mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.

Gene regulatory programs in distinct cell types are maintained in large part through the cell-type-specific binding of transcription factors (TFs). The determinants of TF binding include direct DNA sequence preferences, DNA sequence preferences of cofactors, and the local cell-dependent chromatin context. To explore the contribution of DNA sequence signal, histone modifications, and DNase accessibility to cell-type-specific binding, we analyzed 286 ChIP-seq experiments performed by the ENCODE Consortium. This analysis included experiments for 67 transcriptional regulators, 15 of which were profiled in both the GM12878 (lymphoblastoid) and K562 (erythroleukemic) human hematopoietic cell lines. To model TF-bound regions, we trained support vector machines (SVMs) that use flexible k-mer patterns to capture DNA sequence signals more accurately than traditional motif approaches. In addition, we trained SVM spatial chromatin signatures to model local histone modifications and DNase accessibility, obtaining significantly more accurate TF occupancy predictions than simpler approaches. Consistent with previous studies, we find that DNase accessibility can explain cellline-specific binding for many factors. However, we also find that of the 10 factors with prominent cell-type-specific binding patterns, four display distinct cell-type-specific DNA sequence preferences according to our models. Moreover, for two factors we identify cell-specific binding sites that are accessible in both cell types but bound only in one. For these sites, cell-type-specific sequence models, rather than DNase accessibility, are better able to explain differential binding. Our results suggest that using a single motif for each TF and filtering for chromatin accessible loci is not always sufficient to accurately account for cell-type-specific binding profiles.

Anti-tumor immunity is driven by self vs. non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole genome sequencing (WGS) and RNA sequencing (RNA-seq), we identified a novel gene fusion, and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration, and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens, and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.

Objective In this study, we sought to refine histologic scoring of rheumatoid arthritis (RA) synovial tissue by training with gene expression data and machine learning. Methods Twenty histologic features were assessed in 129 synovial tissue samples (n = 123 RA patients and n = 6 osteoarthritis [OA] patients). Consensus clustering was performed on gene expression data from a subset of 45 synovial samples. Support vector machine learning was used to predict gene expression subtypes, using histologic data as the input. Corresponding clinical data were compared across subtypes. Results Consensus clustering of gene expression data revealed 3 distinct synovial subtypes, including a high inflammatory subtype characterized by extensive infiltration of leukocytes, a low inflammatory subtype characterized by enrichment in pathways including transforming growth factor β, glycoproteins, and neuronal genes, and a mixed subtype. Machine learning applied to histologic features, with gene expression subtypes serving as labels, generated an algorithm for the scoring of histologic features. Patients with the high inflammatory synovial subtype exhibited higher levels of markers of systemic inflammation and autoantibodies. C‐reactive protein (CRP) levels were significantly correlated with the severity of pain in the high inflammatory subgroup but not in the others. Conclusion Gene expression analysis of RA and OA synovial tissue revealed 3 distinct synovial subtypes. These labels were used to generate a histologic scoring algorithm in which the histologic scores were found to be associated with parameters of systemic inflammation, including the erythrocyte sedimentation rate, CRP level, and autoantibody levels. Comparison of gene expression patterns to clinical features revealed a potentially clinically important distinction: mechanisms of pain may differ in patients with different synovial subtypes.

CTNNB1 mutations or APC abnormalities have been observed in ~85% of desmoids examined by Sanger sequencing and are associated with Wnt/β-catenin activation. We sought to identify molecular aberrations in 'wild-type' tumors (those without CTNNB1 or APC alteration) and to determine their prognostic relevance. CTNNB1 was examined by Sanger sequencing in 117 desmoids; a mutation was observed in 101 (86%) and 16 were 'wild-type'. 'Wild-type' status did not associate with tumor recurrence. Moreover, in unsupervised clustering based on U133A-derived gene expression profiles, 'wild-type' and mutated tumors clustered together. Wholeexome sequencing of eight of the 'wild-type' desmoids revealed that three had a CTNNB1 mutation that had been undetected by Sanger sequencing. The mutation was found in a mean 16% of reads (vs 37% for mutations identified by Sanger). Of the other five 'wild-type' tumors sequenced, two had APC loss, two had chromosome 6 loss, and one had mutation of BMI1. The finding of low-frequency CTNNB1 mutation or APC loss in 'wild-type' desmoids was validated in the remaining eight 'wild-type' desmoids; directed miSeq identified low-frequency CTNNB1 mutation in four and comparative genomic hybridization identified APC loss in one. These results HHS Public Access

Integration of expression, copy number, methylation, and regulatory sequence information identifies miRNAs and transcription factors that drive the global expression changes associated with different glioblastoma subtypes.

Mirtrons are intronic hairpin substrates of the dicing machinery that generate functional microRNAs. In this study, we describe experimental assays that defined the essential requirements for entry of introns into the mirtron pathway. These data informed a bioinformatic screen that effectively identified functional mirtrons from the Drosophila melanogaster transcriptome. These included 17 known and six confident novel mirtrons among the top 51 candidates, and additional candidates had limited read evidence in available small RNA data. Our computational model also proved effective on Caenorhabditis elegans, for which the identification of 14 cloned mirtrons among the top 22 candidates more than tripled the number of validated mirtrons in this species. A few low-scoring introns generated mirtron-like read patterns from atypical RNA structures, but their paucity suggests that relatively few such loci were not captured by our model. Unexpectedly, we uncovered examples of clustered mirtrons in both fly and worm genomes, including a <8-kb region in C. elegans harboring eight distinct mirtrons. Altogether, we demonstrate that discovery of functional mirtrons, unlike canonical miRNAs, is amenable to computational methods independent of evolutionary constraint.[Supplemental material is available for this article. Small RNA data have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). A full list of accession numbers can be found in Supplemental Table S1.]Canonical microRNAs (miRNAs) are ;22-nucleotide (nt) regulatory RNAs derived from inverted repeat transcripts whose biogenesis involves a defined series of processing events (Kim et al. 2009). Primary-miRNA (pri-miRNA) transcripts are first cleaved by the nuclear RNase III enzyme Drosha (also known as RNASEN) to yield pre-miRNA hairpins. Following their cytoplasmic export via exportin 5, pre-miRNAs are cleaved on their terminal loop side by a Dicer-class RNase III enzyme to release miRNA/miRNA* duplexes. One side of the duplex, designated the mature miRNA, is preferentially transferred into an Argonaute protein and guides it to regulate target transcripts. Its partner miRNA* strand is inferred to be preferentially degraded on account of its lower steady-state accumulation, although miRNA* species may still be transferred into Argonaute proteins and have regulatory activities. Since RNase III enzymes typically cleave substrates leaving signature 2-nt 39 overhangs, an appropriate geometry of cloned small RNA duplex ends provides evidence for their transit via a Drosha-Dicer pathway (Ambros et al. 2003;Friedlander et al. 2008;Berezikov et al. 2010;Chiang et al. 2010).Since thousands of miRNAs are now known (Griffiths-Jones et al. 2008), one might presume that sufficient information exists to segregate bona fide miRNA genes from bulk genomic hairpins. Although bioinformatic strategies can enrich for genuine miRNA genes, the number of plausible pri-miRNA hairpins in a typical animal genome exceeds the number of confirmed miRNA hairpins by se...

Myxofibrosarcoma is a common mesenchymal malignancy with complex genomics and heterogeneous clinical outcomes. Through gene-expression profiling of 64 primary high-grade myxofibrosarcomas, we defined an expression signature associated with clinical outcome. The gene most significantly associated with disease-specific death and distant metastasis was ITGA10 (integrin-α10). Functional studies revealed that myxofibrosarcoma cells strongly depended on integrin-α10, whereas normal mesenchymal cells did not. Integrin-α10 transmitted its tumor-specific signal via TRIO and RICTOR, two oncoproteins that are frequently co-overexpressed through gene amplification on chromosome 5p. TRIO and RICTOR activated RAC/PAK and AKT/mTOR to promote sarcoma cell survival. Inhibition of these proteins with EHop-016 (RAC inhibitor) and INK128 (mTOR inhibitor) had anti-tumor effects in tumor-derived cell lines and mouse xenografts, and combining the drugs enhanced the effects. Our results demonstrate the importance of integrin-α10/TRIO/RICTOR signaling for driving myxofibrosarcoma progression and provide the basis for promising targeted treatment strategies for patients with high-risk disease.