extraction-histones-from-clinical-specimens-for-epigenetic-profiling

Extraction of Histones from Clinical Specimens for Epigenetic Profiling by Mass Spectrometry

Extensive experimental evidence supports the role of epigenetic mechanisms in cancer onset, progression, and recurrence. Among these, histone variants and ...

European Institute of Oncology IRCCS

In the last 15 years, increasing evidence linking epigenetics to various aspects of cancer biology has prompted the investigation of histone post-translational modifications (PTMs) and histone variants in the context of clinical samples. The studies performed so far demonstrated the potential of this type of investigations for the discovery of both potential epigenetic biomarkers for patient stratification and novel epigenetic mechanisms potentially targetable for cancer therapy. Although traditionally the analysis of histones in clinical samples was performed through antibody-based methods, mass spectrometry (MS) has emerged as a more powerful tool for the unbiased, comprehensive, and quantitative investigation of histone PTMs and variants. MS has been extensively used for the analysis of epigenetic marks in cell lines and animal tissue and, thanks to recent technological advances, is now ready to be applied also to clinical samples. In this review, we will provide an overview on the quantitative MS-based analysis of histones, their PTMs and their variants in cancer clinical samples, highlighting current achievements and future perspectives for this novel field of research. Among the different MS-based approaches currently available for histone PTM profiling, we will focus on the 'bottom-up' strategy, namely the analysis of short proteolytic peptides, as it has been already successfully employed for the analysis of clinical samples.

Mass spectrometry-based characterization of histones in clinical samples: applications, progress, and challenges.

Epigenetics includes a complex set of processes that alter gene activity without modifying the DNA sequence, which ultimately determines how the genetic information common to all the cells of an organism is used to generate different cell types. Dysregulation in the deposition and maintenance of epigenetic features, which include histone posttranslational modifications (PTMs) and histone variants, can result in the inappropriate expression or silencing of genes, often leading to diseased states, including cancer. The investigation of histone PTMs and variants in the context of clinical samples has highlighted their importance as biomarkers for patient stratification and as key players in aberrant epigenetic mechanisms potentially targetable for therapy. Mass spectrometry (MS) has emerged as the most powerful and versatile tool for the comprehensive, unbiased and quantitative analysis of histone proteoforms. In recent years, these approaches-which we refer to as "epi-proteomics"-have demonstrated their usefulness for the investigation of epigenetic mechanisms in pathological conditions, offering a number of advantages compared with the antibody-based methods traditionally used to profile clinical samples. In this review article, we will provide a critical overview of the MS-based approaches that can be employed to study histone PTMs and variants in clinical samples, with a strong focus on the latest advances in this area, such as the analysis of uncommon modifications and the integration of epi-proteomics data into multi-OMICs approaches, as well as the challenges to be addressed to fully exploit the potential of this novel field of research.

Investigating pathological epigenetic aberrations by epi-proteomics.

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 methylation and induces derepression of a gene cluster, including the tumor suppressor Cav1 and several genes involved in the interferon-alpha and -gamma response. We discuss the relevance of this finding considering the hypothesis that KDM5B may possess regulatory roles independent of its catalytic activity.

A truncated and catalytically inactive isoform of KDM5B histone demethylase accumulates in breast cancer cells and regulates H3K4 tri-methylation and gene expression.

Altered histone post-translational modifications are frequently associated with cancer. Here, we apply mass-spectrometry to study the epigenetic landscapes of breast cancer subtypes, with a particular focus on triple-negative breast cancers (TNBCs), a heterogeneous group lacking well-defined molecular targets and effective therapies. The analysis of over 200 tumors reveals epigenetic signatures that discriminate TNBCs from the other BC subtypes, and that distinguish TNBC patients with different prognoses. Employing a multi-OMICs approach integrating epigenomics, transcriptomics, and proteomics data, we investigate the mechanistic role of increased H3K4 methylation in TNBCs, demonstrating that H3K4me2 sustains the expression of genes associated with the TNBC phenotype. Through CRISPR-mediated editing, we establish a causal relationship between H3K4me2 and gene expression for several targets. Furthermore, treatment with H3K4 methyltransferase inhibitors reduce TNBC cell growth in vitro and in vivo. Collectively, our results unravel a novel epigenetic pathway implicated in TNBC pathogenesis and suggest new opportunities for targeted therapy.

Article	Year
Mass spectrometry-based characterization of histones in clinical samples: applications, progress, and challenges. The FEBS journal\| PubMed ID: 33415821	2022
Investigating pathological epigenetic aberrations by epi-proteomics. Clinical epigenetics\| PubMed ID: 36371348	2022
A truncated and catalytically inactive isoform of KDM5B histone demethylase accumulates in breast cancer cells and regulates H3K4 tri-methylation and gene expression. Cancer gene therapy\| PubMed ID: 36697763	2023
A histone-centric multi-omics study shows that increased H3K4 methylation sustains triple-negative breast cancer phenotypes. Nature communications\| PubMed ID: 41028718	2025

Giulia Robusti

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