rMAPS2: an update of the RNA map analysis and plotting server for alternative splicing regulation

The rMAPS2 (RNA Map Analysis and Plotting Server 2) web server, freely available at http://rmaps.cecsresearch.org/, has provided the high-throughput sequencing data research community with curated tools for the identification of RNA binding protein sites. rMAPS2 analyzes differential alternative splicing or CLIP peak data obtained from high-throughput sequencing data analysis tools like MISO, rMATS, Piranha, PIPE-CLIP and PARalyzer, and then, graphically displays enriched RNA-binding protein target sites. The initial release of rMAPS focused only on the most common alternative splicing event, skipped exon or exon skipping. However, there was a high demand for the analysis of other major types of alternative splicing events, especially for retained intron events since this is the most common type of alternative splicing in plants, such as Arabidopsis thaliana. Here, we expanded the implementation of rMAPS2 to facilitate analyses for all five major types of alternative splicing events: skipped exon, mutually exclusive exons, alternative 5′ splice site, alternative 3′ splice site and retained intron. In addition, by employing multi-threading, rMAPS2 has vastly improved the user experience with significant reductions in running time, ∼3.5 min for the analysis of all five major alternative splicing types at once.

Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients

Background: Skin cancer represents the most common human malignancy, and it includes BCC, SCC, and melanoma. Since melanoma is one of the most aggressive types of cancer, we have herein attempted to develop a gene-specific intron retention signature that can distinguish BCC and SCC from melanoma biopsy tumors. Methods: Intron retention events were examined through RT-sqPCR protocols, using total RNA preparations derived from BCC, SCC, and melanoma Greek biopsy specimens. Intron-hosted miRNA species and their target transcripts were predicted via the miRbase and miRDB bioinformatics platforms, respectively. Ιntronic ORFs were recognized through the ORF Finder application. Generation and visualization of protein interactomes were achieved by the IntAct and Cytoscape softwares, while tertiary protein structures were produced by using the I-TASSER online server. Results: c-MYC and Sestrin-1 genes proved to undergo intron retention specifically in melanoma. Interaction maps of proteins encoded by genes being potentially targeted by retained intron-accommodated miRNAs were generated and SRPX2 was additionally delivered to our melanoma-specific signature. Novel ORFs were identified in MCT4 and Sestrin-1 introns, with potentially critical roles in melanoma development. Conclusions: The property of c-MYC, Sestrin-1, and SRPX2 genes to retain specific introns could be clinically used to molecularly differentiate non-melanoma from melanoma tumors.

Intron retention as a novel source of cancer neoantigens

Personalized cancer vaccine strategies directed at tumor neoantigens derived from somatic mutations in the DNA are currently under prospective evaluation1, 2. Alterations in tumor RNA, rather than DNA, may also represent a previously-unexplored source of neoantigens. Here, we show that intron retention, a widespread feature of cancer transcriptomes3, 4, represents a novel source of tumor neoantigens. We developed an in silico approach to identify retained intron neoantigens from RNA sequencing data and applied this methodology to tumor samples from patients with melanoma treated with immune checkpoint blockade5, 6, discovering that the retained intron neoantigen burden in these samples augments the DNA-derived, somatic neoantigen burden. We validated the existence of retained intron derived neoantigens by implementing this technique on cancer cell lines with mass spectrometry-derived immunopeptidome data7, 8, revealing that retained intron neoantigens were complexed with MHC I experimentally. Unexpectedly, we observed a trend toward lack of clinical benefit from immune checkpoint blockade in high retained intron load-tumors, which harbored transcriptional signatures consistent with cell cycle dysregulation and DNA damage repair. Our results demonstrate the contribution of transcriptional dysregulation to the overall burden of tumor neoantigens, provide a foundation for augmenting personalized cancer vaccine development with a new class of tumor neoantigens, and demonstrate how global transcriptional dysregulation may impact selective response to immune checkpoint blockade.Statement of significanceWe developed and experimentally validated a computational pipeline to identify a novel class of tumor neoantigens derived from RNA-based intron retention, which is prevalent throughout cancer transcriptomes. The discovery of transcriptionally-derived tumor neoantigens expands the tumor immunopeptidome and contributes potential substrates for personalized cancer vaccine development.

A Detailed Alternate Splicing Landscape in Multiple Myeloma with Significant Potential Biological and Clinical Implications

Alternative splicing (AS) is a critical post-transcriptional event, which affects number of cellular processes including cell growth and survival. Our group has previously shown that aberrant splicing of numerous genes in multiple myeloma (MM) affects the disease biology. Dysregulated expression or mutations in several splicing factors (SFs), including SF3B1, Fox2, SRSF1, NONO, in MM compared to normal plasma cells have been reported with impact on outcome, highlighting significant role of splicing factors in myeloma. We have previously reported global alternative splicing events in MM to understand biology. Here, we report, using a deep RNAseq data, a detailed analysis of the splicing events, including retained intron, cassette exon, Alternative 3' / 5' site, mutually exclusive exons, mutually alternative 3' / 5' exons and analyze significance of AS in predicting prognosis. We purified myeloma cells from 420 newly-diagnosed patients treated homogeneously with lenalidomide, bortezomib and dexamethsone (RVD) with or without high-dose melphalan followed by lenalidomide maintenance in the IFM/DFCI 2009 study. RNA isolated from these purified CD138+ MM cells and from 18 normal donor plasma cells were processed by RNAseq (80-100 million paired end reads on Illumina HiSeq) and analyzed using a custom computational and statistical pipeline. Our results showed 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). Although we observed 1288 (~84%) of these 1534 genes with only one type of AS, remaining 16% of the genes had 2 or more AS events. Within the AS types, we observed retained intron (495 - ~32%) and cassette exons (727 - ~47%) as the most frequent events, whereas mutually exclusive exons were observed in less than 1% of 1534 genes. We found that 158 genes had same type of AS in more than 50% of the patients and 445 genes showed same AS in more than 30% of patients suggesting significant biological role for the spliced isoforms. Importantly, we have evaluated impact of each AS event with clinical outcome and observed that 43 AS events showed significant correlation with event free survival (EFS) (p value < 0.01), 27 AS events predicted overall survival (OS) and 5 AS events predicting both EFS and OS. We have also observed that although overall expression of number of these genes does not predict overall survival outcome, an isoform level expression predicts survival. We are now developing a composite model using gene expression and alternative splicing events that in our preliminary analysis has provided increased prediction power compared to gene expression model only which shows that alternative splicing events may carry independent significance for outcome prediction over overall gene expression alone. In summary, this study for the first time reports a detailed splicing landscape in myeloma and highlights the biological and clinical importance of alternative splicing events. It suggests the need to further integrate study of spliced isoform in our biological studies, in understanding disease behaviors as well as develop targeted therapeutics. Disclosures Richardson: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Anderson:Acetylon: Other: Scientific Founder; Onyx: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: Scientific Founder; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Attal:celgene: Consultancy, Research Funding; sanofi: Consultancy; janssen: Consultancy, Research Funding; amgen: Consultancy, Research Funding. Moreau:Takeda: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau. Avet-Loiseau:celgene: Consultancy; amgen: Consultancy; sanofi: Consultancy; janssen: Consultancy.

Morpholino-Mediated Knockdown of ERα, ERβa, and ERβb mRNAs in Zebrafish (Danio rerio) Embryos Reveals Differential Regulation of Estrogen-Inducible Genes

Genetically distinct estrogen receptor (ER) subtypes (ERα and ERβ) play a major role in mediating estrogen actions in vertebrates, but their unique and overlapping functions are not entirely clear. Although mammals have 1 gene of each subtype (ESR1 and ESR2), teleost fish have a single esr1 (ERα) and 2 esr2 (ERβa and ERβb) genes. To determine the in vivo role of different ER isoforms in regulating estrogen-inducible transcription targets, zebrafish (Danio rerio) embryos were microinjected with esr-specific morpholino (MO) oligonucleotides to disrupt splicing of the exon III/intron III junction in the DNA-binding domain. Each MO knocked down its respective normal transcript and increased production of variants with a retained intron III (esr1 MO) or a deleted or mis-spliced exon III (esr2a and esr2b MOs). Both esr1 and esr2b MOs blocked estradiol induction of vitellogenin and ERα mRNAs, predominant hepatic genes, but esr2b was the only MO that blocked induction of cytochrome P450 aromatase B mRNA, a predominant brain gene. Knockdown of ERβa with the esr2a MO had no effect on estrogen induction of the 3 mRNAs but, when coinjected with esr1 MO, attenuated the effect of ERα knockdown. Results indicate that ERα and ERβb, acting separately or cooperatively on specific gene targets, are positive transcriptional regulators of estrogen action, but the role of ERβa, if any, is unclear. We conclude that MO technology in zebrafish embryos is an advantageous approach for investigating the interplay of ER subtypes in a true physiological context.