Abstract 4841: DNA methylation status classifies pleural mesothelioma cells according to their immune profile: implication for precision epigenetic therapy

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 a 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 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 and of 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 and methylated were then selected for gene ontology analysis. Results: the CIMP index stratified PM cell lines into two distinct classes, CIMP (hypermethylated; n=7) and LOW (hypomethylated; 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 hypermethylated, 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 supports its potential role in clinical trials of precision epigenetic therapy combined with ICI. Citation Format: Maria Fortunata Lofiego, Rossella Tufano, Emma Bello, Laura Solmonese, Francesco Marzani, Francesca Piazzini, Fabrizio Celesti, Francesca Pia Caruso, Teresa Maria Rosaria Noviello, Roberta Mortarini, Andrea Anichini, Michele Ceccarelli, Luana Calabrò, Michele Maio, Sandra Coral, Anna Maria Di Giacomo, Alessia Covre. DNA methylation status classifies pleural mesothelioma cells according to their immune profile: implication for precision epigenetic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4841.