Publications by authors named "Tiziana Bonaldi"

80 Publications

Long non-coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation.

EMBO Rep 2021 Feb 15:e50852. Epub 2021 Feb 15.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

Transition from proliferative-to-invasive phenotypes promotes metastasis and therapy resistance in melanoma. Reversion of the invasive phenotype, however, is challenged by the poor understanding of mechanisms underlying its maintenance. Here, we report that the lncRNA TINCR is down-regulated in metastatic melanoma and its silencing increases the expression levels of invasive markers, in vitro migration, in vivo tumor growth, and resistance to BRAF and MEK inhibitors. The critical mediator is ATF4, a central player of the integrated stress response (ISR), which is activated in TINCR-depleted cells in the absence of starvation and eIF2α phosphorylation. TINCR depletion increases global protein synthesis and induces translational reprogramming, leading to increased translation of mRNAs encoding ATF4 and other ISR proteins. Strikingly, re-expression of TINCR in metastatic melanoma suppresses the invasive phenotype, reduces numbers of tumor-initiating cells and metastasis formation, and increases drug sensitivity. Mechanistically, TINCR interacts with mRNAs associated with the invasive phenotype, including ATF4, preventing their binding to ribosomes. Thus, TINCR is a suppressor of the melanoma invasive phenotype, which functions in nutrient-rich conditions by repressing translation of selected ISR RNAs.
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http://dx.doi.org/10.15252/embr.202050852DOI Listing
February 2021

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

FEBS J 2021 Jan 8. Epub 2021 Jan 8.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

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.
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http://dx.doi.org/10.1111/febs.15707DOI Listing
January 2021

Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases.

Nucleic Acids Res 2021 01;49(2):791-804

IEO European Institute of Oncology IRCCS, Department of Experimental Oncology, Via Adamello 16, 20139 Milan, Italy.

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.
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http://dx.doi.org/10.1093/nar/gkaa1228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826276PMC
January 2021

Label-Free Mass Spectrometry-Based Quantification of Linker Histone H1 Variants in Clinical Samples.

Int J Mol Sci 2020 Oct 4;21(19). Epub 2020 Oct 4.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, 20139 Milan, Italy.

Epigenetic aberrations have been recognized as important contributors to cancer onset and development, and increasing evidence suggests that linker histone H1 variants may serve as biomarkers useful for patient stratification, as well as play an important role as drivers in cancer. Although traditionally histone H1 levels have been studied using antibody-based methods and RNA expression, these approaches suffer from limitations. Mass spectrometry (MS)-based proteomics represents the ideal tool to accurately quantify relative changes in protein abundance within complex samples. In this study, we used a label-free quantification approach to simultaneously analyze all somatic histone H1 variants in clinical samples and verified its applicability to laser micro-dissected tissue areas containing as low as 1000 cells. We then applied it to breast cancer patient samples, identifying differences in linker histone variants patters in primary triple-negative breast tumors with and without relapse after chemotherapy. This study highlights how label-free quantitation by MS is a valuable option to accurately quantitate histone H1 levels in different types of clinical samples, including very low-abundance patient tissues.
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http://dx.doi.org/10.3390/ijms21197330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582528PMC
October 2020

FAM46C and FNDC3A Are Multiple Myeloma Tumor Suppressors That Act in Concert to Impair Clearing of Protein Aggregates and Autophagy.

Cancer Res 2020 11 22;80(21):4693-4706. Epub 2020 Sep 22.

INGM, National Institute of Molecular Genetics, "Fondazione Romeo ed Enrica Invernizzi," Milan, Italy.

Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1357DOI Listing
November 2020

Dual role of PRMT1-dependent arginine methylation in cellular responses to genotoxic stress.

Mol Cell Oncol 2020 7;7(4):1743808. Epub 2020 Apr 7.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

We have recently shown that arginine methylation by protein arginine N-methyltransferase 1 (PRMT1) controls the response to cisplatin in ovarian cancer cells. In addition to increased methylation of chromatin proteins that favors senescence-associated secretory phenotype (SASP) activation, our study unraveled global hypo-methylation of RNA-binding proteins, which - we speculate - may promote their phase separation and stress granules formation.
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http://dx.doi.org/10.1080/23723556.2020.1743808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469493PMC
April 2020

Clinical Application of Mass Spectrometry-Based Proteomics in Lung Cancer Early Diagnosis.

Proteomics Clin Appl 2020 09 22;14(5):e1900138. Epub 2020 Jun 22.

Department of Thoracic Surgery, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, Milan, 20141, Italy.

The current knowledge on proteomic biomarker analysis for the early diagnosis of lung cancer is summarized, underlining the diversity among the results and the current interest in translating research results into clinical practice. A MEDLINE/PubMed literature search to retrieve all the papers published in the last 10 years is performed. Proteomics studies on lung cancer have gathered evidence on the potential role of biomarkers in early diagnosis. Although promising, none of them have proved to be sufficiently reliable to achieve validation. Future research should evolve toward a multipanel analysis of proteins, considering the possibility that individual biomarkers might not be specific enough to diagnose lung cancer, but could be related to oncological conditions.
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http://dx.doi.org/10.1002/prca.201900138DOI Listing
September 2020

Biochemical and Computational Approaches for the Large-Scale Analysis of Protein Arginine Methylation by Mass Spectrometry.

Curr Protein Pept Sci 2020 ;21(7):725-739

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan 20139, Italy.

The absence of efficient mass spectrometry-based approaches for the large-scale analysis of protein arginine methylation has hindered the understanding of its biological role, beyond the transcriptional regulation occurring through histone modification. In the last decade, however, several technological advances of both the biochemical methods for methylated polypeptide enrichment and the computational pipelines for MS data analysis have considerably boosted this research field, generating novel insights about the extent and role of this post-translational modification. Here, we offer an overview of state-of-the-art approaches for the high-confidence identification and accurate quantification of protein arginine methylation by high-resolution mass spectrometry methods, which comprise the development of both biochemical and bioinformatics methods. The further optimization and systematic application of these analytical solutions will lead to ground-breaking discoveries on the role of protein methylation in biological processes.
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http://dx.doi.org/10.2174/1389203721666200426232531DOI Listing
February 2021

Targeting the scaffolding role of LSD1 (KDM1A) poises acute myeloid leukemia cells for retinoic acid-induced differentiation.

Sci Adv 2020 04 8;6(15):eaax2746. Epub 2020 Apr 8.

Department of Experimental Oncology, European Institute of Oncology (IEO), IRCCS, Via Adamello 16, Milan 20139, Italy.

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.
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http://dx.doi.org/10.1126/sciadv.aax2746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141832PMC
April 2020

PRMT1 Is Recruited via DNA-PK to Chromatin Where It Sustains the Senescence-Associated Secretory Phenotype in Response to Cisplatin.

Cell Rep 2020 01;30(4):1208-1222.e9

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan 20139, Italy. Electronic address:

Protein arginine methyltransferase 1 (PRMT1) is overexpressed in various human cancers and linked to poor response to chemotherapy. Various PRMT1 inhibitors are currently under development; yet, we do not fully understand the mechanisms underpinning PRMT1 involvement in tumorigenesis and chemoresistance. Using mass spectrometry-based proteomics, we identified PRMT1 as regulator of arginine methylation in ovarian cancer cells treated with cisplatin. We showed that DNA-dependent protein kinase (DNA-PK) binds to and phosphorylates PRMT1 in response to cisplatin, inducing its chromatin recruitment and redirecting its enzymatic activity toward Arg3 of histone H4 (H4R3). On chromatin, the DNA-PK/PRMT1 axis induces senescence-associated secretory phenotype through H4R3me2a deposition at pro-inflammatory gene promoters. Finally, PRMT1 inhibition reduces the clonogenic growth of cancer cells exposed to low doses of cisplatin, sensitizing them to apoptosis. While unravelling the role of PRMT1 in response to genotoxic agents, our findings indicate the possibility of targeting PRMT1 to overcome chemoresistance in cancer.
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http://dx.doi.org/10.1016/j.celrep.2019.12.061DOI Listing
January 2020

PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis.

Nucleic Acids Res 2020 01;48(1):96-115

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17-92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.
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http://dx.doi.org/10.1093/nar/gkz1051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943135PMC
January 2020

Enrichment of histones from patient samples for mass spectrometry-based analysis of post-translational modifications.

Methods 2020 12 9;184:19-28. Epub 2019 Oct 9.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy. Electronic address:

Aberrations in histone post-translational modifications (PTMs) have been implicated with the development of numerous pathologies, including cancer. Therefore, profiling histone PTMs in patient samples could provide information useful for the identification of epigenetic biomarkers, as well as for the discovery of potential novel targets. While antibody-based methods have been traditionally employed to analyze histone PTM in clinical samples, mass spectrometry (MS) can provide a more comprehensive, unbiased and quantitative view on histones and their PTMs. To combine the power of MS-based methods and the potential offered by histone PTM profiling of clinical samples, we have recently developed a series of methods for the extraction and enrichment of histones from different types of patient samples, including formalin-fixed paraffin-embedded tissues, fresh- and optimal cutting temperature-frozen tissues, and primary cells. Here, we provide a detailed description of these protocols, together with indications on the expected results and the most suitable workflow to be used downstream of each procedure.
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http://dx.doi.org/10.1016/j.ymeth.2019.10.001DOI Listing
December 2020

Epigenetic drug target deconvolution by mass spectrometry-based technologies.

Nat Struct Mol Biol 2019 10 3;26(10):854-857. Epub 2019 Oct 3.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milano, Italy.

The identification of the full target spectrum of active molecules, known as target deconvolution, has become an indispensable step during the drug discovery process. It is now achievable thanks to mass spectrometry-based technologies. Here we discuss these approaches in the context of epigenetic drug discovery.
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http://dx.doi.org/10.1038/s41594-019-0279-xDOI Listing
October 2019

hSWATH: Unlocking SWATH's Full Potential for an Untargeted Histone Perspective.

J Proteome Res 2019 11 29;18(11):3840-3849. Epub 2019 Aug 29.

ProGenTomics, Laboratory of Pharmaceutical Biotechnology , Ghent University , 9000 Ghent , Belgium.

Mass spectrometry (MS) has become the technique of choice for large-scale analysis of histone post-translational modifications (hPTMs) and their combinatorial patterns, especially in untargeted settings where novel discovery-driven hypotheses are being generated. However, MS-based histone analysis requires a distinct sample preparation, acquisition, and data analysis workflow when compared to traditional MS-based approaches. To this end, sequential window acquisition of all theoretical fragment ion spectra (SWATH) has great potential, as it allows for untargeted accurate identification and quantification of hPTMs. Here, we present a complete SWATH workflow specifically adapted for the untargeted study of histones (hSWATH). We assess its validity on a technical dataset of time-lapse deacetylation of a commercial histone extract using HDAC1, which contains a ground truth, i.e., acetylated substrate peptides reduce in intensity. We successfully apply this workflow in a biological setting and subsequently investigate the differential response to HDAC inhibition in different breast cancer cell lines.
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http://dx.doi.org/10.1021/acs.jproteome.9b00214DOI Listing
November 2019

Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation.

Cancer Cell 2019 08;36(2):194-209.e9

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A(∗)STAR), Singapore 138673, Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119077, Singapore; Department of Oncological Sciences and Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Pharmacological Sciences and Mount Sinai Center for Therapeutics Discovery, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.
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http://dx.doi.org/10.1016/j.ccell.2019.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194031PMC
August 2019

Profiling of Epigenetic Features in Clinical Samples Reveals Novel Widespread Changes in Cancer.

Cancers (Basel) 2019 May 24;11(5). Epub 2019 May 24.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, 20139 Milan, Italy.

Aberrations in histone post-translational modifications (PTMs), as well as in the histone modifying enzymes (HMEs) that catalyze their deposition and removal, have been reported in many tumors and many epigenetic inhibitors are currently under investigation for cancer treatment. Therefore, profiling epigenetic features in cancer could have important implications for the discovery of both biomarkers for patient stratification and novel epigenetic targets. In this study, we employed mass spectrometry-based approaches to comprehensively profile histone H3 PTMs in a panel of normal and tumoral tissues for different cancer types, identifying various changes, some of which appear to be a consequence of the increased proliferation rate of tumors, while others are cell-cycle independent. Histone PTM changes found in tumors partially correlate with alterations of the gene expression profiles of HMEs obtained from publicly available data and are generally lost in culture conditions. Through this analysis, we identified tumor- and subtype-specific histone PTM changes, but also widespread changes in the levels of histone H3 K9me3 and K14ac marks. In particular, H3K14ac showed a cell-cycle independent decrease in all the seven tumor/tumor subtype models tested and could represent a novel epigenetic hallmark of cancer. .
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http://dx.doi.org/10.3390/cancers11050723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562406PMC
May 2019

Functional Landscape of PCGF Proteins Reveals Both RING1A/B-Dependent-and RING1A/B-Independent-Specific Activities.

Mol Cell 2019 06 24;74(5):1037-1052.e7. Epub 2019 Apr 24.

IEO European Institute of Oncology IRCCS, Department of Experimental Oncology, Via Adamello 16, 20139 Milan, Italy; University of Milan, Department of Health Sciences, Via A. di Rudinì, 8, 20142 Milan, Italy. Electronic address:

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.
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http://dx.doi.org/10.1016/j.molcel.2019.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561742PMC
June 2019

Proteomics profiling of arginine methylation defines PRMT5 substrate specificity.

Sci Signal 2019 04 2;12(575). Epub 2019 Apr 2.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

Protein arginine methyltransferases (PRMTs) catalyze arginine methylation on both chromatin-bound and cytoplasmic proteins. Accumulating evidence supports the involvement of PRMT5, the major type II PRMT, in cell survival and differentiation pathways that are important during development and in tumorigenesis. PRMT5 is an attractive drug target in various cancers, and inhibitors are currently in oncological clinical trials. Nonetheless, given the complex biology of PRMT5 and its multiple nonhistone substrates, it is paramount to fully characterize these dynamic changes in methylation and to link them to the observed anticancer effects to fully understand the functions of PRMT5 and the consequences of its inhibition. Here, we used a newly established pipeline coupling stable isotope labeling with amino acids in cell culture (SILAC) with immunoenriched methyl peptides to globally profile arginine monomethylation and symmetric dimethylation after PRMT5 inhibition by a selective inhibitor. We adopted heavy methyl SILAC as an orthogonal validation method to reduce the false discovery rate. Through in vitro methylation assays, we validated a set of PRMT5 targets identified by mass spectrometry and provided previously unknown mechanistic insights into the preference of the enzyme to methylate arginine sandwiched between two neighboring glycines (a Gly-Arg-Gly, or "GRG," sequence). Our analysis led to the identification of previously unknown PRMT5 substrates, thus both providing insight into the global effects of PRMT5 and its inhibition in live cells, beyond chromatin, and refining our knowledge of its substrate specificity.
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http://dx.doi.org/10.1126/scisignal.aat8388DOI Listing
April 2019

hmSEEKER: Identification of hmSILAC Doublets in MaxQuant Output Data.

Proteomics 2019 03 18;19(5):e1800300. Epub 2019 Feb 18.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic labeling strategy employed in proteomics to increase the confidence of global identification of methylated peptides by MS. However, to this day, the automatic and robust identification of heavy and light peak doublets from MS-raw data of hmSILAC experiments is a challenging task, for which the choice of computational methods is very limited. Here, hmSEEKER, a software designed to work downstream of a MaxQuant analysis for in-depth search of MS peak pairs that correspond to light and heavy methyl-peptide within MaxQuant-generated tables is described with good sensitivity and specificity. The software is written in Perl, and its code and user manual are freely available at Bitbucket (https://bit.ly/2scCT9u).
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http://dx.doi.org/10.1002/pmic.201800300DOI Listing
March 2019

Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples.

Proteomics Clin Appl 2019 01 6;13(1):e1700166. Epub 2018 Dec 6.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

Purpose: Profiling histone posttranslational modifications (PTMs) in clinical samples holds great potential for the identification of epigenetic biomarkers and the discovery of novel epigenetic targets. MS-based approaches to analyze histone PTMs in clinical samples usually rely on SDS-PAGE separation following histone enrichment in order to eliminate detergents and further isolate histones. However, this limits the digestions options and hence the modification coverage.

Experimental Design And Results: The aim of this study is the implementation of a procedure involving acetone protein precipitation followed by histone enrichment through a C18 StageTip column to obtain histone preparations suitable for various in-solution digestion protocols. Among them, the Arg-C digestion, which allows profiling histone H4 modifications, and the Prop-PIC method, which improves the detection of short and hydrophilic peptides, are tested. This approach is validated on different types of samples, including formalin-fixed paraffin-embedded pathology tissues, and employed to profile histone H4 modifications in cancer samples and normal tissues, identifying previously reported differences, as well as novel ones.

Conclusions And Clinical Relevance: This protocol widens the number of applications available in the toolbox of clinical epigenomics, allowing the investigation of a larger spectrum of histone marks in patient samples.
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http://dx.doi.org/10.1002/prca.201700166DOI Listing
January 2019

Mass Spectrometry-Based Proteomics to Unveil the Non-coding RNA World.

Front Mol Biosci 2018 8;5:90. Epub 2018 Nov 8.

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.

The interaction between non-coding RNAs (ncRNAs) and proteins is crucial for the stability, localization and function of the different classes of ncRNAs. Although ncRNAs, when embedded in various ribonucleoprotein (RNP) complexes, control the fundamental processes of gene expression, their biological functions and mechanisms of action are still largely unexplored. Mass Spectrometry (MS)-based proteomics has emerged as powerful tool to study the ncRNA world: on the one hand, by identifying the proteins interacting with distinct ncRNAs; on the other hand, by measuring the impact of ncRNAs on global protein levels. Here, we will first provide a concise overview on the basic principles of MS-based proteomics for systematic protein identification and quantification; then, we will recapitulate the main approaches that have been implemented for the screening of ncRNA interactors and the dissection of ncRNA-protein complex composition. Finally, we will describe examples of various proteomics strategies developed to characterize the effect of ncRNAs on gene expression, with a focus on the systematic identification of microRNA (miRNA) targets.
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http://dx.doi.org/10.3389/fmolb.2018.00090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6236024PMC
November 2018

The kinetochore module Okp1/Ame1 is a reader for N-terminal modifications on the centromeric histone Cse4.

EMBO J 2019 01 2;38(1). Epub 2018 Nov 2.

Department of Molecular Cell Biology, Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany

Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. defects in were suppressed by mutations in and , and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin.
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http://dx.doi.org/10.15252/embj.201898991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315295PMC
January 2019

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

Nucleic Acids Res 2018 05;46(8):3817-3832

Department of Experimental Oncology, European Institute of Oncology, Milan 20139, Italy.

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.
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http://dx.doi.org/10.1093/nar/gky224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934616PMC
May 2018

Zc3h10 is a novel mitochondrial regulator.

EMBO Rep 2018 04 5;19(4). Epub 2018 Mar 5.

DiSFeB, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy

Mitochondria are the energy-generating hubs of the cell. In spite of considerable advances, our understanding of the factors that regulate the molecular circuits that govern mitochondrial function remains incomplete. Using a genome-wide functional screen, we identify the poorly characterized protein Zinc finger CCCH-type containing 10 (Zc3h10) as regulator of mitochondrial physiology. We show that Zc3h10 is upregulated during physiological mitochondriogenesis as it occurs during the differentiation of myoblasts into myotubes. Zc3h10 overexpression boosts mitochondrial function and promotes myoblast differentiation, while the depletion of Zc3h10 results in impaired myoblast differentiation, mitochondrial dysfunction, reduced expression of electron transport chain (ETC) subunits, and blunted TCA cycle flux. Notably, we have identified a loss-of-function mutation of Zc3h10 in humans (Tyr105 to Cys105) that is associated with increased body mass index, fat mass, fasting glucose, and triglycerides. Isolated peripheral blood mononuclear cells from individuals homozygotic for Cys105 display reduced oxygen consumption rate, diminished expression of some ETC subunits, and decreased levels of some TCA cycle metabolites, which all together derive in mitochondrial dysfunction. Taken together, our study identifies Zc3h10 as a novel mitochondrial regulator.
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http://dx.doi.org/10.15252/embr.201745531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891430PMC
April 2018

PARP14 Controls the Nuclear Accumulation of a Subset of Type I IFN-Inducible Proteins.

J Immunol 2018 04 2;200(7):2439-2454. Epub 2018 Mar 2.

Department of Experimental Oncology, European Institute of Oncology, 20139 Milan, Italy;

The enzymes of the poly-ADP-ribose polymerase (PARP) superfamily control many relevant cellular processes, but a precise understanding of their activities in different physiological or disease contexts is largely incomplete. We found that transcription of several genes was dynamically regulated upon murine macrophage activation by endotoxin. PARP14 was strongly induced by several inflammatory stimuli and translocated into the nucleus of stimulated cells. Quantitative mass spectrometry analysis showed that PARP14 bound to a group of IFN-stimulated gene (ISG)-encoded proteins, most with an unknown function, and it was required for their nuclear accumulation. Moreover, PARP14 depletion attenuated transcription of primary antiviral response genes regulated by the IFN regulatory transcription factor 3, including , thus reducing IFN-β production and activation of ISGs involved in the secondary antiviral response. In agreement with the above-mentioned data, PARP14 hindered proliferation in murine macrophages. Overall, these data hint at a role of PARP14 in the control of antimicrobial responses and specifically in nuclear activities of a subgroup of ISG-encoded proteins.
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http://dx.doi.org/10.4049/jimmunol.1701117DOI Listing
April 2018

Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases.

Nat Commun 2018 01 9;9(1):105. Epub 2018 Jan 9.

Nuclear Dynamics, Babraham Institute, Cambridge, CB22 3AT, UK.

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.
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http://dx.doi.org/10.1038/s41467-017-02651-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760624PMC
January 2018

A role for CENP-A/Cse4 phosphorylation on serine 33 in deposition at the centromere.

FEMS Yeast Res 2018 02;18(1)

Institut für Biologie, Humboldt-Universität zu Berlin, 10099 Berlin, Germany.

Centromeres are the sites of assembly of the kinetochore, which connect the chromatids to the microtubules for sister chromatid segregation during cell division. Centromeres are characterized by the presence of the histone H3 variant CENP-A (termed Cse4 in Saccharomyces cerevisiae). Here, we investigated the function of serine 33 phosphorylation of Cse4 (Cse4-S33ph) in S. cerevisiae, which lies within the essential N-terminal domain (END) of the extended Cse4 N-terminus. Significantly, we identified histone H4-K5, 8, 12R to cause a temperature-sensitive growth defect with mutations in Cse4-S33 and sensitivity to nocodazole and hydroxyurea. Furthermore, the absence of Cse4-S33ph reduced the levels of Cse4 at centromeric sequences, suggesting that Cse4 deposition is defective in the absence of S33 phosphorylation. We furthermore identified synthetic genetic interactions with histone H2A-E57A and H2A-L66A, which both cause a reduced interaction with the histone chaperone FACT and reduced H2A/H2B levels in chromatin, again supporting the notion that a combined defect of H2A/H2B and Cse4 deposition causes centromeric defects. Altogether, our data highlight the importance of correct histone deposition in building a functional centromeric nucleosome and suggests a role for Cse4-S33ph in this process.
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http://dx.doi.org/10.1093/femsyr/fox094DOI Listing
February 2018

Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers.

Nucleic Acids Res 2017 Dec;45(21):12195-12213

Department of Experimental Oncology, European Institute of Oncology, Milan 20139, Italy.

The integrated activity of cis-regulatory elements fine-tunes transcriptional programs of mammalian cells by recruiting cell type-specific as well as ubiquitous transcription factors (TFs). Despite their key role in modulating transcription, enhancers are still poorly characterized at the molecular level, and their limited DNA sequence conservation in evolution and variable distance from target genes make their unbiased identification challenging. The coexistence of high mono-methylation and low tri-methylation levels of lysine 4 of histone H3 is considered a signature of enhancers, but a comprehensive view of histone modifications associated to enhancers is still lacking. By combining chromatin immunoprecipitation (ChIP) with mass spectrometry, we investigated cis-regulatory regions in macrophages to comprehensively identify histone marks specifically associated with enhancers, and to profile their dynamics after transcriptional activation elicited by an inflammatory stimulation. The intersection of the proteomics data with ChIP-seq and RNA-seq analyses revealed the existence of novel subpopulations of enhancers, marked by specific histone modification signatures: specifically, H3K4me1/K36me2 marks transcribed enhancers, while H3K4me1/K36me3 and H3K4me1/K79me2 combinations mark distinct classes of intronic enhancers. Thus, our MS analysis of functionally distinct genomic regions revealed the combinatorial code of histone modifications, highlighting the potential of proteomics in addressing fundamental questions in epigenetics.
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http://dx.doi.org/10.1093/nar/gkx821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716071PMC
December 2017

Design of KDM4 Inhibitors with Antiproliferative Effects in Cancer Models.

ACS Med Chem Lett 2017 Aug 27;8(8):869-874. Epub 2017 Jul 27.

Celgene Quanticel Research, 10300 Campus Point Drive, Suite 100, San Diego, California 92121, United States.

Histone lysine demethylases (KDMs) play a vital role in the regulation of chromatin-related processes. Herein, we describe our discovery of a series of potent KDM4 inhibitors that are both cell permeable and antiproliferative in cancer models. The modulation of histone H3K9me3 and H3K36me3 upon compound treatment was verified by homogeneous time-resolved fluorescence assay and by mass spectroscopy detection. Optimization of the series using structure-based drug design led to compound (QC6352), a potent KDM4 family inhibitor that is efficacious in breast and colon cancer PDX models.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554903PMC
August 2017

PAT-H-MS coupled with laser microdissection to study histone post-translational modifications in selected cell populations from pathology samples.

Clin Epigenetics 2017 11;9:69. Epub 2017 Jul 11.

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy.

Background: Aberrations in histone post-translational modifications (hPTMs) have been linked with various pathologies, including cancer, and could not only represent useful biomarkers but also suggest possible targetable epigenetic mechanisms. We have recently developed an approach, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS), that allows performing a comprehensive and quantitative analysis of histone PTMs from formalin-fixed paraffin-embedded pathology samples. Despite its great potential, the application of this technique is limited by tissue heterogeneity.

Methods: In this study, we further implemented the PAT-H-MS approach by coupling it with techniques aimed at reducing sample heterogeneity and selecting specific portions or cell populations within the samples, such as manual macrodissection and laser microdissection (LMD).

Results: When applied to the analysis of a small set of breast cancer samples, LMD-PAT-H-MS allowed detecting more marked changes between luminal A-like and triple negative patients as compared with the classical approach. These changes included not only the already known H3 K27me3 and K9me3 marks, but also H3 K36me1, which was found increased in triple negative samples and validated on a larger cohort of patients, and could represent a potential novel marker distinguishing breast cancer subtypes.

Conclusions: These results show the feasibility of applying techniques to reduce sample heterogeneity, including laser microdissection, to the PAT-H-MS protocol, providing new tools in clinical epigenetics and opening new avenues for the comprehensive analysis of histone post-translational modifications in selected cell populations.
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http://dx.doi.org/10.1186/s13148-017-0369-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504751PMC
April 2018