DNA methylation status classifies pleural mesothelioma cells according to their immune profile: implication for precision epigenetic therapy
Authors: Lofiego Maria Fortunata , Tufano Rossella , Bello Emma , Solmonese Laura , Marzani Francesco , Piazzini Francesca , Celesti Fabrizio , Caruso Francesca Pia , Noviello Teresa Maria Rosaria , Mortarini Roberta , Anichini Andrea , Ceccarelli Michele , Calabrò Luana , Maio Michele , Coral Sandra , Di Giacomo Anna Maria , Covre Alessia ,   on behalf of the EPigenetic Immune-oncology Consortium Airc (EPICA) investigators
Published: February 2025

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.