MiRLog and dbmiR: Prioritization and functional annotation tools to study human microRNA sequence variants.

The recent identification of noncoding variants with pathogenic effects suggests that these variations could underlie a significant number of undiagnosed cases. Several computational methods have been developed to predict the functional impact of noncoding variants, but they exhibit only partial concordance and are not integrated with functional annotation resources, making the interpretation of these variants still challenging. MicroRNAs (miRNAs) are small noncoding RNA molecules that act as fine regulators of gene expression and play crucial functions in several biological processes, such as cell proliferation and differentiation. An increasing number of studies demonstrate a significant impact of miRNA single nucleotide variants (SNVs) both in Mendelian diseases and complex traits. To predict the functional effect of miRNA SNVs, we implemented a new meta-predictor, MiRLog, and we integrated it into a comprehensive database, dbmiR, which includes a precompiled list of all possible miRNA allelic SNVs, providing their biological annotations at nucleotide and miRNA levels. MiRLog and dbmiR were used to explore the genetic variability of miRNAs in 15,708 human genomes included in the gnomAD project, finding several ultra-rare SNVs with a potentially deleterious effect on miRNA biogenesis and function representing putative contributors to human phenotypes.

MicroRNAs as Modulators of the Immune Response in T-Cell Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukaemia (ALL) is an aggressive haematological tumour driven by the malignant transformation and expansion of B-cell (B-ALL) or T-cell (T-ALL) progenitors. The evolution of T-ALL pathogenesis encompasses different master developmental pathways, including the main role played by Notch in cell fate choices during tissue differentiation. Recently, a growing body of evidence has highlighted epigenetic changes, particularly the altered expression of microRNAs (miRNAs), as a critical molecular mechanism to sustain T-ALL. The immune response is emerging as key factor in the complex multistep process of cancer but the role of miRNAs in anti-leukaemia response remains elusive. In this review we analyse the available literature on miRNAs as tuners of the immune response in T-ALL, focusing on their role in Natural Killer, T, T-regulatory and Myeloid-derived suppressor cells. A better understanding of this molecular crosstalk may provide the basis for the development of potential immunotherapeutic strategies in the leukemia field.

Exploring non-coding genetic variability in ACE2: Functional annotation and in vitro validation of regulatory variants.

The surge in human whole-genome sequencing data has facilitated the study of non-coding region variations, yet understanding their biological significance remains a challenge. We used a computational workflow to assess the regulatory potential of non-coding variants, with a particular focus on the Angiotensin Converting Enzyme 2 (ACE2) gene. This gene is crucial in physiological processes and serves as the entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 19 (COVID-19). In our analysis, using data from the gnomAD population database and functional annotation, we identified 17 significant Single Nucleotide Variants (SNVs) in ACE2, particularly in its enhancers, promoters, and 3' untranslated regions (UTRs). We found preliminary evidence supporting the regulatory impact of some of these variants on ACE2 expression. Our detailed examination of two SNVs, rs147718775 and rs140394675, in the ACE2 promoter revealed that these co-occurring SNVs, when mutated, significantly enhance promoter activity, suggesting a possible increase in specific ACE2 isoform expression. This method proves effective in identifying and interpreting impactful non-coding variants, aiding in further studies and enhancing understanding of molecular bases of monogenic and complex traits.

Textbook Outcome of Laparoscopic Microwave Ablation for Hepatocellular Carcinoma.

In the context of spreading interest in textbook outcome (TO) evaluation in different fields, we aimed to investigate an uncharted procedure, that is, laparoscopic microwave ablation (MWA) for hepatocellular carcinoma (HCC). Absence of post-MWA complications, a hospital stay of three days, no mortality nor readmission within 30 days, and complete response of the target lesion at post-MWA CT scan defined TO achievement. Patients treated between January 2014 and March 2021 were retrospectively reviewed, and of the 521 patients eligible for the study, 337 (64.7%) fulfilled all the quality indicators to achieve the TO. The absence of complications was the main limiting factor for accomplishing TO. At multivariable analysis, Child-Pugh B cirrhosis, age of more than 70 years old, three nodules, and MELD score ≥ 15 were associated with decreased probabilities of TO achievement. A score based on these factors was derived from multivariable analysis, and patients were divided into three risk groups for TO achievement. At survival analysis, overall survival (OS) was significantly (p = 0.001) higher in patients who achieved TO than those who did not. Moreover, OS evaluation in the three risk groups showed a trend coherent with TO achievement probability. The present study, having assessed the first TO for laparoscopic MWA for HCC, encourages further broader consensus on its definition and, on its basis, on the development of clinically relevant tools for managing treatment allocation.

Potassium Channel KCNH1 Activating Variants Cause Altered Functional and Morphological Ciliogenesis.

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP_002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging.

Identification of a Novel Curcumin Derivative Influencing Notch Pathway and DNA Damage as a Potential Therapeutic Agent in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy considered curable by modern clinical management. Nevertheless, the prognosis for T-ALL high-risk cases or patients with relapsed and refractory disease is still dismal. Therefore, there is a keen interest in developing more efficient and less toxic therapeutic approaches. T-ALL pathogenesis is associated with Notch signaling alterations, making this pathway a highly promising target in the fight against T-ALL. Here, by exploring the anti-leukemic capacity of the natural polyphenol curcumin and its derivatives, we found that curcumin exposure impacts T-ALL cell line viability and decreases Notch signaling in a dose- and time-dependent fashion. However, our findings indicated that curcumin-mediated cell outcomes did not depend exclusively on Notch signaling inhibition, but might be mainly related to compound-induced DNA-damage-associated cell death. Furthermore, we identified a novel curcumin-based compound named CD2066, endowed with potentiated anti-proliferative activity in T-ALL compared to the parent molecule curcumin. At nanomolar concentrations, CD2066 antagonized Notch signaling, favored DNA damage, and acted synergistically with the CDK1 inhibitor Ro3306 in T-ALL cells, thus representing a promising novel candidate for developing therapeutic agents against Notch-dependent T-ALL.

Targeting Notch to Maximize Chemotherapeutic Benefits: Rationale, Advanced Strategies, and Future Perspectives.

Notch signaling guides cell fate decisions by affecting proliferation, apoptosis, stem cell self-renewal, and differentiation depending on cell and tissue context. Given its multifaceted function during tissue development, both overactivation and loss of Notch signaling have been linked to tumorigenesis in ways that are either oncogenic or oncosuppressive, but always context-dependent. Notch signaling is critical for several mechanisms of chemoresistance including cancer stem cell maintenance, epithelial-mesenchymal transition, tumor-stroma interaction, and malignant neovascularization that makes its targeting an appealing strategy against tumor growth and recurrence. During the last decades, numerous Notch-interfering agents have been developed, and the abundant preclinical evidence has been transformed in orphan drug approval for few rare diseases. However, the majority of Notch-dependent malignancies remain untargeted, even if the application of Notch inhibitors alone or in combination with common chemotherapeutic drugs is being evaluated in clinical trials. The modest clinical success of current Notch-targeting strategies is mostly due to their limited efficacy and severe on-target toxicity in Notch-controlled healthy tissues. Here, we review the available preclinical and clinical evidence on combinatorial treatment between different Notch signaling inhibitors and existent chemotherapeutic drugs, providing a comprehensive picture of molecular mechanisms explaining the potential or lacking success of these combinations.

Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer caused by the deregulation of key T-cell developmental pathways, including Notch signaling. Aberrant Notch signaling in T-ALL occurs by NOTCH1 gain-of-function mutations and by NOTCH3 overexpression. Although NOTCH3 is assumed as a Notch1 target, machinery driving its transcription in T-ALL is undefined in leukemia subsets lacking Notch1 activation. Here, we found that the binding of the intracellular Notch3 domain, as well as of the activated Notch1 fragment, to the NOTCH3 gene locus led to the recruitment of the H3K27 modifiers JMJD3 and p300, and it was required to preserve transcriptional permissive/active H3K27 marks and to sustain NOTCH3 gene expression levels. Consistently, pharmacological inhibition of JMJD3 by GSKJ4 treatment or of p300 by A-485 decreased the levels of expression of NOTCH3, NOTCH1 and of the Notch target genes DELTEX1 and c-Myc and abrogated cell viability in both Notch1- and Notch3-dependent T-cell contexts. Notably, re-introduction of exogenous Notch1, Notch3 as well as c-Myc partially rescued cells from anti-growth effects induced by either treatment. Overall our findings indicate JMJD3 and p300 as general Notch1 and Notch3 signaling co-activators in T-ALL and suggest further investigation on the potential therapeutic anti-leukemic efficacy of their enzymatic inhibition in Notch/c-Myc axis-related cancers and diseases.