Altered RNA export by SF3B1 mutants confers sensitivity to nuclear export inhibition

SF3B1 mutations are the most frequent spliceosomal alterations across cancers, yet no successful therapy exists to target this pathway. Previous findings from a phase 2 clinical trial of the XPO1 inhibitor selinexor in patients with high-risk myelodysplastic neoplasms (MDS) relapsed or refractory to hypomethylating agents (HMA) revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) is responsible for the export of over 200 proteins, but also plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and select messenger RNAs (mRNAs) out of the nucleus. We therefore hypothesized that XPO1 inhibition perturbs RNA export and may preferentially affect SF3B1 mutants via altered splicing, given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome. To evaluate the mechanism underlying preferentially sensitivity of SF3B1-mutants to XPO1 inhibition, we performed nuclear and cytoplasmic fraction followed by RNA sequencing before and after XPO1 inhibition in SF3B1 wildtype and SF3B1 K666N cells (subcellular RNA-seq). Whole transcriptomic analysis of subcellular RNA-seq data revealed more nuclear retention of global RNA transcripts after XPO1 inhibition in the SF3B1 mutant cell line. Similarly, we performed subcellular RNA-seq for small RNAs and found snRNAs to be increased in the nucleus after XPO1 inhibition in the SF3B1 mutant cells. We then performed total cellular RNA sequencing to understand the effect of XPO1 inhibition on global RNA expression and RNA splicing. Differential gene expression analysis identified that XPO1 inhibition had the greatest effect on cell cycle in SF3B1 wildtype cells but in SF3B1-mutant cells differentiation pathways were more significantly affected. Alternative splicing analysis showed increased 3' alternative splicing events in SF3B1 mutant after XPO1 inhibition. These results signify the mechanistic basis for preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through nuclear retention of spliceosomal snRNAs and select mRNAs that result in perturbation of differentiation pathways. Overall design: To determine the effect XPO1 inhibition has on RNA export in SF3B1 mutant cells we treated K562 SF3B1 wildtype and K562 SF3B1 K666N for 24 hours with vehicle and 200nM selinexor (first-genetation XPO1 inhibitor). We then isolated the RNA to get 1) total RNA 2) nuclear and cytoplasmic fractions of long RNAs/mRNAs 3) nuclear and cytoplasmic fractions small RNAs. All samples were collected in triplicates. Additionally we performed RNA sequencing the combination of XPO1 inhibitors with BCL-family of proteins (venetoclax, A1331852, navitoclax) in duplicates. Cells were either treated with A) vehicle or B) 200nM selinexor for 24 hours. Followed by one of three RNA sequencing 1) total RNA sequecing 2) total RNA sequecning of nuclear cytoplasmic fractions of long RNAs 3) small RNA sequencing of of nuclear cytoplasmic fractions of short RNAs. The combination treatment RNA sequencing was of PolyA selected RNA sequencing.