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Chromatin Alterations in Tumorigenesis

Director

Saverio Minucci

Activities

Cancer cells show global changes in chromatin structure (DNA methylation and histone post-translational modifications), that lead to stable alterations in gene expression and potentially other nuclear functions (such as DNA replication and repair). Unlike genetic lesions, those alterations are reversible since the underlying DNA sequence is unchanged: this fundamental difference between genetic and epigenetic alterations makes the epigenome much more amenable to the development of therapeutic strategies. Indeed, small molecules with the capacity to interfere with chromatin modifying enzymes have antitumor activity. The concept of epigenetic therapy has been clinically validated with the approval by regulatory authorities of a small number of drugs for use in selected forms of cancer. In our view, however, drugs interfering with epigenetic enzymes (such as DNA methyltransferases and histone deacetylases, the most advanced targets in the epigenetic arena) have been used in the vast majority of cases rather aspecifically, without taking into account the context of chromatin alterations occurring in cancer cells. We surmise therefore that one of the major goals of both basic and applied research in this area should be the search of a set of epigenetic alterations in tumor cells, that dictate sensitivity or resistance to epigenetic drugs.

  • Research projects

    We have focused therefore our activities on the study of deregulation of chromatin structure/function in cancer with the goals of:

    • Identifying sistematically epigenetic alterations in cancer cells;
    • To exploit this knowledge to optimize epigenetic therapies towards a more targeted approach.

    To fulfill these goals, we have adopted a combination of experimental strategies:

    • Mechanistical analysis of chromatin alterations in cancer. We have developed new technologies for the study of epigenetic alterations in cancer patients, to reduce the amounts of material required, and to allow access to paraffin-embedded pathology samples: NASeq, and PAT-ChIP. Thanks to these new approaches, we are studying acute myeloid leukemias and breast cancer (where mechanistical insights on how epigenetic deregulation takes place are partially available) as a paradigm of the cancer epigenome.
    • Functional dissection of the role of chromatin modifiers in leukemogenesis. In parallel, we are undertaking the systematic dissection of the role of individual chromatin modifiers in tumorigesis in murine models of acute myeloid leukemia. By the use of knock-down and conditional knock-out approaches, we are studying the role of histone deacetylases, Polycomb complexes, histone demethylases in both tumor initiation and tumor maintenance.
    • Epigenetic therapy of cancer. In the same disease model, we are studying the biological and mechanistical effects of epigenetic drugs (histone deacetylase and demethylase inhibitors, DNA demethylating agents). In particular, we have developed new assays for the study of th contribution of different subpopulations of tumor cells to cancer growth, focusing on the role of leukemic stem cells.
    • Optimization of anticancer therapies. The know-how and results gained above are being increasingly useful in other settings, to try to exploit the epigenome and its manipulation for the optimization of anticancer therapies. With this goal, we are:
      - Using yeast as a model system (in collaboration with M. Foiani, Project “TYM”) studying systematically the synthetic lethal interactions of anticancer and epigenetic drugs, and subsequently validating them in mammals;
      - By quantitative chemical proteomics (in collaboration with T. Bonaldi), identifying systematically the cell interactors of anticancer and epigenetic drugs;
      - In collaboration with the Drug Discovery Program of the IEO (TIV), conducting in vivo screenings to identify and validate epigenetic targets in leukemias (in collaboration with PG Pelicci), and analyzing the effect of novel epigenetic drugs being developed against chromatin-associated proteins.

    Thus, there is an extremely appealing opportunity to perform a mechanistically oriented analysis (“to understand how things happen”) that can immediately be applied to better treat the patients (“to try to change things, when they have gone bad”). The ultimate goal: to go towards a group that considers Man as the primary model system.

    Methods
    A fundamental part of the lab’s activities consists in the development of new technologies and new assays for the study of epigenetic alterations in cancer.

    The main methods resulting from the last 10 years are summarized as follows:

    • Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue (1-2).
    • NA-Seq (combination of restriction enzymes and next-generation sequencing) for a genome-wide analysis of chromatin accessibility (3).
    • Detection of histone acetylation levels by Flow Cytometry (4).
    • Targeting protein inactivation through oligomerization (5).


    Methods 2 (details)

    1. Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue.


    FANELLI M, AMATORI S, BAROZZI I, MINUCCI S (2011). Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue. NATURE PROTOCOLS, vol. 6, p. 1905-1919, ISSN: 1754-2189, doi: 10.1038/nprot.2011.406

    Abstract
    Formalin-fixed, paraffin-embedded (FFPE) samples represent the gold standard for storage of pathology samples. Here we describe pathology tissue chromatin immunoprecipitation (PAT-ChIP), a technique for extraction and high-throughput analysis, by techniques such as ChIP-seq, of chromatin derived from FFPE samples. Technically, the main challenge of PAT-ChIP is the preparation of good-quality chromatin from FFPE samples. Here we provide a detailed explanation of the methodology used, the choice of reagents and the troubleshooting steps required to establish a robust chromatin preparation procedure. Other steps have also been adapted from existing techniques to optimize their use for PAT-ChIP-seq. The protocol requires 4 d from the start to the end of the PAT-ChIP procedure. PAT-ChIP provides, for the first time, the chance to perform analyses of histone modifications and transcription factor binding on a genome-wide scale using patient-derived FFPE samples. This technique therefore allows the immediate use of pathology archives (even those that are several years old) for epigenetic analyses and the identification of candidate epigenetic biomarkers or targets.

  • Publications

    • THALER F, VARASI M, ABATE A, CARENZI G, COLOMBO A, BIGOGNO C, BOGGIO R, DAL ZUFFO R, RAPETTI D, RESCONI A, REGALIA N, VULTAGGIO S, DONDIO G, GAGLIARDI S, MINUCCI S, MERCURIO C (2013). Synthesis and biological characterization of spiro[2H-(1,3)-benzoxazine-2,4'-piperidine] based histone deacetylase inhibitors. European journal of medicinal chemistry 04/2013; 64C:273-284.
    • SANTORO F, BOTRUGNO OA, DAL ZUFFO R, PALLAVICINI I, MATTHEWS GM, CLUSE L, BAROZZI I, SENESE S, FORNASARI L, MORETTI S, ALTUCCI S, PELICCI PG, CHIOCCA S, JOHNSTONE RW, MINUCCI S (2013). A dual role for Hdac1: oncosuppressor in tumorigenesis, oncogene in tumor maintenance. Blood, 121 (17): 3459-68. doi:10.1182/blood-2012-10-461988
    • SONCINI M, SANTORO F, GUTIERREZ A, FRIGÈ G, ROMANENGHI M, BOTRUGNO OA, PALLAVICINI I, PELICCI P, DI CROCE L, MINUCCI S (2013). The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia. BIOCHIMICA ET BIOPHYSICA ACTA, 1832 (1): 114-20. ISSN: 0006-3002
    • KLIONSKY DJ et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy.Autophagy, 8: 4, 445-544, April 2012.
    • SAEED S, LOGIE C, FRANCOIJS KJ, FRIGÈ G, ROMANENGHI M, NIELSEN FG, RAATS L, SHAHHOSEINI M, HUYNEN M, ALTUCCI L, MINUCCI S, MARTENS JH, STUNNENBERG HG (2012). Chromatin accessibility, p300 and histone acetylation define PML-RARa and AML1-ETO binding sites in acute myeloid leukemia. BLOOD, vol. 120, p. 3058-3068, ISSN: 0006-4971

     

    All publications

  • Funding

    Saverio Minucci has been Unit Responsible, Partner, Principal Investigator or Coordinator in more than 20 Peer-reviewed projects, including EU Framework 6 and Framework 7 projects (Epitron, Blueprint, 4D), PRIN, FIRB, CNR (Epigen Flagship Project), AIRC (Investigator and 5x1000 projects), Cariplo foundation and the Ministry of Health.

A SMALL GESTURE IS A GREAT HELP FOR US

PARTNERSHIP

Università degli Studi di Milano Ecancer Medical Science IFOM-IEO Campus

CREDITS

Ministero della Salute Joint Commission International Breastcertification bollinirosa

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IRCCS - ISTITUTO DI RICOVERO E CURA A CARATTERE SCIENTIFICO

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