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

View ORCID ProfileAndrea Anichini, Alessandra Molla, Gabriella Nicolini, Valentina E. Perotti, Francesco Sgambelluri, Alessia Covre, Carolina Fazio, Maria Fortunata Lofiego, Anna Maria di Giacomo, Sandra Coral, Antonella Manca, Maria Cristina Sini, Marina Pisano, Teresa Noviello, Francesca Caruso, Silvia Brich, Giancarlo Pruneri, Andrea Maurichi, Mario Santinami, View ORCID ProfileMichele Ceccarelli, Giuseppe Palmieri, Michele Maio, Roberta Mortarini the EPigenetic Immune-oncology Consortium AIRC (EPICA) investigators
doi: https://doi.org/10.1101/2022.04.13.488140
Andrea Anichini
1Human Tumors Immunobiology Unit, Dept. of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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  • ORCID record for Andrea Anichini
  • For correspondence: andrea.anichini@istitutotumori.mi.it
Alessandra Molla
1Human Tumors Immunobiology Unit, Dept. of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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Gabriella Nicolini
1Human Tumors Immunobiology Unit, Dept. of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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Valentina E. Perotti
1Human Tumors Immunobiology Unit, Dept. of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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Francesco Sgambelluri
1Human Tumors Immunobiology Unit, Dept. of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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Alessia Covre
2Center for Immuno-Oncology, University Hospital of Siena, Siena, Italy
3University of Siena, Siena, Italy
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Carolina Fazio
2Center for Immuno-Oncology, University Hospital of Siena, Siena, Italy
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Maria Fortunata Lofiego
2Center for Immuno-Oncology, University Hospital of Siena, Siena, Italy
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Anna Maria di Giacomo
2Center for Immuno-Oncology, University Hospital of Siena, Siena, Italy
3University of Siena, Siena, Italy
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Sandra Coral
2Center for Immuno-Oncology, University Hospital of Siena, Siena, Italy
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Antonella Manca
4Unit of Cancer Genetics, National Research Council (CNR), Sassari, Italy
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Maria Cristina Sini
4Unit of Cancer Genetics, National Research Council (CNR), Sassari, Italy
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Marina Pisano
4Unit of Cancer Genetics, National Research Council (CNR), Sassari, Italy
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Teresa Noviello
5Department of Electrical Engineering and Information Technology (DIETI), University of Naples “Federico II”, Naples, Italy
6BIOGEM Institute of Molecular Biology and Genetics, Ariano Irpino, Italy
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Francesca Caruso
5Department of Electrical Engineering and Information Technology (DIETI), University of Naples “Federico II”, Naples, Italy
6BIOGEM Institute of Molecular Biology and Genetics, Ariano Irpino, Italy
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Silvia Brich
7Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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Giancarlo Pruneri
7Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
8University of Milan, School of Medicine, Italy
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Andrea Maurichi
9Melanoma and Sarcoma Unit, Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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Mario Santinami
9Melanoma and Sarcoma Unit, Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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Michele Ceccarelli
5Department of Electrical Engineering and Information Technology (DIETI), University of Naples “Federico II”, Naples, Italy
6BIOGEM Institute of Molecular Biology and Genetics, Ariano Irpino, Italy
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Giuseppe Palmieri
4Unit of Cancer Genetics, National Research Council (CNR), Sassari, Italy
10University of Sassari, Sassari, Italy
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Michele Maio
2Center for Immuno-Oncology, University Hospital of Siena, Siena, Italy
3University of Siena, Siena, Italy
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Roberta Mortarini
1Human Tumors Immunobiology Unit, Dept. of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
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  • Abstract
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Abstract

Background Innovative 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.

Methods Human 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.

Results Drug-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.

Conclusions Guadecitabine 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.

Competing Interest Statement

A.Anichini has received compensated educational activities from Bristol-Myers Squibb. A.M.Di Giacomo has served as a consultant and/or advisor to Incyte, Pierre Fabre, Glaxo Smith Kline, Bristol-Myers Squibb, Merck Sharp Dohme, and Sanofi and has received compensated educational activities from Bristol Myers Squibb, Merck Sharp Dohme, Pierre Fabre and Sanofi. M.Maio has served as a consultant and/or advisor to Roche, Bristol-Myers Squibb, Merck Sharp Dohme, Incyte, AstraZeneca, Amgen, Pierre Fabre, Eli Lilly, Glaxo Smith Kline, Sciclone, Sanofi, Alfasigma, and Merck Serono; and owns shares in Epigen Therapeutics. S.Coral and A.Covre own shares in Epigen Therapeutics. G.Palmieri has advisory role for Bristol-Myers Squibb, Merck Sharp Dohme, Roche, Novartis, Pierre-Fabre, Incyte. A.Maurichi has received compensated educational activities from Novartis. G.Pruneri has received meetings honoraria from from AstraZeneca, Novartis, Exact Sciences, Roche and Illumina and is advisory board member of ADS Biotec. A.Molla, G.Nicolini, V.E.Perotti, F.Sgambelluri, C.Fazio, M.F.Lofiego, A.Manca, M.C.Sini, M.Pisano, S.Brich, T.Noviello, F.Caruso, M.Santinami and R.Mortarini declare no conflicts of interests.

  • Abbreviations

    APM
    antigen processing machinery
    BET
    bromodomain and extra-terminal domain
    CT
    cancer testis
    DNMT
    DNA methyltransferases
    EZH2
    enhancer of zeste homolog 2
    HDAC
    histone deacetylases
    ICI
    immune checkpoint inhibitor
    IPA
    Ingenuity Pathway Analysis
    LSD-1
    Lysine-specific demethylase 1
    MTT
    3-(4,5)dimethylthiazol-2,5-diphenyltetrazolium bromide
    NIBIT
    Italian Network for Tumor Biotherapy
    NGS
    next generation sequencing
    TAC
    Transcriptomic Analysis Console
    UR
    Upstream Regulator.
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    Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy
    Andrea Anichini, Alessandra Molla, Gabriella Nicolini, Valentina E. Perotti, Francesco Sgambelluri, Alessia Covre, Carolina Fazio, Maria Fortunata Lofiego, Anna Maria di Giacomo, Sandra Coral, Antonella Manca, Maria Cristina Sini, Marina Pisano, Teresa Noviello, Francesca Caruso, Silvia Brich, Giancarlo Pruneri, Andrea Maurichi, Mario Santinami, Michele Ceccarelli, Giuseppe Palmieri, Michele Maio, Roberta Mortarini
    bioRxiv 2022.04.13.488140; doi: https://doi.org/10.1101/2022.04.13.488140
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    Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy
    Andrea Anichini, Alessandra Molla, Gabriella Nicolini, Valentina E. Perotti, Francesco Sgambelluri, Alessia Covre, Carolina Fazio, Maria Fortunata Lofiego, Anna Maria di Giacomo, Sandra Coral, Antonella Manca, Maria Cristina Sini, Marina Pisano, Teresa Noviello, Francesca Caruso, Silvia Brich, Giancarlo Pruneri, Andrea Maurichi, Mario Santinami, Michele Ceccarelli, Giuseppe Palmieri, Michele Maio, Roberta Mortarini
    bioRxiv 2022.04.13.488140; doi: https://doi.org/10.1101/2022.04.13.488140

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