Human papilloma virus circulating tumor DNA assay predicts treatment response in recurrent/metastatic head and neck squamous cell carcinoma.

Despite the rising incidence of human papillomavirus related (HPV+) oropharyngeal squamous cell carcinoma (OPSCC), treatment of metastatic disease remains palliative. Even with new treatments such as immunotherapy, response rates are low and can be delayed, while even mild tumor progression in the face of an ineffective therapy can lead to rapid death. Real-time biomarkers of response to therapy could improve outcomes by guiding early change of therapy in the metastatic setting. Herein, we developed and analytically validated a new droplet digital PCR (ddPCR)-based assay for HPV16 circulating tumor DNA (ctDNA) and evaluated plasma HPV16 ctDNA for predicting treatment response in metastatic HPV+ OPSCC. We found that longitudinal changes HPV16 ctDNA correlate with treatment response and that ctDNA responses are observed earlier than conventional imaging (average 70 days, range: 35-166). With additional validation in multi-site studies, this assay may enable early identification of treatment failure, allowing patients to be directed promptly toward clinical trials or alternative therapies.

Phospho-RNA-seq: a modified small RNA-seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma.

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non-coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA-seq protocols due to lack of 5' phosphate or presence of 3' phosphate. These fragments were revealed using a modified protocol ("phospho-RNA-seq") incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5' and 3' ends, as well as human plasma exRNA Analyzing phospho-RNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow- and liver-enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-RNA-seq opens up new possibilities for plasma transcriptomic biomarker development.

The long non-coding RNA NEAT1 is elevated in polyglutamine repeat expansion diseases and protects from disease gene-dependent toxicities.

Polyglutamine (polyQ) repeat diseases are a class of neurodegenerative disorders caused by CAG-repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1 (Neat1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7 and Huntington's disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock-in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored Neat1L to normal levels. In additional studies, we found that Neat1L is repressed by methyl CpG binding protein 2 (MeCP2) by RNA-protein interaction but not by occupancy of MeCP2 at its promoter. We also found that NEAT1L overexpression protects from mHTT-induced cytotoxicity, while reducing it enhanced mHTT-dependent toxicity. Gene set enrichment analysis of previously published RNA sequencing data from mouse embryonic fibroblasts and cells derived from HD patients shows that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD-induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that it may play a protective role in CAG-repeat expansion diseases.

Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics.

RNA interference (RNAi) is a major, powerful platform for gene perturbations, but is restricted by off-target mechanisms. Communication between RNAs, small RNAs, and RNA-binding proteins (RBPs) is a pervasive feature of cellular RNA networks. We present a crosstalk scenario, designated as crosstalk with endogenous RBPs' (ceRBP), in which small interfering RNAs or microRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing endogenous RBP activity. Systematic analysis of small interfering RNA (siRNA) off-target data and genome-wide RNAi cancer lethality screens using 501 human cancer cell lines, a cancer dependency map, identified that seed-to-RBP crosstalk is widespread, contributes to off-target activity, and affects RNAi performance. Specifically, deconvolution of the interactions between gene knockdown and seed-mediated silencing effects in the cancer dependency map showed widespread contributions of seed-to-RBP crosstalk to growth-phenotype modulation. These findings suggest a novel aspect of microRNA biology and offer a basis for improvement of RNAi agents and RNAi-based functional genomics.

Human-specific microRNA regulation of FOXO1: implications for microRNA recognition element evolution.

MicroRNAs (miRNAs) have been established as important negative post-transcriptional regulators for gene expression. Within the past decade, miRNAs targeting transcription factors (TFs) has emerged as an important mechanism for gene expression regulation. Here, we tested the hypothesis that in TF 3'UTRs, human-specific single nucleotide change(s) that create novel miRNA recognition elements (MREs) contribute to species-specific differences in TF expression. From several potential human-specific TF MREs, one candidate, a member of the Forkhead Box O (FOXO) subclass in the Forkhead family known as Forkhead Box O1 (FOXO1; FKHR; NM_002015) was tested further. Human FOXO1 contains two sites predicted to confer miR-183-mediated post-transcriptional regulation: one specific to humans and the other conserved. Utilizing dual luciferase expression reporters, we show that only the human FOXO1 3'UTR contains a functional miR-183 site, not found in chimpanzee or mouse 3'untranslated regions (UTRs). Site-directed mutagenesis supports functionality of the human-specific miR-183 site, but not the conserved miR-183 site. Via overexpression and target site protection assays, we show that human FOXO1 is regulated by miR-183, but mouse FOXO1 is not. Finally, FOXO1-regulated cellular phenotypes, including cell invasion and proliferation, are impacted by miR-183 targeting only in human cells. These results provide strong evidence for human-specific gain of TF MREs, a process that may underlie evolutionary differences between phylogenic groups.

Rational design of therapeutic siRNAs: minimizing off-targeting potential to improve the safety of RNAi therapy for Huntington's disease.

RNA interference (RNAi) provides an approach for the treatment of many human diseases. However, the safety of RNAi-based therapies can be hampered by the ability of small inhibitory RNAs (siRNAs) to bind to unintended mRNAs and reduce their expression, an effect known as off-target gene silencing. Off-targeting primarily occurs when the seed region (nucleotides 2-8 of the small RNA) pairs with sequences in 3'-UTRs of unintended mRNAs and directs translational repression and destabilization of those transcripts. To date, most therapeutic RNAi sequences are selected primarily for gene silencing efficacy, and later evaluated for safety. Here, in designing siRNAs to treat Huntington's disease (HD), a dominant neurodegenerative disorder, we prioritized selection of sequences with minimal off-targeting potentials (i.e., those with a scarcity of seed complements within all known human 3'-UTRs). We identified new promising therapeutic candidate sequences which show potent silencing in cell culture and mouse brain. Furthermore, we present microarray data demonstrating that off-targeting is significantly minimized by using siRNAs that contain "safe" seeds, an important strategy to consider during preclinical development of RNAi-based therapeutics.

Structure and activity of putative intronic miRNA promoters.

MicroRNAs (miRNAs) are RNA sequences of approximately 22 nucleotides that mediate post-transcriptional regulation of specific mRNAs. miRNA sequences are dispersed throughout the genome and are classified as intergenic (between genes) or intronic (embedded into a gene). Intergenic miRNAs are expressed by their own promoter, and until recently, it was supposed that intronic miRNAs are transcribed from their host gene. Here, we performed a genomic analysis of currently known intronic miRNA regions and observed that approximately 35% of intronic miRNAs have upstream regulatory elements consistent with promoter function. Among all intronic miRNAs, 30% have associated Pol II regulatory elements, including transcription start sites, CpG islands, expression sequence tags, and conserved transcription factor binding sites, while 5% contain RNA Pol III regulatory elements (A/B box sequences). We cloned intronic regions encompassing miRNAs and their upstream Pol II (miR-107, miR-126, miR-208b, miR-548f-2, miR-569, and miR-590) or Pol III (miR-566 and miR-128-2) sequences into a promoterless plasmid, and confirmed that miRNA expression occurs independent of host gene transcription. For miR-128-2, a miRNA overexpressed in acute lymphoblastic leukemia, ChIP analysis suggests dual regulation by both intronic (Pol III) and host gene (Pol II) promoters. These data support complex regulation of intronic miRNA expression, and have relevance to disregulation in disease settings.

Adenosine deamination in human transcripts generates novel microRNA binding sites.

Animals regulate gene expression at multiple levels, contributing to the complexity of the proteome. Among these regulatory events are post-transcriptional gene silencing, mediated by small non-coding RNAs (e.g. microRNAs), and adenosine-to-inosine (A-to-I) editing, generated by adenosine deaminases that act on double-stranded RNA (ADAR). Recent data suggest that these regulatory processes are connected at a fundamental level. A-to-I editing can affect Drosha processing or directly alter the microRNA (miRNA) sequences responsible for mRNA targeting. Here, we analyzed the previously reported adenosine deaminations occurring in human cDNAs, and asked if there was a relationship between A-to-I editing events in the mRNA 3' untranslated regions (UTRs) and mRNA:miRNA binding. We find significant correlations between A-to-I editing and changes in miRNA complementarities. In all, over 3000 of the 12 723 distinct adenosine deaminations assessed were found to form 7-mer complementarities (known as seed matches) to a subset of human miRNAs. In 200 of the ESTs, we also noted editing within a specific 13 nucleotide motif. Strikingly, deamination of this motif simultaneously creates seed matches to three (otherwise unrelated) miRNAs. Our results suggest the creation of miRNA regulatory sites as a novel function for ADAR activity. Consequently, many miRNA target sites may only be identifiable through examining expressed sequences.

Rational design leads to more potent RNA interference against hepatitis B virus: factors effecting silencing efficiency.

RNA interference (RNAi) can be an effective antiviral agent; however, overexpression of RNAi can be toxic through competition with the endogenous microRNA (miRNA) machinery. We used rational design to identify highly potent RNAi that is effective at nontoxic doses. A statistical analysis was conducted to pinpoint thermodynamic characteristics correlated with activity. Sequences were selected that conformed to a consensus internal stability profile (ISP) associated with active RNAi, and RNAi triggers were expressed in the context of an endogenous miRNA. These approaches yielded highly active hepatitis B virus (HBV) RNAi. A statistical analysis found a correlation between activity and nucleation by binding within the seed sequence to accessible regions in the target RNA. Guide strands were selected for favorable strand biasing, but increased strand biasing did not correlate with potency, suggesting a threshold effect. Exogenous short hairpin RNAs (shRNAs), but not miRNAs were previously reported to compete with miRNAs for the miRNA/RNAi machinery. In contrast, we show that exogenous Polymerase III- but not Polymerase II-driven miRNAs compete with exogenous miRNAs, at multiple steps in the miRNA pathway. Exogenous miRNAs also compete with endogenous miR-21. Thus, competition with endogenous miRNAs should be monitored even when using miRNA-based therapeutics. However, potent silencing was achieved at doses where competition was not observed.