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.

Dual-Targeting of the HER2 Cancer Receptor with an Antibody-Directed Enzyme and a Nanobody-Guided MMAE Prodrug Scaffold.

Antibody-enzyme conjugates have shown potential as tissue-specific prodrug activators by antibody-directed enzyme prodrug therapy (ADEPT), but the approach met challenges clinically due to systemic drug release. Here, we report a novel dual-targeting ADEPT system (DuADEPT) which is based on active cancer receptor targeting of both a trastuzumab-sialidase conjugate (Tz-Sia) and a highly potent sialidase-activated monomethyl auristatin E (MMAE) prodrug scaffold. The scaffold is based on a four-way junction of the artificial nucleic acid analog acyclic (L)-threoninol nucleic acid ((L)-aTNA) which at the ends of its four arms carries one nanobody targeting HER2 and three copies of the prodrug. Dual-targeting of the constructs to two proximal epitopes of HER2 was shown by flow cytometry, and a dual-targeted enzymatic drug release assay revealed cytotoxicity upon prodrug activation specifically for HER2-positive cancer cells. The specific delivery and activation of prodrugs in this way could potentially be used to decrease systemic side effects and increase drug efficacy, and utilization of Tz-Sia provides an opportunity to combine the local chemotherapeutic effect of the DuADEPT with an anticancer immune response.

The Transcriptional Landscape of Coding and Noncoding RNAs in Recurrent and Nonrecurrent Colon Cancer.

A number of patients with colon cancer with local or local advanced disease suffer from recurrence and there is an urgent need for better prognostic biomarkers in this setting. Here, the transcriptomic landscape of mRNAs, long noncoding RNAs, snRNAs, small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs, pseudogenes, and circular RNAs, as well as RNAs denoted as miscellaneous RNAs, was profiled by total RNA sequencing. In addition to well-known coding and noncoding RNAs, differential expression analysis also uncovered transcripts that have not been implicated previously in colon cancer, such as RNA5SP149, RNU4-2, and SNORD3A. Moreover, there was a profound global up-regulation of snRNA pseudogenes, snoRNAs, and rRNA pseudogenes in more advanced tumors. A global down-regulation of circular RNAs in tumors relative to normal tissues was observed, although only a few were expressed differentially between tumor stages. Many previously undescribed transcripts, including RNU6-620P, RNU2-20P, VTRNA1-3, and RNA5SP60, indicated strong prognostic biomarker potential in receiver operating characteristics analyses. In summary, this study unveiled numerous differentially expressed RNAs across various classes between recurrent and nonrecurrent colon cancer. Notably, there was a significant global up-regulation of snRNA pseudogenes, snoRNAs, and rRNA pseudogenes in advanced tumors. Many of these newly discovered candidates demonstrate a strong prognostic potential for stage II colon cancer.

RNA nanostructures for targeted drug delivery and imaging.

The RNA molecule plays a pivotal role in many biological processes by relaying genetic information, regulating gene expression, and serving as molecular machines and catalyzers. This inherent versatility of RNA has fueled significant advancements in the field of RNA nanotechnology, driving the engineering of complex nanoscale architectures toward biomedical applications, including targeted drug delivery and bioimaging. RNA polymers, serving as building blocks, offer programmability and predictability of Watson-Crick base pairing, as well as non-canonical base pairing, for the construction of nanostructures with high precision and stoichiometry. Leveraging the ease of chemical modifications to protect the RNA from degradation, researchers have developed highly functional and biocompatible RNA architectures and integrated them into preclinical studies for the delivery of payloads and imaging agents. This review offers an educational introduction to the use of RNA as a biopolymer in the design of multifunctional nanostructures applied to targeted delivery in vivo, summarizing physical and biological barriers along with strategies to overcome them. Furthermore, we highlight the most recent progress in the development of both small and larger RNA nanostructures, with a particular focus on imaging reagents and targeted cancer therapeutics in pre-clinical models and provide insights into the prospects of this rapidly evolving field.

An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Differential microRNA editing may drive target pathway switching in human temporal lobe epilepsy.

MicroRNAs have emerged as important regulators of the gene expression landscape in temporal lobe epilepsy. The mechanisms that control microRNA levels and influence target choice remain, however, poorly understood. RNA editing is a post-transcriptional mechanism mediated by the adenosine acting on RNA (ADAR) family of proteins that introduces base modification that diversifies the gene expression landscape. RNA editing has been studied for the mRNA landscape but the extent to which microRNA editing occurs in human temporal lobe epilepsy is unknown. Here, we used small RNA-sequencing data to characterize the identity and extent of microRNA editing in human temporal lobe epilepsy brain samples. This detected low-to-high editing in over 40 of the identified microRNAs. Among microRNA exhibiting the highest editing was miR-376a-3p, which was edited in the seed region and this was predicted to significantly change the target pool. The edited form was expressed at lower levels in human temporal lobe epilepsy samples. We modelled the shift in editing levels of miR-376a-3p in human-induced pluripotent stem cell-derived neurons. Reducing levels of the edited form of miR-376a-3p using antisense oligonucleotides resulted in extensive gene expression changes, including upregulation of mitochondrial and metabolism-associated pathways. Together, these results show that differential editing of microRNAs may re-direct targeting and result in altered functions relevant to the pathophysiology of temporal lobe epilepsy and perhaps other disorders of neuronal hyperexcitability.

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.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells.

Circular RNAs (circRNA) are covalently closed molecules that can play important roles in cancer development and progression. Hundreds of differentially expressed circRNAs between tumors and adjacent normal tissues have been identified in studies using RNA sequencing or microarrays, emphasizing a strong translational potential. Most previous studies have been performed using RNA from bulk tissues and lack information on the spatial expression patterns of circRNAs. Here, we showed that the majority of differentially expressed circRNAs from bulk tissue analyses of colon tumors relative to adjacent normal tissues were surprisingly not differentially expressed when comparing cancer cells directly with normal epithelial cells. Manipulating the proliferation rates of cells grown in culture revealed that these discrepancies were explained by circRNAs accumulating to high levels in quiescent muscle cells due to their high stability; on the contrary, circRNAs were diluted to low levels in the fast-proliferating cancer cells due to their slow biogenesis rates. Thus, different subcompartments of colon tumors and adjacent normal tissues exhibited striking differences in circRNA expression patterns. Likewise, the high circRNA content in muscle cells was also a strong confounding factor in bulk analyses of circRNAs in bladder and prostate cancers. Together, these findings emphasize the limitations of using bulk tissues for studying differential circRNA expression in cancer and highlight a particular need for spatial analysis in this field of research. SIGNIFICANCE: The abundance of circRNAs varies systematically between subcompartments of solid tumors and adjacent tissues, implying that differentially expressed circRNAs discovered in bulk tissue analyses may reflect differences in cell type composition between samples.

Biased activation of the receptor tyrosine kinase HER2.

HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which has so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGß1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGß1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.

Impact of U2AF1 mutations on circular RNA expression in myelodysplastic neoplasms.

Mutations in U2AF1 are relatively common in myelodysplastic neoplasms (MDS) and are associated with an inferior prognosis, but the molecular mechanisms underlying this are not fully elucidated. Circular RNAs (circRNAs) have been implicated in cancer, but it is unknown how mutations in splicing factors may impact on circRNA biogenesis. Here, we used RNA-sequencing to investigate the effects of U2AF1 mutations on circRNA expression in K562 cells with a doxycycline-inducible U2AF1S34 mutation, in a mouse model with a doxycycline-inducible U2AF1S34 mutation, and in FACS-sorted CD34+ bone marrow cells from MDS patients with either U2AF1S34 or U2AF1Q157 mutations. In all contexts, we found an increase in global circRNA levels in the U2AF1-mutated setting, which was independent of expression changes in the cognate linear host genes. In patients, the U2AF1S34 and U2AF1Q157 mutations were both associated with an overall increased expression of circRNAs. circRNAs generated by a non-Alu-mediated mechanism generally showed the largest increase in expression levels. Several well-described cancer-associated circRNAs, including circZNF609 and circCSNK1G3, were upregulated in MDS patients with U2AF1 mutations compared to U2AF1-wildtype MDS controls. In conclusion, high circRNA expression is observed in association with U2AF1 mutations in three biological systems, presenting an interesting possibility for biomarker and therapeutic investigation.

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.

Differential RNA aptamer affinity profiling on plasma as a potential diagnostic tool for bladder cancer.

The molecular composition of blood is a signature of human health, reflected in the thousands of blood biomarkers known for human diseases. However, establishing robust disease markers is challenging due to the diversity of individual samples. New sequencing methods have simplified biomarker discovery for circulating DNA and RNA while protein profiling is still laborious and costly. To harness the power of high-throughput sequencing to profile the protein content of a biological sample, we developed a method termed APTASHAPE that uses oligonucleotide aptamers to recognize proteins in complex biofluids. We selected a large pool of 2'Fluoro protected RNA sequences to recognize proteins in human plasma and identified a set of 33 cancer-specific aptamers. Differential enrichment of these aptamers after selection against 1 µl of plasma from individual patients allowed us to differentiate between healthy controls and bladder cancer-diagnosed patients (91% accuracy) and between early non-invasive tumors and late stage tumors (83% accuracy). Affinity purification and mass spectrometry of proteins bound to the predictive aptamers showed the main target proteins to be C4b-binding protein, Complement C3, Fibrinogen, Complement factor H and IgG. The APTASHAPE method thus provides a general, automated and highly sensitive platform for discovering potential new disease biomarkers.

Expression of Circ_Satb1 Is Decreased in Mesial Temporal Lobe Epilepsy and Regulates Dendritic Spine Morphology.

Mesial temporal lobe epilepsy (mTLE) is a chronic disease characterized by recurrent seizures that originate in the temporal lobes of the brain. Anti-epileptic drugs (AEDs) are the standard treatment for managing seizures in mTLE patients, but are frequently ineffective. Resective surgery is an option for some patients, but does not guarantee a postoperative seizure-free period. Therefore, further insight is needed into the pathogenesis of mTLE to enable the design of new therapeutic strategies. Circular RNAs (circRNAs) have been identified as important regulators of neuronal function and have been implicated in epilepsy. However, the mechanisms through which circRNAs contribute to epileptogenesis remain unknown. Here, we determine the circRNA transcriptome of the hippocampus and cortex of mTLE patients by using RNA-seq. We report 333 differentially expressed (DE) circRNAs between healthy individuals and mTLE patients, of which 23 circRNAs displayed significant adjusted p-values following multiple testing correction. Interestingly, hippocampal expression of circ_Satb1, a circRNA derived from special AT-rich sequence binding protein 1 (SATB1), is decreased in both mTLE patients and in experimental epilepsy. Our work shows that circ_Satb1 displays dynamic patterns of neuronal expression in vitro and in vivo. Further, circ_Satb1-specific knockdown using CRISPR/CasRx approaches in hippocampal cultures leads to defects in dendritic spine morphology, a cellular hallmark of mTLE. Overall, our results identify a novel epilepsy-associated circRNA with disease-specific expression and previously unidentified cellular effects that are relevant for epileptogenesis.

Functionalized Acyclic (l)-Threoninol Nucleic Acid Four-Way Junction with High Stability In Vitro and In Vivo.

Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well-defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non-toxic, and non-immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l-threoninol nucleic acid (aTNA) to form a four-way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum-stable, shows no non-targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer-targeting and a serum half-life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.

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.

The emerging roles of circRNAs in cancer and oncology.

Over the past decade, circular RNAs (circRNAs) have emerged as a large class of primarily non-coding RNA molecules, many of which have key roles in cancer development and progression through diverse mechanisms of action. CircRNAs often have tissue-restricted and cancer-specific expression patterns, and accumulating data suggest that these molecules are of potential clinical relevance and utility. In particular, circRNAs have strong potential as diagnostic, prognostic and predictive biomarkers, which is underscored by their detectability in liquid biopsy samples such as in plasma, saliva and urine. However, technical issues in the detection and assessment of circRNAs as well as biological knowledge gaps need to be addressed to move this relatively young field of research forward and bring circRNAs to the forefront of clinical practice. Herein, we review the current knowledge regarding circRNA biogenesis, regulation and functions in cancer as well as their clinical potential as biomarkers, therapeutic agents and drug targets.

Expression patterns and prognostic potential of circular RNAs in mantle cell lymphoma: a study of younger patients from the MCL2 and MCL3 clinical trials.

Mantle cell lymphoma (MCL) is characterized by marked differences in outcome, emphasizing the need for strong prognostic biomarkers. Here, we explore expression patterns and prognostic relevance of circular RNAs (circRNAs), a group of endogenous non-coding RNA molecules, in MCL. We profiled the circRNA expression landscape using RNA-sequencing and explored the prognostic potential of 40 abundant circRNAs in samples from the Nordic MCL2 and MCL3 clinical trials, using NanoString nCounter Technology. We report a circRNA-based signature (circSCORE) developed in the training cohort MCL2 that is highly predictive of time to progression (TTP) and lymphoma-specific survival (LSS). The dismal outcome observed in the large proportion of patients assigned to the circSCORE high-risk group was confirmed in the independent validation cohort MCL3, both in terms of TTP (HR 3.0; P = 0.0004) and LSS (HR 3.6; P = 0.001). In Cox multiple regression analysis incorporating MIPI, Ki67 index, blastoid morphology and presence of TP53 mutations, circSCORE retained prognostic significance for TTP (HR 3.2; P = 0.01) and LSS (HR 4.6; P = 0.01). In conclusion, circRNAs are promising prognostic biomarkers in MCL and circSCORE improves identification of high-risk disease among younger patients treated with cytarabine-containing chemoimmunotherapy and autologous stem cell transplant.

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.