Targeting Nonsense-Mediated RNA Decay in Splicing Factor Mutant Myeloid Malignancies.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by peripheral blood cytopenias, bone marrow dysplasia, and ineffective hematopoiesis. Approximately 50% of MDS, 60% chronic myelomonocytic leukemia (CMML), 20% of acute myeloid leukemia

(AML) harbor heterozygous mutations in spliceosome factor genes such as SF3B1, U2AF1, SRSF2 and ZRSR2. Although many studies have shown that mutations in splicing factor genes lead to distinct patterns of aberrant splicing, no specific alternatively spliced isoform has been demonstrated to directly cause MDS. However,

aberrations in splicing induced by splicing factor gene mutations create a vulnerability in MDS cells. Our group and others showed that cells harboring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome by splicing modulator drugs. The sensitivity of spliceosome

mutant cells to further splicing perturbations raises the possibility that they are vulnerable to accumulation of misspliced transcripts. A large portion of the misspliced RNAs caused by spliceosome 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 mutant cells will be more sensitive to NMD attenuation due

to the role of NMD in the clearance of nonsense mRNAs that can be detrimental. Preliminary data from our group indicate that NMD disruption (using a SMG1 inhibitor [SMG1i]) preferentially kills cancer cells expressing different splicing factor gene mutations. This cell death is associated with the induction of R-loops and DNA damage.

Building on preliminary studies, we propose to test the therapeutic potential of NMD inhibition in selective killing of spliceosome mutant cancer cells using in vivo models and define the underlying mechanism for the hypersensitivity of spliceosome mutant cells to NMD attenuation. In Specific Aim 1, we will test the therapeutic

potential of NMD inhibition to selectively kill spliceosome mutant cancer cells using in vivo models. We will engraft primary mouse AML cells in congenic mice and test whether in vivo treatment with a highly specific inhibitor of SMG1 (SMG1i), the only known protein kinase that regulates the NMD pathway, can selectively kill cancer cells

with spliceosome mutations. We will further establish the therapeutic potential of targeting NMD by combining SMG1i with ATR or PARP inhibitors, DNA damage repair proteins that are activated in splicing factor mutant cells. In Specific Aim 2, we will define the molecular mechanism for the sensitivity of spliceosome mutant cells

to NMD attenuation. We will identify candidate NMD targets whose levels are modulated by SMG1i or mutant splicing factors, potentially nominating downstream functional targets that could be modulated for cancer treatment. Collectively, this project will nominate therapies to treat MDS and AML with splicing factor mutations.