than the KC mice which is usually indicative of further tumour progression that can occur in p53 null tumours. Due to the further progression of the tumour in the KPC mice, drugs such as nutlin-3a may have inhibitory effect on PANIN formation and nutlin results might therefore not be indicative of p53 dependence. This should at least be mentioned and discussed.__

    A: We appreciate the reviewer’s insightful comment. Indeed, in the KPC model, where PDAC progression is accelerated, Nutlin-3a may exhibit p53-independent effects. We now address this consideration in the revised manuscript on page 6, when describing our PanIN and Alcian blue results in KPC mice.

    __ When looking at how p53 controls the expression of acinar cell identity genes the authors look at MEFs when performing their GSEA (Figure 4a, b). The MEFs are used as a model for neoplastic cells but it would also be beneficial to test this in pancreatic cell lines. When looking at Bhlha15 expression (figure 4c) there is a decrease observed in the p53 containing vs knockout mice, this is from a data set GSE94566, it would be beneficial to test this in the KC and KPC mice the authors generated to see if the results can be validated. Results in 4d re mist expression should also be evaluated in KPC mice to prove p53 dependence. Finally, murine and human fibroblasts are treated with doxorubicin (figure 4e-f; figure s4b) to show Bhlha15 is upregulated, it would also be useful to show this either in pancreatic cell lines with/without p53 or from the murine tissue of the KC and KPC mice.__

    A: We appreciate the reviewer’s insightful comment. We recognize that the way we originally described the GSEA analysis may have inadvertently suggested that it was performed in RNA-seq from MEFs. To clarify, the GSEA analysis in Figure 4a is derived from RNA-seq of sorted precursor lesions from p53-proficient (KC) and p53-deficient (KPC) mice, not MEFs. We have revised the Results section that describes __Figure 4 __to more clearly reflect this distinction. Additionally, we acknowledge the importance of confirming p53’s regulatory role over Bhlha15 expression in the pancreas. To support the findings from the GSE94566 dataset, which was generated using sorted precursor lesions from KC and KPC mice (Mello et al., 2017), we present a boxplot of Bhlha15 expression (Figure 4c).In response to the reviewer’s suggestion, we incorporated Mist1 immunohistochemistry and quantification in KPC mice treated with Nutlin-3a or vehicle control (Figure 4b) to further validate the p53-dependent regulation of Mist1 expression. To strengthen the conclusions from Figures 4c–d, we also conducted complementary experiments in mouse-derived pancreatic cancer cell lines either proficient (KIC1 and KIC2, derived from Kras+/G12D; Pdx1-Cre; Cdkn2afl/fl mice) or deficient (KPC, derived from Kras+/G12D; Pdx1-Cre; Trp53fl/fl mice) for p53. These experiments aim to further substantiate the regulatory role of p53 in controlling Mist1 expression.

    __ It is unclear why varying numbers of mice have been used. For the majority of experiments, the authors use n=6 mock treated mice and n=3 or 4 nutlin-3a treated mice. For KPC mice n=4 and n=4 was used. N=3 for mock and nutlin-3a treated mice were used. Did some mice die unexpectedly during the experiment? It would be good to report this. Also, the smaller amount of animal models used even though it was n=3/the disparity between the control and nutlin treated may raise some question. Usually, 6 vs 3. Possibly testing this with some lesson common Kras mutants and increasing the time in which nutlin-3a is studied in the pancreatic tumours, can it constantly prevent tumour formation.__

    A: We appreciate the reviewer’s concern regarding the variation in cohort sizes across experiments. Several factors contributed to these differences. In some cases, mice were excluded due to health issues such as malocclusion and poor general condition. Additionally, a subset of animals was misgenotyped and later confirmed to lack the KrasG12D allele, necessitating their exclusion from the study. The KPC model in particular is challenging to breed due to the requirement for four specific alleles and its rapid progression toward pancreatic cancer, which can limit survival and experimental flexibility. Despite these limitations, our key experimental groups, such as those evaluating Amylase rescue upon Nutlin-3a treatment, Mist1 induction in ADM, and lineage tracing studies, maintained a statistical power of at least 80% based on our cohort sizes. We have now clarified these details in the Supplemental Materials and Methods to ensure transparency regarding animal exclusions and sample size variability. We also agree with the reviewer that assessing Nutlin-3a at later stages of tumorigenesis would strengthen our findings. To this end, we treated aging KC mice (6 months old), which accumulate ADM and PanINs due to chronic Kras activation (rather than pancreatitis), with Nutlin-3a for a week and analyzed them at 8 months. Treated mice showed increased normal acinar tissue and reduced high-grade PanINs. This new data, presented in Figure 5, highlights the sustained tumor-suppressive effect of p53 activation and suggests that it could delay or prevent PDAC onset.

    Minor comments:

    __ The text is mostly clear, apart from the results section around figure 4, where it is not always clear which material has been used for analysis when referring to a previous paper. The quantification graphs should be wider as they seem squished sometimes. Changing the colours to be darker would make these more easily identifiable as the pale blue/red are sometimes difficult to see.__

    A: We appreciate the reviewer’s feedback and agree with the reviewer that the results section for Figure 4 leaves some margin for misinterpretation. To address this, we have revised the text to improve clarity and ensure that the source of each dataset, condition, and cell line is explicitly stated. Additionally, we have added a Supplementary Materials and Methods section that provides detailed information about the datasets and experimental conditions used in Figure 4. We have also adjusted the quantification graphs to be wider, preventing them from appearing compressed, and modified the color scheme, using gray and white tones to improve visibility and contrast, making the data easier to interpret.

    __ a) Figure 4a and 4b can be moved to the supplementary figures. b). For the figure legend of S1 on the separate file for the supplemental figures there is no S1e mentioned but it is in the paper. c) Figure S1a needs a scale bar.__

    A: We appreciate the reviewer’s suggestions and have implemented all the requested changes:

    1. a) Figures 4a and 4b have been moved to the Supplementary Figures section.
    2. b) The Figure S1 legend in the supplemental file has been updated to include S1e, ensuring consistency with the main text.
    3. c) A scale bar has been added to Figure S1a for clarity.

    Responses to Reviewer 2

    Major comments

    __ The study implies that pharmacologically engaging wild-type p53 (for example, through Nutlin-3a) may serve as a strategy to prevent or significantly delay the onset of PDAC by preserving the normal acinar cell phenotype and blocking early metaplastic changes. Doing a search in Pubmed search, no such findings has been previously published. It is a very important findings as it paves the way to clinical trial. The data are of excellent quality and would support the conclusions but the experiments need additional control experiments to strengthen the conclusions.__

    A: We thank the reviewer for the positive assessment of our work.

    __ For all immunohistochemistry quantification: The authors should explain better how the scoring was performed. - the authors should present a range of positive staining (negative, Weak, medium, high). The authors should state the number of sections analysed and how many cells or nuclei in total were counted per section or ROI to define the percentage of positive cells/nuclei.__

    A: We appreciate the reviewer’s suggestion and have addressed this point by adding a detailed description of our immunohistochemistry quantification methodology in the Supplementary Materials and Methods. This includes a clear explanation of how positive staining was defined. Specifically, we did not use a categorical intensity scoring system (e.g., weak, medium, strong); instead, positive staining was determined based on signal levels clearly distinguishable from background noise, enabling reliable automated detection by the analysis software that we employed. Regarding sample size and quantification scope, we analyzed one representative section per individual in the cohort. For each section, either the entire tissue or specific ROIs, such as ADM or PanIN lesions, were annotated and quantified. The number of nuclei or cells evaluated per section varied depending on tissue size and ROI, and this is now described in the Supplementary Materials and Methods.

    __ In material methods: the antibodies concentration must be indicated in ug/ml.__

    A: We appreciate the reviewer’s suggestion and have updated the Materials and Methods section to include antibody concentrations in µg/ml.

    __ a) Figure1b, 1c must present the following control staining in addition to presented data: i) staining of non-treated pancreas (as negative control); ii) staining of pancreas treated with Nutlin only (not-treated with cerulein) to assess the effect of Nutlin in absence of Cerulein. b) Figure 4d: the authors should repeat the experiment in p53fl/fl mice to assess nutlin off-target effect. c) Figure S1 e) there is no legend for it. d) Figure S4: which p53 exon has been deleted by CRISPr. The sequences of the sgRNA are not indicated.__

    A: We appreciate the reviewer’s suggestions and have addressed all the requested changes. For Figures 1b and 1c, we have added the necessary control stainings, including (a) staining of non-treated pancreas as a negative control and (b) staining of pancreas treated with Nutlin-3a only (without cerulein) to assess the effect of Nutlin-3a in the absence of Cerulein (Figure S1c). For Figure 4d (now Figure 4b), we have included sections from p53-deficient (KPC) mice stained for Mist1 to evaluate potential off-target effects of Nutlin-3a. Our results show no Mist1 expression in the absence of p53, suggesting that Nutlin-3a-mediated upregulation of Mist1 in ADM is p53-dependent. Additionally, we have added a legend for Figure S1e. For Figure S4, we clarified that CRISPR interference (CRISPRi) was used in this experiment rather than gene deletion. As such, the sgRNA is not designed against a specific exon, but instead targets the promoter region of the TP53 gene to suppress its transcription. We have now included the sgRNA sequence used in Figure S4d for clarity.

    Responses to Reviewer 3

    __ The authors suggested, based on their data, that Mist1 may be transactivated by p53 "presumably directly, across distinct cell types and in different contexts, such as oncogenic stress and DNA damage." This statement is too speculative and that is noteworthy because the experiments to get at those potential functional details (including, e.g., gene interference, biochemical assays) are not particularly difficult and would significantly improve the manuscript.__

    A: We appreciate the reviewer’s feedback. Our original statement aimed to accurately reflect our findings without overinterpretation, as we identified a conserved p53 binding site in the Bhlha15 locus, observed p53 occupancy in published ChIP-seq datasets, and demonstrated p53-dependent expression of Mist1 at both RNA and protein levels. To further support this relationship, we expanded our analysis to include p53-proficient and p53-deficient mouse PDAC cell lines, confirming the dependency of Mist1 expression on p53 (Figure 4e). Additionally, we now show that Mist1 protein was detected in lesions of Nutlin-3a–treated KC mice, but not in KPC mice, further indicating that Mist1 induction is p53-dependent in vivo (Figure 4b). While we acknowledge that direct functional testing of the p53 binding site would further strengthen the mechanistic insight, the Bhlha15 locus contains multiple p53 ChIP-seq peaks, making it difficult to isolate the contribution of individual sites. For this reason, we believe that dissecting the precise binding events underlying p53-mediated regulation of Bhlha15 goes beyond the scope of the current study, but we agree it is a valuable direction for future work.

    __ The study did not explore a novel concept beside showing that ADM can be reversed by inhibiting p53, which though may sound novel is intuitive (the focus on Mist1 alone appear narrow too).__

    A: We respectfully disagree with the reviewer’s assessment. The prevailing view in the literature is that p53 suppresses pancreatic cancer primarily by preventing the progression from PanINs to PDAC, largely through the induction of senescence in precursor lesions (Caldwell et al. Oncogene 2012; Morton et al. PNAS 2010). However, whether p53 also plays a tumor-suppressive role at earlier stages, particularly in ADM, remains unclear. Our study provides evidence that p53 regulates ADM plasticity and acinar cell identity, expanding its known functions beyond senescence induction. Additionally, the role of p53 in maintaining tissue homeostasis through the regulation of differentiation programs is an emerging and underexplored concept. Our focus on Mist1 is well justified, as we observed a significant overlap between gene expression changes in p53-proficient (KC) and p53-deficient (KPC) precursor lesions and known Mist1-regulated genes (Fig. 4a), highlighting its potential as a key mediator of p53-dependent acinar cell identity maintenance. While Mist1 is a focal point of our study, the broader implication is that p53 plays an active role in controlling acinar cell fate, which challenges the conventional view of its function solely in later-stage tumor suppression.

    __ The RNA-seq and ChIP data may provide several opportunities to get at how p53 mediates the proposed effect on ADM and it would be worthwhile to leverage those data.__

    A: We appreciate the reviewer’s suggestion. Like the reviewer, we recognize that p53 has a vast downstream network, and while additional pathways may contribute to p53-mediated cell differentiation, we believe that investigations of other mechanisms involved in this process extends beyond the scope of this manuscript and would dilute the central message rather than strengthen it.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study, Twardowski et al. showed that the treatment of pancreatic cancer mice models with Nutlin-3a, a drug that allows p53 stabilization, limits tumor initiation by reversing acinar-to-ductal metaplasia (ADM), which is an early event in pancreatic cancer. This study is well done, and the figures are quite convincing. It also uses a combination of state-of-the-art mouse models, most notably the inducible model that allowed cell lineage tracing. However, it is generally descriptive and provides no mechanistic detail beside suggesting that p53 potentially drives the upregulation of the transcription factor Mist1 (Bhlha15) as part of the ADM to acinar differentiation process. The authors also suggested, based on their data, that Mist1 may be transactivated by p53 "presumably directly, across distinct cell types and in different contexts, such as oncogenic stress and DNA damage." This statement is too speculative and that is noteworthy because the experiments to get at those potential functional details (including, e.g., gene interference, biochemical assays) are not particularly difficult and would significantly improve the manuscript. Besides the above comments, the study did not explore a novel concept beside showing that ADM can be reversed by inhibiting p53, which though may sound novel is intuitive (the focus on Mist1 alone appear narrow too). The RNA-seq and ChIP data may provide several opportunities to get at how p53 mediates the proposed effect on ADM and it would be worthwhile to leverage those data. The study in its current state is descriptive and appears too preliminary.

    Significance

    Besides the above comments, the study did not explore a novel concept beside showing that ADM can be reversed by inhibiting p53, which though may sound novel is intuitive (the focus on Mist1 alone appear narrow too). The RNA-seq and ChIP data may provide several opportunities to get at how p53 mediates the proposed effect on ADM and it would be worthwhile to leverage those data. The study in its current state is descriptive and appears too preliminary.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript entitled:" Drug-Induced p53 Activation Limits Pancreatic Cancer Initiation. " Twardowski et al., investigate using mouse animal models the impact of pharmacological stabilization of the wild-type p53 protein on the formation of acinar-to-ductal metaplasia (ADM) in a KrasG12D-driven mouse model of Pancreatic ductal adenocarcinoma (PDAC). The authors mostly performed immunohistochemistry to assess the differentiation status in response to treatment. The authors claims that they demonstrate that p53 stabilisation via Nutlin-3a treatment, an inhibitor of its ubiquitin ligase MDM2, significantly reduces both ADM and the formation of precursor lesions, such as pancreatic intraepithelial neoplasia (PanIN) by promoting the differentiation of ADM into acinar cells. The authors claim that the differentiation is concomitant with p53-dependent induction of the transcription factor Mist1 (also named Bhlha15), a critical inducer of acinar cell formation. The authors conclude that their data reveal a role for p53 in promoting the re-differentiation of ductal metaplasia in healthy acinar cells, preventing ductal-metaplasia to progress to Pancreatic ductal adenocarcinoma.

    Significance

    The study implies that pharmacologically engaging wild-type p53 (for example, through Nutlin-3a) may serve as a strategy to prevent or significantly delay the onset of PDAC by preserving the normal acinar cell phenotype and blocking early metaplastic changes. Doing a search in Pubmed search, no such findings has been previously published. It is a very important findings as it paves the way to clinical trial.

    The data are of excellent quality and would support the conclusions but the experiments need additional control experiments to strengthen the conclusions.

    Here are the major points that preclude publication as it is

    • For all immunohistochemistry quantification: The authors should explain better how the scoring was performed. - the authors should present a range of positive staining (negative, Weak, medium, high). The authors should state the number of sections analysed and how many cells or nuclei in total were counted per section or ROI to define the percentage of positive cells/nuclei.
    • In material methods: the antibodies concentration must be indicated in ug/ml.
    • Figure1b, 1c must present the following control staining in addition to presented data

    a. staining of non-treated pancreas (as negative control)

    b. staining of pancreas treated with Nutlin only (not-treated with cerulein) to assess the effect of Nutlin in absence of Cerulein

    • Figure 4d: the authors should repeat the experiment in p53fl/fl mice to assess nutlin off-target effect
    • Figure S1 e) there is no legend for it
    • Figure S4: which p53 exon has been deleted by CRISPr. The sequences of the sgRNA are not indicated.
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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The key findings in this paper are that using nutlin-3a to stabilise p53 a reduction of the formation of ADM and PanIN in the KrasG12D driven mouse model of PDAC are observed. They show that as p53 is stabilised by nutlin-3a ADM cells are differentiated into acinar cells, corresponding with a p53 dependent upregulation of Mist1. To show these results the authors utilised multiple mouse models and induced pancreatic damage/oncogenic stress via the injection of cerulein. Histological sections of the pancreas in the various mouse models were stained and quantified to allow the authors to come to their conclusions. The methods are presented sufficiently. The statistical analysis was adequate for this work. References are appropriate.

    Major comments:

    1. In the supplementary figures when looking at the KC vs KPC mice and the trichrome staining (S2) or looking at the muc5a and mucin levels in figure 2, the KPC mice appear to have a larger amount of PANIN formation than the KC mice which is usually indicative of further tumour progression that can occur in p53 null tumours. Due to the further progression of the tumour in the KPC mice, drugs such as nutlin-3a may have inhibitory effect on PANIN formation and nutlin results might therefore not be indicative of p53 dependence. This should at least be mentioned and discussed.
    2. When looking at how p53 controls the expression of acinar cell identity genes the authors look at MEFs when performing their GSEA (Figure 4a, b). The MEFs are used as a model for neoplastic cells but it would also be beneficial to test this in pancreatic cell lines. When looking at Bhlha15 expression (figure 4c) there is a decrease observed in the p53 containing vs knockout mice, this is from a data set GSE94566, it would be beneficial to test this in the KC and KPC mice the authors generated to see if the results can be validated. Results in 4d re mist expression should also be evaluated in KPC mice to prove p53 dependence. Finally, murine and human fibroblasts are treated with doxorubicin (figure 4e-f; figure s4b) to show Bhlha15 is upregulated, it would also be useful to show this either in pancreatic cell lines with/without p53 or from the murine tissue of the KC and KPC mice.
    3. It is unclear why varying numbers of mice have been used. For the majority of experiments, the authors use n=6 mock treated mice and n=3 or 4 nutlin-3a treated mice. For KPC mice n=4 and n=4 was used. N=3 for mock and nutlin-3a treated mice were used. Did some mice die unexpectedly during the experiment? It would be good to report this.

    Minor comments:

    1. The text is mostly clear, apart from the results section around figure 4, where it is not always clear which material has been used for analysis when referring to a previous paper. The quantification graphs should be wider as they seem squished sometimes. Changing the colours to be darker would make these more easily identifiable as the pale blue/red are sometimes difficult to see.
    2. Figure 4a and 4b can be moved to the supplementary figures.
    3. For the figure legend of S1 on the separate file for the supplemental figures there is no S1e mentioned but it is in the paper.
    4. Figure S1a needs a scale bar.

    Significance

    The study is a conscience investigation into how p53 is involved in pancreatic cancer initiation and how this can be reduced by over activation of p53. A strong point of the study is the generation of the genetic lineage mouse model. This allowed the authors to persistently label ADM cells and trace their progeny. This experiment provided strong evidence that nutlin-3a treatment can indeed reverse acini to ADM formation and prevent PanIN formation. Some of the limitations of the study involve relying on mainly immunohistochemistry to show changes in protein level in mouse tissue, western blotting could be used in conjunction with this to further validate the claims put forward in the paper. Also, the smaller amount of animal models used even though it was n=3/the disparity between the control and nutlin treated may raise some question. Usually, 6 vs 3. Possibly testing this with some lesson common Kras mutants and increasing the time in which nutlin-3a is studied in the pancreatic tumours, can it constantly prevent tumour formation.

    Other work in this area has looked at nutlin-3a and its effect on NSCLC with a Kras mutant (https://pubmed.ncbi.nlm.nih.gov/38093368/) and has shown that nutlin-3a is able to induce cell death in Kras mutant NSCLC cells. This paper also builds on work by (https://pmc.ncbi.nlm.nih.gov/articles/PMC5730340/#S9) who looks at NRF2-mediated induction of MDM2 and accumulation of p62 leading to PDAC and how inhibition of MDM2 by nutlin-3a may reduce this progression and this is shown in the present paper. The study for review advances using MDM2 inhibitors such at nutlin-3a in a clinical manner by looking at how it affects the progression of PDAC in mice and starts to elucidate the interactions which cause this to happen.

    The research present is specialised research that will hopefully be able to be translated to the clinic, if the use of nutlin-3a is able to prevent progression to PDAC in a mouse it would be useful to see if this also possible in patient derived primary cell lines to further elucidate the mechanism of this work.

    My expertise: P53, mouse work, lung cancer.

  5. Version published to 10.1101/2024.05.29.595146v2 on bioRxiv
  6. Version published to 10.1101/2024.05.29.595146v1 on bioRxiv