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.