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Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins

Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns ...

Hellenic Open University

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin. We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human -mutant NSCLC cell lines A549 and H460 and mouse -mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the transcription factor (TF) LRF, a known epigenetic silencer, was examined as a modulator of non-coding RNAs expression. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060-109,747,960 mouse chromosomal region to micro-RNA upregulated species. Furthermore, the novel epigenetic miR-148b-3p/lnc axis was identified, which differentiates human cell line of lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes. The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease progression, NSCLC subtypes discrimination and advancement to patient-tailored treatments.

"Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer".

Patients with higher-risk (HR) myelodysplastic syndrome (MDS), ineligible for allogeneic hematopoietic stem cell transplantation (alloHSCT), require prompt therapeutic interventions, such as treatment with hypomethylating agents (HMAs) to restore normal DNA methylation patterns, mainly of oncosuppressor genes, and consequently to delay disease progression and increase overall survival (OS). However, response assessment to HMA treatment relies on conventional methods with limited capacity to uncover a wide spectrum of underlying molecular events.

Integrating advanced analytical methods to assess epigenetic marks affecting response to hypomethylating agents in higher risk myelodysplastic syndrome.

The intracellular topography of RNA molecules, encompassing ribonucleotides with biochemical modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), adenosine to inosine (A → I) editing, and isomerization of uridine to pseudouridine (Ψ), as well as of non-coding RNA molecules, is currently studied within the frame of the epigenome. Circulating RNA molecules in the intracellular space that have incorporated information by carrying specific modifications depend on the balanced activity and correct subcellular installation of their modifying enzymes, the "writers", "readers" and "erasers". Modifications are critical for RNA translocation from the nucleus to the cytoplasm, for stability and translation efficiency, and for other, still-uncovered functions. Moreover, trafficking of non-coding RNA molecules depends on membrane transporters capable of recognizing signal sequences and RNA recognition-binding proteins that can facilitate their transport to different intracellular locations, guiding the establishment of interconnection possibilities with different macromolecular networks. The potential of long non-coding RNAs to form multilayer molecular connections, as well as the differential topology of micro-RNAs in cell nuclei, compared to cytoplasm, has been recognized by several studies. The study of the intercellular compartmentalization of these molecules has recently become feasible thanks to technological progress; however, a wealth of information has not yet been produced that would lead to safe conclusions regarding non-coding RNA's contributions to the early steps of pathogenesis and disease progression in hematological malignancies. Both, the bone marrow, as the main hematopoietic tissue, and the lymphoid tissues are composed of cells with highly reactive potential to signals affecting the epigenome and initiating cascade pathways in response. Independently or in combination with coexistent driver genetic mutations, especially mutations of enzymes involved in epigenomic surveillance, intracellular microenvironmental alterations within the cell nuclear, cytoplasmic, and mitochondrial compartments can lead to disorganization of hematopoietic stem cells' epigenomes, promoting the generation of hematological malignancies. In this review, we discuss the various intracellular processes that, when disrupted, may result in the ectopic placement of RNA molecules, either inducing specific modifications or non-coding molecules or promoting hematological malignant phenotypes. The crosstalk between mitochondrial and nuclear genomes and the complex regulatory effects of mis-localized RNA molecules are highlighted. This research approach may constitute a field for new, more specifically targeted therapies in hematology based on RNA technology.

Article	Year
"Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer". Non-coding RNA research\| PubMed ID: 38577020	2024
Integrating advanced analytical methods to assess epigenetic marks affecting response to hypomethylating agents in higher risk myelodysplastic syndrome. Molecular medicine (Cambridge, Mass.)\| PubMed ID: 39953389	2025
Intracellular Mis-Localization of Modified RNA Molecules and Non-Coding RNAs: Facts from Hematologic Malignancies. Current issues in molecular biology\| PubMed ID: 41020880	2025

Irene Dereki

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