The microdissected gene expression landscape of nasopharyngeal cancer reveals vulnerabilities in FGF and noncanonical NF-κB signaling.

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-positive epithelial malignancy with an extensive inflammatory infiltrate. Traditional RNA-sequencing techniques uncovered only microenvironment signatures, while the gene expression of the tumor epithelial compartment has remained a mystery. Here, we use Smart-3SEQ to prepare transcriptome-wide gene expression profiles from microdissected NPC tumors, dysplasia, and normal controls. We describe changes in biological pathways across the normal to tumor spectrum and show that fibroblast growth factor (FGF) ligands are overexpressed in NPC tumors, while negative regulators of FGF signaling, including SPRY1, SPRY2, and LGALS3, are down-regulated early in carcinogenesis. Within the NF-κB signaling pathway, the critical noncanonical transcription factors, RELB and NFKB2, are enriched in the majority of NPC tumors. We confirm the responsiveness of EBV-positive NPC cell lines to targeted inhibition of these pathways, reflecting the heterogeneity in NPC patient tumors. Our data comprehensively describe the gene expression landscape of NPC and unravel the mysteries of receptor tyrosine kinase and NF-κB pathways in NPC.

Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ.

RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.

Genomic analysis of benign prostatic hyperplasia implicates cellular re-landscaping in disease pathogenesis.

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.

Transcription-factor occupancy at HOT regions quantitatively predicts RNA polymerase recruitment in five human cell lines.

BACKGROUND: High-occupancy target (HOT) regions are compact genome loci occupied by many different transcription factors (TFs). HOT regions were initially defined in invertebrate model organisms, and we here show that they are a ubiquitous feature of the human gene-regulation landscape. RESULTS: We identified HOT regions by a comprehensive analysis of ChIP-seq data from 96 DNA-associated proteins in 5 human cell lines. Most HOT regions co-localize with RNA polymerase II binding sites, but many are not near the promoters of annotated genes. At HOT promoters, TF occupancy is strongly predictive of transcription preinitiation complex recruitment and moderately predictive of initiating Pol II recruitment, but only weakly predictive of elongating Pol II and RNA transcript abundance. TF occupancy varies quantitatively within human HOT regions; we used this variation to discover novel associations between TFs. The sequence motif associated with any given TF's direct DNA binding is somewhat predictive of its empirical occupancy, but a great deal of occupancy occurs at sites without the TF's motif, implying indirect recruitment by another TF whose motif is present. CONCLUSIONS: Mammalian HOT regions are regulatory hubs that integrate the signals from diverse regulatory pathways to quantitatively tune the promoter for RNA polymerase II recruitment.

Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers.

BACKGROUND: Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported. RESULTS: We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker. CONCLUSIONS: This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research.

Preexisting immunity and low expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy.

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.

Evaluation of systemic follistatin as an adjuvant to stimulate muscle repair and improve motor function in Pompe mice.

Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.

3'-end sequencing for expression quantification (3SEQ) from archival tumor samples.

Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET). Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3'-end sequencing for expression quantification (3SEQ)) for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF) and solitary fibrous tumor (SFT)) (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations). Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01) on both the frozen tissue (approximately 9.6K genes) and FFPET (approximately 8.1K genes). Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (approximately 4.64K), and analysis of microarray data on FFPET revealed very few (69) differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research.

Combination brain and systemic injections of AAV provide maximal functional and survival benefits in the Niemann-Pick mouse.

Niemann-Pick disease (NPD) is caused by the loss of acid sphingomyelinase (ASM) activity, which results in widespread accumulation of undegraded lipids in cells of the viscera and CNS. In this study, we tested the effect of combination brain and systemic injections of recombinant adeno-associated viral vectors encoding human ASM (hASM) in a mouse model of NPD. Animals treated by combination therapy exhibited high levels of hASM in the viscera and brain, which resulted in near-complete correction of storage throughout the body. This global reversal of pathology translated to normal weight gain and superior recovery of motor and cognitive functions compared to animals treated by either brain or systemic injection alone. Furthermore, animals in the combination group did not generate antibodies to hASM, demonstrating the first application of systemic-mediated tolerization to improve the efficacy of brain injections. All of the animals treated by combination therapy survived in good health to an investigator-selected 54 weeks, whereas the median lifespans of the systemic-alone, brain-alone, or untreated ASM knockout groups were 47, 48, and 34 weeks, respectively. These data demonstrate that combination therapy is a promising therapeutic modality for treating NPD and suggest a potential strategy for treating disease indications that cause both visceral and CNS pathologies.

Fas ligand-induced murine pulmonary inflammation is reduced by a stable decoy receptor 3 analogue.

Fas ligand (FasL)-induced lung inflammation has recently been suggested to play an important role in the pathogenesis of acute respiratory disease syndrome (ARDS). In order to further explore this connection, we established a FasL-induced murine model of pulmonary inflammation. Instillation of recombinant FasL (rFasL) into the lung induced neutrophil infiltration and increased pulmonary permeability, as evidenced by increased total protein in the airspace; both occur in patients with ARDS. These effects were accompanied with a rapid induction of proinflammatory mediators: cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and the chemokines macrophage inflammatory protein-2 (MIP-2) and KC. Pretreatment with a FasL antagonist, a decoy receptor 3 analogue (DcR3 analogue), reduced neutrophil infiltration into the airspace and resulted in a highly significant reduction in the levels of GM-CSF, MIP-2 and KC in bronchoalveolar lavage (BAL) fluid. We postulate that rFasL may be responsible for induction of proinflammatory chemokines and cytokines in the lung, which in turn attract neutrophil infiltration into the airspace. This proinflammatory process and the associated pulmonary permeability may, in part, explain the association of FasL with severe pulmonary inflammation, such as ARDS, and shed new light on FasL and its role in lung injury.