Nonsense-Mediated RNA Decay Disruption Induces Altered Splicing in Spliceosome Mutant Cells

Introduction: Expression of mutant splicing factors U2AF1(S34F), SF3B1(K700E), or SRSF2(P95H) alter RNA splicing in myeloid malignancies, including increased production of nonsense transcripts that rely on nonsense-mediated RNA decay (NMD) for clearance. Cell lines expressing spliceosome mutants are more sensitive to NMD inhibition than wild-type cells and the differential sensitivity is partially dependent on R-loop formation. The impact of NMD disruption on the viability of primary splicing factor mutant hematopoietic cells and the consequence on downstream RNA splicing are not known.

Methods: Cell viability of primary mouse hematopoietic cells (untransformed and AML cells) and a human AML cell line that expresses mutant or wild-type U2AF1 after treatment with a highly-specific inhibitor of SMG1 (SMG1i), the only known protein kinase in the NMD pathway, was measured using a CellTiter-Glo assay. Gene expression and RNA splicing changes induced by mutant U2AF1 and/or SMG1 inhibition were identified by short and long-read RNA sequencing (RNA-seq).

Results: We treated primary untransformed mouse U2AF1^S34F or U2AF1^WT expressing cKit+ hematopoietic cells ex vivo with SMG1 inhibitor and monitored cell viability. We found that SMG1i reduced the viability of primary mouse cells expressing mutant U2AF1^S34F compared to U2AF1^WT (IC50 52.6 nM vs 173.5 nM, respectively, p=<0.0001). Next, we tested the sensitivity of primary mouse AML cells expressing a doxycycline-inducible U2AF1^S34F or U2AF1^WT transgene to SMG1i that were generated using retroviral expression of the fusion oncogene MLL-AF9 to induce AML. We found that NMD disruption using SMG1i also reduced the ex vivo viability of primary U2AF1^S34F compared to U2AF1^WT expressing mouse AML cells (IC50 40.9 nM vs 102.2 nM, respectively, p=<0.0001), highlighting the potential utility of SMG1i treatment for spliceosome mutant myeloid malignancies. To explore the underlying mechanism for the sensitivity of spliceosome mutant cells to NMD inhibition, we generated, K562 cell lines expressing doxycycline-inducible U2AF1^S34F or U2AF1^WT and confirmed that NMD disruption using SMG1i reduced cell viability of mutant expressing cells in vitro (IC50 128.1nM vs 578.5nM at 72 hours, respectively, p≤0.0001). Next, we performed RNA-seq on K562 cells expressing U2AF1^WT or U2AF1^S34F that were treated with vehicle or 1uM SMG1i for 24 hours (n=3 replicates). We identified reduced expression of genes involved in the cell cycle, DNA replication/repair, telomere maintenance and mRNA splicing in SMG1i treated U2AF1^S34F compared to U2AF1^WT cells (GSEA; FDR<0.008). Next, we identified 3,393 RNA splicing alterations induced by SMG1i treatment of U2AF1^S34F compared to U2AF1^WT cells (rMATS; FDR<0.1, percent spliced-in >10%). Genes with altered splicing were enriched in similar pathways, including the cell cycle, DNA replication, and RNA splicing and processing pathways (Cluster Profiler; FDR≤0.02). Finally, we performed long-read RNA-seq (Nanopore) on RNA isolated from K562 cells (not expressing spliceosome mutants) that were treated with vehicle or SMG1i (0.5uM, 24hours, n=2) to directly identify full length transcripts containing premature termination codons (PTC) that are normal targets of NMD. Our preliminary data indicate that SMG1i significantly increases the number of detectable PTC containing transcripts (known and novel) compared to vehicle treatment (mean 19,291 vs 5,621 PTCs, respectively; p=0.008). In addition, PTC-containing transcripts that were overexpressed in SMG1i vs vehicle treated cells also occur in genes involved in the cell cycle, DNA repair and mRNA splicing (FDR£0.001), suggesting they are regulated by NMD under basal conditions. These gene pathways may be particularly vulnerable to further dysregulation in mutant treated cells because they are also altered by mutant U2AF1^S34F expression.

Conclusion: A vulnerability of primary hematopoietic cancer cells with spliceosome mutations to NMD inhibition suggests the possibility that NMD can be targeted for treating MDS with aberrant splicing. Excessive R-loop accumulation in SMG1i treated U2AF1^S34F cells may contribute to the downstream gene expression and splicing alterations and reduced viability of mutant cells. Ongoing pre-clinical in vivo efficacy studies will test the potential for SMG1i to treat hematologic malignancies that have splicing factor mutations.

Bailis:Amgen: Current Employment.