Exploring the prognostic value of circular RNAs in pancreatic ductal adenocarcinoma using genome-wide expression profiling.

BACKGROUND/OBJECTIVES: Median survival of pancreatic ductal adenocarcinoma (PDAC) is around eight months and new prognostic tools are needed. Circular RNAs (circRNAs) have gained interest in different types of cancer. However, only a few studies have evaluated their potential in PDAC. We aimed to identify the most differentially expressed circRNAs in PDAC compared to controls and to explore their potential as prognostic markers. METHODS: Using frozen specimens with PDAC and controls, we performed RNA sequencing and identified 20,440 unique circRNAs. A custom code set of capture- and reporter probes for NanoString nCounter analysis was designed to target 152 circRNAs, based on abundancy, differential expression and a literature study. Expression of these 152 circRNAs was examined in 108 formalin-fixed and paraffin-embedded surgical PDAC specimens and controls. The spatial expression of one of the most promising candidates, ciRS-7 (hsa_circ_0001946), was evaluated by chromogenic in situ hybridization (CISH) using multi-punch tissue microarrays (TMAs) and digital imaging analysis. RESULTS: Based on circRNA expression profiles, we identified different PDAC subclusters. The 30 most differentially expressed circRNAs showed log2 fold changes from -3.43 to 0.94, where circNRIP1 (hsa_circ_0004771), circMBOAT2 (hsa_circ_0007334) and circRUNX1 (hsa_circ_0002360) held significant prognostic value in multivariate analysis. CiRS-7 was absent in PDAC cells but highly expressed in the tumor microenvironment. CONCLUSIONS: We identified several new circRNAs with biomarker potential in surgically treated PDAC, three of which showed an independent prognostic value. We also found that ciRS-7 is absent in cancer cells but abundant in tumor microenvironment and may hold potential as marker of activated stroma.

circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Profiling of circRNAs using an enzyme-free digital counting method.

Circular RNAs (circRNAs) have recently gained substantial attention as potential biomarkers in several human diseases, most prominently in cancer. However, the detection and quantification of circRNA biomarkers pose specific challenges owing to their circular nature. In particular, the use of enzymes, such as reverse transcriptases, may lead to impaired quantification, poor interlaboratory reproducibility and even false-positive results. Therefore, the development of methods for accurate quantification of circRNA biomarkers is a high priority. An enzyme-free and digital quantification method, named NanoString nCounter, was recently adapted for circRNA detection. In this review, I describe the advantages of using this technology for circRNA quantification as well as methodological considerations, potential pitfalls and disadvantages.

Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies.

Circular RNAs (circRNAs) are covalently closed endogenous molecules with tissue- and disease-specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The molecules are formed by a backsplicing event linking the 3'-end of an exon to the 5'-end of the same or an upstream exon, and they exert diverse regulatory functions important in carcinogenesis. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Here, we demonstrate that circRNAs can be accurately quantified without enzymatic reactions or bias using color-coded probes (NanoString technology). First, we performed high-throughput RNA sequencing (RNA-seq) of several mantle cell lymphoma and multiple myeloma cell lines to profile the genome-wide landscape of circRNA expression. We detected several circRNAs known to be deregulated in other cancers and identified a novel circRNA from the IKZF3 gene. Based on these data, we selected 52 unique circRNAs for which we designed color-coded probes spanning their specific backsplicing junctions. These circRNAs were quantified in cell lines and patient samples from several different B-cell malignancies (mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia) simultaneously using the NanoString technology. The circRNA expression profiles obtained could distinguish different B-cell malignancies, and confirmed the presence of the novel circRNA derived from IKZF3. The NanoString assays were specific for circRNA detection and data were more reproducible and quantitatively more accurate than RNA-seq data. In addition, we obtained high-quality data on severely degraded RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. Together, we provide a map of circRNA expression in B-cell malignancies and present an enzyme-free digital counting methodology, which has the potential to become a new gold standard for circRNA quantification.

Circular RNAs are abundantly expressed and upregulated during human epidermal stem cell differentiation.

The expression patterns of endogenous circular RNA (circRNA) molecules during epidermal stem cell (EpSC) differentiation have not previously been explored. Here, we show that circRNAs are abundantly expressed in EpSCs and that their expression change dramatically during differentiation in a coordinated manner. Overall, circRNAs are expressed at higher levels in the differentiated cells, and many upregulated circRNAs are derived from developmental genes, including four different circRNAs from DLG1. The observed changes in circRNA expression were largely independent of host gene expression, and circRNAs independently upregulated upon differentiation are more prone to AGO2 binding and have more predicted miRNA binding sites compared to stably expressed circRNAs. In particular, upregulated circRNAs from the HECTD1 and ZNF91 genes have exceptionally high numbers of AGO2 binding sites and predicted miRNA target sites, and circZNF91 contains 24 target sites for miR-23b-3p, which is known to play important roles in keratinocyte differentiation. We also observed that upregulated circRNAs are less likely to be flanked by homologues inverted Alu repeats compared to stably expressed circRNAs. This coincide with DHX9 being upregulated in the differentiated keratinocytes. Finally, none of the circRNAs upregulated upon differentiation were also upregulated upon DNMT3A or DNMT3B knockdown, making it unlikely that epigenetic mechanisms are governing the observed circRNA expression changes. Together, we provide a map of circRNA expression in EpSCs and their differentiated counterparts and shed light on potential function and regulation of differentially expressed circRNAs.

Long Non-Coding RNAs Guide the Fine-Tuning of Gene Regulation in B-Cell Development and Malignancy.

With the introduction of next generation sequencing methods, such as RNA sequencing, it has become apparent that alterations in the non-coding regions of our genome are important in the development of cancer. Particularly interesting is the class of long non-coding RNAs (lncRNAs), including the recently described subclass of circular RNAs (circRNAs), which display tissue- and cell-type specific expression patterns and exert diverse regulatory functions in the cells. B-cells undergo complex and tightly regulated processes in order to develop from antigen naïve cells residing in the bone marrow to the highly diverse and competent effector cells circulating in peripheral blood. These processes include V(D)J recombination, rapid proliferation, somatic hypermutation and clonal selection, posing a risk of malignant transformation at each step. The aim of this review is to provide insight into how lncRNAs including circRNAs, participate in normal B-cell differentiation, and how deregulation of these molecules is involved in the development of B-cell malignancies. We describe the prognostic value and functional significance of specific deregulated lncRNAs in diseases such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma and multiple myeloma, and we provide an overview of the current knowledge on the role of circRNAs in these diseases.

DNA Methylation Levels of the ELMO Gene Promoter CpG Islands in Human Glioblastomas.

Complete surgical resection of glioblastoma is difficult due to the invasive nature of this primary brain tumor, for which the molecular mechanisms behind remain poorly understood. The three human ELMO genes play key roles in cellular motility, and have been linked to metastasis and poor prognosis in other cancer types. The aim of this study was to investigate methylation levels of the ELMO genes and their correlation to clinical characteristics and outcome in patients diagnosed with glioblastoma. To measure DNA methylation levels we designed pyrosequencing assays targeting the promoter CpG island of each the ELMO genes. These were applied to diagnostic tumor specimens from a well-characterized cohort of 121 patients who received standard treatment consisting of surgery, radiation therapy, plus concomitant and adjuvant chemotherapy. The promoter methylation levels of ELMO1 and ELMO2 were generally low, whereas ELMO3 methylation levels were high, in the tumor biopsies. Thirteen, six, and 18 biopsies were defined as aberrantly methylated for ELMO1, ELMO2, and ELMO3, respectively. There were no significant associations between the methylation status of any of the ELMO gene promoter CpG islands and overall survival, progression-free survival, and clinical characteristics of the patients including intracranial tumor location. Therefore, the methylation status of the ELMO gene promoter CpG islands is unlikely to have prognostic value in glioblastoma.

Global hypomethylation is an independent prognostic factor in diffuse large B cell lymphoma.

Global hypomethylation has been linked to disease progression in several cancers, but has not been reported for Diffuse Large B Cell Lymphoma (DLBCL). This study aimed to assess global methylation in DLBCL and describe its prognostic value. Mean LINE1 methylation, a validated surrogate measure for global methylation, was measured in DNA from 67 tumor biopsies. Additionally, cell-free circulating DNA (cfDNA) in plasma samples from 74 patients was tested to assess the feasibility of global hypomethylation as a biomarker in liquid biopsies. LINE1 methylation was assessed using a commercially available kit, based on pyrosequencing of PCR amplified bisulfite-treated DNA. Global hypomethylation was detected in a subset of cases and was associated with poor overall survival in both tumor biopsies (P = .001) and cfDNA (P = .009). It was the strongest risk factor in multivariate analysis in both biopsies (HR: 10.65, CI: 2.03-55.81, P = .005) and cfDNA (HR: 11.87, CI: 2.80-50.20, P = .001), outperforming conventional clinical risk factors. Finally, hierarchical cluster analyses were performed for the cfDNA samples using previously published gene-specific methylation data. This analysis shows that global hypomethylation co-occurs with other epigenetic abnormalities, including DAPK1 promoter hypermethylation. In conclusion, we have shown that global hypomethylation is strongly associated with poor survival in DLBCL both when present in tumor biopsy DNA and when detected in plasma cfDNA, and has potential for clinical application as a prognostic biomarker.

Mutations in idiopathic cytopenia of undetermined significance assist diagnostics and correlate to dysplastic changes.

Cytopenia is common in the elderly population and etiology may be difficult to assess. Here, we investigated the occurrence of mutations in patients with idiopathic cytopenia of undetermined significance and the usefulness in improving diagnostics. We included 60 patients with persistent cytopenia > 6 months without definite diagnosis of hematological neoplasm after routine assessment. Bone marrow material underwent a blinded morphology review and DNA was sequenced with a targeted 20 gene panel representing the most commonly mutated genes in myelodysplastic syndrome. Thirty seven (62%) patients carried at least one mutation at inclusion, and of these 95% carried a mutation in TET2, ASXL1, SRSF2, or DNMT3A. The most commonly mutated gene was TET2 observed in 43% of all patients. During one to eight years follow-up seven patients progressed to a myeloid neoplasm and six of these had a detectable mutation at study entry. Median time to progression was 53 months (range 10-78), and at time of progression each patient had at least two mutations detected. Mutations in TP53 and NRAS were not present in patients at inclusion, but identified as secondary hits triggering progression. The morphology review was concordant in 68% of all cases, and 93% of the cases reclassified into the group "highly suspicious for MDS" had a mutation. All patients who had a concordant review "highly suspicious for MDS" had at least two mutations detected. Overall, we show that morphology examination is challenging in this heterogeneous group and targeted sequencing helps identify patients at risk of progression. Am. J. Hematol. 91:1234-1238, 2016. © 2016 Wiley Periodicals, Inc.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and thereby STAT3 mRNA levels. However, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.

Concurrent Inhibition of Akt and ERK Using TIC-10 Can Overcome Venetoclax Resistance in Mantle Cell Lymphoma.

Venetoclax, a BCL-2 inhibitor, has proven to be effective in several hematological malignancies, including mantle cell lymphoma (MCL). However, development of venetoclax resistance is inevitable and understanding its underlying molecular mechanisms can optimize treatment response. We performed a thorough genetic, epigenetic and transcriptomic analysis of venetoclax-sensitive and resistant MCL cell lines, also evaluating the role of the stromal microenvironment using human and murine co-cultures. In our model, venetoclax resistance was associated with abrogated TP53 activity through an acquired mutation and transcriptional downregulation leading to a diminished apoptotic response. Venetoclax-resistant cells also exhibited an upregulation of the PI3K/Akt pathway, and pharmacological inhibition of Akt and ERK with TIC-10 led to cell death in all venetoclax-resistant cell lines. Overall, we highlight the importance of targeted therapies, such as TIC-10, against venetoclax resistance-related pathways, which might represent future therapeutic prospects.

A Circular RNA Expressed from the FAT3 Locus Regulates Neural Development.

Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, using RNA-sequencing, we show that circRNA levels increased substantially over the course of differentiation of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in minor transcriptional alterations in bulk RNA analysis. However, single-cell transcriptomics of 30 and 90 days differentiated cerebral organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell types, neuronal differentiation, or migration.

Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer.

BACKGROUND: Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC) a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR). Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. METHODS: We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA). The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. RESULTS: The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. CONCLUSIONS: The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

Non-coding RNAs and epithelial mesenchymal transition in cancer: molecular mechanisms and clinical implications.

Epithelial-mesenchymal transition (EMT) is a fundamental process for embryonic development during which epithelial cells acquire mesenchymal characteristics, and the underlying mechanisms confer malignant features to carcinoma cells such as dissemination throughout the organism and resistance to anticancer treatments. During the past decades, an entire class of molecules, called non-coding RNA (ncRNA), has been characterized as a key regulator of almost every cellular process, including EMT. Like protein-coding genes, ncRNAs can be deregulated in cancer, acting as oncogenes or tumor suppressors. The various forms of ncRNAs, including microRNAs, PIWI-interacting RNAs, small nucleolar RNAs, transfer RNA-derived RNA fragments, long non-coding RNAs, and circular RNAs can orchestrate the complex regulatory networks of EMT at multiple levels. Understanding the molecular mechanism underlying ncRNAs in EMT can provide fundamental insights into cancer metastasis and may lead to novel therapeutic approaches. In this review, we describe recent advances in the understanding of ncRNAs in EMT and provide an overview of recent ncRNA applications in the clinic.

The transcriptional landscape and biomarker potential of circular RNAs in prostate cancer.

BACKGROUND: Circular RNAs (circRNAs) constitute a largely unexplored source for biomarker discovery in prostate cancer (PC). Here, we characterize the biomarker potential of circRNAs in PC, where the need for novel diagnostic and prognostic tools to facilitate more personalized management is pressing. METHODS: We profiled the transcriptomic landscape of circRNAs in PC by total RNA sequencing of 31 adjacent-normal and 143 tumor samples from localized (radical prostatectomy (RP)) and metastatic PC patients (cohort 1, training). Diagnostic and prognostic potential was evaluated in cohort 1, and 39 top circRNA candidates were selected for validation in two additional PC cohorts (cohort 2, n = 111; RP cohort 3, n = 191) by NanoString-based expression analysis. Biochemical recurrence (BCR)-free survival was assessed using Kaplan-Meier, univariate, and multivariate Cox regression analyses. The circRNA candidates were further detected in extracellular vesicle (EV)-enriched plasma samples from PC patients and controls (cohort 4, n = 54). RESULTS: Expression of circABCC4, circFAT3, circATRNL1, and circITGA7 was highly cancer-specific (area under the curve 0.71-0.86), while low circITGA7 expression was significantly (P < 0.05) associated with BCR in univariate analysis in two RP cohorts. Moreover, we successfully trained and validated a novel 5-circRNA prognostic signature (circKMD1A/circTULP4/circZNF532/circSUMF1/circMKLN1) significantly associated with BCR beyond routine clinicopathological variables (RP cohort 1: P = 0.02, hazard ratio = 2.1; RP cohort 3: P < 0.001, hazard ratio = 2.1). Lastly, we provide proof-of-principle for detection of candidate circRNAs in EV-enriched plasma samples from PC patients. CONCLUSIONS: circRNAs hold great biomarker potential in PC and display both high cancer specificity and association to disease progression.

VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy.

Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA-targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.

Genome-Wide Circular RNA Expression Patterns Reflect Resistance to Immunomodulatory Drugs in Multiple Myeloma Cells.

Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, may induce significant remissions in multiple myeloma (MM) patients, but relapses are frequently observed and the underlying molecular mechanisms for this are not completely understood. Circular RNAs (circRNAs) constitute an emerging class of non-coding RNAs with important roles in cancer. Here, we profiled genome-wide expression patterns of circRNAs in IMiD-sensitive MM cells and their resistant counterparts as well as in IMiD-resistant cells treated with specific epigenetic drugs alone or in combination. We found that genome-wide circRNA expression patterns reflect IMiD sensitivity and ciRS-7 (also known as CDR1as) was the most downregulated circRNA upon acquired resistance. The depletion of ciRS-7 correlated with increased methylation levels of the promoter CpG island of its host gene, LINC00632. Expression of LINC00632 and ciRS-7 was partly restored by treatment with a combination of an EZH2 inhibitor (EPZ-6438) and a DNA methyl transferase inhibitor (5-azacytidine), which also restores the IMiD sensitivity of the cells. However, knockdown of ciRS-7 did not affect IMiD sensitivity and we found that ciRS-7 also becomes epigenetically silenced after prolonged cell culture without drug-exposure. In conclusion, we found that genome-wide circRNA expression patterns reflect IMiD sensitivity in an in vitro model of acquired resistance.

A comprehensive analysis of coding and non-coding transcriptomic changes in cutaneous squamous cell carcinoma.

Cutaneous Squamous Cell Carcinoma (cSCC) is the most common and fastest-increasing cancer with metastatic potential. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are novel regulators of gene expression. To identify mRNAs, lncRNAs and circRNAs, which can be involved in cSCC, RNA-seq was performed on nine cSCCs and seven healthy skin samples. Representative transcripts were validated by NanoString nCounter assays using an extended cohort, which also included samples from pre-cancerous skin lesions (actinic keratosis). 5,352 protein-coding genes, 908 lncRNAs and 55 circular RNAs were identified to be differentially expressed in cSCC. Targets of 519 transcription factors were enriched among differentially expressed genes, 105 of which displayed altered level in cSCCs, including fundamental regulators of skin development (MYC, RELA, ETS1, TP63). Pathways related to cell cycle, apoptosis, inflammation and epidermal differentiation were enriched. In addition to known oncogenic lncRNAs (PVT1, LUCAT1, CASC9), a set of skin-specific lncRNAs were were identified to be dysregulated. A global downregulation of circRNAs was observed in cSCC, and novel skin-enriched circRNAs, circ_IFFO2 and circ_POF1B, were identified and validated. In conclusion, a reference set of coding and non-coding transcripts were identified in cSCC, which may become potential therapeutic targets or biomarkers.

Defects in LC3B2 and ATG4A underlie HSV2 meningitis and reveal a critical role for autophagy in antiviral defense in humans.

Recurrent herpesvirus infections can manifest in different forms of disease, including cold sores, genital herpes, and encephalitis. There is an incomplete understanding of the genetic and immunological factors conferring susceptibility to recurrent herpes simplex virus 2 (HSV2) infection in the central nervous system (CNS). Here, we describe two adult patients with recurrent HSV2 lymphocytic Mollaret's meningitis that each carry a rare monoallelic variant in the autophagy proteins ATG4A or LC3B2. HSV2-activated autophagy was abrogated in patient primary fibroblasts, which also exhibited significantly increased viral replication and enhanced cell death. HSV2 antigen was captured in autophagosomes of infected cells, and genetic inhibition of autophagy by disruption of autophagy genes, including ATG4A and LC3B2, led to enhanced viral replication and cell death in primary fibroblasts and a neuroblastoma cell line. Activation of autophagy by HSV2 was sensitive to ultraviolet (UV) irradiation of the virus and inhibited in the presence of acyclovir, but HSV2-induced autophagy was independent of the DNA-activated STING pathway. Reconstitution of wild-type ATG4A and LC3B2 expression using lentiviral gene delivery or electroporation of in vitro transcribed mRNA into patient cells restored virus-induced autophagy and the ability to control HSV2 replication. This study describes a previously unknown link between defective autophagy and an inborn error of immunity that can lead to increased susceptibility to HSV2 infection, suggesting an important role for autophagy in antiviral immunity in the CNS.