Publications

The tumor microenvironment (TME) is a heterogeneous ecosystem containing cancer cells, immune cells, stromal cells, cytokines, and chemokines which together govern tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), a core catalytic subunit for RNA N6-methyladenosine (m6A) modification, plays a crucial role in regulating various physiological and pathological processes. Whether and how METTL3 regulates the TME and anti-tumor immunity in non-small-cell lung cancer (NSCLC) remain poorly understood. Here, we report that METTL3 elevates expression of pro-tumorigenic chemokines including CXCL1, CXCL5, and CCL20, and destabilizes PD-L1 mRNA in an m6A-dependent manner, thereby shaping a non-inflamed TME. Thus, inhibiting METTL3 reprograms a more inflamed TME that renders anti-PD-1 therapy more effective in several murine lung tumor models. Clinically, NSCLC patients who exhibit low-METTL3 expression have a better prognosis when receiving anti-PD-1 therapy. Collectively, our study highlights targeting METTL3 as a promising strategy to improve immunotherapy in NSCLC patients.

Severe COVID-19 leads to widespread transcriptomic changes in the human brain, mimicking diminished cognitive performance. As long noncoding RNAs (lncRNAs) play crucial roles in the regulation of gene expression, identification of the lncRNAs differentially expressed upon COVID-19 may nominate key regulatory nodes underpinning cognitive changes. Here we identify hundreds of lncRNAs differentially expressed in the brains of COVID-19 patients relative to uninfected age/sex-matched controls, many of which are associated with decreased cognitive performance and inflammatory cytokine response. Our analyses reveal pervasive transcriptomic changes in lncRNA expression upon severe COVID-19, which may serve as key regulators of neurocognitive changes in the brain.

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.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of human malignancies. PDAC is characterized by dense fibrous stroma which obstructs drug delivery and plays complex tumor-promoting roles. Photodynamic therapy (PDT) is a light-based modality which has been demonstrated to be clinically feasible and effective for tumors of the pancreas. Here, we use in vitro heterocellular 3D co-culture models in conjunction with imaging, bulk rheology and microrheology to investigate photodegradation of non-cellular components of PDAC stroma (photodynamic stromal depletion, PSD). By measuring the rheology of extracellular matrix (ECM) before and after PDT we find that softening of ECM is concomitant with increased transport of nanoparticles (NPs). At the same time, as shown by us previously, photodestruction of stromal fibroblasts leads to enhanced tumor response to PDT. Here we specifically evaluate the capability of PSD to enhance RNA nanomedicine delivery, using a NP carrying an inhibitor of miR-21-5P, a PDAC oncomiR. We confirm improved delivery of this therapeutic NP after PSD by observation of increased expression of PDCD4, a protein target of miR-21-5P. Collectively, these results in 3D tumor models suggest that PSD could be developed to enhance delivery of other cancer therapeutics and improve tumor response to treatment.

RNA therapeutics have emerged as next-generation therapy for the treatment of many diseases. Unlike small molecules, RNA targeted drugs are not limited by the availability of binding pockets on the protein, but rather utilize Watson-Crick (WC) base-pairing rules to recognize the target RNA and modulate gene expression. Antisense oligonucleotides (ASOs) present a powerful therapeutic approach to treat disorders triggered by genetic alterations. ASOs recognize the cognate site on the target RNA to alter gene expression. Nine single-stranded ASOs have been approved for clinical use and several candidates are in late-stage clinical trials for both rare and common diseases. Several chemical modifications, including phosphorothioates, locked nucleic acid, phosphorodiamidate, morpholino, and peptide nucleic acids (PNAs), have been investigated for efficient RNA targeting. PNAs are synthetic DNA mimics where the deoxyribose phosphate backbone is replaced by N-(2-aminoethyl)-glycine units. The neutral pseudopeptide backbone of PNAs contributes to enhanced binding affinity and high biological stability. PNAs hybridize with the complementary site in the target RNA and act by a steric hindrance–based mechanism. In the last three decades, various PNA designs, chemical modifications, and delivery strategies have been explored to demonstrate their potential as an effective and safe RNA-targeting platform. This review covers the advances in PNA-mediated targeting of coding and noncoding RNAs for a myriad of therapeutic applications.

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.

Circular RNAs (circRNA) are a recently described class of RNA molecules that have attracted substantial attention as new components of disease mechanisms and as potential biomarkers in multiple diseases, including cancer. CircRNAs are often highly conserved and exhibit developmental stage- and disease-specific expression. Several studies have reported circRNA expression patterns that are associated with specific cancer types and with patient prognosis. Here, we overview the active registered clinical trials that investigate the value of circRNAs as cancer biomarkers and discuss the potential of circRNAs in clinical cancer care. Taken together, circRNAs are actively being investigated as diagnostic, predictive, and prognostic biomarkers, and their potential to serve as therapeutic intervention points motivates ongoing translational and clinical research.

The Keystone Symposium 'Small Regulatory RNAs: From Bench to Bedside' was held in Santa Fe, New Mexico from May 1-4, 2022. The symposium was organized by Frank J. Slack, Jörg Vogel, Ivan Martinez and Karyn Schmidt, and brought together scientists working in noncoding RNA biology, therapeutics, and technologies to address mechanistic questions about small regulatory RNAs and facilitate translation of these findings into clinical applications. The conference addressed four specific aims: Aim 1. Focus on the exciting biology of small regulatory RNAs, highlighting the best current research into the role that small RNAs play in fundamental biological processes; Aim 2. Focus on the latest efforts to harness the power of these RNAs as agents in the fight against disease and provide the basic understanding that will drive the invention of powerful clinical tools; Aim 3. Attract leaders from both academia and industry working in small RNAs to one place for critical discussions that will advance the field and accelerate the bench to bedside use of this technology; Aim 4. Provide a stimulating environment where students, postdoctoral researchers and junior investigators, along with scientists from Biotechnology and Pharmaceutical companies specializing in small regulatory RNAs, can present and discuss their research with the best minds in the field.

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.

Many diseases, especially cancer, are caused by the abnormal expression of non-coding microRNAs (miRNAs), which regulate gene expression, leading to the development of miRNA-based therapeutics. Synthetic miRNA inhibitors have shown promising efficacy in blocking the activity of aberrant miRNAs that are upregulated in disease-specific pathologies. On the other hand, miRNAs that aid in preventing certain diseases and are reduced in expression in the disease state need different strategies. To tackle this, miRNA mimics, which mimic the activity of endogenous miRNAs, can be delivered for those miRNAs downregulated in different disease states. However, the delivery of miRNA mimics remains a challenge. Here, we report a cationic polylactic-co-glycolic acid (PLGA)-poly-L-histidine delivery system to deliver miRNA mimics. We chose miR-34a mimics as a proof of concept for miRNA delivery. miR-34a-loaded PLGA-poly-L-histidine nanoparticles (NPs) were formulated and biophysically characterized to analyze the structural properties of miRNA mimic-loaded NPs. In vitro efficacy was determined by investigating miR-34a and downstream target levels and performing cell viability and apoptosis assays. We confirmed in vivo efficacy through prolonged survival of miR-34a NP-treated A549-derived xenograft mice treated intratumorally. The results of these studies establish PLGA-poly-L-histidine NPs as an effective delivery system for miRNA mimics for treating diseases characterized by downregulated miRNAs.