Aberrant Splicing Events Research Articles

Competing Endogenous RNA (ceRNA) Networks and Splicing Switches in Cervical Cancer: HPV Oncogenesis, Clinical Significance and Therapeutic Opportunities.

Cervical cancer (CC) is the primary cause of female cancer fatalities in low-middle-income countries (LMICs). Persistent infections from the human papillomavirus (HPV) can result in cervical cancer. However, numerous different factors influence the development and progression of cervical cancer. Transcriptomic knowledge of the mechanisms with which HPV causes cervical cancer pathogenesis is growing. Nonetheless, there is an existing gap hindering the development of therapeutic approaches and the improvement of patient outcomes. Alternative splicing allows for the production of numerous RNA transcripts and protein isoforms from a single gene, increasing the transcriptome and protein diversity in eukaryotes. Cancer cells exhibit astounding transcriptome modifications by expressing cancer-specific splicing isoforms. High-risk HPV uses cellular alternative splicing events to produce viral and host splice variants and proteins that drive cancer progression or contribute to distinct cancer hallmarks. Understanding how viruses utilize alternative splicing to drive pathogenesis and tumorigenesis is essential. Although research into the role of miRNAs in tumorigenesis is advancing, the function of other non-coding RNAs, including lncRNA and circRNA, has been understudied. Through their interaction with mRNA, non-coding RNAs form a network of competing endogenous RNAs (ceRNAs), which regulate gene expression and promote cervical cancer development and advancement. The dysregulated expression of non-coding RNAs is an understudied and tangled process that promotes cervical cancer development. This review will present the role of aberrant alternative splicing and immunosuppression events in HPV-mediated cervical tumorigenesis, and ceRNA network regulation in cervical cancer pathogenesis will also be discussed. Furthermore, the therapeutic potential of splicing disruptor drugs in cervical cancer will be deliberated.

Multi-omics analysis reveals RNA splicing alterations and their biological and clinical implications in lung adenocarcinoma

Alternative RNA splicing is one of the most important mechanisms of posttranscriptional gene regulation, which contributes to protein diversity in eukaryotes. It is well known that RNA splicing dysregulation is a critical mechanism in tumor pathogenesis and the rationale for the promising splice-switching therapeutics for cancer treatment. Although we have a comprehensive understanding of DNA mutations, abnormal gene expression profiles, epigenomics, and proteomics in lung adenocarcinoma (LUAD), little is known about its aberrant alternative splicing profiles. Here, based on the multi-omics data generated from over 1000 samples, we systematically studied the RNA splicing alterations in LUAD and revealed their biological and clinical implications. We identified 3688 aberrant alternative splicing events (AASEs) in LUAD, most of which were alternative promoter and exon skip. The specific regulatory roles of RNA binding proteins, somatic mutations, and DNA methylations on AASEs were comprehensively interrogated. We dissected the functional implications of AASEs and concluded that AASEs mainly affected biological processes related to tumor proliferation and metastasis. We also found that one subtype of LUAD with a particular AASEs pattern was immunogenic and had a better prognosis and response rate to immunotherapy. These findings revealed novel events related to tumorigenesis and tumor immune microenvironment and laid the foundation for the development of splice-switching therapies for LUAD.

Comprehensive analysis of prognosis-related alternative splicing events in ovarian cancer

ABSTRACT Ovarian cancer (OV) is characterized by high incidence and poor prognosis. Increasing evidence indicates that aberrant alternative splicing (AS) events are associated with the pathogenesis of cancer. We examined prognosis-related alternative splicing events and constructed a clinically applicable model to predict patients’ outcomes. Public database including The Cancer Genome Atlas (TCGA), TCGA SpliceSeq, and the Genomics of Drug Sensitivity in Cancer databases were used to detect the AS expression, immune cell infiltration and IC50. The prognosis-related AS model was constructed and validated by using Cox regression, LASSO regression, C-index, calibration plots, and ROC curves. A total of eight AS events (including FLT3LG|50942|AP) were selected to establish the prognosis-related AS model. Compared with high-risk group, low-risk group had a better outcome (P = 1.794e-06), was more sensitive to paclitaxel (P = 0.022), and higher proportions of plasma cells. We explored the upstream regulatory mechanisms of prognosis-related AS and found that two splicing factor and 156 tag single nucleotide polymorphisms may be involved in the regulation of prognosis-related AS. In order to assess patient prognosis more comprehensively, we constructed a clinically applicable model combining risk score and clinicopathological features, and the 1 -, and 3-year AUCs of the clinically applicable model were 0.812, and 0.726, which were 7.5% and 3.3% higher than that of the risk score. We constructed a prognostic signature for OV patients and comprehensively analysed the regulatory characteristics of the prognostic AS events in OV.

Emerging Therapies and Novel Targets for TDP-43 Proteinopathy in ALS/FTD.

Nuclear clearance and cytoplasmic mislocalization of the essential RNA binding protein, TDP-43, is a pathologic hallmark of amyotrophic lateral sclerosis, frontotemporal dementia, and related neurodegenerative disorders collectively termed "TDP-43 proteinopathies." TDP-43 mislocalization causes neurodegeneration through both loss and gain of function mechanisms. Loss of TDP-43 nuclear RNA processing function destabilizes the transcriptome by multiple mechanisms including disruption of pre-mRNA splicing, the failure of repression of cryptic exons, and retrotransposon activation. The accumulation of cytoplasmic TDP-43, which is prone to aberrant liquid-liquid phase separation and aggregation, traps TDP-43 in the cytoplasm and disrupts a host of downstream processes including the trafficking of RNA granules, local translation within axons, and mitochondrial function. In this review, we will discuss the TDP-43 therapy development pipeline, beginning with therapies in current and upcoming clinical trials, which are primarily focused on accelerating the clearance of TDP-43 aggregates. Then, we will look ahead to emerging strategies from preclinical studies, first from high-throughput genetic and pharmacologic screens, and finally from mechanistic studies focused on the upstream cause(s) of TDP-43 disruption in ALS/FTD. These include modulation of stress granule dynamics, TDP-43 nucleocytoplasmic shuttling, RNA metabolism, and correction of aberrant splicing events.

Abstract 2725: Molecular mechanisms of aberrant splicing in pediatric high grade gliomas

Abstract Pediatric brain cancer is the number one cause of disease-related death in children. While much of the somatic mutation variation in these tumors have been profiled, there have been few studies exploring their transcriptional splicing programs. Here, we characterize aberrant alternative splicing across pediatric brain tumors. We identified three splicing-based clusters that spanned across seven broad histological tumor classifications. Patients in clusters 1 and 2 had significantly lower overall survival (p < 0.001) than those in cluster 3. High-grade gliomas (HGGs) displayed the greatest splicing heterogeneity, spanning all three clusters. We focused on midline HGGs (n = 84), identifying 19,275 recurrent and significant (20% change from control, P < 0.05, FDR < 0.05) aberrant splicing events in 8,587 genes compared to non-diseased brainstem controls. We also identified several splicing factor genes, including SRSF11, that were differentially expressed (p < 0.05), suggesting a potential tumor-specific novel mechanism of splicing dysregulation. Strikingly, 27% (5,157) of these splicing events resulted in either a gain or loss of a functional site in 3,294 genes: disulfide bonding (n = 391), localization signaling (n = 216), modifications, such as phosphorylation sites (n = 1,670), and other, such as ion-binding sites (n = 4,245). We prioritized splice variants affecting targetable kinases and found that mRNAs encoding CDC-like kinase 1 (CLK1), a known modulator of the SR family of master splicing regulators, exhibit significantly increased exon 4 inclusion. This leads to a gain of two known phosphorylation sites in CLK1, increased protein expression, and hyper-phosphorylation of SR proteins, inducing cooperative binding of SR proteins to RNA. To assess the impact of this CLK1 event, we compared splicing in tumors with high exon 4 inclusion (n = 5) to those with low exon 4 inclusion (n = 5) and discovered 862 differential splicing events (DSE). Samples with high CLK1 exon 4 inclusion had significantly lower percent spliced in events, suggesting that increased activity of CLK1 resulting from the gain of the exon 4 promotes overall exon skipping. Additionally, more than 80% of the DSE (n = 695), including those in known brain tumor oncogenes such as CDC27 and E2H2, were recurrently mis-spliced in midline HGG tumors, suggesting that CLK1 splicing dysregulation leads to a global aberrant splicing phenotype in HGGs. Finally, we determined a subset of events significantly associated with prolonged survival, suggesting that stratification of patients based on splicing phenotypes can be utilized as a prognostic indicator. In summary, we demonstrate aberrant splicing in pediatric HGGs as an alternative mechanism driving tumorigenesis and future work will focus on urgently needed therapeutic targeting of these pathways toward our overall goal of improving survival outcomes in children with brain tumors. Citation Format: Ammar S. Naqvi, Brian Ennis, Run Jin, Karina L. Conkrite, Komal S. Rathi, Adam A. Kraya, Poonam Sonawane, Phillip B. Storm, Adam C. Resnick, Jo Lynne Rokita. Molecular mechanisms of aberrant splicing in pediatric high grade gliomas [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 2725.

Abstract 778: Dysregulation of alternative mRNA splicing by oncogenic KRAS in lung adenocarcinoma

Abstract Alternative mRNA splicing is dysregulated in many cancers including lung adenocarcinoma. These aberrant splicing events can sometimes be explained by mutations in splice sites or splicing factors. However, the majority of mis-splicing in cancers remains unexplained. We hypothesize that oncogenic signaling pathways play a role in regulating alternative splicing. In this study, we focus on mRNA splicing activity regulated by the Ras signaling cascade, which is frequently mutated in cancers. These pathways offer avenues for therapeutic modulation through small molecule inhibitors.We took a global proteomic and transcriptomic approach to study the effects of oncogenic Ras signaling on RNA splicing in vitro. Specifically, we performed LC-MS/MS and RNA-seq profiling on normal human lung airway epithelial (AALE) cells overexpressing wild-type or mutant KRAS or RIT1 (N = 3 replicates per allele). Phosphorylation of splicing factors was preferentially downregulated in KRASmut cells compared to KRASWT cells, suggesting that oncogenic KRAS signaling regulates splicing factor activity. Additionally, of the 2227 and 2452 skipped exon (SE) events in KRASG12V and KRASQ61H cells, respectively, compared to KRASWT overexpressing cells, 1013 events were shared between the two mutants. This statistically significant overlap (p < 0.001, hypergeometric test) indicates that KRAS has a role in splicing regulation that is disrupted when the protein is mutated. To determine how KRAS-regulated splicing activity compared to other signaling proteins and oncogenes, we also profiled the whole transcriptomes of isogenic A549 lung adenocarcinoma cell lines overexpressing 86 wild-type or variant alleles across 27 genes implicated in lung cancers (N = 4 to 8 replicates per allele). Of all pairs of mutant and wild-type alleles tested, KRASmut cells compared to KRASWT overexpressing cells exhibited the second highest levels of alternative splicing, behind only RNA binding protein RBM45 and its mutant allele RBM45M126I. In particular, ten SE events were observed to be differentially regulated in a KRAS dependent manner in both AALE and A549 cells. One of these 10 events is the differential splicing of the Myc Associated Zinc Finger (MAZ) protein to increase the expression of its splice variant that acts as a negative regulator of transcription. As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of KRAS mutants. Our proteomic and transcriptomic profiling in lung epithelial and lung adenocarcinoma cells uncovers splicing factor activity and mRNA splicing events regulated by oncogenic KRAS. With further studies of the alternative splicing events described, it will be possible to design or repurpose therapies in order to target aberrant splicing caused by disrupted Ras signaling and its downstream effectors. Citation Format: April Lo, Maria McSharry, Alice H. Berger. Dysregulation of alternative mRNA splicing by oncogenic KRAS in lung adenocarcinoma [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 778.

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 < 0.05, FDR < 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.

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.

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.

Transcriptome-based variant calling and aberrant mRNA discovery enhance diagnostic efficiency for neuromuscular diseases

BackgroundWhole-exome sequencing-based diagnosis of rare diseases typically yields 40%–50% of success rate. Precise diagnosis of the patients with neuromuscular disorders (NMDs) has been hampered by locus heterogeneity or phenotypic heterogeneity....

Comprehensive Transcriptome Analysis of Patients With Keratoconus Highlights the Regulation of Immune Responses and Inflammatory Processes

Keratoconus (KTCN), characterized by the steeper curvature of the cornea and the thinner central corneal thickness, was a degenerative corneal ectasia with ambiguous etiology and mechanism. We aim to reveal underlying pathological mechanisms of KTCN by multi-level transcriptomic, integrative omics analyses. We performed RNA-sequencing on corneal epithelial samples from seven patients and seven control donors, as well as peripheral matched blood samples from three of the patients and three control donors. After RNA extraction, library construction, and sequencing, differentially expressed genes and splicing events were identified, followed by over-representation analysis. In total, 547 differential expressed genes were identified in KTCN corneal epithelial samples, which were mainly enriched in immune responses and inflammatory processes. WGCNA module analysis, the transcriptomic analysis of peripheral blood samples, multiple omics data, and the meta-analysis of GEO samples confirmed the involvement of immune and inflammatory factors. Besides, 190 and 1,163 aberrant splicing events were identified by rMATS combined with CASH methods in corneal epithelial and blood samples with KCTN. In conclusion, this comprehensive transcriptome analysis of KTCN patients based on patients’ tissue and blood samples uncovered a significant association between immune-inflammatory genes and pathways with KCTN, highlighting the contribution of the perturbed immune signaling to the pathogenesis of KCTN. Our study suggested the importance of measures to control inflammation in the treatment of KCTN.

Characterization of aberrant alternative splicing landscape in patients with renal cell carcinoma (RCC).

386 Background: Aberrant alternative splicing (AS) events have been implicated in the initiation and progression of various cancers; however, the detailed nature of their role in RCC is yet to be fully elucidated. Our study aims to characterize AS events in RCC tumors using a novel AS pipeline (Bisbee). Methods: We retrospectively identified patients (pts) with RCC who had tumor-normal whole exome sequencing and tumor whole transcriptome sequencing (GEMExtra, Ashion Analytics) performed as part of their routine clinical care. AS events from RNA sequencing data were identified and further characterized as (1) alternative splice 3’ site (A3), (2) alternative splice 5’ site (A5), (3) exon skipping (ES), (4) intron retention (IR), and (5) mutually exclusive exons splice events (MUT). The Bisbee outlier analysis was performed against normal kidney tissues from the GTEx tissue library to further identify tumor-associated splice events. Outlier splice events were categorized as either non-coding/protein loss/silent, isoform switch, novel, or unknown. Results: Overall, 147 RCC pts (77% male) with RNA sequencing data were included in this analysis. Median age at diagnosis was 60 (range 31-94) and 97% of pts had metastatic RCC. The distribution of histology was 85% clear cell RCC followed by 11% papillary RCC. The AS analysis identified 25,928 outlier splice events. Approximately 60% of these were predicted to be protein-coding events, with the majority arising from IR and ES. These were followed by A3, A5, and MUT, in descending order of frequency. We also examined tumor-associated novel outlier events where 70% of analyzed RCC tumor samples noted 34 tumor-associated novel events were present, shared in most of the cohort and found an enrichment for IR events leading to frame disruptions. Data of splice variants will be presented at the meeting. Conclusions: In depth examination of this large cohort suggests that IR resulting from AS events occur frequently within RCC. Further efforts to investigate the association of AS events and clinical outcomes are underway.

Modulation of RNA Splicing by Oligonucleotides: Mechanisms of Action and Therapeutic Implications.

Dysregulation of RNA splicing causes many diseases and disorders. Several therapeutic approaches have been developed to correct aberrant alternative splicing events for the treatment of cancers and hereditary diseases, including gene therapy and redirecting splicing, using small molecules or splice switching oligonucleotides (SSO). Significant advances in the chemistry and pharmacology of nucleic acid have led to the development of clinically approved SSO drugs for the treatment of spinal muscular dystrophy and Duchenne muscular dystrophy (DMD). In this review, we discuss the mechanisms of SSO action with emphasis on "less common" approaches to modulate alternative splicing, including bipartite and bifunctional SSO, oligonucleotide decoys for splice factors and SSO-mediated mRNA degradation via AS-NMD and NGD pathways. We briefly discuss the current progress and future perspectives of SSO therapy for rare and ultrarare diseases.

Aberrant splicing events caused by insertion of genes of interest into expression vectors.

Background: Expression of genes of interest from plasmids or lentiviral vectors is one of the most common tools in molecular and gene therapy. Aberrant splicing between the inserted gene of interest and downstream vector sequence has not been systematically analyzed.Methods: Formation of aberrant fusion transcripts and proteins was detected by RT-PCR, sequencing, Western blotting and mass spectrometry. Bioinformatic analysis was performed to identify all human and mouse genes prone to vector-dependent aberrant splicing. Selected genes were experimentally validated.Results: When we expressed human FACI in cultured cells, an aberrant splicing event was found to occur between FACI transcript and downstream plasmid sequence through one exon-exon junction in FACI that accidentally contributes a splice donor site. To explore whether this could be a general phenomenon, we searched the whole human and mouse genomes for protein-coding genes that harbor an exon-exon junction resembling a splice donor site. Almost all genes prone to this type of aberrant splicing were identified. A total of 17 genes among the hits were randomly selected for experimental validation. RT-PCR and sequencing results verified that 13 genes were aberrantly spliced on the identified exon-exon junctions. In addition, all 17 genes were aberrantly spliced on their V5 tag sequence. Aberrant fusion protein expression from all 17 genes was validated by immunoblotting. Aberrant splicing was prevented by recoding the V5 tag or the splice sites.Conclusions: Our study revealed an unexpectedly high frequency of vector-dependent aberrant splicing events. Aberrant formation of the resulting fusion proteins could undermine the accuracy of gain-of-function studies and might cause potential side effects when the therapeutic gene is expressed in vivo. Our work has implications in improving vector construction and epitope tagging for gene expression and therapy.

Immunotherapeutic Significance of a Prognostic Alternative Splicing Signature in Bladder Cancer.

Objectives: Bladder cancer is the fourth most common malignancy in men in the United States. Aberrant alternative splicing (AS) events are involved in the carcinogenesis, but the association between AS and bladder cancer remains unclear. This study aimed to construct an AS-based prognostic signature and elucidate the role of the tumor immune microenvironment (TIME) and the response to immunotherapy and chemotherapy in bladder cancer. Methods: Univariate Cox regression analysis was performed to detect prognosis-related AS events. The least absolute shrinkage and selection operator (LASSO) and multivariate Cox analyses were employed to build prognostic signatures. Kaplan–Meier survival analysis, multivariate Cox regression analysis, and receiver operating characteristic (ROC) curves were conducted to validate the prognostic signatures. Then, the Estimation of Stromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) and tumor immune estimation resource (TIMER) databases were searched and the single-sample gene set enrichment analysis (ssGSEA) algorithm and CIBERSORT method were performed to uncover the context of TIME in bladder cancer. The Tumor Immune Dysfunction and Exclusion (TIDE) web tool and pRRophetic algorithm were used to predict the response to immunotherapy and chemotherapy. Finally, we constructed a correlation network between splicing factors (SFs) and survival-related AS events. Results: A total of 4684 AS events were significantly associated with overall survival in patients with bladder cancer. Eight prognostic signatures of bladder cancer were established, and a clinical survival prediction model was built. In addition, the consolidated prognostic signature was closely related to immune infiltration and the response to immunotherapy and chemotherapy. Furthermore, the correlation identified EIF3A, DDX21, SDE2, TNPO1, and RNF40 as hub SFs, and function analysis found ubiquitin-mediated proteolysis is correlated most significantly with survival-associated AS events. Conclusion: Our findings highlight the prognostic value of AS for patients with bladder cancer and reveal pivotal players of AS events in the context of TIME and the response to immunotherapy and chemotherapy, which may be important for patient management and treatment.

MicroRNA and Alternative mRNA Splicing Events in Cancer Drug Response/Resistance: Potent Therapeutic Targets.

Cancer is a multifaceted disease that involves several molecular mechanisms including changes in gene expression. Two important processes altered in cancer that lead to changes in gene expression include altered microRNA (miRNA) expression and aberrant splicing events. MiRNAs are short non-coding RNAs that play a central role in regulating RNA silencing and gene expression. Alternative splicing increases the diversity of the proteome by producing several different spliced mRNAs from a single gene for translation. MiRNA expression and alternative splicing events are rigorously regulated processes. Dysregulation of miRNA and splicing events promote carcinogenesis and drug resistance in cancers including breast, cervical, prostate, colorectal, ovarian and leukemia. Alternative splicing may change the target mRNA 3′UTR binding site. This alteration can affect the produced protein and may ultimately affect the drug affinity of target proteins, eventually leading to drug resistance. Drug resistance can be caused by intrinsic and extrinsic factors. The interplay between miRNA and alternative splicing is largely due to splicing resulting in altered 3′UTR targeted binding of miRNAs. This can result in the altered targeting of these isoforms and altered drug targets and drug resistance. Furthermore, the increasing prevalence of cancer drug resistance poses a substantial challenge in the management of the disease. Henceforth, molecular alterations have become highly attractive drug targets to reverse the aberrant effects of miRNAs and splicing events that promote malignancy and drug resistance. While the miRNA–mRNA splicing interplay in cancer drug resistance remains largely to be elucidated, this review focuses on miRNA and alternative mRNA splicing (AS) events in breast, cervical, prostate, colorectal and ovarian cancer, as well as leukemia, and the role these events play in drug resistance. MiRNA induced cancer drug resistance; alternative mRNA splicing (AS) in cancer drug resistance; the interplay between AS and miRNA in chemoresistance will be discussed. Despite this great potential, the interplay between aberrant splicing events and miRNA is understudied but holds great potential in deciphering miRNA-mediated drug resistance.

Transcriptome Based Classification Model and Co-Expression Networks Analysis Identify CXCR4alt Subtype and Blnkaf in B-Cell Precursor Acute Lymphoblastic Leukemia

BackgroundB-cell precursor acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of acute leukemia with stage-specific phenotypes and cytogenetic features. Although the research on the molecular profile of B-ALL benefits diagnosis and risk stratification, the idiographic leukemogenesis beyond the transcriptome remains unknown. Genomic lesions in B-ALL frequently involve genes belonging to transcription factors, such as TCF3, EBF1, PAX5, and IKZF1. The investigation of dysregulated transcriptional networks behind various B-ALL subtypes may help unravel the specific process of leukemogenesis.MethodsA random forest model was trained on a well-defined molecular subtype B-ALL cohort (n = 504) to improve the molecular classification. The subtype-specific transcriptional network was constructed by weighted correlation network analysis (WGCNA) once the B-ALL subtypes were genetically determined by the random forest model. Additionally, alternative splicing analysis from RNA-seq was emphasized since aberrant splicing events could lead to abnormalities in transcription factors or tumor suppressor genes.ResultsThe random forest model performs well for the classification of most B-ALL subtypes (Figure 1A). It also benefits the classification of Ph-like B-ALL, which displays a gene expression profile similar to BCR-ABL1 B-ALL, as it achieves 100% accuracy on well-known Ph-like cases characterized by ABL-class gene fusions, PAX5-JAK2, EBF1-PDGFRB, and IGH-EPOR.We successfully separated a candidate molecular subtype characterized by CXCR4 alteration (CXCR4alt) for the first time, through our novel classification model (Figure 1B). This newly identified CXCR4alt subtype accounts for 2% of B-ALL cases (11/504), characterized by CXCR4 C-terminal mutation R334X or FLNA overexpression. Both C-terminal mutation and upregulated FLNA contribute to delayed CXCR4 receptor internalization, enhanced CXCL12-CXCR4 signaling, and then continuously activates the downstream MAPK pathway. It is further supported by the high expression of the two oncogenic MAPK signaling pathway genes KIAA1549 and KIAA1549L from the co-expression network of CXCR4alt in these cases.Transcriptional co-expression networks constructed by WGCNA and network hub genes for most B-ALL subtypes also help to elucidate the mechanism of leukemogenesis (Figure 2). We identified an alternative first exon of BLNK (BLNKaf) that leads to loss of function as a shared event in specific subtypes, such as BCR-ABL1, BCR-ABL1-like, and PAX5alt; while in pre-BCR signaling positive subtypes, such as TCF3-PBX1 and MEF2D-r, only express normal BLNK transcripts.DiscussionBy comprehensive transcriptome-based classification model and co-expression networks analysis, we identified a novel defined CXCR4alt subtype with an incidence of 2% in B-ALL. We also observed that BLNKaf might supply a practical marker for monitoring pre-BCR signaling. Our report emphasizes the role of transcriptome-based machine learning and WGCNA in mining the molecular mechanism of B-ALL. The molecular pathogenesis and clinical significance of these newly identified molecular subtypes and molecular abnormalities are worthy of further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CTX-712, a Novel Clk Inhibitor Targeting Myeloid Neoplasms with SRSF2 Mutation

Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. SF mutations are largely mutually exclusive and except for ZRSR2 mutations, heterozygous, suggesting synthetic lethality of homozygous or multiple mutations. Thus, SF functions might be a plausible target of therapy for MDS/AML. Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases resulted in reduction of phosphorylation levels of SR proteins, induction of splicing alterations and protein depletion for multiple genes. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression. Thus, CLK inhibitors might have a role in the therapeutics of SF-mutated MDS/AML, by further compromising RNA splicing.We have recently developed an orally available and highly potent CLK inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. When tested in human myeloid cell lines (K562 and MV-4-11), CTX-712 showed a strong inhibitory effect on cell proliferation (IC 50=0.15 and 0.036 μM, respectively). The anti-leukemic effect was also confirmed by survival assay using a total of 79 primary AML cells (the average of IC 50 was 0.078 μM). In addition, CTX-712 suppressed phosphorylation of multiple SR proteins including SRSF3/4/6, all of which bind to SRSF2. RNA-seq analysis revealed that CTX-712 induced global splicing changes, which typically resulted in skipped exon. Notably, the degree of splicing (percent spliced-in value) in skipped exon events induced by the drug was positively correlated with the sensitivity to the drug (IC 50) in primary AML cells (n=32, R=0.61, P=0.00018).To further investigate the effect of CTX-712 on tumor growth in vivo, we established 13 MDS/AML-derived xenografts (PDXs), which were treated with varying doses of CTX-712. Among the 13 PDX models, SRSF2 mutation was found in 2 cases, which had the SRSF2 P95H or P95L mutation. The SRSF2 P95H PDX showed a significant response to CTX-712 in a dose-dependent manner. Of note, 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two weeks after treatment, tumor volumes (mm 3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.0395), and 39 ± 39 (high dose, P=0.0064) (N=5 each, mean ± SEM). Interestingly CLK inhibition induced aberrant splicing events including skipped exons in vivo, which were more strongly affected in the SRSF2-mutated model. In addition, CTX-712 efficacy was also confirmed in the model with the SRSF2 P95L mutation. The SRSF2 P95L model showed a significant reduction in tumor volumes (mm 3) 2 weeks after CTX-712 treatment; 406 ± 94 (vehicle) and 75 ± 17 (high dose, P=0.0162) (N=6 each, mean ± SEM). CTX-712 also significantly improved the survival of the SRSF2 P95L-mutated model (high dose, P=0.0030) (N=6 each).Overall, 10 out of 13 PDX AML/MDS models, including 2 SRSF2-mutated models, showed anti-tumor effect of CTX-712. Complete disappearances of tumors were obtained in the SRSF2 P95H mutation model. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of the novel CLK inhibitor in MDS/AML. DisclosuresYoda: Chordia Therapeutics Inc.: Research Funding. Morishita: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Mizutani: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Tozaki: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Satoh: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Miyake: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.

The Catastrophic HPV/HIV Dual Viral Oncogenomics in Concert with Dysregulated Alternative Splicing in Cervical Cancer.

Cervical cancer is a public health problem and has devastating effects in low-to-middle-income countries (LTMICs) such as the sub-Saharan African (SSA) countries. Infection by the human papillomavirus (HPV) is the main cause of cervical cancer. HIV positive women have higher HPV prevalence and cervical cancer incidence than their HIV negative counterparts do. Concurrent HPV/HIV infection is catastrophic, particularly to African women due to the high prevalence of HIV infections. Although various studies show a relationship between HPV, HIV and cervical cancer, there is still a gap in the knowledge concerning the precise nature of this tripartite association. Firstly, most studies show the relationship between HPV and cervical cancer at genomic and epigenetic levels, while the transcriptomic landscape of this relationship remains to be elucidated. Even though many studies have shown HPV/HIV dual viral pathogenesis, the dual molecular oncoviral effects on the development of cervical cancer remains largely uncertain. Furthermore, the effect of highly active antiretroviral therapy (HAART) on the cellular splicing machinery is unclear. Emerging evidence indicates the vital role played by host splicing events in both HPV and HIV infection in the development and progression to cervical cancer. Therefore, decoding the transcriptome landscape of this tripartite relationship holds promising therapeutic potential. This review will focus on the link between cellular splicing machinery, HPV, HIV infection and the aberrant alternative splicing events that take place in HIV/HPV-associated cervical cancer. Finally, we will investigate how these aberrant splicing events can be targeted for the development of new therapeutic strategies against HPV/HIV-associated cervical cancer.

Alternative Splicing: A New Cause and Potential Therapeutic Target in Autoimmune Disease.

Alternative splicing (AS) is a complex coordinated transcriptional regulatory mechanism. It affects nearly 95% of all protein-coding genes and occurs in nearly all human organs. Aberrant alternative splicing can lead to various neurological diseases and cancers and is responsible for aging, infection, inflammation, immune and metabolic disorders, and so on. Though aberrant alternative splicing events and their regulatory mechanisms are widely recognized, the association between autoimmune disease and alternative splicing has not been extensively examined. Autoimmune diseases are characterized by the loss of tolerance of the immune system towards self-antigens and organ-specific or systemic inflammation and subsequent tissue damage. In the present review, we summarized the most recent reports on splicing events that occur in the immunopathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and attempted to clarify the role that splicing events play in regulating autoimmune disease progression. We also identified the changes that occur in splicing factor expression. The foregoing information might improve our understanding of autoimmune diseases and help develop new diagnostic and therapeutic tools for them.