Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma

Abstract

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.

Figure 1. Identification of proteins required for EWS-FLI1 activity by genome-wide RNAi screening

(A) A genome-wide RNAi screen of EWS-FLI1 activity. Data are shown as the median Z-score value for the 3 siRNAs corresponding to each gene (~21,000 genes) except for POL2RA (light blue circles), EWSR1(magenta diamonds) and FLI1 (dark blue squares) for which the Z-score value of each of the 3 siRNAs targeting each gene are shown. (B) The Z_diff (Z_NR0B1-Z_CMV) for the EWSR1 and FLI1 siRNAs included in the screen and a schematic of the sites in EWSR1, FLI1, and, EWS-FLI1 targeted by these siRNAs. (C) The median Z_diff (Z_NR0B1-Z_CMV) (3 siRNAs per gene) for the 139 genes, selected for follow up analysis, that exhibited a selective decrease in the TC32-NR0B1-luc reporter when silenced. (D) Ratio of the TC32-NR0B1-luc and TC32-CMV-luc reporter signals, 72 h post siRNA-transfection (mean ± standard deviation (SD), n=5). ** p<0.01; *** p<0.001 compared to siNeg. See also Figure S1 and Table S1.

Figure 2. EWS-FLI1 driven expression in TC32 cells is altered by the silencing of SF3B1, SUPT6H, or HNRNPH1

(A) GSEA of the transcriptome of SF3B1, SUPT6H, or HNRNPH1-silenced TC32 cells (48 h) using a set of genes up-regulated (751) or down-regulated (494) in ES. NES = normalized enrichment score, FDR = false discovery rate, FWER = family-wise error rate. (B) Heat-map representation of the fold-change (Log2) in the expression of genes deregulated in ES following silencing of SF3B1, SUPT6H, or HNRNPH1 in TC32 cells (48 h). (C) qPCR assessment of EWS-FLI1 expression in HNRNPH1, SF3B1, or SUPT6H–silenced TC32 cells (48 h). Data are expressed relative to siNeg-transfected cells (mean ± SD, n=3). (D) Immunoblot analysis of whole-cell lysates prepared from ES cells 48 h post transfection of HNRNPH1, SF3B1, or SUPT6H siRNAs using antibodies against the proteins indicated. * p<0.05; ** p<0.01; *** p<0.001compared to siNeg. (E) Relative viability of TC32 cells 48 and 72 h post-siRNA transfection (siNeg median normalized, mean ± SD, n=5). See also Figure S2 and Tables S2 and S3.

Figure 3. HNRNPH1 is required for the splicing of EWS-FLI1 in Ewing sarcoma cell lines with a genomic breakpoint that retains EWSR1 exon 8

(A) Schematic of the organization of the EWSR1 and FLI1 genes and EWS-FLI1 fusion transcripts in ES cell lines. EWSR1 exons are indicated in magenta and FLI1 exons in blue. The EWSR1 and FLI1 exon count are based on NM_05243 and NM_002017 reference sequences. (B) Expression of SF3B1 and EWS-FLI1 (E7/F7 primer pair) in SF3B1-silenced ES cells (48 h). Data are expressed relative to siNeg-transfected cells (mean ± SD, n=3). (C) Expression of HNRNPH1 and EWS-FLI1 (E7/F7 primer pair) in HNRNPH1-silenced ES cells (48 h). Data are expressed relative to siNeg-transfected cells (mean ± SD, n=3). (D) PCR analysis of the splicing of EWS-FLI1 using E6/F6 primer pair and sequence analysis of the amplified products labeled a and b focusing on the junction of EWSR1 exons 7 and 8 and EWSR1 exon 8 and FLI1 exon 7. (E) Immunoblot analysis of whole-cell lysates prepared from TC32 cells 48 h post siRNA-transfection using antibodies against the proteins indicated. ** p<0.01; *** p<0.001compared to siNeg. See also Figure S3.

Figure 4. HNRNPH1 directly binds the EWS-FLI1 pre-mRNA expressed in TC32 cells

(A) Expression of EWS-FLI1 regulated genes in HNRNPH1-silenced SKNMC or TC71 cells. Data are expressed relative to untransfected cells (mean ± SD, n=3). * p<0.05; ** p<0.01; *** p<0.001 compared to siNeg. (B) Immunoblot analysis of whole-cell lysates prepared from ES cells 48 h post siRNA-transfection using antibodies against the proteins indicated. (C) The viability of TC32 and TC71 cells 72 h post siRNA-transfection (siNeg median normalized, mean ± SD, n=10). p-values are compared to siNeg. (D) UV-cross-linked RNA from TC32 cells (nuclear fraction) were subjected to RNA immunoprecipitation using an HNRNPH1 antibody or a control IgG isotype control. The fold-enrichment of co-precipitating RNA (HNRNPH1-bound versus IgG control) was determined by qPCR across regions of the EWS-FLI1 transcript (see Figure S4A). Data are shown as the mean ± standard error of the mean (SEM) of 3 experiments. (E) Sequence of EWSR1 exon 8 indicating putative G-rich HNRNPH1 binding sites. (F) The fold-enrichment for HNRNPH1-bound RNA oligomers determined by comparison of chemiluminiscent signals from the pull-down performed using a HNRNPH1 antibody and an IgG isotype control. Data are shown as the mean ± SEM of 3 experiments. p-values are compared to no oligomer/no antibody controls. See also Figure S4.

Figure 5. Depletion of SF3B1 results in mis-splicing of EWS-FLI1

(A) Immunoblot analysis of whole cell lysates prepared from ES cells 48 h post siRNA-transfection using antibodies against the proteins indicated. (B)EWS-FLI1 expression (E7/F7 or E7/F9 primer pairs) following silencing of HNRNPH1 or SF3B1 in SKNMC cells. Data are expressed relative to siNeg-transfected cells (mean ± SD, n=3). Expression of RPL27 is shown as a control for assessing non-specific splicing effects. * p<0.05; ** p<0.01; *** p<0.001 compared to siNeg. (C) PCR analysis of the splicing of EWS-FLI1 using primers corresponding to EWSR1 exon 7 and FLI1 exon 8 and representative sequence chromatograms for the PCR products indicated in each gel (a - f). (D) Expression of EWS-FLI1 in TC32 cells treated with increasing concentrations of PlaB (1.25 – 10 nM, 6 h). Data are expressed relative to 0.1% DMSO treated cells (mean ± SD, n=3). * p<0.05; ** p<0.01; *** p<0.001 compared to V. (E) PCR analysis of the splicing of EWS-FLI1 using primers corresponding to EWSR1 exon 7 and FLI1 exon 8 and representative sequence chromatograms for the PCR product indicated *. TC32 cells were treated with 5 nM PlaB for 6 h. (F) Immunoblot analysis of whole-cell lysates prepared from TC32 cells treated with increasing concentrations of PlaB for 24 h using antibodies against the proteins indicated. (G) HCD Fragmentation spectra of peptide ([M+2H]²⁺-H₂O, m/z 657.62) from SKMNC cells treated with 5 nM PlaB for 24 h. The protein band corresponding to EWS-FLI1 was excised and digested with trypsin and the resulting peptides were analyzed on a Thermo Fusion Orbitrap. (H) PCR analysis of the splicing of EWS-FLI1 using a FLI1 exon 6 forward and a FL11 exon 9 reverse primer and representative sequences of the FLI1 exon 6/8 junction for the indicated product *. See also Figure S5.

Figure 6. Inhibition of SF3b function reverses expression of genes activated by EWS-FLI1

(A) The viability of Ewing sarcoma (TC32, SKNMC, TC71, and RD-ES) and prostate cancer (PC3 and LNCaP) cell lines exposed to increasing concentrations of the spliceosome inhibitor Pladienolide B (PlaB) for 48 h, mean ± SEM, n=3 at each PlaB concentration normalized to vehicle (V, 0.1% DMSO); CI = Confidence Interval. (B) GSEA of the transcriptome of PlaB treated ES cells (5 nM, 24 h) using a set of 751 genes up-regulated in ES and a set of ~100 genes activated by EWS-FLI1 (Riggi et al., 2014). (C) Heat-map of the fold-change (Log₂) in the expression of EWS-FLI1 regulated genes (Kauer et al., 2009) and genes considered direct targets of EWS-FLI1 (Riggi et al., 2014) following exposure of ES cells to PlaB (5 nM for 24 h). (D) Expression of genes activated by EWS-FLI1 in 5 nM PlaB treated (24 h) TC32 or SKNMC cells. Data are expressed relative to DMSO treated cells (mean ± SD, n=3). * p<0.05; ** p<0.01; *** p<0.001 compared to siNeg or V. (E) Expression of genes activated by EWS-FLI1 in SF3B1-silenced TC32 or SKNMC cells (48 h). Data are expressed relative to siNeg-transfected cells (mean ± SD, n=3). * p<0.05; ** p<0.01; *** p<0.001 compared to siNeg. (F) Immunoblot analysis of whole-cell lysates prepared from ES cells treated with either 0.1% DMSO or 5 nm PlaB (24 h) or siNeg or SF3B1-silenced (48 h) ES cells using antibodies against the proteins indicated. (G) ChIP analysis of DNA from TC32 cells (DMSO 0.1% or 5nM PlaB treated, 24 h) using either a FLI1 antibody or an IgG antibody. Fold-enrichment of co-precipitating DNA (PlaB compared to DMSO) was determined by qPCR for the indicated EWS-FLI1 bound GGAA microsatellites; the regions targeted and the PCR primers used are detailed in Supplemental Experimental Procedures, mean ± SEM, 3 independent experiments (triplicate samples for each treatment in each experiment). See also Figure S6 and Tables S2 and S4

Figure 7. The altered splicing of EWS-FLI1 results in disruption of its activity as an oncogenic transcription factor

Schematic illustrating the splicing of the EWS-FLI1 pre-mRNA by SF3B1 and HNRNPH1.