Aberrant Splicing Research Articles

Abstract 6248: Modulation of CD22 protein expression in childhood leukemia by pervasive splicing aberrations: Implications for CD22-directed immunotherapies

Abstract Downregulation of surface epitopes causes post-immunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma-membrane-bound CD22 Δex5-6 splice isoform resistant to CAR-T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein; therefore, this event is rate-limiting for epitope presentation. Indeed, forcing exon 2 skipping with Morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab in vitro. Furthermore, among inotuzumab-treated pediatric B-ALL patients, we identified one non-responder in whose blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy. Citation Format: Sisi Zheng, Elisabeth Gillespie, Ammar S. Naqvi, Katharina E. Hayer, Zhiwei Ang, Manuel Torres-Diz, Mathieu Quesnel-Vallières, David A. Hottman, Asen Bagashev, John A. Chukinas, Carolin Schmidt, Mukta Mukta Asnani, Rawan Shraim, Deanne M. Taylor, Susan R. Rheingold, Maureen M. O’Brien, Nathan Singh, Kristen W. Lynch, Marco Ruella, Yoseph Barash, Sarah K. Tasian, Andrei Thomas-Tikhonenko. Modulation of CD22 protein expression in childhood leukemia by pervasive splicing aberrations: Implications for CD22-directed immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6248.

Abstract 986: RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness

Abstract NGS studies implicate dysregulation of the splicing machinery in the development of diverse cancers. The X-chromosome RBM10 gene encodes an RNA-binding protein that modulates transcriptome-wide cassette exon splicing. Truncation and missense RBM10 mutations are enriched (11%) in non-anaplastic thyroid cancers of patients who died of metastatic disease. MSK-MET, an integrated pan-cancer cohort of tumor genomic and clinical outcome data showed RBM10 alterations associate with metastatic burden in thyroid cancer. Within the MSK-IMPACT thyroid cancer cohort, 44% of RBM10 alterations co-occur with RAS mutations. We developed a murine Rbm10 floxed allele, which results in a non-functional transcript. Thyroid-specific Rbm10 inactivation through Tpo-Cre did not induce a phenotype. When crossed with FR-HrasG12V mice, which upon recombination generate endogenous expression of HrasG12V, Hras/Rbm10 mice developed thyroid cancers, ~ 20% of which were metastatic to lung. Cell lines derived from these tumors showed high penetrance of lung metastases after tail vein or orthotopic implantation into the thyroid. We identified splicing targets of RBM10 by high depth RNAseq of 5 isogenic human thyroid cancer cell lines (2 RBM10-null with dox-induced RBM10; 3 RBM10 WT with RBM10 shRNA). The common abnormalities associated with RBM10 loss were exon inclusion events. Ingenuity Pathway Analysis of global transcriptomes in RBM10 isogenic human thyroid cancer cell lines showed enrichment in pro-migratory, aberrant integrin and RHO/RAC signaling expression signatures. Enriched GO analysis confined to genes subject to aberrant splicing by RBM10 loss extended these findings, with the top terms being cell adhesion, cytoskeleton, cadherin and integrin binding. RBM10 loss was associated with increased cell migration velocity in vitro as measured by time lapse imaging. Key cytoskeletal and extracellular matrix genes subject to exon inclusion events included vinculin (VCL), tenascin C (TNC), CD44, fibronectin (FN1) and tropomyosin 1 (TPM1). Isoform-specific knockdown of the VCL splice inclusion cassette exon that leads to illegitimate expression of metavinculin (mVCL) reduced cell migration, whereas isoform-specific knockdown of TNC and CD44 exon inclusion events reduced cell invasiveness in vitro. Rho GTPases transduce signals from the ECM to integrin receptors to regulate cell adhesion, migration, and invasiveness. Consistent with this, RBM10 overexpression in RBM10 null cells reduced RAC1-GTP levels. Finally, RBM10 re-expression in RBM10 null cells reversed metastatic competency in vivo. In conclusion, RBM10 loss alters the ratio of cassette exon inclusion events in a subset of transcripts that regulate interactions between the ECM and the cytoskeleton, leading to RHO/RAC activation and governing a process favoring increased cell movement and metastatic competence. Citation Format: Gnana P. Krishnamoorthy, Anthony Glover, Brian Untch, Mahesh Saqcena, Dina Vukel, Katherine Berman, Omar Abdel-Wahab, Robert K. Bradley, Jeffrey A. Knauf, James A. Fagin. RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 986.

Abstract 883: Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer

Abstract SF3B1 is the most commonly mutated splicing factor in cancer, occurring in a broad variety of hematologic and solid malignancies, including: breast, melanoma, pancreatic, bladder, colorectal, leukemia, and more. SF3B1 is a protein component of the spliceosome responsible for recognizing intronic splice sites and stabilizing the interaction between the spliceosome and pre-mRNA transcripts. Hotspot mutations in SF3B1 result in a neomorphic protein that causes aberrant splicing of hundreds of transcripts. These transcriptional alterations have several proposed impacts on known cancer pathways; however, the mechanisms by which SF3B1 mutations contribute to tumorigenesis are incompletely understood. Interestingly, systematic analysis of online cancer databases shows SF3B1 mutations and TP53 alterations are mutually exclusive in cancers with predominant SF3B1 hotspots, suggesting either synthetic lethality or overlapping roles in promoting tumorigenesis. To address this question, TP53 was knocked-out using CRISPR-Cas9 in AAV-generated SF3B1 K700E mutant knock-in and wild type MCF-10A breast epithelial cells. Successful generation of TP53 knock-outs in both the SF3B1 mutant and wild type groups suggests the mutations are unlikely to be synthetic lethal. Instead, this finding may direct us to novel mechanisms by which SF3B1 mutations contribute to cancer development and progression. Two overarching areas of interest based on preliminary data include changes in mitochondrial metabolism and metabolic reprogramming and alterations in DNA damage, repair, and genomic stability. Further defining the relationship between mutant SF3B1 and TP53 may reveal new mechanisms of tumorigenesis and reveal therapeutic vulnerabilities that can be additionally leveraged against the large subset of cancers with TP53 mutations. Citation Format: Riley Bergman, Ben Park, Sarah Croessmann. Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 883.

Genetic Manipulation Strategies for β-Thalassemia: A Review.

Thalassemias are monogenic hematologic diseases that are classified as α- or β-thalassemia according to its quantitative abnormalities of adult α- or β-globin chains. β-thalassemia has widely spread throughout the world especially in Mediterranean countries, the Middle East, Central Asia, India, Southern China, and the Far East as well as countries along the north coast of Africa and in South America. The one and the only cure for β-thalassemia is allogenic hematopoietic stem cell transplantations (HSCT). Nevertheless, the difficulty to find matched donors has hindered the availability of this therapeutic option. Therefore, this present review explored the alternatives for β-thalassemia treatment such as RNA manipulation therapy, splice-switching, genome editing and generation of corrected induced pluripotent stem cells (iPSCs). Manipulation of β-globin RNA is mediated by antisense oligonucleotides (ASOs) or splice-switching oligonucleotides (SSOs), which redirect pre-mRNA splicing to significantly restore correct β-globin pre-mRNA splicing and gene product in cultured erythropoietic cells. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) are designer proteins that can alter the genome precisely by creating specific DNA double-strand breaks. The treatment of β-thalassemia patient-derived iPSCs with TALENs have been found to correct the β-globin gene mutations, implying that TALENs could be used as a therapy option for β-thalassemia. Additionally, CRISPR technologies using Cas9 have been used to fix mutations in the β-globin gene in cultured cells as well as induction of hereditary persistence of fetal hemoglobin (HPFH), and α-globin gene deletions have proposed a possible therapeutic option for β-thalassemia. Overall, the accumulated research evidence demonstrated the potential of ASOs-mediated aberrant splicing correction of β-thalassemia mutations and the advancements of genome therapy approaches using ZFNs, TALENs, and CRISPR/Cas9 that provided insights in finding the permanent cure of β-thalassemia.

Abstract 1462: Oncogenic activation of FOXR2 driven by somatic acquisition of a LINE-1 promoter in pediatric high-grade glioma

Abstract Long Interspersed Element-1 (LINE-1 or L1) is the only autonomously active mobile genetic element in the human genome. It has been shown to mobilize (i.e., retrotranspose) in cancers, causing de novo somatic L1 insertions. Somatic L1 retrotransposition can cause aberrant splicing of tumor suppressor genes by utilizing cryptic splice donor (SD) sites such as the nucleotide 97 SD in the L1 5’UTR or disrupting repressive regulatory regions of proto-oncogenes, inducing aberrant activation, leading to cancer. In contrast to these reported mechanisms, here we report the first evidence of a driver oncogene activation due to somatic acquisition of an L1 promoter in a pediatric high-grade glioma (HGG). While investigating activation of FOXR2 (forkhead box R2), a known oncogene in CNS embryonal tumors, in a recurrent pediatric diffuse HGG that showed a methylation profile of CNS FOXR2-activated tumors, we identified overexpression of a non-canonical FOXR2 isoform. Transcription of FOXR2 initiated from ~11kb upstream of exon 2 at a site harboring a somatic structural variation (SV) reminiscent of an L1 insertion. Targeted PCR amplification and subsequent PacBio sequencing revealed a 5’ inverted/deleted L1 insertion (~3kb sequence) containing an intact L1 5’UTR, presence of a 3’ transduction sequence, two poly-A tails, and target site duplications. The 3’ transduction sequence matches the sequence downstream an intact polymorphic full-length L1 element at 6p24.1, the likely source L1 element that initiated the FOXR2 somatic insertion. Strikingly, tumor RNA-seq data reveals FOXR2 transcription initiated from the intact sense pol-II promoter of the inserted L1 5’UTR, generating a fusion transcript joining the L1 5’UTR 97 SD to a canonical splice acceptor within exon 2 of FOXR2. Targeted bisulfite sequencing of the FOXR2 L1 5’UTR revealed a hypomethylated state, further supporting its role in FOXR2 transcription initiation. FOXR2 activation is likely a tumor initiating event as the following was observed in the primary tumor that occurred two years prior: a matching recurrent tumor methylation profile, the presence of L1/FOXR2 fusion transcripts, and overexpression of FOXR2. Additional driver mutations, clonal TP53 R175H mutation and PDGFRA amplification, identified in the recurrent tumor but absent in the primary tumor, support the somatic L1 insertion as a tumor initiating event occurring prior to later acquired alterations. Our data represent the first example of oncogenic activation by L1 “promoter donation” via somatic retrotransposition as a novel tumor initiating mechanism. Future large-scale research and development of new computational methods for detecting rearranged L1 insertions are required to determine the prevalence of L1 “promoter donation” in cancer or other diseases. Citation Format: Xiaolong Chen, Diane A. Flasch, Bensheng Ju, Heather L. Mulder, John Easton, Lu Wang, Suzanne J. Baker, Jason Chiang, Jinghui Zhang. Oncogenic activation of FOXR2 driven by somatic acquisition of a LINE-1 promoter in pediatric high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1462.

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C.

Simple SummaryLoss-of-function variants of the RAD51C gene are known to confer a risk of breast and ovarian cancers. In this study, we analyzed the impact of RAD51C variants on splicing, a highly regulated gene expression step by which introns are removed and exons are sequentially joined. Exon recognition is guided by specific sequences, the 3′ and 5′ splice sites, which define the exon boundaries. Variants of these sequences of susceptibility genes may lead to aberrant splicing and abnormal transcripts that may trigger a disease. Splicing can be tested using a biotechnological tool called minigenes, which mimic the human gene of interest. Thus, we checked 20 RAD51C splice-site variants using the minigene mgR51C_ex2-8. We found that they all disrupted the splicing mechanism, and 16 variants could be classified as likely pathogenic. Our findings are clinically actionable, and variant carriers may benefit from tailored prevention protocols and therapies.RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1–4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants.

Unique Ataxia-Oculomotor Apraxia 2 (AOA2) in Israel with Novel Variants, Atypical Late Presentation, and Possible Identification of a Poison Exon.

AOA2 is a rare progressive adolescent-onset disease characterised by cerebellar vermis atrophy, peripheral neuropathy and elevated serum alpha-fetoprotein (AFP) caused by pathogenic bi-allelic variants in SETX, encoding senataxin, involved in DNA repair and RNA maturation. Sanger sequencing of genomic DNA, co-segregation and oxidative stress functional studies were performed in Family 1. Trio whole-exome sequencing (WES), followed by SETX RNA and qRT-PCR analysis, were performed in Family 2. Sanger sequencing in Family 1 revealed two novel in-frame SETX deletion and duplication variants in trans (c.7009_7011del; p.Val2337del and c.7369_7371dup; p.His2457dup). Patients had increased induced chromosomal aberrations at baseline and following exposure to higher mitomycin-C concentration and increased sensitivity to oxidative stress at the lower mitomycin-C concentration in cell viability test. Trio WES in Family 2 revealed two novel SETX variants in trans, a nonsense variant (c.568C > T; p.Gln190*), and a deep intronic variant (c.5549-107A > G). Intronic variant analysis and SETX mRNA expression revealed activation of a cryptic exon introducing a premature stop codon (p.Met1850Lysfs*18) and resulting in aberrant splicing, as shown by qRT-PCR analysis, thus leading to higher levels of cryptic exon activation. Along with a second deleterious allele, this variant leads to low levels of SETX mRNA and disease manifestations. Our report expands the phenotypic spectrum of AOA2. Results provide initial support for the hypomorphic nature of the novel in-frame deletion and duplication variants in Family 1. Deep-intronic variant analysis of Family 2 variants potentially reveals a previously undescribed poison exon in the SETX gene, which may contribute to tailored therapy development.

HGG-54. CLK1 aberrant splicing in pediatric high-grade gliomas disrupts key oncogenic transcriptional programs

Abstract While much of the somatic coding variation underlying the oncogenic transformation of pediatric high-grade gliomas (HGGs) has been profiled, transcriptional splicing programs of these tumors remain under-explored. Here, we characterize aberrant alternative splicing in pediatric midline HGGs (n = 84). We identified 19,275 recurrent and significant (20% change from control, P &amp;lt; 0.05, FDR &amp;lt; 0.05) aberrant splicing events in 8,587 genes compared to non-diseased brainstem controls. Of those, 27% (n = 5,157) resulted in either a gain or loss of a known protein functional site within 3,294 genes. We prioritized splice variants affecting targetable kinases and found that mRNAs encoding CDC-like kinase 1 (CLK1), a known modulator of master splicing regulators, exhibit significantly increased exon 4 inclusion in midline HGGs. This leads to a gain of two known phosphorylation sites in CLK1, increased CLK1 protein expression and hyper-phosphorylation of Serine-rich splicing factors. To assess the impact of this event, we performed differential splicing and expression analyses, comparing tumors with the highest (n= 5) and lowest (n = 5) exon 4 inclusion. We discovered 3,037 genes to be differentially up-regulated in high exon 4 inclusion tumors with an enrichment of cancer-related pathways, including DNA repair, mitotic spindle, myogenesis and EMT. We next integrated these gene signatures with protein-protein interaction networks of kinase and transcription factors and show that increased CLK1 exon 4 inclusion disrupts critical regulatory networks, such as those involving FOXM1, which is implicated in cell cycle and proliferation processes. In summary, we describe aberrant splicing in pediatric HGGs as an additional mechanism that could drive tumorigenesis. Future work will focus on molecular validation and therapeutic targeting of CLK1 in available HGG models. Characterizing tumor-specific splicing variation has the potential to open new therapeutic strategies and understand mechanisms of treatment resistance in children with central nervous system tumors.

Targeted RNAseq Improves Clinical Diagnosis of Very Early-Onset Pediatric Immune Dysregulation

Despite increased use of whole exome sequencing (WES) for the clinical analysis of rare disease, overall diagnostic yield for most disorders hovers around 30%. Previous studies of mRNA have succeeded in increasing diagnoses for clearly defined disorders of monogenic inheritance. We asked if targeted RNA sequencing could provide similar benefits for primary immunodeficiencies (PIDs) and very early-onset inflammatory bowel disease (VEOIBD), both of which are difficult to diagnose due to high heterogeneity and variable severity. We performed targeted RNA sequencing of a panel of 260 immune-related genes for a cohort of 13 patients (seven suspected PID cases and six VEOIBD) and analyzed variants, splicing, and exon usage. Exonic variants were identified in seven cases, some of which had been previously prioritized by exome sequencing. For four cases, allele specific expression or lack thereof provided additional insights into possible disease mechanisms. In addition, we identified five instances of aberrant splicing associated with four variants. Three of these variants had been previously classified as benign in ClinVar based on population frequency. Digenic or oligogenic inheritance is suggested for at least two patients. In addition to validating the use of targeted RNA sequencing, our results show that rare disease research will benefit from incorporating contributing genetic factors into the diagnostic approach.

A Chemical Biology Perspective to Therapeutic Regulation of RNA Splicing in Spinal Muscular Atrophy (SMA).

Manipulation of RNA splicing machinery has emerged as a drug modality. Here, we illustrate the potential of this novel paradigm to correct aberrant splicing events focused on the recent therapeutic advances in spinal muscular atrophy (SMA). SMA is an incurable neuromuscular disorder and at present the primary genetic cause of early infant death. This Review summarizes the exciting journey from the first reported SMA cases to the currently approved splicing-switching treatments, i.e., antisense oligonucleotides and small-molecule modifiers. We emphasize both chemical structures and molecular bases for recognition. We briefly discuss the advantages and disadvantages of these treatments and include the remaining challenges and future directions. Finally, we also predict that these success stories will contribute to further therapies for human diseases by RNA-splicing control.

Diminished Rbfox1 increases vascular constriction by dynamically regulating alternative splicing of CaV1.2 calcium channel in hypertension.

Calcium influx from depolarized CaV1.2 calcium channels triggers the contraction of vascular smooth muscle cells (VSMCs), which is important for maintaining vascular myogenic tone and blood pressure. The function of CaV1.2 channel can be subtly modulated by alternative splicing (AS), and its aberrant splicing involves in the pathogenesis of multiple cardiovascular diseases. The RNA-binding protein Rbfox1 is reported to regulate the AS events of CaV1.2 channel in the neuronal development, but its potential roles in vascular CaV1.2 channels and vasoconstriction remain undefined. Here, we detect Rbfox1 is expressed in rat vascular smooth muscles. Moreover, the protein level of Rbfox1 is dramatically decreased in the hypertensive small arteries from spontaneously hypertensive rats in comparison with normotensive ones from Wistar-Kyoto rats. In VSMCs, Rbfox1 could dynamically regulate the AS of CaV1.2 exons 9* and 33. By whole-cell patch clamp, we identify knockdown of Rbfox1 induces the hyperpolarization of CaV1.2 current-voltage relationship curve in VSMCs. Furthermore, siRNA-mediated knockdown of Rbfox1 increases the K+-induced constriction of rat mesenteric arteries. In summary, our results indicate Rbfox1 modulates vascular constriction by dynamically regulating CaV1.2 alternative exons 9* and 33. Therefore, our work elucidates the underlying mechanisms for CaV1.2 channels regulation and provides a potential therapeutic target for hypertension.

Exon junction complex-associated multi-adapter RNPS1 nucleates splicing regulatory complexes to maintain transcriptome surveillance.

The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing and nonsense-mediated mRNA decay (NMD) via various interacting proteins. The peripheral EJC-binding protein RNPS1 was reported to serve two insufficiently characterized functions: suppressing mis-splicing of cryptic splice sites and activating NMD in the cytoplasm. The analysis of transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines supports the conclusion that RNPS1 can moderately influence NMD activity, but is not a globally essential NMD factor. However, numerous aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that the RRM and C-terminal domain of RNPS1 both contribute partially to regulate RNPS1-dependent splicing events. We defined the RNPS1 core interactome using complementary immunoprecipitations and proximity labeling, which identified interactions with splicing-regulatory factors that are dependent on the C-terminus or the RRM domain of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.

Correction of Beta-Thalassemia IVS-II-654 Mutation in a Mouse Model Using Prime Editing.

Prime editing was used to insert and correct various pathogenic mutations except for beta-thalassemia variants, which disrupt functional beta-globin and prevent hemoglobin assembly in erythrocytes. This study investigated the effect of gene correction using prime editor version 3 (PE3) in a mouse model with the human beta-thalassemia IVS-II-654 mutation (C > T). The T conversion generates a 5′ donor site at intron 2 of the beta-globin gene resulting in aberrant splicing of pre-mRNA, which affects beta-globin expression. We microinjected PE3 components (pegRNA, nick sgRNA, and PE2 mRNA) into the zygotes from IVS-II-654 mice to generate mutation-edited mice. Genome sequencing of the IVS-II-654 site showed that PE3 installed the correction (T > C), with an editing efficiency of 14.29%. Reverse transcription-PCR analysis showed that the PE3-induced conversion restored normal splicing of beta-globin mRNA. Subsequent comprehensive phenotypic analysis of thalassemia symptoms, including anemic hematological parameters, anisocytosis, splenomegaly, cardiac hypertrophy, extramedullary hematopoiesis, and iron overload, showed that the corrected IVS-II-654 mice had a normal phenotype identical to the wild type mice. Off-target analysis of pegRNA and nick sgRNA additionally showed the genomic safety of PE3. These results suggest that correction of beta-thalassemia mutation by PE3 may be a straightforward therapeutic strategy for this disease.

Calpain-2 Mediates MBNL2 Degradation and a Developmental RNA Processing Program in Neurodegeneration.

Increasing loss of structure and function of neurons and decline in cognitive function is commonly seen during the progression of neurologic diseases, although the causes and initial symptoms of individual diseases are distinct. This observation suggests a convergence of common degenerative features. In myotonic dystrophy type 1 (DM1), the expression of expanded CUG RNA induces neurotransmission dysfunction before axon and dendrite degeneration and reduced MBNL2 expression associated with aberrant alternative splicing. The role of loss of function of MBNL2 in the pathogenesis of neurodegeneration and the causal mechanism of neurodegeneration-reduced expression of MBNL2 remain elusive. Here, we show that increased MBNL2 expression is associated with neuronal maturation and required for neuronal morphogenesis and the fetal to adult developmental transition of RNA processing. Neurodegenerative conditions including NMDA receptor (NMDAR)-mediated excitotoxicity and dysregulated calcium homeostasis triggered nuclear translocation of calpain-2, thus resulting in MBNL2 degradation and reversal of MBNL2-regulated RNA processing to developmental patterns. Nuclear expression of calpain-2 resembled its developmental pattern and was associated with MBNL2 degradation. Knock-down of calpain-2 expression or inhibition of calpain-2 nuclear translocation prevented neurodegeneration-reduced MBNL2 expression and dysregulated RNA processing. Increased calpain-2 nuclear translocation associated with reduced MBNL2 expression and aberrant RNA processing occurred in models for DM1 and Alzheimer's disease (AD) including EpA960/CaMKII-Cre mice of either sex and female APP/PS1 and THY-Tau22 mice. Our results identify a regulatory mechanism for MBNL2 downregulation and suggest that calpain-2-mediated MBNL2 degradation accompanied by re-induction of a developmental RNA processing program may be a converging pathway to neurodegeneration.SIGNIFICANCE STATEMENT Neurologic diseases share many features during disease progression, such as cognitive decline and brain atrophy, which suggests a common pathway for developing degenerative features. Here, we show that the neurodegenerative conditions glutamate-induced excitotoxicity and dysregulated calcium homeostasis induced translocation of the cysteine protease calpain-2 into the nucleus, resulting in MBNL2 degradation and reversal of MBNL2-regulated RNA processing to an embryonic pattern. Knock-down or inhibition of nuclear translocation of calpain-2 prevented MBNL2 degradation and maintained MBNL2-regulated RNA processing in the adult pattern. Models of myotonic dystrophy and Alzheimer's disease (AD) also showed calpain-2-mediated MBNL2 degradation and a developmental RNA processing program. Our studies suggest MBNL2 function disrupted by calpain-2 as a common pathway, thus providing an alternative therapeutic strategy for neurodegeneration.

Identifying common transcriptome signatures of cancer by interpreting deep learning models

BackgroundCancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified. It is not clear, however, whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types.ResultsHere, we agnostically identify transcriptomic features that are commonly shared between cancer types using 13,461 RNA-seq samples from 19 normal tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression, or splice junction use, to distinguish between normal and tumor samples. All three models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints. Importantly, we find that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer.ConclusionsOur results highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features on which core cancer pathways might converge across a large array of solid tumor types.

Characterization and clustering of kinase isoform expression in metastatic melanoma.

Mutations to the human kinome are known to play causal roles in cancer. The kinome regulates numerous cell processes including growth, proliferation, differentiation, and apoptosis. In addition to aberrant expression, aberrant alternative splicing of cancer-driver genes is receiving increased attention as it could lead to loss or gain of functional domains, altering a kinase's downstream impact. The present study quantifies changes in gene expression and isoform ratios in the kinome of metastatic melanoma cells relative to primary tumors. We contrast 538 total kinases and 3,040 known kinase isoforms between 103 primary tumor and 367 metastatic samples from The Cancer Genome Atlas (TCGA). We find strong evidence of differential expression (DE) at the gene level in 123 kinases (23%). Additionally, of the 468 kinases with alternative isoforms, 60 (13%) had significant difference in isoform ratios (DIR). Notably, DE and DIR have little correlation; for instance, although DE highlights enrichment in receptor tyrosine kinases (RTKs), DIR identifies altered splicing in non-receptor tyrosine kinases (nRTKs). Using exon junction mapping, we identify five examples of splicing events favored in metastatic samples. We demonstrate differential apoptosis and protein localization between SLK isoforms in metastatic melanoma. We cluster isoform expression data and identify subgroups that correlate with genomic subtypes and anatomic tumor locations. Notably, distinct DE and DIR patterns separate samples with BRAF hotspot mutations and (N/K/H)RAS hotspot mutations, the latter of which lacks effective kinase inhibitor treatments. DE in RAS mutants concentrates in CMGC kinases (a group including cell cycle and splicing regulators) rather than RTKs as in BRAF mutants. Furthermore, isoforms in the RAS kinase subgroup show enrichment for cancer-related processes such as angiogenesis and cell migration. Our results reveal a new approach to therapeutic target identification and demonstrate how different mutational subtypes may respond differently to treatments highlighting possible new driver events in cancer.

Transcriptome analysis of clock disrupted cancer cells reveals differential alternative splicing of cancer hallmarks genes

Emerging evidence points towards a regulatory role of the circadian clock in alternative splicing (AS). Whether alterations in core-clock components may contribute to differential AS events is largely unknown. To address this, we carried out a computational analysis on recently generated time-series RNA-seq datasets from three core-clock knockout (KO) genes (ARNTL, NR1D1, PER2) and WT of a colorectal cancer (CRC) cell line, and time-series RNA-seq datasets for additional CRC and Hodgkin’s lymphoma (HL) cells, murine WT, Arntl KO, and Nr1d1/2 KO, and murine SCN WT tissue. The deletion of individual core-clock genes resulted in the loss of circadian expression in crucial spliceosome components such as SF3A1 (in ARNTLKO), SNW1 (in NR1D1KO), and HNRNPC (in PER2KO), which led to a differential pattern of KO-specific AS events. All HCT116KO cells showed a rhythmicity loss of a crucial spliceosome gene U2AF1, which was also not rhythmic in higher progression stage CRC and HL cancer cells. AS analysis revealed an increase in alternative first exon events specific to PER2 and NR1D1 KO in HCT116 cells, and a KO-specific change in expression and rhythmicity pattern of AS transcripts related to cancer hallmarks genes including FGFR2 in HCT116_ARNTLKO, CD44 in HCT116_NR1D1KO, and MET in HCT116_PER2KO. KO-specific changes in rhythmic properties of known spliced variants of these genes (e.g. FGFR2 IIIb/FGFR2 IIIc) correlated with epithelial-mesenchymal-transition signalling. Altogether, our bioinformatic analysis highlights a role for the circadian clock in the regulation of AS, and reveals a potential impact of clock disruption in aberrant splicing in cancer hallmark genes.

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

Silent but Not Harmless: A Synonymous SLC5A5 Gene Variant Leading to Dyshormonogenic Congenital Hypothyroidism.

BackgroundCongenital iodide transport defect (ITD) is an uncommon cause of dyshormonogenic congenital hypothyroidism characterized by the absence of active iodide accumulation in the thyroid gland. ITD is an autosomal recessive disorder caused by loss-of-function variants in the sodium/iodide symporter (NIS)-coding SLC5A5 gene.ObjectiveWe aimed to identify, and if so to functionally characterize, novel ITD-causing SLC5A5 gene variants in a cohort of five unrelated pediatric patients diagnosed with dyshormonogenic congenital hypothyroidism with minimal to absent 99mTc-pertechnetate accumulation in the thyroid gland.MethodsThe coding region of the SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of a novel synonymous variant were performed.ResultsSanger sequencing revealed a novel homozygous synonymous SLC5A5 gene variant (c.1326A>C in exon 11). In silico analysis revealed that the c.1326A>C variant is potentially deleterious for NIS pre-mRNA splicing. The c.1326A>C variant was predicted to lie within a putative exonic splicing enhancer reducing the binding of splicing regulatory trans-acting protein SRSF5. Splicing minigene reporter assay revealed that c.1326A>C causes exon 11 or exon 11 and 12 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variants p.G415_P443del and p.G415Lfs*32, respectively. Significantly, the frameshift variant p.G415Lfs*32 is predicted to be subjected to degradation by nonsense-mediated decay.ConclusionsWe identified the first exonic synonymous SLC5A5 gene variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape of the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.

RNA analysis of the GALNS transcript reveals novel pathogenic mechanisms associated with Morquio syndrome A

Morquio syndrome A (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficiency of N-acetyl-galactosamine-6-sulfatase (GALNS) which catabolizes the glycosaminoglycans (GAG), keratan sulfate and chondroitin-6-sulfate. Homozygous or compound heterozygous pathogenic variants in the GALNS result in the deficiency of the enzyme and consequent GAG accumulations. DNA sequence and copy number analysis of the GALNS coding region fails to identify biallelic causative pathogenic variants in up to 15% of patients with Morquio syndrome A. RNA transcript analysis was performed to identify pathogenic alterations in two unrelated families with Morquio syndrome A in whom a single heterozygous or no pathogenic alteration was detected by standard analysis of the GALNS gene. RNA sequencing and quantitative expression analysis identified the overabundance of an aberrant GALNS transcript isoform and a reduction of the clinically relevant isoform (NM_000512.4) in the Morquio syndrome A patients from both families. The aberrant isoform (ENST00000568613.1) was produced by alternative splicing and contained intronic sequence that was likely a cryptic exon predicted to result in a reading frame shift and generation of a premature termination codon. These findings indicated that the aberrant splicing is likely the novel molecular defect in our patients. RNA transcript analysis could be useful to identify pathogenic alterations and increase the yield of molecular diagnosis in patients with Morquio syndrome A whose genetic variants are not found by standard sequencing or gene dosage analysis.