Splicing Of Gene Research Articles

Lung Transcriptomics Links Emphysema to Barrier Dysfunction and Macrophage Subpopulations.

While many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient non-imaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort. Our goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung samples. Using RNA-sequencing data from 446 samples in the Lung Tissue Research Consortium (LTRC) and 3,606 blood samples from the COPDGene study, we examined the transcriptomic features of chest computed tomography-quantified emphysema. We also leveraged publicly available lung single-cell RNA-sequencing data to identify cell types showing COPD-associated differential expression of the emphysema pathways found in the bulk analyses. In the bulk lung RNA-seq analysis, 1,087 differentially expressed genes and 34 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of several genes and increased activity in pluripotency and cell barrier function pathways. Lung tissue and blood samples shared differentially expressed genes and biological pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and distinct pathway activities were observed among various macrophage subpopulations. This study identified emphysema-related changes in gene expression and alternative splicing, cell-type specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung.

KHSRP has oncogenic functions and regulates the expression and alternative splicing of DNA repair genes in breast cancer MDA-MB-231 cells

Breast cancer has become the most common type of cancers worldwide. Its high prevalence and malignant features are associated with various environmental factors and molecules. The KH-type splicing regulatory protein (KHSRP) participates in the development of breast cancer, while the underlying mechanisms are largely unknown. In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then assessed its effects on cellular features. Finally, we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP, and validated their changed pattern using quantitative polymerase chain reaction. We found KHSRP showed higher expression level and was associated with worse prognosis in breast cancer patients. In siKHSRP samples, the proliferation, invasion, and migration abilities were significantly repressed compared with negative control (NC) samples, while the apoptosis level was increased. By investigating the RNA-seq data, we found KHSRP globally regulates the expression and alternative splicing profiles of MDA-MB-231 cells by identifying 1632 differentially expressed genes (DEGs) and 1630 HKSRP-regulated AS events (RASEs). Functional enriched analysis of DEGs demonstrated that cilium assembly and movement and extracellular matrix organization pathways were specifically enriched in up DEGs, consistent with the repressed migration and invasion abilities in siKHSRP cells. Interestingly, the cell cycle and DNA damage and repair associated pathways were enriched in both down DEGs and RASE genes, suggesting that KHSRP may modulate cell proliferation by regulating genes in these pathways. Finally, we validated the changed expression and AS patterns of genes in cell cycle and DNA damage/repair pathways. Expression levels of BIRC5, CCNA2, CDK1, FEN1, FOXM1, PTTG1, and UHRF1 were downregulated in siKHSRP samples. The AS patterns of PARK7, ERCC1, CENPX, and UBE2A were also dysregulated in siKHSRP samples and confirmed PCR experiments. In summary, our study comprehensively explored the downstream targets and their functions of KHSRP in breast cancer cells, highlighting the molecular mechanisms of KHSRP on the oncogenic features of breast cancer. The identified molecular targets could be served as potential therapeutic targets for breast cancer in future.

PredGCN: a Pruning-enabled Gene-Cell Net for automatic cell annotation of single cell transcriptome data.

The annotation of cell types from single-cell transcriptomics is essential for understanding the biological identity and functionality of cellular populations. Although manual annotation remains the gold standard, the advent of automatic pipelines has become crucial for scalable, unbiased, and cost-effective annotations. Nonetheless, the effectiveness of these automatic methods, particularly those employing deep learning, significantly depends on the architecture of the classifier and the quality and diversity of the training datasets. To address these limitations, we present a Pruning-enabled Gene-Cell Net (PredGCN) incorporating a Coupled Gene-Cell Net (CGCN) to enable representation learning and information storage. PredGCN integrates a Gene Splicing Net (GSN) and a Cell Stratification Net (CSN), employing a pruning operation (PrO) to dynamically tackle the complexity of heterogeneous cell identification. Among them, GSN leverages multiple statistical and hypothesis-driven feature extraction methods to selectively assemble genes with specificity for scRNA-seq data while CSN unifies elements based on diverse region demarcation principles, exploiting the representations from GSN and precise identification from different regional homogeneity perspectives. Furthermore, we develop a multi-objective Pareto pruning operation (Pareto PrO) to expand the dynamic capabilities of CGCN, optimizing the sub-network structure for accurate cell type annotation. Multiple comparison experiments on real scRNA-seq datasets from various species have demonstrated that PredGCN surpasses existing state-of-the-art methods, including its scalability to cross-species datasets. Moreover, PredGCN can uncover unknown cell types and provide functional genomic analysis by quantifying the influence of genes on cell clusters, bringing new insights into cell type identification and characterizing scRNA-seq data from different perspectives. The source code is available at https://github.com/IrisQi7/PredGCN and test data is available at https://figshare.com/articles/dataset/PredGCN/25251163.

CWF19L2 is Essential for Male Fertility and Spermatogenesis by Regulating Alternative Splicing.

The progression of spermatogenesis along specific developmental trajectories depends on the coordinated regulation of pre-mRNA alternative splicing (AS) at the post-transcriptional level. However, the fundamental mechanism of AS in spermatogenesis remains to be investigated. Here, it is demonstrated that CWF19L2 plays a pivotal role in spermatogenesis and male fertility. In germline conditional Cwf19l2 knockout mice exhibiting male sterility, impaired spermatogenesis characterized by increased apoptosis and decreased differentiated spermatogonia and spermatocytes is observed. That CWF19L2 interacted with several spliceosome proteins to participate in the proper assembly and stability of the spliceosome is discovered. By integrating RNA-seq and LACE-seq data, it is further confirmed CWF19L2 directly bound and regulated the splicing of genes related to spermatogenesis (Znhit1, Btrc, and Fbxw7) and RNA splicing (Rbfox1, Celf1, and Rbm10). Additionally, CWF19L2 can indirectly amplify its effect on splicing regulation through modulating RBFOX1. Collectively, this research establishes that CWF19L2 orchestrates a splicing factor network to ensure accurate pre-mRNA splicing during the early steps of spermatogenesis.

Frameshift variants in C10orf71 cause dilated cardiomyopathy in human, mouse, and organoid models.

Research advances over the past 30 years have confirmed a critical role for genetics in the etiology of dilated cardiomyopathies (DCMs). However, full knowledge of the genetic architecture of DCM remains incomplete. We identified candidate DCM causal gene, C10orf71, in a large family with 8 patients with DCM by whole-exome sequencing. Four loss-of-function variants of C10orf71 were subsequently identified in an additional group of492 patients with sporadic DCM from 2 independent cohorts. C10orf71 was found to be an intrinsically disordered protein specifically expressed in cardiomyocytes. C10orf71-KO mice had abnormal heart morphogenesis during embryonic development and cardiac dysfunction as adults with altered expression and splicing of contractile cardiac genes. C10orf71-null cardiomyocytes exhibited impaired contractile function with unaffected sarcomere structure. Cardiomyocytes and heart organoids derived from human induced pluripotent stem cells with C10orf71 frameshift variants also had contractile defects with normal electrophysiological activity. A rescue study using a cardiac myosin activator, omecamtiv mecarbil, restored contractile function in C10orf71-KO mice. These data support C10orf71 as a causal gene for DCM by contributing to the contractile function of cardiomyocytes. Mutation-specific pathophysiology may suggest therapeutic targets and more individualized therapy.

Co-transcriptional splicing facilitates transcription of gigantic genes.

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.

The Study of SRSF1 Regulates Abnormal Alternative Splicing of BCL2L11 and the Role in Refractory Acute Myeloid Leukemia.

Abnormalities in splicing factors, such as mutations or deregulated expression, can lead to aberrant splicing of target genes, potentially contributing to the pathogenesis of acute myeloid leukemia (AML). Despite this, the precise mechanism underlying the abnormal alternative splicing (AS) induced by SRSF1, a splicing factor associated with poor AML prognosis, remains elusive. Using strict splicing criteria, we globally screened for AS events in NPMc-positive and NPMc-negative AML samples from TCGA. An AS network associated with AML prognosis was then established. Functional assays, including CCK-8, flow cytometry, and Western blot, were conducted on K562 and THP-1 cells overexpressing SRSF1. Cell viability following 72-h Omipalisib treatment was also assessed. To explore the mechanism of SRSF1-induced AS, we created a BCL2L11 miniGene with a site-specific mutation at its branch point. The AS patterns of both wild-type and mutant miniGenes were analyzed following SRSF1 overexpression in HEK-293T, along with the subcellular localization of different spliceosomes. SRSF1 was significantly associated with AML prognosis. Notably, its expression was markedly upregulated in refractory AML patients compared to those with a favorable chemotherapy response. Overexpression of SRSF1 promoted THP-1 cell proliferation, suppressed apoptosis, and reduced sensitivity to Omipalisib. Mechanistically, SRSF1 recognized an aberrant branch point within the BCL2L11 intron, promoting the inclusion of a cryptic exon 3, which in turn led to apoptosis arrest. Overexpression of SRSF1 and the resulting abnormal splicing of BCL2L11 are associated with drug resistance and poor prognosis in AML.

Abstract IA005: Therapeutic vulnerabilities of cohesin-mutant myeloid malignancies

Abstract Splicing modulation is a promising treatment strategy pursued to date only in splicing-factor mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in myeloid malignancies, including 15-20% of myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. I will discuss our recent findings identifying cohesin mutations as biomarkers of sensitivity to drugs targeting splicing-factor SF3B1 (H3B-8800 and E-7107) and describe the mechanism by which drug-induced alterations in splicing of DNA repair genes, such as BRCA1 and BRCA2, underlie this sensitivity. We have demonstrated that treatment of cohesin-mutant cells with SF3B1 modulators results in impaired DNA damage response, accumulation of DNA damage, and increased sensitivity to PARP inhibitors and a panel of chemotherapeutic agents in vitro and in vivo, using AML cell lines and patient-derived xenograft models. Furthermore, we identified RAD51 foci formation as a predictive biomarker of sensitivity to SF3B1 splicing modulation alone or followed by sequential treatment with PARP inhibition and chemotherapy, and have identified additional non-cohesin mutant subtypes of MDS/AML and ovarian and breast cancer which are sensitive to this therapeutic approach. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS/AML and we propose this as broader strategy for therapeutic targeting of other DNA damage-repair deficient cancers. Citation Format: Zuzana Tothova. Therapeutic vulnerabilities of cohesin-mutant myeloid malignancies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA005.

Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock‐related protein Rbm20 in female mdx mice

AbstractFemale carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT‐labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA‐binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post‐menopausal DMD carriers.

Using expression data to fine map QTL associated with fertility in dairy cattle

BackgroundFemale fertility is an important trait in dairy cattle. Identifying putative causal variants associated with fertility may help to improve the accuracy of genomic prediction of fertility. Combining expression data (eQTL) of genes, exons, gene splicing and allele specific expression is a promising approach to fine map QTL to get closer to the causal mutations. Another approach is to identify genomic differences between cows selected for high and low fertility and a selection experiment in New Zealand has created exactly this resource. Our objective was to combine multiple types of expression data, fertility traits and allele frequency in high- (POS) and low-fertility (NEG) cows with a genome-wide association study (GWAS) on calving interval in Australian cows to fine-map QTL associated with fertility in both Australia and New Zealand dairy cattle populations.ResultsVariants that were significantly associated with calving interval (CI) were strongly enriched for variants associated with gene, exon, gene splicing and allele-specific expression, indicating that there is substantial overlap between QTL associated with CI and eQTL. We identified 671 genes with significant differential expression between POS and NEG cows, with the largest fold change detected for the CCDC196 gene on chromosome 10. Our results provide numerous candidate genes associated with female fertility in dairy cattle, including GYS2 and TIGAR on chromosome 5 and SYT3 and HSD17B14 on chromosome 18. Multiple QTL regions were located in regions with large numbers of copy number variants (CNV). To identify the causal mutations for these variants, long read sequencing may be useful.ConclusionsVariants that were significantly associated with CI were highly enriched for eQTL. We detected 671 genes that were differentially expressed between POS and NEG cows. Several QTL detected for CI overlapped with eQTL, providing candidate genes for fertility in dairy cattle.

Transcriptomic Analysis of the Spatiotemporal Axis of Oogenesis and Fertilization in C. elegans.

The oocyte germline of the C. elegans hermaphrodite presents a unique model to study the formation of oocytes. However, the size of the model animal and difficulties in retrieval of specific stages of the germline have obviated closer systematic studies of this process throughout the years. Here, we present a transcriptomic level analysis into the oogenesis of C. elegans hermaphrodites. We dissected a hermaphrodite gonad into seven sections corresponding to the mitotic distal region, the pachytene, the diplotene, the early diakinesis region and the 3 most proximal oocytes, and deeply sequenced the transcriptome of each of them along with that of the fertilized egg using a single-cell RNA-seq protocol. We identified specific gene expression events as well as gene splicing events in finer detail along the oocyte germline and provided novel insights into underlying mechanisms of the oogenesis process. Furthermore, through careful review of relevant research literature coupled with patterns observed in our analysis, we attempt to delineate transcripts that may serve functions in the interaction between the germline and cells of the somatic gonad. These results expand our knowledge of the transcriptomic space of the C. elegans germline and lay a foundation on which future studies of the germline can be based upon.

Expression of alternatively spliced BCMA RNA with skipping of transmembrane domain.

7514 Background: The TNFRSF17 gene, which encodes for the B cell maturation antigen (BCMA), is a small gene expressed mainly on the surface of plasma cells and some DLBCL cells. This gene is composed of three exons. Exon 1 is responsible for the extracellular domain, exon 2 for the transmembrane domain, and exon 3 for the TRAF binding domain. The BCMA is currently targeted by various types of immunotherapies as a major therapeutic approach for the treatment of multiple myeloma (MM). This includes CAR-T cells, bi-specific antibodies, and antibody-drug conjugates (ADC). However, emerging data indicates that alternative splicing in genes is an important mechanism for expression of different isoforms that may influence antibody-based therapies. We explored the potential of the presence of alternative splicing in BCMA transcripts via sequencing of BCMA RNA in patients with lymphoma or multiple myeloma. Methods: RNA was extracted from 587 fresh bone marrow samples with lymphoid/plasma cell neoplasms or from FFPE samples with lymphoma. In addition, cfRNA was extracted from 260 peripheral blood plasma samples from patients with lymphoma or multiple myeloma. RNA was sequenced using a hybrid capture-targeted RNA panel with analysis focused on TNFRSF17 (BCMA) gene transcript. Quantification of RNA transcript was done using Salmon algorithm. Results: Of the 587 lymphoma/plasma cell samples, 161 (27%) samples showed alternative splicing involving deletion of exon 2 (BCMA∆Ex2). Of the 260 cfRNA samples, 14 (6%) showed BCMA∆Ex2. The median percentage of BCMA∆Ex2 transcripts was 0.7% of total BCMA transcripts in cellular samples as compared with 9% in cfRNA samples. In cellular samples, there was a correlation between levels of BCMA and BCMA∆Ex2 (R=0.63). Cases with higher levels of BCMA had significantly higher levels of BCMA∆Ex2 (P<0.0001, Kruskal-Wallis). In contrast, cfRNA showed no correlation between levels of BCMA and levels of BCMA∆Ex2 (R=021, Spearman) and mildly higher level of BCMA∆Ex2 in cases with higher levels of BCMA (P=0.002, Kruskal-Wallis). Conclusions: BCMA exon 2 skipping is detected in a significant number of patients with multiple myeloma and lymphoma. The percentage of skipping transcripts is low in cells and relatively higher in cfRNA. Since exon 2 skipping deletes the transmembrane domain, this BCMA protein may remain in the cytoplasm or perhaps is secreted as cell-free BCMA protein. The demonstration of relatively higher levels of BCMA∆Ex2 in cfRNA is likely reflecting higher turnover of cells and raises the possibility that cells with this isoform of BCMA are more aggressive than cells without the expression of this isoform. Further studies are needed to explore the clinical relevance of the expression of such abnormal BCMA protein on treatment with the various forms of anti-body-based therapy or CAR-T.

Expression of alternatively spliced PD-L1 RNA with skipping of transmembrane domain.

e14596 Background: Programmed death-ligand 1 (PD-L1) is a transmembrane ligand for the programmed cell death protein 1 (PD-1), a receptor that inhibits T-cell activity. The PD-L1/PD-1 immune checkpoint axis has been successfully targeted to enhance antitumor immune responses. This gene is composed of seven exons. Exons 1-4 are responsible for the extracellular domain, exon 5 for the transmembrane domain, and exons 6-7 for the intracellular domain. Emerging data indicates that alternative splicing in genes is an important mechanism for expression of different isoforms that may influence antibody-based therapies. We explored the potential of the presence of alternative splicing in PD-L1 transcripts via sequencing of PD-L1 RNA in patients with various type of solid tumor cancers. Methods: RNA was extracted from 3443 FFPE solid tumor samples including lung, colon, pancreas, breast, and other cancer types. In addition, cfRNA was extracted from 532 peripheral blood plasma samples from patients with solid tumor cancers. RNA was sequenced using a hybrid capture targeted RNA panel on FFPE samples and peripheral blood with analysis focused on PD-L1 gene transcript. Quantification of RNA transcript was done using Salmon algorithm. Results: Of the 3443 FFPE samples, 38 (1.2%) samples showed alternative splicing involving deletion of exon 5 (PD-L1∆Ex5). The alternative splicing was in 2.10% of lung cancer, 2% of colon cancer, 0.70% of pancreatic cancer, 1.40% of gastric cancer, 0.38% of breast cancer, 0.30% of ovarian cancer, 1.10% of sarcoma cases, 1.50% of glioblastomas, 3.30% of thyroid cancer and 2% of urothelial cancer. Of the 532 cfRNA samples, 20 (3.7%) showed PD-L1∆Ex5. The median percentage of PD-L1∆Ex5 transcripts was 2.2% of total PD-L1 transcripts in tissue samples as compared with 6.6% in cfDNA samples. In tissue samples, there was a correlation between levels of PD-L1 and PD-L1∆Ex5 (r = .567, p < .001). In contrast, cfRNA showed no significant correlation between levels of PD-L1 and levels of PD-L1∆Ex5 (r = .268). Conclusions: PD-L1 exon 5 skipping was detected in a number of patients with different types of solid tumor cancers. The percentage of skipping transcripts is low in cells and relatively higher in cfRNA. Since exon 5 skipping deletes the transmembrane domain, the PD-L1 protein may remain in the cytoplasm or perhaps is secreted as cell-free PD-L1 protein. The demonstration of relatively higher levels of PD-L1∆Ex5 in cfRNA is likely reflecting higher turnover of cells and raises the possibility that cells with this isoform of PD-L1 are more aggressive than cells without the expression of this isoform. Further studies are needed to explore the clinical relevance of the expression of such abnormal PD-L1 protein on treatment with immune checkpoint inhibitors.

Survey of actionable driver mutations KRAS and EGFR in non–small cell lung cancer to evaluate coexisting splicing gene and pathway mutations: A CBioportal database analysis.

8561 Background: Molecularly targeted therapies have advanced treatment for certain cancers, such as lung cancer with specific EGFR or KRAS mutations. However, oftentimes the tumor becomes eventually treatment resistant. This may be partly because cancers driven by EGFR/KRAS mutations may also harbor additional co-occurring genetic alterations. In non-small cell lung cancer (NSCLC), aberrations in RNA splicing pathways have shown substantial implications for tumor behavior and therapeutic responsiveness. Our study focuses on elucidating the interplay between RNA splicing defects and prominent NSCLC mutations. Methods: In this retrospective, cross-sectional study, we analyzed data from 20,719 NSCLC patients enrolled in participating institutions between 2017 and 2022. The data were sourced from the American Association for Cancer Research Genomics Evidence Neoplasia Information Exchange (version 13.0). EGFR L858R, EGFR E746_750del, KRAS G12C, KRAS G12D and other KRAS G12 mutations cases were identified and individually analyzed to abstract co-occurring RNA splicing genomic alterations. Results: Among 20,719 patients with NSCLC, we identified 1309 patients with EGFR L 858R mutation, 1121 patients with EGFR E746_750del mutation, 2446 with KRAS 12C, 852 with KRAS G12D, 1107 with KRAS G12V, 452 with KRAS G12A, 173 with KRAS G12F, and 109 with KRAS G12S. RBM 10 was the most common co-occurring RNA splicing gene for EGFR L 858R (178, 13.6%), followed by CDK12 (31, 2.3%) and SPEN (27, 2 %). Similarly, the most prevalent co-occurring RNA splicing genes with EGFR E746_750del were RBM 10 (38, 3.4%) and SPEN (23, 2 %). RBM 10 was the most prevalent co-existing RNA splicing gene for KRAS G12C (211, 8.6 %), KRAS G12D (63, 7.4 %), KRAS G12V (80, 7.2%), KRAS G12A (59, 13.1%), KRAS G12F (12, 7.0%), KRAS G12S (11, 10.1%) and KRAS G12R (6, 8%). Conclusions: Our study uncovers a crucial link between EGFR and KRAS mutations in NSCLC and RNA splicing gene dysregulation, particularly involving RBM10. Further research in this field is warranted as targeting RNA splicing, alone or alongside existing targeted inhibitors, could substantially enhance treatment efficacy for EGFR+ or KRAS+ NSCLC patients. [Table: see text]

Alternative splicing of immune-related genes identifies breast cancer subtypes with differential immune cell infiltration

Alternative splicing of immune-related genes identifies breast cancer subtypes with differential immune cell infiltration

The Many Roads from Alternative Splicing to Cancer: Molecular Mechanisms Involving Driver Genes.

Cancer driver genes are either oncogenes or tumour suppressor genes that are classically activated or inactivated, respectively, by driver mutations. Alternative splicing-which produces various mature mRNAs and, eventually, protein variants from a single gene-may also result in driving neoplastic transformation because of the different and often opposed functions of the variants of driver genes. The present review analyses the different alternative splicing events that result in driving neoplastic transformation, with an emphasis on their molecular mechanisms. To do this, we collected a list of 568 gene drivers of cancer and revised the literature to select those involved in the alternative splicing of other genes as well as those in which its pre-mRNA is subject to alternative splicing, with the result, in both cases, of producing an oncogenic isoform. Thirty-one genes fall into the first category, which includes splicing factors and components of the spliceosome and splicing regulators. In the second category, namely that comprising driver genes in which alternative splicing produces the oncogenic isoform, 168 genes were found. Then, we grouped them according to the molecular mechanisms responsible for alternative splicing yielding oncogenic isoforms, namely, mutations in cis splicing-determining elements, other causes involving non-mutated cis elements, changes in splicing factors, and epigenetic and chromatin-related changes. The data given in the present review substantiate the idea that aberrant splicing may regulate the activation of proto-oncogenes or inactivation of tumour suppressor genes and details on the mechanisms involved are given for more than 40 driver genes.

The Role of Alternative Splicing of Immune Response Genes in Cancer Aggression

The Role of Alternative Splicing of Immune Response Genes in Cancer Aggression

Deciphering the Role of Exosomal Non-Coding RNA (ncRNA) in Drug Resistance of Gastrointestinal Tumors; an Updated Review.

One of the most prevalent types of cancer worldwide today is gastric intestinal (GI) tumors. To guarantee their lives, people with a developed GI require palliative care. This covers the application of targeted medicines in addition to chemotherapy treatments including cisplatin, 5-fluorouracil, oxaliplatin, paclitaxel, and pemetrexed. Because of the evidence of drug resistance emerging in poor patient outcomes and prognoses, determining the exact process of medication resistance is motivated. Besides, it is noteworthy that exosomes and noncoding RNAs, like microRNAs and long non-coding RNAs (lncRNAs), produced from tumor cells are implicated in both GI medication resistance and the carcinogenesis and development of GI disease. Biochemical events related to the cell cycle, differentiation of cells, growth, and pluripotency, in addition to gene transcription, splicing, and epigenetics, are all regulated by noncoding RNAs (ncRNAs). Therefore, it should come as a wonder that several ncRNAs have been connected in recent years to drug susceptibility and resistance as well as tumorigenesis. Additionally, through communicating directly with medications, altering the transcriptome of tumor cells, and affecting the immune system, exosomes may govern treatment resistance. Because of this, exosomal lncRNAs often act as a competitive endogenous RNA (ceRNA) of miRNAs to carry out its role in modifying drug resistance. In light of this, we provide an overview of the roles and processes of ncRNA-enriched exosomes in GI medication resistance.

Pharmacodynamics of venetoclax plus cirtuvivint a first-in-class splice variant regulator.

e15093 Background: NSC835563 (SM08502, cirtuvivint) is a first-in-class pan CDC-like kinase (CLK) and dual specificity tyrosine kinase (DYRK) inhibitor that targets alternative splicing of genes in multiple pathways and modifies their protein expression. Preclinical screening of hematologic malignancy models has shown sensitivity to cirtuvivint, leading to proposed clinical trials in AML in combinations with venetoclax. We performed pharmacodynamic studies to identify probable mechanisms of combination activity in AML models. Methods: In vivo tolerability, efficacy and pharmacodynamic (PD) studies were performed in two AML xenograft models, MV411 and KG1a in NSG mice, at 6.25-25 mg/kg PO cirtuvivint and 25 mg/kg PO venetoclax. Body weights (BW) and tumor volumes were recorded periodically to assess toxicity and anti-tumor efficacy. Tumor tissue samples were collected at 6 and 24 h post venetoclax to assess PD response. Multiplexed analysis of apoptosis pathway proteins/protein-protein complexes was performed by clinically validated assays (PMID 26446940). Results: Cirtuvivint alone at high doses (25 mg/kg) caused tumor regressions in MV411 but only a modest tumor growth inhibition (TGI) in KG1a, a resistant AM model. Importantly, the lowest dose of 6.25 mg/kg cirtuvivint combined with 25 mg/kg venetoclax led to tumor regression in the MV411 model and a moderate TGI in KG1a. Cirtuvivint alone caused a transient 25% reduction (p<0.05) in Mcl-1 levels at 6 h post dose in MV411. The venetoclax combination triggered a sustained (for 24 h) 50-75% disruption of Bcl-2 and Mcl-1 complexes with Bax and Bim, resulting in >20-fold increased mitochondrial Bax/Bak heterodimer and >10-fold increased cytosolic cleaved caspase-3. In KG1a, combination treatment resulted in only modest (<20%) changes in biomarkers. Overall, biomarkers provided evidence of the combination MoA. Conclusions: Venetoclax and cirtuvivint in combination effectively induced tumor regressions at low tolerable doses in an AML model. Mitochondrial Bax/Bak heterodimer provided a sensitive biomarker of combination activity. Cirtuvivint contributed its combinatorial effect primarily through disruption of Bim complexes and inhibition of Mcl-1. PD biomarkers identified in this study can confirm MoA of drug combination in ongoing clinical trials.

GRN knockdown regulates the expression and alternative splicing of genes associated with aphasia-related diseases in PC12 cells

BackgroundPrior research has shown that granulin precursor (GRN, also termed PGRN) is closely linked to aphasia. However, there has been little research on the mechanism of action of GRN in post-stroke aphasia (PSA). MethodsIn this study, RT-qPCR was used to identify variations in gene expression, while RNA sequencing (RNA-seq) was utilized to acquire transcriptional profiles. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were employed for bioinformatics analysis. ResultsGRN was considerably more active in PSA subjects. After silencing the GRN, 197 transcripts had differential expression, and 237 alternative splicing events (ASEs) were substantially affected. The analysis of differentially expressed genes (DEGs) using GO and KEGG approaches showed that these genes have various molecular functions and are significantly enriched in metabolic signaling pathways. Regarding Alternative Splicing (AS), the GO and KEGG analyses revealed numerous functional genes involved in transcription and metabolism. ConclusionsThe knockdown of GRN has been shown to be associated with alterations in transcription, metabolism, and ASEs, potentially impacting transcriptional and metabolic pathways through its involvement in AS. Furthermore, GRN knockdown is associated with nervous system disease-related gene transcription and AS processes, as well as its involvement in G protein-coupled receptor (GPCR) and wingless/integrated (Wnt) signaling pathways, which impact the initiation and resolution of PSA.