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

SF3B1 mutations frequently occur in cancer yet lack targeted therapies. Clinical trials of XPO1 inhibitors, selinexor and eltanexor, in high-risk myelodysplastic neoplasms (MDS) revealed responders were enriched with SF3B1 mutations. Given that XPO1 (Exportin-1) is a nuclear exporter responsible for the export of proteins and multiple RNA species, this led to the hypothesis that SF3B1-mutant cells are sensitive to XPO1 inhibition, potentially due to altered splicing. Subsequent RNA sequencing after XPO1 inhibition in SF3B1 wildtype and mutant cells showed increased nuclear retention of RNA transcripts and increased alternative splicing in the SF3B1 mutant cells particularly of genes that impact apoptotic pathways. To identify novel drug combinations that synergize with XPO1 inhibition, a forward genetic screen was performed with eltanexor treatment implicating anti-apoptotic targets BCL2 and BCLXL, which were validated by functional testing in vitro and in vivo. These targets were tested in vivo using Sf3b1K700E conditional knock-in mice, which showed that the combination of eltanexor and venetoclax (BCL2 inhibitor) had a preferential sensitivity for SF3B1 mutant cells without excessive toxicity. In this study, we unveil the mechanisms underlying sensitization to XPO1 inhibition in SF3B1-mutant MDS and preclinically rationalize the combination of eltanexor and venetoclax for high-risk MDS.


Dynamic BH3 profiling
DynamicBH3 profiling was performed as described previously (56).Briefly, this involves the cell lines (NALM6 and K562) to be exposed to eltanexor or vehicle (DMSO) for 16 hours followed by BH3 profiling involving BH3 peptides and mimetics.The delta priming is calculated as cytochrome c loss in eltanexor-cytochrome c loss in vehicle.

Genome-wide CRISPR Screen
CRISPR screens were performed as previously described (57).MOLM-13 and U937 Cas9expressing cells were transduced with whole genome Brunello sgRNA library at a low multiplicity of infection (~0.3) to obtain 500 cells per sgRNA.Genomic DNA was harvested on day 4 and day 20 post-transduction using the QIAamp DNA mini kit (51306, QIAGEN).The CRISPR score were calculated by normalized using (sgRNA read count/total read count) x 100,000.Following this, log2 fold change was calculated using normalized read count eltanexor/normalized read count DMSO.

Knockdown studies
Cells were transfected with Silencer Select siRNAs targeting DDX19A (AM16708 and 4392420, Thermo Fisher) using Lipofectamine 3000 transfection reagent (L3000, Invitrogen).Silencer Select Negative Control No. 1 siRNA (A390843, Thermo Fisher) was used as a negative control.Cells were transfected for 48 hours after which RT-qPCR and cell viability assays were performed.

RT-qPCR Gene Expression analysis
Total RNA was extracted from whole cells using the RNEasy mini kit (74106, QIAGEN) according to the manufacturer's instructions.RNA quality and quantity was measured using the Nanodrop spectrophotometer.Total RNA (500ng) was reverse-transcribed into double stranded cDNA using SuperScript III First-Strand Synthesis System (18080051, Invitrogen).Quantitative RT-PCR amplifications were performed using the following Taqman Gene Expression probes: GAPDH (Hs02758991_g1), XPO1 (Hs_00185645_m1), DDX19A (Hs01035515_m1), BCL2L1 (Hs00236329_m1), SIK1 (Hs00545020_m1), SLC25A48 (Hs00415075_m1), and CT45A2 (Hs04190919_mH).The expression level of the target genes was calculated using the Delta-Delta Ct method.GAPDH was used as the internal control.

Apoptosis analysis
Cells were stained with FITC-conjugated Annexin V in Annexin V-binding buffer and propidium iodide according to manufacturer's instructions (556547, BD Biosciences).Data was acquired on a CytoFlex LX Flow Cytometer and data analysis was performed using FlowJo software.

Colony forming assay
Total mouse bone marrow cells were plated at 2,000 cells per well in triplicates with methylcellulose media (M3434, StemCell Technologies) supplemented with 1% P/S.Cells were treated with vehicle, eltanexor (200nM), venetoclax (1µM), or the combination.After 14 days, the colonies were counted.
Immunoprecipitation 30 million cells were treated with either vehicle (DMSO) or 200nM of eltanexor for 24 hours at 37°C.Cells were harvested and washed with ice cold 1X PBS.Cells were lysed in IP lysis buffer containing protease and phosphatase inhibitors.Cell extracts were incubated overnight at 4°C with 50µl of protein-A Dynabeads crosslinked with anti-BIM antibody (2819, Cell Signaling, RRID:AB_10692515).Proteins were then eluted with glycine HCl and 2X Laemelli sample buffer and boiled for 10 minutes at 70°C.Supernatant was taken, and western blot analysis was performed.
Nuclear and cytoplasmic RNA extraction and messenger RNA sequencing K562 wildtype and SF3B1 mutant cells were treated with 200nM selinexor and without selinexor for 24 hours with three replicates each.To separate nuclear and cytoplasmic fractions, pellet the cells via centrifugation for 5 minutes at 1500RPM and discard the supernatant.The pellet was then resuspended into 300µL of ice cold 0.25x lysis buffer (Tris-Cl pH 8.0, NaCl, MgCl2, Nonidet P-40).The pellet was kept on ice for two minutes and rotate the tube every 45 seconds to ensure proper lysis followed by centrifugation at highest speed at 4°C for two minutes.After centrifugation, the supernatant was carefully removed and placed in a new tube as this was the cytoplasmic fraction.For the remaining nuclear fraction pellet, 500µL of ice-cold PBS was added and centrifuged at 1500RPM for 5 minutes.The QIAGEN RNeasy mini kit was then used for purified RNA.To confirm the separation of the nuclear and cytoplasmic fractions, RT-qPCR was performed as described above using MALAT1 (Hs00273907_s1) as the nuclear marker and TUG1 (Hs00215501_m1) as the cytoplasmic marker.To identify the mRNAs that are differentially exported by XPO1, ribosomal RNA depletion method with the TruSeq stranded Total RNA library was used with 50 million paired end reads.
For alternative splicing (AS) events detection, reads were mapped to the human genome hg19 using STAR aligner (v2.7.9a).The Selinexor replicate bam files and their control (DMSO) replicate bam files with the Gencode (v41) annotation file were used as input for rMATS (v4.1.2) (62), to report five types of the differential AS events.SE (skipped exon), MXE (mutually exclusive exons), A3SS (alternative 3′ splice site), A5SS (alternative 5′ splice site) and RI (retained intron).Events with |inclusion level difference| > 0.05, P < 0.05 and FDR < 0.05 were identified as significantly differentially expressed AS events.Custom R code using the ggplot graph library was used to generate additional volcanos plots from the analysis results.
The gene ontology (GO) analysis were performed with ShinyGO (RRID:SCR_019213) (63) using the differentially expressed genes or differentially alternative splicing genes.

Small RNA extraction and sequencing
A similar nuclear cytoplasmic fractionation method as described above was performed but instead with the miRNeasy Tissue/Cells Advanced Micro Kit (217684, QIAGEN) to identify differentially exported small nuclear RNAs.The small RNA sequencing was done with a size selection step with the Perkin Elmer Next Flex Small RNA library preparation kit on the Illumina sequencing platform with 50 million single end reads.
Small RNA sequencing data analysis Data analysis was performed as described in (64).Briefly, sequencing reads were trimmed with Trimmomatic (v0.32) (58) to remove low-quality and short reads (shorter than 17 bp).Reads were aligned against human elements in RepBase (v23.08) with STAR (v2.7.9a) (59), and the unmapped output was then mapped against the human genome (hg19), allowing three mismatches and keeping all uniquely aligned reads.
Combination treatment mRNA sequencing K562 wildtype and SF3B1 K666N cells were treated with either vehicle (DMSO), 200nM eltanexor, 1µM BCL-family inhibitor (venetoclax, A1331852, or navitoclax) and the combinations (eltanexor + venetoclax, eltanexor + A1331852, and eltanexor + navitoclax) for 24 hours with two technical replicates.After 24 hours, RNA extraction was done using the QIAGEN RNeasy mini kit.RNA library preparation was done with poly(A) selection on the Illumina HiSeq 2x150bp.The sequence reads were trimmed using Trimmomatic and mapped using GRCh38 to STAR aligner.Feature counts were calculated using subhead package v.1.5.2.DESeq2 was used for differential expression analysis with genes with adjusted p-value < 0.05 and log2 fold change > 1 were considered differentially expressed genes.

Statistical analysis
Statistical analyses for these experimental results was performed using GraphPad Prism and R. Specific statistical analysis details are located in the figure legends.Data were analyzed by unpaired two-tailed Student's t-test or analysis of variance (ANOVA) for more than two groups comparison.For unequal distribution, Welch's t-test was performed where appropriate.Kaplan-Meier survival curve used the log-rank statistics in R, with p <0.05 considered significant.Error bars represent the standard error of the mean (SEM) and the data is shown as significant when p <0.05 with a 95% confidence interval.All experiments are reported as at least mean triplicate ± SEM unless otherwise indicated in the figure legend.Investigators were not blinded to any of the experiments and the outcome assessment.
showing significantly increased levels of NOXA in the SF3B1 mutant after XPO1 inhibition.Data is shown as the mean unpaired two-tailed t-test (n=3 replicates, NOXA p=0.0076).(E) Validation via RT-qPCR of the upregulation of genes, SIK1 and SLC24A48, in the nucleus of SF3B1 mutant cells after XPO1 inhibition (n ³ 3 replicates).Data is mean ± standard deviation, two-way ANOVA p<0.0001.(F) Heat map of small nuclear RNAs before and after XPO1 inhibition shown as the average of 3 independent biological replicates.(G) Expression levels of each small nuclear RNAs (U1, U2, U3, U4, U5, and U6) in K562 cells relative to control primers (5S rRNA, RN7SL1, and RN7SK) (n=4 replicates).

Figure S2 .
SF3B1 mutations have BCLXL dependence.(A) Delta priming responses of the indicated BH3 peptides and mimetics in NALM6 SF3B1 WT and mutant cells (B) K562 SF3B1 WT and mutant cells after 16 hours of 1µM eltanexor treatment compared to DMSO.**** p<0.0001 (n= 3 replicates).(C) BH3 profiling of K562 SF3B1 WT and mutant cells after 16 hours of eltanexor treatment in a heatmap with the delta priming responses.