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Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1 , which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. SIGNIFICANCE: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations., (©2021 American Association for Cancer Research.)

Purpose of Review: Mutations in components of the spliceosome are the most common acquired lesions in myelodysplastic syndromes (MDS) and are frequently identified in other myeloid malignancies with a high rate of progression to acute myeloid leukemia (AML) including chronic myelomonocytic leukemia and primary myelofibrosis. The only curative option for these disorders remains allogeneic stem-cell transplantation, which is associated with high morbidity and mortality in these patients. The purpose of this review is to highlight the recent therapeutic developments and strategies being pursued for clinical benefit in splicing factor mutant myeloid malignancies., Recent Findings: Cells harboring splicing factor mutations have increased aberrant splicing leading to R-loop formation and cell cycle stalling that create dependencies on Checkpoint kinase 1 (CHK1) activation and canonical splicing maintained by protein arginine methyltransferase activity. Both targeting of the spliceosome and targeting of the downstream consequences of splicing factor mutation expression show promise as selective strategies for the treatment of splicing factor-mutant myeloid malignancies., Summary: An improved understanding of the therapeutic vulnerabilities in splicing factor-mutant MDS and AML has led to the development of clinical trials of small molecule inhibitors that target the spliceosome, ataxia telangectasia and Rad3 related (ATR)-CHK1 pathway, and methylation of splicing components., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five-amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis., (© 2019 Abdulhay et al.)

Mutations in components of the 3' mRNA splicing machinery are found in almost 50% of myelodysplastic syndrome (MDS) cases. In this issue of Cancer Cell, Kim and colleagues, Colla and colleagues, and Shirai and colleagues report on the impact of mutated or dysregulated splicing factors to hematopoiesis, mRNA splicing, and MDS pathogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hematopoietic stem and progenitor cells (HSPCs) maintain hematopoiesis throughout the lifetime of an organism. HSPC dysfunction can lead to disorders such as myelodysplastic syndrome (MDS). Spliceosome mutations are prevalent in MDS, but how splicing regulates HSPCs remains poorly understood. Using a zebrafish loss-of-function mutant for the top mutated splicing factor in MDS, splicing factor 3b, subunit 1 (sf3b1), we found that impaired splicing hindered HSPC specification. To identify Sf3b1-regulated transcripts, we performed RNA-sequencing on kdrl:gfp+ endothelial cells from sf3b1 mutant and unaffected siblings at 24hpf. ∼900 genes were mis-spliced with significant enrichment for Signaling Transducer and Activator of Transcription 3 (Stat3) pathway components. Mis-spliced Stat3 signaling components observed in sf3b1 mutants were predicted to dampen protein levels or inhibit functionality, and Stat3 target genes were significantly downregulated in sf3b1 mutants. Constitutively-active Stat3 overexpression in sf3b1 mutants partially suppressed the HSPC defect. Induced mis-splicing of stat3 using morpholinos caused a significant HSPC decrease in sf3b1 heterozygotes at 28hpf, but elicited no effect in wildtype embryos, demonstrating a synthetic lethal interaction. Inhibiting Stat3 with small molecule STATTIC similarly reduced HSPCs in heterozygous but not wildtype embryos. We next tested if STAT3 inhibition selectively affected SF3B1-mutated human cells. K562 cells expressing the MDS-associated SF3B1-K666N mutation were more sensitive to STAT3 inhibition with STATTIC than wildtype cells. Similarly, primary SF3B1-mutant MDS patient cells produced fewer hematopoietic colonies when treated with STATTIC than controls. Together, STAT3 inhibition is a conserved synthetic lethal vulnerability for SF3B1-mutated cells with potential to treat splicing factor-mutated MDS. [ABSTRACT FROM AUTHOR]