MDM2 stabilization of Notch intracellular domain upon DNA damage plays a major role in non-small cell lung carcinoma response to platinum chemotherapy

  1. Sara Bernardo
  2. Quentin-Dominique Thomas
  3. Maicol Mancini
  4. Alba Santos
  5. Sylvia Fenosoa Rasamizafy
  6. Amina-Milissa Maacha
  7. Anais Giry
  8. Emilie Bousquet-Mur
  9. Laura Papon
  10. Marion Goussard
  11. Christophe Fremin
  12. Andrea Pasquier
  13. María Rodríguez
  14. Camille Travert
  15. Jean-Louis Pujol
  16. Laetitia K Linares
  17. Lisa Heron-Milhavet
  18. Alexandre Djiane
  19. Irene Ferrer
  20. Luis Paz-Ares
  21. Xavier Quantin
  22. Luis M Montuenga
  23. Hélène Tourriere
  24. Antonio Maraver

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Despite major advances in lung cancer clinical management, majority of patients suffering non-small cell lung carcinoma (NSCLC) are treated in first line with platinum in combination with immune checkpoint inhibitors. Although platinum compounds normally display an initial therapeutic effect, relapse constitutes a major challenge in the clinical management of NSCLC patients. Therefore, it is fundamental to understand the relapse underlying mechanisms to find new therapeutic opportunities to improve patients’ survival. Here, we found that different DNA damage inducers increase the protein levels of Notch Intracellular Domain (NICD), i.e., the active form of NOTCH1. Mechanistically, we unveiled that upon platinum treatment, there was a concomitant increase of MDM2 together with NICD, and we also observed an MDM2-mediated ubiquitination and stabilization of NICD. Of note, using patient-derived xenografts displaying intrinsic carboplatin resistance, we demonstrated that the combination of carboplatin with MDM2 and NICD inhibitors increased survival and reduced tumor growth compared with carboplatin in monotherapy. Moreover, in patients with NSCLC who received platinum chemotherapy, MDM2 expression level in the tumor was correlated with poor progression-free survival, further validating MDM2 key role in the response to platinum compounds. Our findings open a therapeutic opportunity for NSCLC patients, the main lung cancer subtype of the leading cause of death by cancer worldwide.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Manuscript number: RC- 2024-02497

    Corresponding author(s): Tourriere, Hélene and Maraver, Antonio

    1. General Statements [optional]

    We sincerely thank the Editors and Reviewers for the time devoted to our manuscript. We found their critiques interesting and very helpful. After careful examination and thanks to a large collaborative effort, we will be able to answer to all the reviewers’ comments by adding significantly new experimental data.

    We are also encouraged by the positive comments of the Reviewers:

    “This manuscript will likely engage oncologists who investigate the chemotherapy-resistant mechanisms of platinum compounds in NSCLC treatment” (Reviewer 1);

    “Overall, the authors have conducted experiments that sufficiently elucidate their claims, and the description of the experiments is detailed.”; and “Overall, this work unveils a novel mechanism for Notch activation in response to platinum chemotherapy, providing a renewed outlook on overcoming chemotherapy resistance in NSCLC” (Reviewer 2).

    We are also aware that both reviewers agreed that there is room for improvement, and we are sure that upon accomplishment of all proposed experiments both reviewers will be fully satisfied.

    Please bear in mind that although it was known that platinum-based chemotherapy induced the Notch pathway in lung cancer cells, the underlying molecular mechanism was largely unknown. Thanks to the molecular dissection we performed in our study, we propose an innovative treatment for patients with lung cancer, the main cause of death by cancer in the world. Hence, we agree with both reviewers that our study will be appealing for a large number of cancer researchers, and we feel it will be also the case for those interested in DNA damage, Notch and MDM2 pathways.

    2. Description of the planned revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary: This manuscript from Maraver and co-authors investigates the putative resistance mechanisms that hinder the efficacy of platinum-based therapies (e.g., carboplatin) against non-small cell lung carcinoma (NSCLC). Using in vitro lung cancer cell lines, shRNA-based knockdown, and exogenous overexpression systems, the research describes a DNA damage-induced resistance mechanism involving the NOTCH signaling pathway and the E3 ligase MDM2. The authors show that carboplatin treatment induces DNA damage and promotes ATM activation, which in turn activates the NOTCH signaling pathway via ubiquitination and stabilization of the Notch Intracellular Domain (NICD). New findings include the MDM2-mediated ubiquitination and stabilization of NICD. Using in vivo NSCLC-PDX models, they demonstrate that combining carboplatin with Notch and MDM2 inhibitors can enhance tumor killing, suggesting that targeting the MDM2/NICD axis in conjunction with carboplatin may be a viable therapeutic alternative. Furthermore, they show that NICD and MDM2 levels are elevated among tumor samples from chemo-resistant patients. Consistent with these findings, high MDM2 levels correlate with poor progression-free survival (PFS) in NSCLC patients.

    [Authors] We thank this reviewer for her/his fair summary of our work that highlights our new findings.

    Major comments:

    Some of the key conclusions may not be convincing.

    [Authors] We understand the concerns that reviewer might have and we are sure that upon accomplishment of all experiments detailed below, she/he will be convinced that the manuscript will be ready for publication.

    One significant weakness of the manuscript is the lack of exploration into the underlying mechanism of how MDM2 mediates the stabilization of NICD. While the observation of MDM2-mediated NICD stabilization is intriguing, it is important to provide a more convincing explanation for the reviewers. This could be achieved by offering a detailed molecular mechanism, especially considering that MDM2 typically targets proteins for degradation.

    [Authors] After reading this reviewer’s comment, we realize we did a poor job discussing better the previous study demonstrating that MDM2 induced ubiquitination on NICD but not for degradative purposes (Pettersson et al., 2013). In particular, they performed it using a mutated form of ubiquitin in lysine 48, i.e., the K48R mutant. Like this, the authors of this seminal study demonstrated that MDM2 was still able to induce ubiquitination in NICD, and hence it was not degradative.

    Still, and to confirm that this is the case also upon DNA damage, we will perform experiments using same K48R mutant to formally prove that MDM2 upon DNA damage does not ubiquitinate NICD via lysine 48-linked polymers, and hence it is not degradative. Even more, upon discussion with Laetitia Linares, author of our study and long-lasting expert in ubiquitination (for instance see (Riscal et al., 2016) and (Arena et al., 2018)), we will use another ubiquitin mutant in lysine 63. This different type of ubiquitination does not mark proteins for degradation but promote an association of the targeted protein with DNA helping for DNA repair (Liu et al., 2018). Using a ubiquitin mutated in this lysine, i.e., K63R, this type of ubiquitination cannot occur. Taking into account that we observe NICD increase ubiquitination upon DNA damage, the use of K63R will be very informative.

    Hence, we will repeat experiments of current Figure 3A with the same WT ubiquitin as before, and now also with K48R and K63R mutants. Even more, we will also include mutant forms of ubiquitin which can only form ubiquitin chains on lysine 48 (K48 only) or lysine 63 (K63 only) and we anticipate that in the presence of K48 only mutant, NICD will not be ubiquitinated upon DNA damage, while the use of K63 only mutant will be very useful. All these data will be part of the new Figure 3A.

    Of note, Dr Linares has all tools required to perform these experiments and hence we will start them soon.

    Another weakness lies in the unclear role and the underlying mechanism of ATM in the MDM2-mediated NICD stabilization. While the data presented (Fig. 3B, 3C) suggest that carboplatin could elevate MDM2 levels for NICD stabilization, a more precise method to induce MDM2 overexpression specifically for targeting NICD is required. It appears that ATM plays a crucial role in this regulatory process. The following questions must be addressed: Does ATM induce the phosphorylation of MDM2 for its protein stabilization and/or E3 ligase activity?

    [Authors] There are several points here.

    For the first one, the use of a more precise method to induce MDM2 overexpression, it is exactly what we did in Figure 4A, i.e., ectopic expression of MDM2 to demonstrate that MDM2 is sufficient to increase NICD levels.

    For the second one, i.e., the phosphorylation status of MDM2 by ATM in our system, we will perform different experiments. There are up to six proposed residues in MDM2 to be phosphorylated by ATM upon DNA damage: S386, S395, S407, T419, S425, and S429 (Cheng et al., 2011). Among all of them, S395 is the most well-known and again Dr Linares has interesting tools we will use to answer to this specific reviewer’s point. We will use an MDM2 mutant harboring an aspartate instead of the serine in this position, i.e., S395D, that mimics the serine 395 phosphorylation induced by ATM upon DNA damage. We will use this mutant together with the WT and 464A MDM2 proteins already used, and if this residue is important in our phenotype, total levels of NICD will be even higher and/or localize more in the nuclei when compared with WT MDM2. All these new data will appear as the new Figure 4A and new Figure 4B.

    Furthermore, we will also use an antibody that recognizes this phosphorylation site by WB after carboplatin treatment and it will be part of the new Figure 3B.

    Finally, we will also express WT MDM2 and purify it by immunoprecipitation in different experimental conditions: steady state, upon carboplatin treatment and also in combination of carboplatin and ATM inhibitor, to perform phospho-proteomics analysis upon all these conditions. Of note, and to show the feasibility of this approach, the proteomic platform at Biocampus in Montpellier has experience using this technique (Kassouf et al., 2019).

    The combination therapy of carboplatin with MDM2 and NICD inhibitors may lack compelling rationale (see below).

    [Authors] This is a very important point but we discuss it below, where more information is provided by the reviewer. Still, we anticipate we will perform a new in vivo experiment to answer to this point.

    In lines 275-276, the authors stated that their preclinical data establish the enhancement of carboplatin's therapeutic effect in NSCLC in vivo through MDM2-NICD axis inhibition. However, it's important to note that this finding remains preliminary at this stage.

    [Authors] We consider that our statement is not exaggerated, but we will tone down the message as proposed by the reviewer in the next submission.

    Minor comments:

    The observed loss of NICD during ATMi + carboplatin treatment in Figures 2A and 2B raises the question of whether ATM regulates the gene transcription of NOTCH. In addition to the CHX assay conducted in Figures 2C and 2D, quantifying NOTCH mRNA upon ATM inhibition could provide further insights. Alternatively, referencing relevant studies on this topic may strengthen the discussion.

    [Authors] This is an interesting experiment and we will perform it.

    In Figures 4A and 4B, the noticeable discrepancy between the exogenous expression of wild-type (WT) MDM2 and catalytically inactive MDM2-464A raises concerns. It is essential to consider if the reduced ubiquitination and stability of NICD might be attributed to varying levels of MDM2-464A in the cells rather than its catalytic inactivity. While p53 ubiquitination was utilized as a control, ensuring comparable levels of MDM2 and MDM2-464A expression could enhance the experimental rigor. Compared to the smear poly-ubiquitination bands observed for MDM2 in Figure 4B, the ubiquitination of NICD appears simpler. What distinguishes the feature of MDM2-mediated NICD ubiquitination? Could it potentially involve mono-ubiquitination?

    [Authors] The point of the reviewer is well taken, and importantly, as mentioned above in main point 2, we will repeat these experiments and will appear as new Figure 4A and new Figure 4B.

    Regarding the type of ubiquitination, as explained in detail in major point 1 to same reviewer, we will fully characterize the type of ubiquitination on NICD induced by DNA damage, and we will confirm that MDM2 is required for this specific ubiquitination in future new Figure 4C where we will overexpress the required ubiquitin forms and WT MDM2.

    In Figure 5A, the authors need to consider conducting additional NOTCH-associated factors to definitively demonstrate the activation of NOTCH signaling beyond HES1. Alternatively, in Figure 5B, the NICD Western blot could be complemented by detecting HES1 or other NOTCH-associated factors.

    [Authors] To answer to this particular point, we will test for other downstream targets of Notch as NRARP and it will appear as part of new Figure 5C.

    In Figures 5C and 5D, crucial control groups are missing, specifically mice treated solely with SP141+DBZ, carboplatin+SP141, and SP141+DBZ. It is essential to include these groups to demonstrate that the enhanced tumor killing results from the combination of carboplatin with SP141 and/or DBZ, rather than from SP141 and DBZ alone. Furthermore, in addition to the currently used NSCLC-PDX model harboring the p53 (P151R) mutation, it would be informative to include a NSCLC-PDX model expressing WT p53.

    [Authors] This is a crucial point in this rebuttal as mentioned before in major point 3 and we detail it in here.

    We did only 3 groups because preliminary data indicated that SP141 in combination with carboplatin was not showing any benefit compared to carboplatin alone while upon combination of carboplatin with Notch inhibition there was only a slight increase in therapeutic carboplatin benefit but otherwise not very potent, and for simplicity we preferred to don’t show these data. But, after reading this point from Reviewer 1, even if we will propose later only the triple combination for patients, we clearly need to demonstrate that the other combinations are not potent enough or not at all.

    The reviewer asked to include: “SP141+DBZ, carboplatin+SP141, and SP141+DBZ”. We imagine that she/he meant: SP141+DBZ, carboplatin+SP141, and carboplatin +DBZ, that together with the vehicle, carboplatin and carboplatin+SP141+DBZ makes 6 groups of treatments. Putting together the 8 mice devoted for tumor growth and survival, plus 4 mice for the acute treatment for IHC and WB purposes (for current Figures 5A and 5B) makes a total of 72, that is a substantial number of mice. Of note, since we performed the in vivo experiment presented in the current manuscript, a new Notch inhibitor called nirogacestat, appear in the market being the first in class Notch inhibitor to treat solid cancer patients (desmoid tumors) after demonstrating a significant therapeutic effect in clinical trials (Gounder et al., 2023).

    Hence, we will take advantage of the repetition of this experiment to substitute this new molecule instead of DBZ, that is an interesting molecule for preclinical research, but without any clinical relevance. Therefore, the use of nirogacestat will further increase the medical impact of our data. Importantly, nirogacestat is better tolerated than DBZ, meaning that mice can be treated for longer periods of time and we propose in here to treat up to 12 weeks. Finally, after discussion with Quentin Thomas, author of the manuscript and clinical researcher in the lab, we will provide 4 carboplatin cycles as it is proposed today to NSCLC patients in an attempt of getting closer to the clinical setting. In particular we will provide carboplatin to mice on weeks 1, 4, 7 and 10, while treating with MDM2 inhibitor (SP141) and Notch inhibitor (nirogacestat) from Monday to Friday for the 12 weeks.

    This experiment will be long and will require an important use of resources both human and financial, but we are sure that the effect in tumor growth and survival will be more dramatic than the one presented now.

    On the contrary and as explained in the 4th subheading part of this “revision plan”, including another 72 mice to treat a p53 proficient NSCLC PDX, when we already demonstrated in vitro that p53 is not required for the phenotype described in this study, for us it is totally unfeasible by ethical reasons, i.e., the use of animals in research (please see below for further details).

    All the new data will appear as new Figure 5 (B to E). For new Figure 5A please see below the major comment 2 of Reviewer 2.

    Though beyond the current study's scope, in the discussion section, the authors may want to propose or hypothesize on how MDM2-mediated NICD stabilization contributes to carboplatin resistance. This could provide valuable insights for future research directions.

    [Authors] We will discuss this part as proposed by the reviewer.

    In the Western blot results, the total ATM and ATR controls were absent.

    [Authors] The reviewer is totally right and we will repeat experiments to include all the totals as requested.

    Authors may choose to include a graphical abstract at the end of their study to visually illustrate the mechanisms they have described.

    [Authors] Very good idea thanks, we will do it.

    Reviewer #1 (Significance (Required)):

    Advance: The authors aim to present a novel perspective on the resistance mechanisms to platinum compounds in NSCLC therapy. They explore platinum compounds-induced DNA damage, ATM activation, and MDM2-mediated stabilization of the active form of NOTCH (NICD). However, to strengthen their claims, they must provide more conclusive results.

    Audience: This manuscript will likely engage oncologists who investigate the chemotherapy-resistant mechanisms of platinum compounds in NSCLC treatment, as well as scientists specializing in NOTCH and MDM2 pathways. However, the manuscript's central claims lack robust support from the available data, and the current approaches employed are not sufficiently thoughtful and rigorous; there is room for improvement.

    My expertise is molecular medicine, cancer biology, and epigenetics.

    [Authors] We want to thank again this reviewer for her/his helpful comments that will increase the impact and the relevance of our study while keeping the original message.

    We are also very satisfied when she/he said: “This manuscript will likely engage oncologists who investigate the chemotherapy-resistant mechanisms of platinum compounds in NSCLC treatment”.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Sara Bernardo et al. investigated the molecular mechanisms underlying the activation of the Notch signaling in response to DNA damage induced by platinum-based chemotherapeutic agents in non-small cell lung cancer (NSCLC). They demonstrated that carboplatin treatment induces DNA double-strand breaks (DSBs) and stabilizes NICD, a process dependent on ATM and mediated by MDM2. In vivo experiments in patient-derived xenografts (PDX) showed that inhibition of NICD and MDM2 enhanced platinum effectiveness. Furthermore, clinical analysis revealed a correlation between MDM2 expression and poor prognosis in NSCLC patients treated with platinum compounds, emphasizing the clinical relevance of the MDM2-NICD axis in platinum resistance.

    [Authors] We thank this reviewer for her/his nice synopsis of our study.

    Major comments:

    Overall, the authors have conducted experiments that sufficiently elucidate their claims, and the description of the experiments is detailed. However, there is still room for the improvement.

    [Authors] We are very pleased that reviewer finds our experimental work “…sufficiently elucidate their claims, and the description of the experiments is detailed.” And we are sure that after all the new experiments we are proposing in here, she/he will be fully satisfied.

    1.The finding that MDM2 promoted NICD stability through non degradative ubiquitination is interesting and in line with a previous study. As it is also known that NICD is regulated by various post-translational modifications, including ubiquitination that promotes NICD degradation. It is unclear what's the potential difference between these two types of ubiquitination. For example, do these two differ in specific ubiquitination sites? Can the authors provide some discussion?

    [Authors] We agree with the reviewer and hence we will perform a new set of experiments to determine the role of 2 key lysine residues in the ubiquitin protein promoting either degradation or DNA binding. As explained in detail in major point 1 from reviewer 1, we will determine if DNA damage promotes ubiquitination in position 48, i.e., to degrade, or in position 63, i.e., to facilitate the binding to DNA for repairing upon DNA damage, or in any of these 2 positions. And as mentioned above, we will then confirm that MDM2 is responsible of the specific ubiquitination type we will uncover. We are sure that the reviewer will be satisfied by these new data once is generated.

    As for the specific ubiquitination sites in NICD, there are up to 17 lysine residues susceptible of being ubiquitinated. Hence unveiling what residues are targeted by MDM2 and if they differ from others inducing degradation as those promoted by the E3 ligase FBXW7, we feel is out of the scope of the current manuscript. Still, we will discuss all this part as kindly proposed by the reviewer.

    Could the overexpression of MDM2 or NICD lead to carboplatin resistance in A549 or H358 cells?

    [Authors] This is a very interesting experiment and prompted by the reviewer’s comment we started the subcloning of inducible NICD into lentiviral vectors to generate stable cells and test the carboplatin sensitivity in presence of different levels of NICD. These new data will be the new Figure 5A.

    The trends observed in the western blot data within the manuscript appear inconsistent. While the authors propose that NICD levels increased upon incubation with carboplatin, the discrepancy arises when considering the NICD levels without cycloheximide (CHX) treatment in Figure 1E, where no significant elevation is observed (Lane 6 vs. Lane 1).

    [Authors] The point of the reviewer is well taken. Please bear in mind that in here we are handling several signaling pathways that interact among them while having each one different kinetics. Our finding of increased NICD upon carboplatin treatment is highly consistent in vitro and in vivo, but it is true that in the experiment mentioned by the reviewer is not obvious, probably due to some kinetic issue. We are repeating this experiment to have the increased in NICD upon carboplatin as it is in the rest of the manuscript (up to 9 times only in main figures).

    The quality of western blots needs to be improved, especially Fig. 1C and S1C, also Figure 3B. Moreover, the NICD western blot sometimes appears as one band and sometimes as two bands. Please provide an explanation. If possible, please quantify the bands in western blots.

    [Authors] We agree with the reviewers that not all WB have the same quality and we will repeat some of them to homogenize the quality all over the manuscript, and particularly, we will repeat the ones kindly pointed out by the reviewer.

    The two bands it is something we also noticed and we will pay attention while reproducing the WB, since it might be related to discrepancies in the percentage of acrylamide. If this is not the case, i.e., upon repetition we still observe in some conditions and not in others, we will provide explanations for this in the new submission as kindly proposed by the reviewer.

    Finally, and also as proposed by the reviewer we will quantify the WB bands.

    Please provide a necessary discussion on whether the targeted treatment approach towards the MDM2-NICD axis is applicable to all patients or only to those with high expression of MDM2/NICD.

    [Authors] In the discussion of the current manuscript, we focused into the MDM2 high expression subset of patients for this issue, but in the next submission we will enlarge to patients with high levels of NICD also.

    How to interpret the significance of the simultaneous increase in NICD ubiquitination and stability mediated by MDM2? Please provide a relevant discussion.

    [Authors] We will provide strong experimental data to go beyond discussion (please see above the experiments with ubiquitin mutants), but we will also provide discussion of this particular point.

    In Figure 5B, please also check the level of MDM2. In Figure 5C, carboplatin appears to have little impact on tumor growth. How to explain the increase of Ki-67 in the carboplatin treatment group in Figure 5A?

    [Authors] We will measure also levels of MDM2 in the future new Figure 5C as requested by the reviewer.

    As for the interesting observation of the Ki67, since we will repeat the whole experiment, we will pay special attention to this point if ever it is repeated. Should be this the case, we will elaborate an explanation.

    Minor comments:

    1.Please include scale bars in Figure 1B and Supplemental Figure 1B.

    [Authors] We thank the reviewer for this comment. We will include the scale bars where required.

    2.Figure 5D, the P values of the survival curve should be indicated in the figures.

    [Authors] We will include the P values in the future new Figure 5E.

    3.The presentation of survival curve data in Figures 5D and 6A should be consistent.

    [Authors] The point of the reviewer is well taken and we will use Prism to draw the PFS for patients in Figure 6A as we did for the mice in current Figure 5D.

    4.It seems that supplemental figure 2 is missing.

    [Authors] We actually jumped from supplemental figure 1 to 3 because we do not have any associated supplemental figure to main Figure 2. We will clarify this point in the next submission.

    5.Please carefully check the spelling of the entire text, for example, on page 20, line 426 it should be 'western'. Also, please spell out the abbreviations DDR and ATM.

    [Authors] We will double check all spelling and provide the abbreviations kindly suggested by the reviewer.

    6.The abbreviation for Cleaved caspase 3 should be CC3.

    [Authors] We thank the reviewer for this information, we will use CC3 in the next submission.

    Reviewer #2 (Significance (Required)):

    Notch signaling is associated with the occurrence and development of non-small cell lung cancer (NSCLC). Previous study indicates that the expression of Notch protein is significantly higher in NSCLC tissues compared to normal tissues (PMID: 31170211). Additionally, the upregulation of Notch1 is correlated with higher tumor grades, lymph node metastasis, tumor-node-metastasis (TNM) staging, and poor prognosis (PMID: 25996086). Abnormal activation of Notch signaling pathway is frequently observed in chemotherapy-resistant NSCLC, and some studies have aimed to address NSCLC drug resistance via modulating Notch signaling (PMID: 30087852, 38301911). This manuscript firstly proposes that MDM2-mediated stabilization of NICD upon DNA damage plays a major role in NSCLC response to platinum chemotherapy. It further suggests that targeting the MDM2-NICD axis could prove to be an effective therapeutic strategy. Overall, this work unveils a novel mechanism for Notch activation in response to platinum chemotherapy, providing a renewed outlook on overcoming chemotherapy resistance in NSCLC. This manuscript will attract those interested in the mechanisms of chemotherapy resistance and novel treatment approaches.

    [Authors] We sincerely thank the reviewer for finding that our “…work unveils a novel mechanism for Notch activation in response to platinum chemotherapy, providing a renewed outlook on overcoming chemotherapy resistance in NSCLC”. We are also very satisfied when she/he says: “This manuscript will attract those interested in the mechanisms of chemotherapy resistance and novel treatment approaches.”

    Finally, we are convinced that the reviewer will appreciate all the new proposed experimental data, and also that upon finishing all experiments, she/he will think that the manuscript will be suitable for publication.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    For simplicity, we decided to introduce all changes in next submission upon conclusion of all experimental approaches proposed above.

    4. Description of analyses that authors prefer not to carry out

    While we will perform almost all experiments proposed by reviewers, there is one we feel is not possible to do due to ethical reasons. Reviewer 1 wanted us to perform a new in vivo experiment with the same PDX using up to 6 treatment groups. We use 8 mice per condition (for tumor growth and survival) plus 4 for the “acute” treatment for WB and IHC purposes, hence 12 mice x 6 groups = 72 mice, and we will perform this experiment as indicated above and proposed by the reviewer.

    On the contrary, the reviewer asked us also to repeat the same experiment with a PDX p53 proficient. While we understand the possible interest, since we demonstrated in vitro that p53 is not required for the protective phenotype of MDM2 and Notch upon DNA damage, we honestly believe that using another 72 mice to confirm this aspect in vivo, is against the rational use of animals in research going against the 3Rs rule. Hence, we will not perform this experiment unless Editors believe is strictly required.

    REFERENCES

    Arena, G., Cisse, M. Y., Pyrdziak, S., Chatre, L., Riscal, R., Fuentes, M., Arnold, J. J., Kastner, M., Gayte, L., Bertrand-Gaday, C., et al. (2018). Mitochondrial MDM2 Regulates Respiratory Complex I Activity Independently of p53. Mol Cell* 69*, 594-609 e598.

    Cheng, Q., Cross, B., Li, B., Chen, L., Li, Z., and Chen, J. (2011). Regulation of MDM2 E3 ligase activity by phosphorylation after DNA damage. Mol Cell Biol* 31*, 4951-4963.

    Gounder, M., Ratan, R., Alcindor, T., Schoffski, P., van der Graaf, W. T., Wilky, B. A., Riedel, R. F., Lim, A., Smith, L. M., Moody, S., et al. (2023). Nirogacestat, a gamma-Secretase Inhibitor for Desmoid Tumors. N Engl J Med* 388*, 898-912.

    Kassouf, T., Larive, R. M., Morel, A., Urbach, S., Bettache, N., Marcial Medina, M. C., Merezegue, F., Freiss, G., Peter, M., Boissiere-Michot, F., et al. (2019). The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex. Cancers (Basel)* 11*.

    Liu, P., Gan, W., Su, S., Hauenstein, A. V., Fu, T. M., Brasher, B., Schwerdtfeger, C., Liang, A. C., Xu, M., and Wei, W. (2018). K63-linked polyubiquitin chains bind to DNA to facilitate DNA damage repair. Sci Signal* 11*.

    Pettersson, S., Sczaniecka, M., McLaren, L., Russell, F., Gladstone, K., Hupp, T., and Wallace, M. (2013). Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway. Biochem J* 450*, 523-536.

    Riscal, R., Schrepfer, E., Arena, G., Cisse, M. Y., Bellvert, F., Heuillet, M., Rambow, F., Bonneil, E., Sabourdy, F., Vincent, C., et al. (2016). Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53. Mol Cell* 62*, 890-902.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Sara Bernardo et al. investigated the molecular mechanisms underlying the activation of the Notch signaling in response to DNA damage induced by platinum-based chemotherapeutic agents in non-small cell lung cancer (NSCLC). They demonstrated that carboplatin treatment induces DNA double-strand breaks (DSBs) and stabilizes NICD, a process dependent on ATM and mediated by MDM2. In vivo experiments in patient-derived xenografts (PDX) showed that inhibition of NICD and MDM2 enhanced platinum effectiveness. Furthermore, clinical analysis revealed a correlation between MDM2 expression and poor prognosis in NSCLC patients treated with platinum compounds, emphasizing the clinical relevance of the MDM2-NICD axis in platinum resistance.

    Major comments:

    Overall, the authors have conducted experiments that sufficiently elucidate their claims, and the description of the experiments is detailed. However, there is still room for the improvement.

    1.The finding that MDM2 promoted NICD stability through non degradative ubiquitination is interesting and in line with a previous study. As it is also known that NICD is regulated by various post-translational modifications, including ubiquitination that promotes NICD degradation. It is unclear what's the potential difference between these two types of ubiquitination. For example, do these two differ in specific ubiquitination sites? Can the authors provide some discussion?

    1. Could the overexpression of MDM2 or NICD lead to carboplatin resistance in A549 or H358 cells?
    2. The trends observed in the western blot data within the manuscript appear inconsistent. While the authors propose that NICD levels increased upon incubation with carboplatin, the discrepancy arises when considering the NICD levels without cycloheximide (CHX) treatment in Figure 1E, where no significant elevation is observed (Lane 6 vs. Lane 1).
    3. The quality of western blots needs to be improved, especially Fig. 1C and S1C, also Figure 3B. Moreover, the NICD western blot sometimes appears as one band and sometimes as two bands. Please provide an explanation. If possible, please quantify the bands in western blots.
    4. Please provide a necessary discussion on whether the targeted treatment approach towards the MDM2-NICD axis is applicable to all patients or only to those with high expression of MDM2/NICD.
    5. How to interpret the significance of the simultaneous increase in NICD ubiquitination and stability mediated by MDM2? Please provide a relevant discussion.
    6. In Figure 5B, please also check the level of MDM2. In Figure 5C, carboplatin appears to have little impact on tumor growth. How to explain the increase of Ki-67 in the carboplatin treatment group in Figure 5A?

    Minor comments:

    1.Please include scale bars in Figure 1B and Supplemental Figure 1B. 2.Figure 5D, the P values of the survival curve should be indicated in the figures. 3.The presentation of survival curve data in Figures 5D and 6A should be consistent. 4.It seems that supplemental figure 2 is missing. 5.Please carefully check the spelling of the entire text, for example, on page 20, line 426 it should be 'western'. Also, please spell out the abbreviations DDR and ATM. 6.The abbreviation for Cleaved caspase 3 should be CC3.

    Significance

    Notch signaling is associated with the occurrence and development of non-small cell lung cancer (NSCLC). Previous study indicates that the expression of Notch protein is significantly higher in NSCLC tissues compared to normal tissues (PMID: 31170211). Additionally, the upregulation of Notch1 is correlated with higher tumor grades, lymph node metastasis, tumor-node-metastasis (TNM) staging, and poor prognosis (PMID: 25996086). Abnormal activation of Notch signaling pathway is frequently observed in chemotherapy-resistant NSCLC, and some studies have aimed to address NSCLC drug resistance via modulating Notch signaling (PMID: 30087852, 38301911). This manuscript firstly proposes that MDM2-mediated stabilization of NICD upon DNA damage plays a major role in NSCLC response to platinum chemotherapy. It further suggests that targeting the MDM2-NICD axis could prove to be an effective therapeutic strategy. Overall, this work unveils a novel mechanism for Notch activation in response to platinum chemotherapy, providing a renewed outlook on overcoming chemotherapy resistance in NSCLC. This manuscript will attract those interested in the mechanisms of chemotherapy resistance and novel treatment approaches.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This manuscript from Maraver and co-authors investigates the putative resistance mechanisms that hinder the efficacy of platinum-based therapies (e.g., carboplatin) against non-small cell lung carcinoma (NSCLC). Using in vitro lung cancer cell lines, shRNA-based knockdown, and exogenous overexpression systems, the research describes a DNA damage-induced resistance mechanism involving the NOTCH signaling pathway and the E3 ligase MDM2. The authors show that carboplatin treatment induces DNA damage and promotes ATM activation, which in turn activates the NOTCH signaling pathway via ubiquitination and stabilization of the Notch Intracellular Domain (NICD). New findings include the MDM2-mediated ubiquitination and stabilization of NICD. Using in vivo NSCLC-PDX models, they demonstrate that combining carboplatin with Notch and MDM2 inhibitors can enhance tumor killing, suggesting that targeting the MDM2/NICD axis in conjunction with carboplatin may be a viable therapeutic alternative. Furthermore, they show that NICD and MDM2 levels are elevated among tumor samples from chemo-resistant patients. Consistent with these findings, high MDM2 levels correlate with poor progression-free survival (PFS) in NSCLC patients.

    Major comments:

    Some of the key conclusions may not be convincing.

    1. One significant weakness of the manuscript is the lack of exploration into the underlying mechanism of how MDM2 mediates the stabilization of NICD. While the observation of MDM2-mediated NICD stabilization is intriguing, it is important to provide a more convincing explanation for the reviewers. This could be achieved by offering a detailed molecular mechanism, especially considering that MDM2 typically targets proteins for degradation.
    2. Another weakness lies in the unclear role and the underlying mechanism of ATM in the MDM2-mediated NICD stabilization. While the data presented (Fig. 3B, 3C) suggest that carboplatin could elevate MDM2 levels for NICD stabilization, a more precise method to induce MDM2 overexpression specifically for targeting NICD is required. It appears that ATM plays a crucial role in this regulatory process. The following questions must be addressed: Does ATM induce the phosphorylation of MDM2 for its protein stabilization and/or E3 ligase activity?
    3. The combination therapy of carboplatin with MDM2 and NICD inhibitors may lack compelling rationale (see below).
    4. In lines 275-276, the authors stated that their preclinical data establish the enhancement of carboplatin's therapeutic effect in NSCLC in vivo through MDM2-NICD axis inhibition. However, it's important to note that this finding remains preliminary at this stage.

    Minor comments:

    1. The observed loss of NICD during ATMi + carboplatin treatment in Figures 2A and 2B raises the question of whether ATM regulates the gene transcription of NOTCH. In addition to the CHX assay conducted in Figures 2C and 2D, quantifying NOTCH mRNA upon ATM inhibition could provide further insights. Alternatively, referencing relevant studies on this topic may strengthen the discussion.
    2. In Figures 4A and 4B, the noticeable discrepancy between the exogenous expression of wild-type (WT) MDM2 and catalytically inactive MDM2-464A raises concerns. It is essential to consider if the reduced ubiquitination and stability of NICD might be attributed to varying levels of MDM2-464A in the cells rather than its catalytic inactivity. While p53 ubiquitination was utilized as a control, ensuring comparable levels of MDM2 and MDM2-464A expression could enhance the experimental rigor. Compared to the smear poly-ubiquitination bands observed for MDM2 in Figure 4B, the ubiquitination of NICD appears simpler. What distinguishes the feature of MDM2-mediated NICD ubiquitination? Could it potentially involve mono-ubiquitination?
    3. In Figure 5A, the authors need to consider conducting additional NOTCH-associated factors to definitively demonstrate the activation of NOTCH signaling beyond HES1. Alternatively, in Figure 5B, the NICD Western blot could be complemented by detecting HES1 or other NOTCH-associated factors.
    4. In Figures 5C and 5D, crucial control groups are missing, specifically mice treated solely with SP141+DBZ, carboplatin+SP141, and SP141+DBZ. It is essential to include these groups to demonstrate that the enhanced tumor killing results from the combination of carboplatin with SP141 and/or DBZ, rather than from SP141 and DBZ alone. Furthermore, in addition to the currently used NSCLC-PDX model harboring the p53 (P151R) mutation, it would be informative to include a NSCLC-PDX model expressing WT p53.
    5. Though beyond the current study's scope, in the discussion section, the authors may want to propose or hypothesize on how MDM2-mediated NICD stabilization contributes to carboplatin resistance. This could provide valuable insights for future research directions.
    6. In the Western blot results, the total ATM and ATR controls were absent.
    7. Authors may choose to include a graphical abstract at the end of their study to visually illustrate the mechanisms they have described.

    Significance

    Advance: The authors aim to present a novel perspective on the resistance mechanisms to platinum compounds in NSCLC therapy. They explore platinum compounds-induced DNA damage, ATM activation, and MDM2-mediated stabilization of the active form of NOTCH (NICD). However, to strengthen their claims, they must provide more conclusive results.

    Audience: This manuscript will likely engage oncologists who investigate the chemotherapy-resistant mechanisms of platinum compounds in NSCLC treatment, as well as scientists specializing in NOTCH and MDM2 pathways. However, the manuscript's central claims lack robust support from the available data, and the current approaches employed are not sufficiently thoughtful and rigorous; there is room for improvement."

    My expertise is molecular medicine, cancer biology, and epigenetics.

  4. Version published to 10.1101/2024.04.29.591624v1 on bioRxiv