Abstract

Approximately 50% of patients with myelodysplastic syndromes (MDS) (which frequently progresses to secondary acute myeloid leukemia [AML]), 60% of chronic myelomonocytic leukemia (CMML) and 20% of de novo AML harbor heterozygous somatic mutations in spliceosome genes. We and others have shown that mutations in spliceosome genes U2AF1, SF3B1, SRSF2 and ZRSR2 cause alterations in RNA splicing, leading to production of numerous aberrant RNAs. Interestingly, spliceosome mutant cells are more sensitive to genetic alterations or drugs that further perturb RNA splicing, compared to wild-type cells. The sensitivity of spliceosome mutant cells to further perturbations in RNA splicing raises the possibility that mutant cells may be vulnerable to accumulation of dysregulated transcripts. A large portion of alternatively spliced RNAs caused by spliceosome gene mutations or splicing modulator treatment are nonsense mRNAs that harbor premature termination codons (PTCs). These nonsense mRNAs, which may cause deleterious effects if translated, are normally degraded by a RNA surveillance pathway called nonsense-mediated RNA decay (NMD). The prevalence of nonsense mRNAs in cancer cells with spliceosome mutations leads us to hypothesize that these mutant cells may be more sensitive to NMD attenuation. In support of this idea, we have previously shown that cultured cancer cells expressing mutant spliceosome factors exhibited heightened sensitivity to NMD disruption. Using two pre-clinical mouse models, in my thesis research, we show that transformed and non-transformed primary mouse cells expressing splicing factor mutant U2AF1S34F are preferentially sensitive to inhibition of the kinase SMG1 (SMG1i), a key regulator of NMD. Importantly, we show that SMG1i treatment extended the overall survival of mice harboring splicing factor mutant leukemic cells compared to the vehicle control treated mice. We also demonstrated that SMG1 inhibition in vivo causes R-loop accumulation, a RNA:DNA hybrid with a displaced single strand of DNA, and increased DNA damage in the cells. These findings were also validated in a human cell line-derived AML model in vivo in mice. Importantly, the sensitivity of mouse and human cells that express splicing factor mutant U2AF1S34F could be rescued by RNase H1 overexpression, an enzyme that degrades RNA:DNA hybrids, suggesting that R-loops are a major mechanism of the synthetic lethality between altered RNA splicing and NMD inhibition. In our effort to further explore the mechanisms underlying the synthetic lethality between spliceosome mutations and NMD inhibition, we performed a series of RNA sequencing experiments and found that SMG1i treatment led to distinct alternative pre-mRNA splicing and expression changes in genes involved in the unfolded protein response (UPR) and DNA repair pathways. Interestingly, we also identified that increased expression of PTC-containing isoforms in ATR and RAD51 occurred after U2AF1S34F or SMG1i treatment, resulting in reduced protein expression. This observation nominates these genes as potential vulnerabilities to target in splicing factor mutant cells in combination with SMG1i. Indeed, we found that splicing factor mutant cells were more sensitive to pharmacologic inhibition of ATR or RAD51 (ATRi and RAD51i, respectively) in combination with SMG1i than splicing factor wild-type cells. The combination of SMG1i with either ATRi or RAD51i likely further induces R-loop accumulation, causing increased DNA damage and cell apoptosis. The identification of a unique vulnerability of cancer cells with spliceosome mutations to NMD inhibition suggests a new approach to treat these cancers. Our results have laid a strong foundation for testing the therapeutic potential of SMG1 inhibitors as a monotherapy or in combination with ATRi or RAD51i in cancers with spliceosome gene mutations.

Committee Chair

Matthew Walter

Committee Members

Zhongsheng You; Daniel Link; Laura Schuettpelz; Li Ding

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-6-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0001-5053-6805

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