The CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases as well as the preferential advantages of CRISPR over the antisense technology recently developed targeting the PE of EZH2.
Abstract
Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted a PE of a tumor suppressor (EZH2) from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, escaped AS-NMD, and restored the protein expression. This subsequently reinstated H3K27 histone methylation and rescued defective chromatin regulation associated with impaired hematopoietic stem cell differentiation. Finally, we showed the preferential advantages of CRISPR over the antisense technology we recently developed targeting the PE of EZH2. Therefore, the CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases.
Recurrent mutations in splicing factors (SFs) have been established as crucial drivers of tumorigenesis in several types of blood cancer and are also common in a variety of solid tumors. Mutations change the RNA-binding preferences of SFs, promote global splicing alterations, and often generate erroneous mRNAs that are then degraded by nonsense-mediated mRNA decay (NMD). Consequently, several critical genes linked to hematopoiesis are dysregulated, leading to blood cancer. Although the field has progressed considerably in identifying aberrant genes and affected pathways, effective therapies have not yet emerged. To address this key gap, we instigated a gene-specific targeted strategy by unlocking the regulatory network. As a proof-of-concept, we scrutinized a tumor suppressor gene, EZH2, which is a bona fide target in SRSF2-mutated cancer. We precisely defined splicing cis-elements in EZH2 transcripts and illustrated the dynamic choreography of regulatory proteins in the entire splicing and NMD catalytic pathways. We then designed antisense oligonucleotides (ASOs) targeting important regulatory sites. Our lead ASO successfully corrects aberrant splicing and NMD, restores the expression and function of EZH2, and partially rescues hematopoietic defects and cellular properties. Our study demonstrates that ASO pharmacology is an actionable strategy for clinical development, challenging the existing paradigms in SF-mutated cancers.
ASXL3 patient truncations in neurodevelopmental condition Bainbridge-Ropers syndrome are shown to mediates gain-of-function (GOF) by escaping nonsense-mediated decay and Cullin 4-dependent degradation, resulting in aberrant protein accumulation, widespread transcriptional dysregulation, and altered chromatin accessibility.
Y. Nakamura, T. Nguyen, N. Mor et al.· medRxiv· 0 citations
Transcriptional adaptation (TA) is a regulatory process in which loss or disruption of gene function caused by protein-truncating variants (PTVs) triggers compensatory changes in expression of the healthy allele or in expression of related genes. Nonsense-mediated mRNA decay (NMD), a conserved RNA surveillance pathway that degrades transcripts containing premature termination codons, is required to initiate this process. In natural yeast populations, PTV mutations occur relatively frequently, raising the question of whether and how their effects are mitigated. In this study, we investigated TA and NMD among PTVs occurring in natural yeast populations. We observed a strong reduction in the abundance of PTV-containing transcripts, suggesting efficient recognition and degradation of transcripts containing premature stop codons. However, despite evidence of this mRNA surveillance activity, we did not detect a clear signature of transcriptional adaptation. While compensation may still occur in specific contexts, particularly for dosage-sensitive genes, it does not appear to represent a general response to PTVs in yeast. The transcriptional deregulation caused by PTVs in natural isolates may be causing too little harm to favor the evolution or maintenance of complex mechanisms required for adequate compensation. The ability to resist specific transcriptomic ruptures would thus rely mostly on the general robustness of genetic networks. Overall, our findings suggest that TA is not a universal response to loss-of-function mutations in yeast.
Marzena Marszałek, W. Babik, R. Korona et al.· Molecular biology and evolut...· 0 citations
Alternative pre-mRNA splicing is a central layer of gene regulation that enables a limited number of genes to generate a far larger and more context-dependent transcriptome and proteome. In cancer, splicing is disrupted by mutations in cis-regulatory sequences, recurrent lesions in spliceosome components, altered abundance or activity of RNA-binding proteins, and changes in transcription, chromatin, RNA modification, metabolism and stress signalling. These alterations are not merely by-products of malignant transformation. They can create oncogenic protein isoforms, eliminate tumour-suppressive products, remodel cellular identity, promote metastasis and drug resistance, and generate tumour-restricted peptides that are visible to the immune system. Large pan-cancer datasets, long-read sequencing, single-cell isoform profiling, proteogenomics and functional perturbation screens are now resolving this complexity at unprecedented scale. In parallel, multiple therapeutic strategies are advancing, including modulators of the SF3B complex, molecular glues that degrade RBM39, inhibitors of protein arginine methyltransferases and splicing kinases, splice-switching oligonucleotides, programmable RNA-targeting systems, and vaccines or T-cell receptors directed against splicing-derived neoantigens. This review integrates the molecular logic of splice-site selection with the cancer-specific mechanisms that perturb it, summarizes representative isoform switches across the hallmarks of cancer, evaluates emerging technologies and clinical biomarkers, and discusses the opportunities and constraints of translating splicing biology into precision oncology. Particular emphasis is placed on tumour specificity, intratumoural heterogeneity, proteomic validation, therapeutic windows and rational combination strategies.