Publications

Oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) employs a biphasic life cycle consisting of latency and lytic replication to achieve lifelong infection. Despite its essential role in KSHV persistence and tumorigenicity, much remains unknown about how KSHV lytic reactivation is regulated. Leveraging high-throughput transcriptomics, we identify microRNA-31-5p (miRNA-31-5p) as a key regulator of KSHV lytic reactivation, capable of modulating expression of KSHV lytic genes and progression through the lytic cascade. Mechanistically, miR-31-5p controls the KSHV lytic switch by regulating expression of the RNA-binding protein KHDRBS3. miR-31-5p-mediated KHDRBS3 repression results in decreased nascent transcription of crucial viral lytic genes including the main KSHV transcription factor RTA. We characterize KHDRBS3 as a major host factor that is critical for KSHV lytic replication and uncover its key role in KSHV lytic gene transcription. Our results highlight a pivotal role for the miR-31-5p/KHDRBS3 axis in modulating KSHV reactivation and provide insights into gene-expression regulation of lytic KSHV.

Cutaneous melanoma, a highly aggressive and therapy-resistant skin cancer, is characterized by its remarkable cellular plasticity, enabling tumour cells to switch between different phenotypic states. This plasticity contributes to tumour heterogeneity and is regulated by key transcription factors. Long non-coding RNAs (lncRNAs) are emerging as crucial regulators in melanoma progression, yet much remains to be explored regarding their role in phenotype switching. In this study, we analysed long non-coding RNAs (lncRNAs) across different murine melanoma cell lines, identifying a set of lncRNAs potentially involved in regulating melanoma phenotypic state through cis-regulation of neighbouring protein-coding genes. We demonstrated that the lncRNA Dlx4os regulates genes associated with melanoma plasticity, favouring a mesenchymal-like, undifferentiated state. Dlx4os knockdown redirected melanoma cells to a more differentiated and less malignant phenotype, confirmed by differential expression of phenotypic state markers (Sox10, Mitf, Tgfβ, Sox6, Mlana), reduced their invasive and migratory potential, and delayed tumour progression in vivo. Furthermore, we identified a human orthologue of Dlx4os. Our findings highlight the potential of Dlx4os as both a biomarker and therapeutic target, capable of modulating melanoma's phenotypic plasticity to influence treatment response and metastasis.

Peptide nucleic acid (PNA) is a synthetic mimic of DNA where the deoxyribose-phosphodiester backbone is replaced with N-(2-aminoethyl) glycine units. The lack of deoxyribose-phosphodiester bonds enhances enzymatic stability and improves binding affinity of PNA with complementary DNA and RNA strands. To enhance target binding, conformational stability, and pharmacological activity, several chemical modifications have been introduced into PNA. Modified PNAs have demonstrated promising preclinical potential as antisense and anti-gene agents, supporting their use in diverse biomedical applications. The limited in vivo biodistribution and cellular uptake of PNA have significantly hindered its clinical development. Enhancing PNA biodistribution using nanoformulations and bioconjugate-based delivery strategies has resulted in substantial in vivo pharmacological effects. Further, with advancements in chemistry and delivery techniques, PNA holds promise in treating genetic diseases, metabolic disorders, cancers, and infectious diseases. This review summarizes PNA's pharmacological mechanisms, chemical modifications, delivery strategies, and therapeutic applications while addressing limitations for clinical translation.

The human prefrontal cortex (PFC), whose laminar organization is essential for cognitive function, is among the first regions to show age-related functional decline1,2. Single-cell sequencing studies revealed cell type-dependent aging effects but lacked spatial specificity3-6. Spatial transcriptomics (ST) advanced our molecular understanding of the human PFC7, yet whether aging-driven changes differ across PFC layers remains unclear. Here, we performed whole-transcriptome ST on postmortem PFC from 37 individuals across the adult lifespan. We mapped cortical layers and revealed aging mechanisms across layers. This represents one of the largest and most comprehensive lifespan ST analysis of the human PFC brain, offering crucial insight into how the brain ages and identifying potential molecular targets to mitigate cognitive aging and extend healthspan.

BRCA1/2 -mutated breast cancers exhibit homologous recombination deficiency (HRD), making them initially sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. However, 40-70% of patients develop resistance, necessitating combination strategies and predictive biomarkers. We first investigated approaches to overcome PARP resistance and then explored spatial microRNA (miRNA) profiling as a prognostic tool. Using the K14-Cre Brca1 f/f Trp53 f/f model with tumors that acquired PARP resistance, we evaluated PARP inhibitor combinations with either PI3K inhibition or Poly(I:C) in vivo . Both combinations improved antitumor activity compared to PARP inhibition alone. Next, to predict resistance we applied a sensitive assay that quantifies and spatially profiles miRNA expression in situ onto FFPE sections from tumors treated for 10 days using nanoliter well arrays with functionalized hydrogel posts. We developed a spatial miRNA analysis framework integrating latent Dirichlet allocation (LDA) and principal component analysis (PCA) to develop "topics" that stratify early tumors as either PARP inhibitor-sensitive or - resistant and distinguish their treatment regimens. We also incorporated immune architecture using Structural Similarity Index Measure (SSIM) maps that revealed co-localization of immune infiltration and miRNA topics. This integrative approach highlights how miRNA-based spatial analysis can predict PARP inhibitor resistance and provide a promising biomarker to inform therapeutic strategies for BRCA1/2- related breast cancers.

Increasing lifespans make health problems in the elderly such as opioid misuse a more prominent concern. Understanding the effects that opioids may have on the aged brain can help us address age-related concerns of opioid exposure. This study aimed to assess potential interactions between aging and opioid exposure. Three-month-old (young adult) and 19-month-old (aged) C57BL/6JN mice were assigned to either a morphine (3 mg/kg, i.p.) or saline group. A conditioned placed preference (CPP) task was used to assess reward sensitivity, while rotarod and beam walk tests were used to assess sensorimotor coordination. To assess for potential age-dependent effects of morphine on gene expression, we performed RNA sequencing in the prefrontal cortex (PFC). We found that morphine induced CPP in both age groups. Our results indicate impaired motor coordination in aged mice; however, morphine did not significantly affect motor coordination in either age group, although a trend toward an increased number of slips was observed in morphine-treated aged mice. Transcriptomic analysis revealed more robust effects of morphine on gene expression in the aged brain compared to the young brain. Interestingly, we found limited overlap between morphine-regulated genes in young and old mice, suggesting that the molecular effects of morphine are age-dependent. Taken together, while we found no significant interactions between morphine (at the tested dose) and aging in the behavioral assays, morphine caused age-dependent gene expression changes. Our findings suggest that age should be considered when prescribing opioids and that age-specific therapeutics may help address opioid use disorder in the elderly.

Metastasis is the leading cause of cancer related deaths, however therapies specifically targeting metastasis are lacking and remain a dire therapeutic need in the clinic. Metastasis is a highly inefficient process that is inhibited by extracellular stress. Therefore, metastasizing cells that ultimately survive and successfully colonize distant organs must undergo molecular rewiring to mitigate stress. Wobble uridine modifications, especially 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U 34 ), have been implicated in stress response and poor prognosis of cancer patients. We use a patient derived xenograft (PDX) model of melanoma metastasis to study the role of the mcm 5 s 2 U 34 modification in the stress response of metastasizing cells. We find that upon depletion of elongator acetyltransferase complex subunit 1 (ELP1)- a component of the mcm 5 s 2 U 34 pathway on , and -codon-biased translation, migration, invasion, and metastatic burden in vivo is reduced. Further, we observe that stress granule components are enriched in a subset of codon-biased genes that are exclusively upregulated at the protein level in metastatic nodules compared to the primary tumor in our PDX model. Additionally, upon knockdown of ELP1, stress granule components have decreased protein expression with no significant change to their mRNA levels. Efficient translation, mediated by the carboxy-methylation arm of the mcm 5 s 2 U 34 modification, is required for metastasizing cancer cells to withstand stress via stress granule formation and increase survival throughout the metastatic cascade. This makes the mcm 5 s 2 U 34 machinery a potentially actionable therapeutic target, specific to metastatic disease.

Oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma, employs a biphasic life cycle consisting of latency and lytic replication to achieve lifelong infection. Despite its essential role in KSHV persistence and tumorigenicity, much remains unknown about how KSHV lytic reactivation is regulated. Leveraging high-throughput transcriptomics, we identify microRNA-31-5p (miR-31-5p) as a key regulator of KSHV lytic reactivation capable of restricting KSHV entry into the lytic replication cycle. Ectopic expression of miR-31-5p impairs KSHV lytic gene transcription and production of lytic viral proteins, culminating in dramatic reduction of infectious virion production during KSHV reactivation. miR-31-5p overexpression also markedly reduces the expression of critical viral early genes, including the master regulator of the latent-lytic switch, KSHV replication and transcription activator (RTA) protein. Through mechanistic studies, we demonstrate that miR-31-5p represses KSHV lytic reactivation by directly targeting the KH domain protein KHDRBS3, an RNA-binding protein known to regulate RNA processing including alternative splicing. Our study highlights KHDRBS3 as an essential proviral host factor that is key to the successful completion of KSHV lytic replication and suggests its novel function in viral lytic gene transcription during KSHV reactivation. Taken together, these findings reveal a previously unrecognized role for the miR-31-5p/KHDRBS3 axis in regulating the KSHV latency-lytic replication switch and provide insights into gene expression regulation of lytic KSHV, which may be leveraged for lytic cycle-targeted therapeutic strategies against KSHV-associated malignancies.

Nucleic acids are a class of drugs that can modulate gene and protein expression by various mechanisms, namely, RNAi, mRNA degradation by RNase H cleavage, splice modulation, and steric blocking of protein binding or mRNA translation, thus exhibiting immense potential to treat various genetic and rare diseases. Unlike protein-targeted therapeutics, the clinical use of nucleic acids relies on Watson-Crick sequence recognition to regulate aberrant gene expression and impede protein translation. Though promising, targeted delivery remains a bottleneck for the clinical adoption of nucleic acid-based therapeutics. To overcome the delivery challenges associated with nucleic acids, various chemical modifications and bioconjugation-based delivery strategies have been explored. Currently, liver targeting by N-acetyl galactosamine (GalNAc) conjugation has been at the forefront for the treatment of rare and various metabolic diseases, which has led to FDA approval of four nucleic acid drugs. In addition, various other bioconjugation strategies have been explored to facilitate active organ and cell-enriched targeting. This review briefly covers the different classes of nucleic acids, their mechanisms of action, and their challenges. We also elaborate on recent advances in bioconjugation strategies in developing a diverse set of ligands for targeted delivery of nucleic acid drugs.

MicroRNAs (miRNAs) have been implicated in human disorders, from cancers to infectious diseases. Targeting miRNAs or their target genes with small molecules offers opportunities to modulate dysregulated cellular processes linked to diseases. Yet, predicting small molecules associated with miRNAs remains challenging due to the small size of small molecule-miRNA datasets. Herein, we develop a generalized deep learning framework, sChemNET, for predicting small molecules affecting miRNA bioactivity based on chemical structure and sequence information. sChemNET overcomes the limitation of sparse chemical information by an objective function that allows the neural network to learn chemical space from a large body of chemical structures yet unknown to affect miRNAs. We experimentally validated small molecules predicted to act on miR-451 or its targets and tested their role in erythrocyte maturation during zebrafish embryogenesis. We also tested small molecules targeting the miR-181 network and other miRNAs using in-vitro and in-vivo experiments. We demonstrate that our machine-learning framework can predict bioactive small molecules targeting miRNAs or their targets in humans and other mammalian organisms.