Biology subjects

Mortarini, R.

Publications and source records attributed to Mortarini, R..

4 recordsLinked to original sources

bioRxiv2025

Transposable Elements and Homotypic Niches Drive Immune Dynamics and Resistance in Melanoma Epigenetic-based immunotherapy

Melanoma plasticity drives immune evasion and therapy resistance through dynamic cell-state transitions beyond genetic alterations. Epigenetic remodeling critically influences such processes, yet its role in reshaping the tumor ecosystem under therapeutic pressure remains unresolved. Here, we profiled longitudinal biopsies from melanoma patients treated in the phase Ib NIBIT-M4 epi-immunotherapy clinical trial (NCT02608437), testing the combination of a DNMT1 inhibitor with anti-CTLA4 using single-cell multiome and high-resolution spatial transcriptomics. Integrated analyses resolved seven malignant meta-programs, including a rare Wnt/{beta}-catenin-driven melanocytic state and a de-differentiated neural crest-like state enriched in non-responders. Spatial modeling revealed that homotypic clustering stabilizes resistant programs, with neural crest-like cells forming compact, centrally localized niches, whereas Wnt/{beta}-catenin subpopulations displayed a bimodal architecture, either cohesive clusters sustained by adhesion or dispersed, transcriptionally plastic cells. Responders exhibited progressive enrichment of an antigen presentation/interferon program and coordinated remodeling of the tumor microenvironment with T and B cell expansion, whereas tumors from non-responder patients maintained stable composition of neural crest-like clusters. Epigenetic therapy reactivated transposable elements, providing both regulatory signals that prime innate immunity within microenvironment and generating antigens that drive immunoediting and immunogenicity of Antigen presentation/interferon cell states in responders. Finally, NFATC2 emerged as a master regulator of neural crests-like transcriptional phenotypes and promoter of resistance to therapeutic interventions in melanoma patients. NFATC2 perturbation was able to shift tumor cells towards more differentiated and immunogenic states. These findings reveal how epigenetic-based immunotherapy reshapes melanoma ecosystems, provide mechanistic insights into how multiple transcriptional programs promote tumor plasticity and resistance to both combinatorial therapies and immune checkpoint blockade, identify spatial clustering as a principle stabilizing resistant niches, and highlight {beta}-catenin and NFATC2 as actionable vulnerabilities to overcome resistance.

genomics↗

bioRxiv2025

Integrated multi-omics profiling reveals the role of the DNA methylation landscape in shaping biological heterogeneity and clinical behaviour of metastatic melanoma

The biological and clinical relevance of the DNA methylation landscape in metastatic melanoma (MM) remains underexplored. In a retrospective cohort of 191 MM lesions from 165 AJCC Stage III and IV patients (EPICA cohort) we identified four tumor subsets (i.e. DEMethylated, LOW, INTermediate and CIMP) with progressively increasing levels of DNA methylation. These findings were validated in the TCGA MM. In EPICA, patients with LOW methylation tumors exhibited a significantly longer survival and a lower progression rate to more advanced AJCC stages, compared to patients with CIMP tumors. Furthermore, in an independent adjuvant immune checkpoint blockade MM cohort, patients with DEM/LOW pre-therapy lesions showed significantly longer relapse-free survival compared to those with INT/CIMP lesions. RNA-seq data analysis revealed that LOW and CIMP EPICA tumors showed opposite activation of master molecules influencing prognostic target genes, and differential expression of immunotherapy response and melanoma differentiation signatures. Compared to CIMP tumors, LOW lesions showed enrichment for pre-exhausted and exhausted T cells and more frequent retention of HLA Class I antigens. The differentiation and immune-related transcriptional features associated with LOW vs CIMP lesions were tumor-intrinsic programs retained in-vitro by melanoma cell lines. Consistently, treatment of differentiated melanoma cell lines with a DNMT inhibitor induced global DNA de-methylation, promoted de-differentiation and upregulated viral mimicry and IFNG predictive signatures of immunotherapy response. These findings underscore the role of DNA methylation in driving MM biological and clinical heterogeneity and support exploration of methylome targeting strategies for precision immunotherapy in melanoma.

cancer biology↗

bioRxiv2024

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

Backgroundco-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 over conventional chemotherapy. The analysis by tumor histotype demonstrated a greater efficacy of ICI therapy 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. Thus, 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. Methodsthe methylome profile (EPIC array) of distinct E (#5) and non-E (#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. Resultsthe CIMP index stratified PM cell lines in two distinct classes, CIMP (hyper-methylated; #7) and LOW (hypo-methylated; #7), regardless of their E or non-E histotype. Integrated analyses of methylome and transcriptome data revealed that CIMP PM cells had 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. Conclusionthe study highlighted the relevance of DNA methylation in shaping the constitutive immune classification of PM cells, that is independent from their histological subtypes. The identified role of DHA in shifting the phenotype of PM cells towards an immune-favorable state supports its role in clinical trials of precision epigenetic therapy combined with ICI.

cancer biology↗

bioRxiv2022

Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy

BackgroundInnovative cancer immunotherapy approaches aim at combining immune checkpoint inhibitors with other immunomodulatory agents. Epigenetic regulators can control immune-related genes, therefore targeting them with specific inhibitors may be a potential way forward. Here we identified immune-related signatures induced by four classes of epigenetic drugs in human melanoma cells to define the most promising agent and to understand its biological activity in-vitro, in-vivo and in clinical samples. MethodsHuman melanoma cell lines were characterized for mutational and differentiation profile and treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), bromodomain and extraterminal domain proteins (JQ1 and OTX-015) and enhancer of zeste homolog 2 (GSK126). Drug-specific gene signatures were identified by Clariom S and Nanostring platforms. Modulation of 14 proteins was determined by quantitative western blot. Ingenuity Pathway Analysis (IPA) identified Upstream Regulator (UR) molecules explaining changes in gene expression and biological activity of drugs. Gene set enrichment and IPA were used to test modulation of guadecitabine-specific gene and UR signatures, respectively, in on-treatment tumor biopsies from melanoma patients enrolled in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. ResultsDrug-specific gene and UR signatures were identified for each of the four inhibitors. Immune-related genes were frequently upregulated by guadecitabine, to a lesser extent by givinostat, but downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Treatment of melanoma cells with combination of two epigenetic drugs revealed a dominant effect of guadecitabine and JQ1 on immune-related gene modulation. Drug-specific modulatory profiles were confirmed at the protein level. The guadecitabine-specific UR signature was characterized by activated molecules of the TLR, NF-kB, and IFN innate immunity pathways and was induced in drug-treated melanoma, mesothelioma, hepatocarcinoma cell lines and human melanoma xenografts. Most of the guadecitabine-specific signature genes (n>160) were upregulated in on-treatment tumor biopsies from NIBIT-M4 trial. Progressive activation of guadecitabine UR signature molecules was observed in on-treatment tumor biopsies from responding compared to non-responding patients. ConclusionsGuadecitabine was the most promising immunomodulatory agent among those investigated. This DNA methyltransferases inhibitor emerged as a strong inducer of innate immunity pathways, supporting the rationale for its use in combinatorial immunotherapy approaches.

immunology↗