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Long-term cultivation using ineffective MDM2 inhibitor concentrations alters the drug sensitivity profiles of PL21 leukaemia cells

Subject: Life Science and Biomedicine

Published online by Cambridge University Press:  05 March 2020


Martin Michaelis
Affiliation:
Industrial Biotechnology Centre and School of Biosciences, University of Kent, CanterburyCT2 7NJ, UK
Florian Rothweiler
Affiliation:
Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul Ehrlich-Str. 40, 60596Frankfurt am Main, Germany
Constanze Schneider
Affiliation:
Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul Ehrlich-Str. 40, 60596Frankfurt am Main, Germany
Tamara Rothenburger
Affiliation:
Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul Ehrlich-Str. 40, 60596Frankfurt am Main, Germany
Marco Mernberger
Affiliation:
Institute of Molecular Oncology, Philipps-University, 35037Marburg, Germany
Andrea Nist
Affiliation:
Genomics Core Facility, Philipps-University, 35037Marburg, Germany
Thorsten Stiewe
Affiliation:
Institute of Molecular Oncology, Philipps-University, 35037Marburg, Germany Genomics Core Facility, Philipps-University, 35037Marburg, Germany
Jindrich Cinatl
Affiliation:
Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul Ehrlich-Str. 40, 60596Frankfurt am Main, Germany
Corresponding
E-mail address:


Abstract

Acquired MDM2 inhibitor resistance is commonly caused by loss-of-function TP53 mutations. In addition to the selection of TP53-mutant cells by MDM2 inhibitors, MDM2 inhibitor-induced DNA damage may promote the formation of TP53 mutations. Here, we cultivated 12 sublines of the intrinsically MDM2 inhibitor-resistant TP53 wild-type acute myeloid leukaemia cell line PL21 for 52 passages in the presence of ineffective concentrations of the MDM2 inhibitor nutlin-3 but did not observe loss-of-function TP53 mutations. This suggests that MDM2 inhibitors select TP53-mutant cells after mutations have occurred, but do not directly promote TP53 mutations. Unexpectedly, many sublines displayed increased sensitivity to the anti-cancer drugs cytarabine, doxorubicin, or gemcitabine. Consequently, therapies can affect the outcome of next-line treatments, even in the absence of a therapy response. This finding is conceptually novel. A better understanding of such processes will inform the design of improved therapy protocols in the future.


Type
Research Article
Information
Creative Commons
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
© The Author(s) 2020

Introduction

MDM2 (Mouse Double Minute 2) inhibitors, which activate p53 by inhibiting MDM2-mediated p53 degradation, are under development for the treatment of TP53 wild-type cancer [Reference Tisato1]. The MDM2 inhibitor idasanutlin is currently investigated in clinical phase II and III trials for acute myeloid leukaemia (AML; NCT02670044, NCT02545283).

Resistance formation of TP53 wild-type cancer cells to MDM2 inhibitors commonly results in the formation of TP53 mutations as resistance mechanism [Reference Aziz2-Reference Jung8]. TP53 mutations may be the consequence of the selection of pre-existing TP53-mutant cell subpopulations or the induction of de novo TP53 mutations [Reference Michaelis3,Reference Drummond5-Reference Michaelis7]. De novo TP53 mutations may be the consequence of the selection of cells in which TP53 mutations have occurred by chance and which would have disappeared in the absence of the selection pressure induced by an MDM2 inhibitor. However, MDM2 inhibitors may also actively promote the formation of TP53 mutations by inducing DNA damage [Reference Vassilev9-Reference Wang12].

Objective

We used the AML cell line PL21 to investigate whether MDM2 inhibitor-induced DNA damage may promote the formation of TP53 mutations in the absence of a selection pressure. PL21 AML cells are TP53 wild-type (Table 1) but intrinsically resistant to nutlin-3 (an MDM2 inhibitor closely related to idasanutlin [Reference Vassilev9]), as indicated by a nutlin-3 IC50 of 20.49 μM (Figure 1, Table 1). Nutlin-3-sensitive cells display nutlin-3 IC50 values in the very low micromolar range, while nutlin-3 concentrations above 20 μM are associated with non-specific, p53-independent effects [Reference Michaelis3,Reference Michaelis7]. Twelve PL21 sublines were cultivated for 52 passages in the presence of nutlin-3 10 μM. The emergence of TP53 mutations in response to nutlin-3 treatment would indicate mutagenic effects that promote the formation of TP53 mutations also in the absence of a selective pressure on p53.

Table 1. Drug concentrations that reduce the viability of PL21 and its sublines cultivated for 52 weeks in the presence of nutlin-3 (20 μM) by 50% (IC50) as indicated by MTT assay after 120 h of incubation.

1 Polymorphism that does not affect p53 function.

2 Fold change relative to PL21.

Figure 1. Drug sensitivity profiles of the AML cell line PL21 and its sublines cultivated in the presence of nutlin-3 (10 μM) for 52 weeks. Concentrations that inhibit cell viability by 50% (IC50, mean ± SD from three independent experiments) as determined by MTT assay after 120 h incubation and IC50 fold changes relative to PL21 were determined for nutlin-3 (A), cytarabine (B), doxorubicin (C), and gemcitabine (D). * P < 0.05 relative to PL21.

Methods

PL21 cells (DSMZ, Braunschweig, Germany) were cultivated in the absence or presence of drug in Iscove’s modified Dulbecco’s medium supplemented with 10% foetal calf serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 °C. Cells were routinely tested for mycoplasma contamination and authenticated by short tandem repeat profiling.

The TP53 status was determined by next generation sequencing, and cell viability was measured using eight drug concentrations by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described [Reference Michaelis3,Reference Michaelis7]. Based on the MTT data, concentrations that inhibit cell viability by 50% (IC50) were determined using CalcuSyn (Biosoft, Cambridge, UK). Nutlin-3 was purchased from Selleck Chemicals via BIOZOL GmbH (Eching, Germany). Cytarabine was obtained from Tocris via Bio-Techne GmbH (Wiesbaden, Germany). Doxorubicin and gemcitabine were purchased from Teva GmbH (Ulm, Germany).

Results

All sublines had retained wild-type TP53 except for PL21rNutlin20XII and PL21rNutlin20XV, which displayed an M66L variant (Table 1). This variant was present in 386 (3.2%) out of 11,945 reads from the parental cell line and, hence not a de novo mutation induced by nutlin-3 treatment. If it had been of functional relevance, it would have been consistently selected by nutlin-3 treatment, as previously shown in other cell lines [5-7]. Thus, this observation does not suggest that nutlin-3 may directly induce TP53 mutations.

The 12 nutlin-3-treated PL21 sublines displayed an up to 3.1-fold variation in their nutlin-3 sensitivity (Figure 1, Table 1) and in their sensitivity to cytarabine (up to 6.7-fold), doxorubicin (up to 7.7-fold), and gemcitabine (up to 40.8-fold). Twelve PL21 sublines that had been cultivated for 52 weeks as control in parallel in the absence of nutlin-3 did not display any changes in their drug sensitivity profiles (Table 2).

Table 2. Drug concentrations that reduce the viability of PL21 sublines cultivated separately for 52 weeks by 50% (IC50) as indicated by MTT assay after 120 h of incubation. Values are represented as means ± S.D. of at least three independent experiments.

1 Fold change relative to PL21.

Discussion

Since treatment of PL21 cells with ineffective nutlin-3 concentrations did not result in loss-of-function TP53 mutations, TP53 mutations in MDM2 inhibitor-adapted cells may be rather the consequence of selection processes than of drug-induced mutations. In agreement, a fraction of MDM2 inhibitor-adapted cell lines retains wild-type TP53 [Reference Michaelis3,Reference Michaelis7]. Unexpectedly, prolonged nutlin-3 treatment resulted in increased sensitivity of a fraction of sublines to cytarabine, doxorubicin, or gemcitabine. In this context, MDM2 inhibition has been shown to increase the cellular reactive oxygen species (ROS) levels [Reference Riscal13,Reference Arena14], and higher ROS levels were associated with increased cytarabine sensitivity [Reference Hosseini15]. Cytarabine and anthracyclines are standard drugs for AML [Reference De Kouchkovsky and Abdul-Hay16], and gemcitabine has recently been suggested as drug candidate for paediatric AML [Reference Drenberg17]. This may be of clinical relevance in AML patients in whom MDM2 inhibitor treatment may modify the efficacy of next-line therapies, even if there is no response to MDM2 inhibitor therapy.

Conclusion

Our data do not provide evidence that MDM2 inhibitors may exert mutagenic effects that would promote the formation of loss-of-function TP53 mutations. MDM2 inhibitors rather seem to select TP53-mutant cells after mutations have occurred. Surprisingly, we found that cultivation of PL21 cells in the presence of ineffective nutlin-3 concentrations resulted in increased drug sensitivity in a substantial fraction of sublines. This is conceptually important, because our findings show that non-effective therapies can affect the outcome of next-line therapies. A better understanding of such processes may inform therapy protocols in the future. Our study also illustrates how cancer cell lines as permanent preclinical model systems can be used to produce findings that cannot be made in the clinics, because different treatment schedules cannot be compared in the same patient.

Author Contributions

J.C. and M. Michaelis designed and conducted the study. C.S., F.R., T.R., M. Mernberger, and A.N. performed experiments. All authors analysed data. M. Michaelis and J.C. wrote the initial manuscript draft. All authors read and approved the final version.

Funding Information

The work was supported by the Hilfe für krebskranke Kinder Frankfurt e.V. (J.C.), the Frankfurter Stiftung für krebskranke Kinder (J.C.), the Deutsche José Carreras Leukämie-Stiftung (J.C., T.S.), and the Kent Cancer Trust (M. Michaelis).

Publishing Ethics

The authors confirm that

  1. 1. the manuscript has been submitted only to the journal – it is not under consideration, accepted for publication or in press elsewhere. Manuscripts may be deposited on pre-print servers;

  2. 2. all listed authors know of and agree to the manuscript being submitted to the journal; and

  3. 3. the manuscript contains nothing that is abusive, defamatory, fraudulent, illegal, libellous, or obscene.

Conflict of Interest

The authors declare none.

Data Availability

All data are included in the manuscript.


References

Tisato, V, et al.MDM2/X inhibitors under clinical evaluation: perspectives for the management of hematological malignancies and pediatric cancer. J Hematol Oncol 2017; 10: 1.CrossRefGoogle ScholarPubMed
Aziz, MH, et al.Acquisition of p53 mutations in response to the non-genotoxic p53 activator Nutlin-3. Oncogene 2011; 30: 4678–10.CrossRefGoogle ScholarPubMed
Michaelis, M, et al.Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells. Cell Death Dis 2011; 2: e243.CrossRefGoogle ScholarPubMed
Hoffman-Luca, CG, et al.Elucidation of acquired resistance to Bcl-2 and MDM2 inhibitors in acute leukemia in vitro and in vivo. Clin Cancer Res 2015; 21: 25582568.CrossRefGoogle ScholarPubMed
Drummond, CJ, et al.TP53 mutant MDM2-amplified cell lines selected for resistance to MDM2-p53 binding antagonists retain sensitivity to ionizing radiation. Oncotarget 2016; 7: 4620346218.CrossRefGoogle ScholarPubMed
Hata, AN, et al.Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers. Oncogene 2017, 36: 65816591.CrossRefGoogle ScholarPubMed
Michaelis, M, et al.TP53 mutations and drug sensitivity in acute myeloid leukaemia cells with acquired MDM2 inhibitor resistance. 2018. Available at https://www.biorxiv.org/content/10.1101/404475v1.CrossRefGoogle Scholar
Jung, J, et al.TP53 mutations emerge with HDM2 inhibitor SAR405838 treatment in de-differentiated liposarcoma. Nat Commun 2016; 7: 12609.CrossRefGoogle ScholarPubMed
Vassilev, LT, et al.In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004; 303: 844848.CrossRefGoogle ScholarPubMed
Bouska, A, Eischen, CMMurine double minute 2: p53-independent roads lead to genome instability or death. Trends Biochem Sci 2009; 34: 279286.CrossRefGoogle ScholarPubMed
Rigatti, MJ, et al.Pharmacological inhibition of Mdm2 triggers growth arrest and promotes DNA breakage in mouse colon tumors and human colon cancer cells. Mol Carcinog 2012; 51: 363378.CrossRefGoogle ScholarPubMed
Wang, H, et al.Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist. J Neurosurg 2017; 126: 446459.CrossRefGoogle Scholar
Riscal, R, et al.Chromatin-bound MDM2 regulates serine metabolism and redox homeostasis independently of p53. Mol Cell 2016; 62: 890902.CrossRefGoogle ScholarPubMed
Arena, G, et al.Mitochondrial MDM2 regulates respiratory complex I activity independently of p53. Mol Cell 2018; 69: 594.e8609.e8.CrossRefGoogle ScholarPubMed
Hosseini, M, et al.Targeting myeloperoxidase disrupts mitochondrial redox balance and overcomes cytarabine resistance in human acute myeloid leukemia. Cancer Res 2019; 79: 51915203.CrossRefGoogle ScholarPubMed
De Kouchkovsky, I, Abdul-Hay, M.Acute myeloid leukemia: a comprehensive review and 2016 update. Blood Cancer J 2016; 6: e441.CrossRefGoogle Scholar
Drenberg, CD, et al.A high-throughput screen indicates gemcitabine and JAK inhibitors may be useful for treating pediatric AML. Nat Commun 2019; 10: 2189.CrossRefGoogle ScholarPubMed
Reviewing editor:  Michael Nevels University of St Andrews, Biomolecular Sciences Building, Fife, United Kingdom of Great Britain and Northern Ireland, KY16 9ST
This article has been accepted because it is deemed to be scientifically sound, has the correct controls, has appropriate methodology and is statistically valid, and met required revisions.

Review 1: Long-term cultivation using ineffective MDM2 inhibitor concentrations alters leukaemia cell drug sensitivity profiles

Conflict of interest statement

Reviewer declares none.

Comments

Comments to the Author: Michaelis and co-workers aim at the verification of the hypothesis of the generation of TP53 mutations upon the treatment of p53wt PL21 cells with MDM2 antagonist, nutlin-3. Also, increased susceptibility of nutlin-treated cells to three anti-cancer drugs is reported. While this study is important, the manuscript suffers from several critical conceptual mistakes which largely decrease its impact.

Presentation

Overall score 3.7 out of 5
Is the article written in clear and proper English? (30%)
4out of5
Is the data presented in the most useful manner? (40%)
4out of5
Does the paper cite relevant and related articles appropriately? (30%)
3out of5

Context

Overall score 4.8 out of 5
Does the title suitably represent the article? (25%)
5out of5
Does the abstract correctly embody the content of the article? (25%)
5out of5
Does the introduction give appropriate context? (25%)
4out of5
Is the objective of the experiment clearly defined? (25%)
5out of5

Analysis

Overall score 2.8 out of 5
Does the discussion adequately interpret the results presented? (40%)
3out of5
Is the conclusion consistent with the results and discussion? (40%)
3out of5
Are the limitations of the experiment as well as the contributions of theexperiment clearly outlined? (20%)
2out of5

Review 2: Long-term cultivation using ineffective MDM2 inhibitor concentrations alters leukaemia cell drug sensitivity profiles

Conflict of interest statement

Reviewer declares none.

Comments

Comments to the Author: The work describes that resistance against nutlin can sensitize leukemic cells to standard chemotherapy. The manuscript is well written and informative. The data are presented clearly and I have only minor criticism.

    Critique:
  1. 1. I would like to have more information on the PL21 cell line. What is known about the driving oncogene(s)? Where are they from? Which type of leukemia? AML, CML, others…?

  2. 2. Line 110

    The authors write

    TP53 mutations MDM2…

    I think that this sentence is incomplete. Was its second part accidentally deleted?

  3. 3. Line 115

    The authors write

    MDM2 inhibition has been shown to increase the cellular reactive oxygen species (ROS) levels…

    Do the PL21 sublines have increased ROS levels?

  4. 4. Figure 1: Which values reach statistical significance in ANOVA analysis?

  5. 5. Title: Maybe

    …alters leukaemia cell drug sensitivity profiles

    is maybe better written as

    …alters the drug sensitivity profiles of PL21 leukaemia cells

    This would be more precise and not 5 nouns in a row (leukaemia cell drug sensitivity profiles), which is not so easy to read and immediately understand.

  6. 6. If the authors wish, they may additionally discuss some recent HDM2 inhibitors that are under investigation, see for example, Conradt, L. … Schneider, G., Int. J. Cancer 2003, May 15.

  7. 7. I suggest that the authors add some speculation/discussion why some of the sublines behave different than the others.

Presentation

Overall score 4.7 out of 5
Is the article written in clear and proper English? (30%)
5out of5
Is the data presented in the most useful manner? (40%)
5out of5
Does the paper cite relevant and related articles appropriately? (30%)
4out of5

Context

Overall score 4.8 out of 5
Does the title suitably represent the article? (25%)
4out of5
Does the abstract correctly embody the content of the article? (25%)
5out of5
Does the introduction give appropriate context? (25%)
5out of5
Is the objective of the experiment clearly defined? (25%)
5out of5

Analysis

Overall score 5 out of 5
Does the discussion adequately interpret the results presented? (40%)
5out of5
Is the conclusion consistent with the results and discussion? (40%)
5out of5
Are the limitations of the experiment as well as the contributions of theexperiment clearly outlined? (20%)
5out of5

Review 3: Long-term cultivation using ineffective MDM2 inhibitor concentrations alters leukaemia cell drug sensitivity profiles

Conflict of interest statement

Reviewer declares none.

Comments

Comments to the Author: The abbreviation MDM-2 should be spelled out when first used.

How many different concentrations were used to determine the IC50-values? This information should be given in the Methods section. The source of the drugs used in the experiments should also be added.

Tables 1 and 2: Drug concentrations given in µM would be better. Please add information if means or median are given, standard deviations or ranges and the number of experiments per value given result.

Presentation

Overall score 4.3 out of 5
Is the article written in clear and proper English? (30%)
5out of5
Is the data presented in the most useful manner? (40%)
4out of5
Does the paper cite relevant and related articles appropriately? (30%)
4out of5

Context

Overall score 4 out of 5
Does the title suitably represent the article? (25%)
4out of5
Does the abstract correctly embody the content of the article? (25%)
4out of5
Does the introduction give appropriate context? (25%)
4out of5
Is the objective of the experiment clearly defined? (25%)
4out of5

Analysis

Overall score 3.8 out of 5
Does the discussion adequately interpret the results presented? (40%)
4out of5
Is the conclusion consistent with the results and discussion? (40%)
4out of5
Are the limitations of the experiment as well as the contributions of theexperiment clearly outlined? (20%)
3out of5

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