DNA methylation status classifies pleural mesothelioma cells according to their immune profile: implication for precision epigenetic therapy

Our plain-English summary of DNA methylation status classifies pleural mesothelioma cells according to their immune profile: implication for precision epigenetic therapy, by M.F. Lofiego, R. Tufano, E. Bello, et al (Journal of Experimental & Clinical Cancer Research, 2025).

The study

• Authors: M.F. Lofiego, R. Tufano, E. Bello, L. Solmonese, F. Marzani, F. Piazzini, F. Celesti, F.P. Caruso, T.M.R. Noviello, R. Mortarini, A. Anichini, M. Ceccarelli, L. Calabrò, M. Maio, S. Coral, A.M. Di Giacomo, A. Covre, EPigenetic Immune-oncology Consortium Airc (EPICA) investigators
• Journal: Journal of Experimental & Clinical Cancer Research
• Published: 2025;44(1):58
• Cited by: 7 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Co-targeting of immune checkpoint inhibitors (ICI) CTLA-4 and PD-1 has recently become the new first-line standard of care therapy of pleural mesothelioma (PM) patients, with a significant improvement of overall survival (OS) over conventional chemotherapy. The analysis by tumor histotype demonstrated greater efficacy of ICI therapy compared to standard chemotherapy in non-epithelioid (non-E) vs. epithelioid (E) PM, although some E PM patients also benefit from ICI treatment. This evidence suggests that molecular tumor features, beyond histotype, could be relevant to improve the efficacy of ICI therapy in PM. Among these, tumor DNA methylation emerges as a promising factor to explore, due to its potential role in driving the immune phenotype of cancer cells. Therefore, we utilized a panel of cultured PM cells of different histotype to provide preclinical evidence supporting the role of the tumor methylation landscape, along with its pharmacologic modulation, to prospectively improve the efficacy of ICI therapy of PM patients.

Methods: The methylome profile (EPIC array) of distinct E (n = 5) and non-E (n = 9) PM cell lines was analyzed, followed by integrated analysis with their associated transcriptomic profile (Clariom S array), before and after in vitro treatment with the DNA hypomethylating agent (DHA) guadecitabine. The most variable methylated probes were selected to calculate the methylation score (CIMP index) for each cell line at baseline. Genes that were differentially expressed (DE) and differentially methylated (DM) were then selected for gene ontology analysis.

Results: The CIMP index stratified PM cell lines into two distinct classes, CIMP (hyper-methylated; n = 7) and LOW (hypo-methylated; n = 7), regardless of their E or non-E histotype. Integrated methylome and transcriptome analyses revealed that CIMP PM cells exhibited a substantial number of hyper-methylated, silenced genes, which negatively impacted their immune phenotype compared to LOW PM cells. Treatment with DHA reverted the methylation-driven immune-compromised profile of CIMP PM cells and enhanced the constitutive immune-favorable profile of LOW PM cells.

Conclusion: The study highlighted the relevance of DNA methylation in shaping the constitutive immune classification of PM cells, independent of their histological subtypes. The identified role of DHA in shifting the phenotype of PM cells towards an immune-favorable state highlights its potential for evaluation in phase I/II clinical trials investigating the efficacy of epigenetic-based ICI combinations to reverse cancer immune resistance mechanisms.

© 2025. The Author(s).

In plain English

This was a laboratory study of 14 pleural mesothelioma cell lines, 5 epithelioid and 9 non-epithelioid. Ten came from patients' pleural fluid in Siena, Italy; four were bought from a cell bank. The study did not look at asbestos exposure. The authors measured DNA methylation (chemical tags that can silence genes) and gene activity. Splitting at the median methylation score gave 7 highly methylated (CIMP) and 7 low-methylated (LOW) lines, regardless of epithelioid or non-epithelioid type. Immune-related gene signatures were higher in the LOW lines. In CIMP lines, 217 genes had lower activity than in LOW lines, linked to heavier methylation of their control regions. In 87 tumors from a public database (TCGA-MESO), computer estimates showed significantly more B cells and neutrophils in LOW tumors. Median survival was 689 days for 36 LOW patients and 459 days for 37 CIMP patients (p = 0.065), not statistically significant. The drug guadecitabine reduced methylation and raised immune-related gene activity in both groups of cell lines. The authors concluded that methylation class could help predict immunotherapy benefit, pending confirmation in patients.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study of cultured pleural mesothelioma cell lines, with an added analysis of public tumor data from the TCGA-MESO cohort
• Who was studied: 14 human pleural mesothelioma cell lines (5 epithelioid, 9 non-epithelioid). Ten were established from pleural effusions of patients at the University Hospital of Siena and four were purchased from the American Type Culture Collection. A separate analysis used 87 tumor samples from the TCGA-MESO cohort (44 CIMP, 43 LOW).
• Exposure: No asbestos exposure was studied. The exposure tested was the DNA hypomethylating drug guadecitabine, given at 1 µM every 12 hours on days 1 and 2, with cells harvested on day 5. Untreated cells served as controls.
• Main results:
  • The methylation score split the cell lines into 7 hyper-methylated (CIMP) and 7 hypo-methylated (LOW) lines. Epithelioid and non-epithelioid lines were spread across both groups.
  • Gene signatures linked to tumor immunogenicity or to response to immune checkpoint inhibitors were enriched in LOW cell lines compared with CIMP cell lines.
  • In CIMP versus LOW cell lines, 217 genes had lower expression associated with hypermethylation of their promoter regions. Pathways predicted to be inhibited included class I MHC-mediated antigen processing and presentation, IL-17 signaling, dendritic cell maturation and B cell receptor signaling.
  • In TCGA-MESO tumors, computational deconvolution estimated a higher density of selected immune cells in LOW lesions than in CIMP lesions. The differences were statistically significant for B cells and neutrophils. They were also significant for cytotoxic lymphocytes and myeloid dendritic cells only when the top 25% of each methylation group were compared.
  • In TCGA-MESO patients, median overall survival was 459 days for the CIMP group (printed 95% CI: 0.95–2.49) and 689 days for the LOW group (printed 95% CI: 0.42–1.05). The p value was 0.065, above the study's significance threshold of p less than 0.05.
  • The survival curve included 37 CIMP and 36 LOW TCGA patients.
  • After guadecitabine, 152,420 probes were hypo-methylated in the CIMP cell lines and 5,765 in the LOW cell lines.
  • Of 21,448 genes examined, guadecitabine significantly changed 2,358 in CIMP cell lines and 3,034 in LOW cell lines.
  • Guadecitabine raised expression of human endogenous retrovirus (HERV) and interferon-stimulated (ISG) genes across the cell lines. The median fold change was higher in LOW than in CIMP lines: 4.44 vs 2.39 for HERV and 1.97 vs 1.71 for ISG. The authors note this difference was not statistically significant.
  • After guadecitabine, levels of the released immune molecules IFN-γ, IL-2, IP-10 and IL-6 rose (fold change greater than 1) in both CIMP and LOW cell lines.
• The authors' conclusion: The authors concluded that classifying mesothelioma by tumor methylation, which was associated with the cells' immune profile, could serve as a better molecular predictor of outcome with immune checkpoint inhibitor therapy, regardless of tumor histotype. They also concluded that guadecitabine-induced immune remodeling could improve checkpoint inhibitor efficacy, supporting its use in future clinical trials.
• Limitations the authors state:
  • The authors state that further confirmatory analyses are needed in the clinical setting.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, checkpoint inhibitors, CTLA-4, Overall Survival, Chemotherapy, Non-epithelioid, Epithelioid, Histological Subtype.

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• Methylation profile landscape in mesothelioma: possible implications in early detection, disease progression, and therapeutic options — X. Zhang, N. Tang, et al. (2015)
• Epigenetic Immune Remodeling of Mesothelioma Cells: A New Strategy to Improve the Efficacy of Immunotherapy — M.F. Lofiego, S. Cannito, et al. (2021)
• DNA methylation status of REIC/Dkk-3 gene in human malignancies — T. Hayashi, H. Asano, et al. (2012)
• Hypomethylation reduced the aggressive potential of human malignant mesothelioma cells — N.Y. Kim, M.C. Kim, et al. (2016)

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