Publications by Author: Frank J Slack

The landscape of non-coding mutations in cancer progression and immune evasion is largely unexplored. Here, we identify transcrptome-wide somatic and germline 3' untranslated region (3'-UTR) variants from 375 gastric cancer patients from The Cancer Genome Atlas. By performing gene expression quantitative trait loci (eQTL) and immune landscape QTL (ilQTL) analysis, we discover 3'-UTR variants with cis effects on expression and immune landscape phenotypes, such as immune cell infiltration and T cell receptor diversity. Using a massively parallel reporter assay, we distinguish between causal and correlative effects of 3'-UTR eQTLs in immune-related genes. Our approach identifies numerous 3'-UTR eQTLs and ilQTLs, providing a unique resource for the identification of immunotherapeutic targets and biomarkers. A prioritized ilQTL variant signature predicts response to immunotherapy better than standard-of-care PD-L1 expression in independent patient cohorts, showcasing the untapped potential of non-coding mutations in cancer.

Several microRNAs have emerged as regulators of pathways that control aging. For example, miR-228 is required for normal lifespan and dietary restriction (DR) mediated longevity through interaction with PHA-4 and SKN-1 transcription factors in Caenorhabditis elegans. miR-229,64,65, and 66, a cluster of microRNAs located adjacent to each other on chromosome III, are in the same family as miR-228, albeit with slight differences in the miR-228 seed sequence. We demonstrate that, in contrast to the anti-longevity role of miR-228, the miR-229-66 cluster is required for normal C. elegans lifespan and for the longevity observed in mir-228 mutants. miR-229-66 is also critical for lifespan extension observed under DR and reduced insulin signaling (IIS) and by constitutive nuclear SKN-1. Both DR and low-IIS upregulate the expression of the miRNA cluster, which is dependent on transcription factors PHA-4, SKN-1, and DAF-16. In turn, the expression of SKN-1 and DAF-16 requires mir-229,64,65,66. miR-229-66 targets the odd-skipped-related transcription factor, odd-2 to regulate lifespan. Knockdown of odd-2 increases lifespan, suppresses the short lifespan of mir-229,64,65,66(nDf63) III mutants, and alters levels of SKN-1 in the ASI neurons. Together with SKN-1, the miRNA cluster also indirectly regulates several genes in the xenobiotic detoxification pathway which increases wild-type lifespan and significantly rescues the short lifespan of mir-229,64,65,66(nDf63) III mutants. Thus, by interacting with SKN-1, miR-229-66 transduces the effects of DR and low-IIS in lifespan extension in C. elegans. Given that this pathway is conserved, it is possible that a similar mechanism regulates aging in more complex organisms.

Aging is associated with the accumulation of damaged and misfolded proteins through a decline in the protein homeostasis (proteostasis) machinery, leading to various age-associated protein misfolding diseases such as Huntington's or Parkinson's. The efficiency of cellular stress response pathways also weakens with age, further contributing to the failure to maintain proteostasis. MicroRNAs (miRNAs or miRs) are a class of small, non-coding RNAs (ncRNAs) that bind target messenger RNAs at their 3'UTR, resulting in the post-transcriptional repression of gene expression. From the discovery of aging roles for lin-4 in C. elegans, the role of numerous miRNAs in controlling the aging process has been uncovered in different organisms. Recent studies have also shown that miRNAs regulate different components of proteostasis machinery as well as cellular response pathways to proteotoxic stress, some of which are very important during aging or in age-related pathologies. Here, we present a review of these findings, highlighting the role of individual miRNAs in age-associated protein folding and degradation across different organisms. We also broadly summarize the relationships between miRNAs and organelle-specific stress response pathways during aging and in various age-associated diseases.

As coronavirus disease 2019 (COVID-19) and aging are both accompanied by cognitive decline, we hypothesized that COVID-19 might lead to molecular signatures similar to aging. We performed whole-transcriptome analysis of the frontal cortex, a critical area for cognitive function, in individuals with COVID-19, age-matched and sex-matched uninfected controls, and uninfected individuals with intensive care unit/ventilator treatment. Our findings indicate that COVID-19 is associated with molecular signatures of brain aging and emphasize the value of neurological follow-up in recovered individuals.

Uveal melanoma (UM) is an intraocular cancer tumor with high metastatic risk. It is considered a rare disease, but 90% of affected patients die within 15 years. Non-coding elements (ncRNAs) such as long non-coding RNAs (lncRNAs) have a crucial role in cellular homeostasis maintenance, taking part in many critical cellular pathways. Their deregulation, therefore, contributes to the induction of cancer and neurodegenerative and metabolic diseases. In cancer, lncRNAs are implicated in apoptosis evasion, proliferation, invasion, drug resistance, and other roles because they affect tumor suppressor genes and oncogenes. For these reasons, lncRNAs are promising targets in personalized medicine and can be used as biomarkers for diseases including UM.

Coronavirus disease 2019 (COVID-19) is predominantly an acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and remains a significant threat to public health. COVID-19 is accompanied by neurological symptoms and cognitive decline, but the molecular mechanisms underlying this effect remain unclear. As aging induces distinct molecular signatures in the brain associated with cognitive decline in healthy populations, we hypothesized that COVID-19 may induce molecular signatures of aging. Here, we performed whole transcriptomic analysis of human frontal cortex, a critical area for cognitive function, in 12 COVID-19 cases and age- and sex-matched uninfected controls. COVID-19 induces profound changes in gene expression, despite the absence of detectable virus in brain tissue. Pathway analysis shows downregulation of genes involved in synaptic function and cognition and upregulation of genes involved in immune processes. Comparison with five independent transcriptomic datasets of aging human frontal cortex reveals striking similarities between aged individuals and severe COVID-19 patients. Critically, individuals below 65 years of age exhibit profound transcriptomic changes not observed among older individuals in our patient cohort. Our data indicate that severe COVID-19 induces molecular signatures of aging in the human brain and emphasize the value of neurological follow-up in recovered individuals.

Cancer immunotherapies targeting the interaction between Programmed death 1 (PD-1) and Programmed death ligand 1 (PD-L1) have recently been approved for the treatment of multiple cancer types, including gastric cancer. However, not all patients respond to these therapies, while some eventually acquire resistance. A partial predictive biomarker for positive response to PD-1/PD-L1 therapy is PD-L1 expression, which has been shown to be under strict post-transcriptional control in cancer. By fractionating the PD-L1 3' untranslated region (3'UTR) into multiple overlapping fragments, we identified a small 100-nucleotide-long cis-acting region as being necessary and sufficient for post-transcriptional repression of PD-L1 expression in gastric cancer. In parallel, we performed a correlation analysis between PD-L1 expression and all host miRNAs in stomach cancer patient samples. A single miRNA, miR-105-5p, was predicted to bind to the identified cis-acting 3'UTR region and to negatively correlate with PD-L1 expression. Overexpression of miR-105-5p in gastric cancer cell lines resulted in decreased expression of PD-L1, both at the total protein and surface expression levels, and induced CD8+ T cell activation in co-culture assays. Finally, we show that expression of miR-105-5p in gastric cancer is partly controlled by DNA methylation of a cancer- and germline-specific promoter of its host gene, GABRA3. Dysregulation of miR-105-5p is observed in many cancer types and this study shows the importance of this miRNA in controlling the immunogenicity of cancer cells, thus highlighting it as a potential biomarker for PD-1/PD-L1 therapy and target for combinatorial immunotherapy.

Selective inhibition of microRNAs (miRNAs) offers a new avenue for cancer therapeutics. While most of the current anti-miRNA (antimiR) reagents target full length miRNAs, here we investigate novel nanoparticle-delivered short PNA probes containing cationic domains targeting the seed region of the miRNA for effective antimiR therapy. For proof of concept, we tested PNAs targeting miRNA-155 and employed poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle formulation for delivery. A comprehensive evaluation of PLGA nanoparticles (NPs) containing short PNA probes showed significantly superior loading, release profile, and uniform size distribution, compared to conventional non-cationic PNA probes. Confocal microscopy and flow cytometry analyses showed efficient transfection efficiency and uniform distribution of PLGA NPs containing short PNA probes in the cytoplasm. Functional analysis also confirmed efficient miRNA-155 inhibition including an effect on its downstream target proteins. Further, reduced tumor growth was observed after systemic delivery of PLGA nanoparticles containing short PNA probes in vivo in a xenograft mouse model following inhibition of miR-155. There was no evidence of acute or chronic toxicity associated with systemic delivery of PLGA NPs containing short PNA probes in the mice. Overall, in this paper we present a novel antimiR strategy based on PLGA nanoparticle delivered short PNA probes for potential cancer therapy.

Gastric cancer (GC) is the fifth most common cancer worldwide. In approximately 10% of GC cases, cancer cells show ubiquitous and monoclonal Epstein-Barr virus (EBV) infection. A significant feature of EBV-associated GC (EBVaGC) is high lymphocytic infiltration and high expression of immune checkpoint proteins, including programmed death-ligand 1 (PD-L1). This highlights EBVaGC as a strong candidate for immune checkpoint blockade therapy. Indeed, several recent studies have shown that EBV positivity in GC correlates with positive response to programmed cell death protein 1 (PD-1)/PD-L1 blockade therapy. Understanding the mechanisms that control PD-L1 expression in EBVaGC can indicate new predictive biomarkers for immunotherapy, as well as therapeutic targets for combination therapy. Various mechanisms have been implicated in PD-L1 expression regulation, including structural variations, post-transcriptional control, oncogenic activation of intrinsic signaling pathways, and increased sensitivity to extrinsic signals. This review provides the most recent updates on the multilayered control of PD-L1 expression in EBVaGC.

Peptide nucleic acids (PNAs) have emerged as one of the most versatile tools with a wide range of biomedical applications including antisense, antimiR, antigene, as well as site-specific gene editing. The application and potential of PNAs has been limited due to low solubility and poor cellular uptake. Several strategies have been employed to overcome the aforementioned challenges like conjugation to cationic peptides or nanotechnology to achieve superior transfection efficiency ex vivo and in vivo. Here, we report a detailed procedure optimized in our lab for synthesis of short cationic PNA probes, which exhibit high purity and yield in comparison to full-length PNA oligomers. We also provide step-by-step details of encapsulating short cationic PNA probes in poly (lactic-co-glycolic acid) nanoparticles by double emulsion solvent evaporation technique. 1.Detailed procedure for synthesis of short cationic PNAs with or without fluorophore (dye) conjugation while ensuring high yield and purity.2.Step-by-step details for encapsulation of short cationic PNAs in PLGA nanoparticles via double emulsion solvent evaporation technique.