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DIR LABORATORY

DNA METHYLATION

DNA methylation is a key epigenetic mechanism implicated in transcriptional regulation, normal cellular development, and function. The addition of methyl groups that occurs mostly within CpG dinucleotides is catalyzed by three major DNA methyltransferase (DNMT) family members: DNMT1, DNMT3a, and DNMT3b.
CpG dinucleotides, which are under-represented in the genome, are concentrated in CpG islands (CGI). While most CpGs in the genome are methylated, CGI are not. Importantly, most CGI are located within promoter and transcription start sites of protein coding genes the expression of which is influenced by the methylation status of those CGI. As a matter of fact, methylated CGI are associated with silenced genes whereas unmethylated CGI are associated with expressed genes.

It is commonly known that inactivation of certain tumor-suppressor genes occurs as a consequence of abnormal DNA methylation within the promoter regions and numerous studies have demonstrated a broad range of genes silenced by DNA methylation in different cancer types. Global hypomethylation, instead, contributes to cell transformation by inducing genomic instability. In addition to alterations in promoter regions and repetitive DNA sequences, DNA methylation is also associated with transcriptional regulation of noncoding RNAs that could play a role in tumor suppression. Unlike genetic alterations, DNA methylation is reversible. The reversible nature of DNA methylation makes it an attractive therapeutic target along with providing a dynamic biomarker for prognostic use to follow disease progression.

DNA METHYLATION

Our goal is to design RNA-based platforms to control DNA methylation

#sciencebringspeopletogether

Our goal is to design RNA-based platforms to control DNA methylation

#sciencebringspeopletogether

ABOUT

Dr. Annalisa Di Ruscio is an Assistant Professor in Medicine in the division of Hematology-Oncology, Beth Israel Deaconess Medical Center, at Harvard Medical School.
She received her MD from the Università Cattolica Sacro Cuore (UCSC), Rome. While completing the Hematology fellowship she moved to Boston to join Prof. Tenen’s group at Harvard Medical School, and started working on long non-coding RNAs. Upon completion of the clinical fellowship she pursued a PhD program in molecular biology and then continued as a post-doctoral fellow in Prof. Tenen’s laboratory. Here, she discovered a class of novel RNAs, termed DNA methyltransferase 1 (DNMT1)-interacting RNAs (DiRs) (Di Ruscio, Ebralidze et al. Nature, 2013), that play a key role in controlling cell type-specific DNA methylation patterns. This discovery defined a paradigm shift on the existing view on DNA methylation establishment offering a trailblazing perspective on the relationship between transcription and DNA methylation.

The main focus of Dr. Di Ruscio laboratory is to understand the impact of transcriptional activity in the establishment of epigenetic marks and to define the translational potential of RNAs as a tool to correct aberrant DNA methylation and other epigenetic marks (Ebralidze AK et al. preprint).
As aberrant DNA methylation is one of the most common molecular lesions in cancer cells, Di Ruscio’s group investigates the functional role of DiRs in myeloid disorders that are characterized by abnormal DNA methylation profile, such as Myelodysplastic Syndromes (MDS) and MDS evolution to Acute Myeloid Leukemia.
Using a pioneering approach, Di Ruscio’s group is developing DiR-mimicking platforms to correct DNA methylation profile globally and selectively in hematological and rare genetic conditions. The overarching goal of their studies is to generate RNA-guided gene-specific demethylating agents and overcome the toxicity and lack of specificity of currently approved hypomethylating therapies, with great benefit for patients’ quality of life and standard of cure (Liu Y et al. preprint, Esposito, CL et al.).
More recently, Dr. Di Ruscio laboratory has started diving into understanding the potential role of nicotinamide adenine dinucleotide (NAD) on DNA methylation and implication on normal and leukaemic hematopoietic differentiation (Ummarino et al. 2021).

OUR PROJECTS

ABOUT

OUR PROJECTS

DiR in blood

Myelodysplastic syndromes (MDS) are a collection of hematopoietic diseases characterized by ineffective hematopoiesis, cytopenia and risk of progression to acute myeloid leukemia (AML) in approximately 30 percent of the cases. MDS are considered the most common acquired bone marrow failure syndrome, primarily among the elderly – the median age at MDS diagnosis is ~70 years. Abnormal DNA methylation is considered the molecular lesion leading to tumor suppressor gene (TSG) silencing, MDS clonal variation, and a primary driver of disease evolution to AML. Since 2006, the FDA-approved hypomethylating agents (HMA) – Azacitidine, Decitabine, and Lenalidomide, have been the frontline therapies for the management of higher-risk to transform MDS, ineligible to more aggressive treatment, such as allogenic transplants and standard chemotherapy. Unfortunately, the high toxicity and global non-specific demethylation effects of these compounds have impaired their clinical application and limited their clinical indications. As the population continues to age (the percentage of people over 65 will double between 2025-2050), MDS incidence is expected to grow, and the development of non-toxic, more specific therapies is highly warranted.

The aim of this project is to link the presence or absence of DiRs to global DNA methylation and gene expression in bone marrow mononuclear cells from patients with MDS and following evolution to AML and preclinical murine models of MDS-AML. By an integrated computational analysis, we plan to identify molecular switches involved in leukemia development and capable of correcting abnormal DNA methylation.
In the long term, this study will lay the foundations of a transformative “personalized medicine” for targeted and precise correction of DNA methylation in MDS, leukemia, and all cancers caused by DNA methylation abnormalities with measurable improvement in patients’ life and standard of cure.

AptaDiR & CRISPR-DiR

Non-covalent and selective inhibition of DNMT1 may be achieved by aptamers. As a single-stranded DNA or RNA, an aptamer is the same type of molecule as the substrates for DNMT1, providing the possibility of competitive binding. Thus, an aptamer that can bind to a specific DNMT stronger than its native substrates, could inhibit its activity selectively via preventing the enzyme-substrate binding. Compared with the protein-based therapeutics, aptameric drugs are easier to be stored, handled, and modified to fit for medical applications; and can penetrate through biological barriers more easily, as well as possess much-reduced immunogenicity with low toxicity, presenting good promises for disease treatment. “Systematic Evolution of Ligands by EXponential enrichment” (SELEX) can help the discover of AptaDiR against DNMT1.

By combining the demethylating features of DiRs with the targeting properties of the CRISPR-dead Cas9 (dCas9) system, we developed a novel platform, namely CRISPR-DiR, to induce precise locus specific demethylation and activation. The incorporation of DiR-baits into the single-guide RNA (sgRNA) scaffold enables the delivery of an RNA DNMT1-interacting domain to a selected location while recruiting dCas9

saRNA

RNA activation is a phenomenon wherein double strand RNAs, known as small activating RNAs (saRNAs) can increase the transcription of a specific target gene. In literature it is reported that short activating RNAs (saRNAs) act in an AGO2 dependent manner. Indeed, they are loaded onto AGO2 and shuttled into the nucleus where they can bind their target gene, activating the transcription. Recently “RNA drugs” have emerged as a potential candidate to treat diseases at gene and RNA levels. However, little is known about their action mechanism and about the effects that they can have on the DNA methylation profile.
In this project we focused on a synthetic saRNA called AW1-51, that bind the coding sequence of CEBPA that is a gene deeply involved in granulopoiesis/monopoiesis, lung development, liver functionality. Aberrant promoter DNA methylation of CEBPA locus and the resultant decreases in gene expression is associated to number of cancers, including lung cancer and acute myeloid leukaemia.

Taking advantage of several leukemia and lung cancer models, we are investigating whether AW1-51 exerts its mechanism of transcriptional activation by acting as a DiR mimicking molecule and modifying the epigenetic profile of CEBPA promoter and nearby regulatory elements.