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Updated: May 27, 2025
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
DNA methylation status classifies pleural mesothelioma cells according to their immune profile: implication for
Maria Fortunata Lofiego1, Rossella Tufano2,3, Emma Bello1
1University of Siena, Siena, Italy.
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.
Insights
DNA methylation patterns influence immune cell profiles in pleural mesothelioma (PM), independent of tumor type. DNA hypomethylating agents can reprogram PM cells towards an immune-favorable state, suggesting potential for novel epigenetic therapies combined with immune checkpoint inhibitors (ICI).
Area of Science:
- Oncology
- Immunology
- Epigenetics
Background:
- Immune checkpoint inhibitors (ICI) targeting CTLA-4 and PD-1 are standard care for pleural mesothelioma (PM), improving survival.
- ICI efficacy varies by tumor histotype, suggesting molecular features beyond histology influence treatment response.
- Tumor DNA methylation is a potential driver of cancer cell immune phenotype, warranting investigation in PM.
Purpose of the Study:
- To investigate the role of the DNA methylation landscape in pleural mesothelioma (PM) and its impact on the tumor immune microenvironment.
- To explore the potential of pharmacologic modulation of DNA methylation to enhance ICI therapy efficacy in PM.
- To provide preclinical evidence for the utility of targeting DNA methylation in PM treatment strategies.
Main Methods:
- Analyzed methylome (EPIC array) and transcriptome (Clariom S array) profiles of 14 cultured PM cell lines (5 epithelioid, 9 non-epithelioid).
- Assessed methylation and gene expression changes before and after in vitro treatment with the DNA hypomethylating agent (DHA) guadecitabine.
- Calculated a methylation score (CIMP index) and identified differentially expressed and methylated genes for ontology analysis.
Main Results:
- The CIMP index stratified PM cell lines into hyper-methylated (CIMP) and hypo-methylated (LOW) groups, irrespective of histotype.
- CIMP PM cells displayed hyper-methylated, silenced genes that compromised their immune phenotype compared to LOW PM cells.
- DHA treatment reversed the immune-compromised profile in CIMP PM cells and enhanced the immune-favorable profile in LOW PM cells.
Conclusions:
- DNA methylation significantly shapes the immune classification of PM cells, independent of histological subtype.
- DNA hypomethylating agents can induce an immune-favorable phenotype in PM cells.
- Epigenetic-based ICI combinations targeting DNA methylation warrant evaluation in clinical trials for PM to overcome immune resistance.
