CircFBXW7 in patients with T-cell ALL: depletion sustains MYC and NOTCH activation and leukemia cell viability.

Circular RNAs (circRNAs) are emerging as new players in leukemogenic mechanisms. In patients with T-cell Acute Lymphoblastic Leukemia (T-ALL), the recent report of a remarkable dysregulation of circRNAs incited further functional investigation. Here we focus on circFBXW7, highly expressed in T-cells, with a notably high abundance of the circular compared to linear transcript of FBXW7. Two T-ALL patient cohorts profiled with RNA-seq were analyzed in comparison with five populations of developing thymocytes as normal counterpart, quantifying circRNA and gene expression. CircFBXW7 expression was very heterogeneous in T-ALL patients allowing their stratification in two groups with low and high expression of this circRNA, not correlated with FBXW7 mutation status and T-ALL molecular subgroups. With a loss-of-function study in T-ALL in vitro, we demonstrate that circFBXW7 depletion increases leukemic cell viability and proliferation. Microarray profiling highlighted the effect of the circFBXW7 silencing on gene expression, with activation of pro-proliferative pathways, supporting a tumor suppressor role of circFBXW7 in T-ALL. Further, MYC and intracellular NOTCH1 protein levels, as well as expression of MYC target and NOTCH signaling genes were elevated after circFBXW7 depletion, suggesting an inhibitory role of circFBXW7 in these oncogenic axes. Plus, low circFBXW7 levels were associated with a particular gene expression profile in T-ALL patients, which was remarkably mirrored by the effects of circFBXW7 loss-of-function in vitro. CircFBXW7 depletion notably emerges as a new factor enhancing a proliferative phenotype and the activation of the MYC signaling pathway, key players in this aggressive malignancy.

Discovery of fusion circular RNAs in leukemia with KMT2A::AFF1 rearrangements by the new software CircFusion.

Chromosomal translocations in cancer genomes, key players in many types of cancers, generate chimeric proteins that drive oncogenesis. Genomes with chromosomal rearrangements can also produce fusion circular RNAs (f-circRNAs) by backsplicing of chimeric transcripts, as first shown in leukemias with PML::RARα and KMT2A::MLLT3 translocations and later in solid cancers. F-circRNAs contribute to the oncogenic processes and reinforce the oncogenic activity of chimeric proteins. In leukemia with KMT2A::AFF1 (MLL::AF4) fusions, we previously reported specific alterations of circRNA expression, but nothing was known about f-circRNAs. Due to the presence of two chimeric sequences, fusion and backsplice junctions, the identification of f-circRNAs with available tools is challenging, possibly resulting in the underestimation of this RNA species, especially when the breakpoint is not known. We developed CircFusion, a new software tool to detect linear fusion transcripts and f-circRNAs from RNA-seq data, both in samples for which the breakpoints are known and when the information about the joined exons is missing. CircFusion can detect linear and circular chimeric transcripts deriving from the main and reciprocal translocations also in the presence of multiple breakpoints, which are common in malignant cells. Benchmarking tests on simulated and real datasets of cancer samples with previously experimentally determined f-circRNAs showed that CircFusion provides reliable predictions and outperforms available methods for f-circRNA detection. We discovered and validated novel f-circRNAs in acute leukemia harboring KMT2A::AFF1 rearrangements, leading the way to future functional studies aimed to unveil their role in this malignancy.

CRAFT: a bioinformatics software for custom prediction of circular RNA functions.

Circular RNAs (circRNAs), transcripts generated by backsplicing, are particularly stable and pleiotropic molecules, whose dysregulation drives human diseases and cancer by modulating gene expression and signaling pathways. CircRNAs can regulate cellular processes by different mechanisms, including interaction with microRNAs (miRNAs) and RNA-binding proteins (RBP), and encoding specific peptides. The prediction of circRNA functions is instrumental to interpret their impact in diseases, and to prioritize circRNAs for functional investigation. Currently, circRNA functional predictions are provided by web databases that do not allow custom analyses, while self-standing circRNA prediction tools are mostly limited to predict only one type of function, mainly focusing on the miRNA sponge activity of circRNAs. To solve these issues, we developed CRAFT (CircRNA Function prediction Tool), a freely available computational pipeline that predicts circRNA sequence and molecular interactions with miRNAs and RBP, along with their coding potential. Analysis of a set of circRNAs with known functions has been used to appraise CRAFT predictions and to optimize its setting. CRAFT provides a comprehensive graphical visualization of the results, links to several knowledge databases, and extensive functional enrichment analysis. Moreover, it originally combines the predictions for different circRNAs. CRAFT is a useful tool to help the user explore the potential regulatory networks involving the circRNAs of interest and generate hypotheses about the cooperation of circRNAs into the modulation of biological processes.

Sensitive, reliable and robust circRNA detection from RNA-seq with CirComPara2.

Circular RNAs (circRNAs) are a large class of covalently closed RNA molecules originating by a process called back-splicing. CircRNAs are emerging as functional RNAs involved in the regulation of biological processes as well as in disease and cancer mechanisms. Current computational methods for circRNA identification from RNA-seq experiments are characterized by low discovery rates and performance dependent on the analysed data set. We developed CirComPara2 (https://github.com/egaffo/CirComPara2), a new automated computational pipeline for circRNA discovery and quantification, which consistently achieves high recall rates without losing precision by combining multiple circRNA detection methods. In our benchmark analysis, CirComPara2 outperformed state-of-the-art circRNA discovery tools and proved to be a reliable and robust method for comprehensive transcriptome characterization.

CircIMPACT: An R Package to Explore Circular RNA Impact on Gene Expression and Pathways.

Circular RNAs (circRNAs) are transcripts generated by back-splicing. CircRNAs might regulate cellular processes by different mechanisms, including interaction with miRNAs and RNA-binding proteins. CircRNAs are pleiotropic molecules whose dysregulation has been linked to human diseases and can drive cancer by impacting gene expression and signaling pathways. The detection of circRNAs aberrantly expressed in disease conditions calls for the investigation of their functions. Here, we propose CircIMPACT, a bioinformatics tool for the integrative analysis of circRNA and gene expression data to facilitate the identification and visualization of the genes whose expression varies according to circRNA expression changes. This tool can highlight regulatory axes potentially governed by circRNAs, which can be prioritized for further experimental study. The usefulness of CircIMPACT is exemplified by a case study analysis of bladder cancer RNA-seq data. The link between circHIPK3 and heparanase (HPSE) expression, due to the circHIPK3-miR558-HPSE regulatory axis previously determined by experimental studies on cell lines, was successfully detected. CircIMPACT is freely available at GitHub.

Bioinformatic Analysis of Circular RNA Expression.

Circular RNAs (circRNAs) are stable RNA molecules generated by backsplicing that play regulatory functions through interaction with other RNA and proteins, as well as by encoding peptides. Dysregulation of circRNA expression can drive cancer development and progression with different mechanisms. CircRNAs are currently regarded as extremely attractive molecules in cancer research for the identification of new and possibly targetable disease regulatory networks and for the development of biomarkers for cancer diagnosis, prognosis definition, and monitoring. Using specific experimental and computational protocols, circRNAs can be identified through RNA-seq by spotting the reads spanning backsplice junctions, which are specific to circular molecules. In this chapter, we report a state-of-the-art computational protocol for a genome-wide analysis of circRNAs from RNA-seq data, which considers circRNA detection, quantification, and differential expression testing. Finally, we indicate how to determine circular transcript sequences and the resources for an in silico functional characterization of circRNAs.

Large-scale circular RNA deregulation in T-ALL: unlocking unique ectopic expression of molecular subtypes.

Circular RNAs (circRNAs) are stable RNA molecules that can drive cancer through interactions with microRNAs and proteins and by the expression of circRNA encoded peptides. The aim of the study was to define the circRNA landscape and potential impact in T-cell acute lymphoblastic leukemia (T-ALL). Analysis by CirComPara of RNA-sequencing data from 25 T-ALL patients, immature, HOXA overexpressing, TLX1, TLX3, TAL1, or LMO2 rearranged, and from thymocyte populations of human healthy donors disclosed 68 554 circRNAs. Study of the top 3447 highly expressed circRNAs identified 944 circRNAs with significant differential expression between malignant T cells and normal counterparts, with most circRNAs displaying increased expression in T-ALL. Next, we defined subtype-specific circRNA signatures in molecular genetic subgroups of human T-ALL. In particular, circZNF609, circPSEN1, circKPNA5, and circCEP70 were upregulated in immature, circTASP1, circZBTB44, and circBACH1 in TLX3, circHACD1, and circSTAM in HOXA, circCAMSAP1 in TLX1, and circCASC15 in TAL-LMO. Backsplice sequences of 14 circRNAs ectopically expressed in T-ALL were confirmed, and overexpression of circRNAs in T-ALL with specific oncogenic lesions was substantiated by quantification in a panel of 13 human cell lines. An oncogenic role of circZNF609 in T-ALL was indicated by decreased cell viability upon silencing in vitro. Furthermore, functional predictions identified circRNA-microRNA gene axes informing modes of circRNA impact in molecular subtypes of human T-ALL.

CircRNAs Dysregulated in Juvenile Myelomonocytic Leukemia: CircMCTP1 Stands Out.

Juvenile myelomonocytic leukemia (JMML), a rare myelodysplastic/myeloproliferative neoplasm of early childhood, is characterized by clonal growth of RAS signaling addicted stem cells. JMML subtypes are defined by specific RAS pathway mutations and display distinct gene, microRNA (miRNA) and long non-coding RNA expression profiles. Here we zoom in on circular RNAs (circRNAs), molecules that, when abnormally expressed, may participate in malignant deviation of cellular processes. CirComPara software was used to annotate and quantify circRNAs in RNA-seq data of a "discovery cohort" comprising 19 JMML patients and 3 healthy donors (HD). In an independent set of 12 JMML patients and 6 HD, expression of 27 circRNAs was analyzed by qRT-PCR. CircRNA-miRNA-gene networks were reconstructed using circRNA function prediction and gene expression data. We identified 119 circRNAs dysregulated in JMML and 59 genes showing an imbalance of the circular and linear products. Our data indicated also circRNA expression differences among molecular subgroups of JMML. Validation of a set of deregulated circRNAs in an independent cohort of JMML patients confirmed the down-regulation of circOXNAD1 and circATM, and a marked up-regulation of circLYN, circAFF2, and circMCTP1. A new finding in JMML links up-regulated circMCTP1 with known tumor suppressor miRNAs. This and other predicted interactions with miRNAs connect dysregulated circRNAs to regulatory networks. In conclusion, this study provides insight into the circRNAome of JMML and paves the path to elucidate new molecular disease mechanisms putting forward circMCTP1 up-regulation as a robust example.

Circular RNA differential expression in blood cell populations and exploration of circRNA deregulation in pediatric acute lymphoblastic leukemia.

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.

CircRNAs Are Here to Stay: A Perspective on the MLL Recombinome.

Chromosomal translocations harbored by cancer genomes are important oncogenic drivers. In MLL rearranged acute leukemia (MLLre) MLL/KMT2A fuses with over 90 partner genes. Mechanistic studies provided clues of MLL fusion protein leukemogenic potential, but models failed to fully recapitulate the disease. Recently, expression of oncogenic fusion circular RNAs (f-circ) by MLL-AF9 fusion was proven. This discovery, together with emerging data on the importance and diversity of circRNAs formed the incentive to study the circRNAs of the MLL recombinome. Through interactions with other RNAs, such as microRNAs, and with proteins, circRNAs regulate cellular processes also related to cancer development. CircRNAs can translate into functional peptides too. MLL and most of the 90 MLL translocation partners do express circRNAs and exploration of our RNA-seq dataset of sorted blood cell populations provided new data on alternative circular isoform generation and expression variability of circRNAs of the MLL recombinome. Further, we provided evidence that rearrangements of MLL and three of the main translocation partner genes can impact circRNA expression, supported also by preliminary observations in leukemic cells. The emerging picture underpins the view that circRNAs are worthwhile to be considered when studying MLLre leukemias and provides a new perspective on the impact of chromosomal translocations in cancer cells at large.

Interhemispheric transfer of spatial and semantic information: electrophysiological evidence.

The goal of this study was to cast light on the existence of functional callosal channels for the interhemispheric transfer (IHT) of spatial and semantic information. To do so, we recorded event-related potentials in healthy humans while performing a primed odd-even discrimination task. Targets were visually presented numbers preceded by single-letter primes signaling the probable presentation of an odd or an even number. Primes and targets could appear either in the same or in different visual fields, thus requiring an IHT in the latter case. The P1 and N2 components were influenced by IHT of spatial information only, whereas the later N400 was influenced by IHT of both spatial and semantic information. This was not the case for the P3b, which was modulated by semantic validity only. These results provide novel evidence of the existence of a temporally separated interhemispheric exchange of spatial and semantic information.