Inhibition of the oncogenic fusion protein EWS-FLI1 causes G2-M cell cycle arrest and enhanced vincristine sensitivity in Ewing's sarcoma

Abstract

Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surrounding tissues mostly affecting adolescents and young adults. A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), which is generated from a chromosomal translocation, is implicated in driving most ES cases by modulation of transcription and alternative splicing. The small-molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis in ES cells. We aimed to identify both the underlying mechanism of the drug and potential combination therapies that might enhance its antitumor activity. We tested 69 anticancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergistic therapeutic effect. YK-4-279 rapidly induced G₂-M arrest, increased the abundance of cyclin B1, and decreased EWS-FLI1-mediated generation of microtubule-associated proteins, which rendered cells more susceptible to microtubule depolymerization by vincristine. YK-4-279 reduced the expression of the EWS-FLI1 target gene encoding the ubiquitin ligase UBE2C, which, in part, contributed to the increase in cyclin B1. YK-4-279 also increased the abundance of proapoptotic isoforms of MCL1 and BCL2, presumably through inhibition of alternative splicing by EWS-FLI1, thus promoting cell death in response to vincristine. Thus, a combination of vincristine and YK-4-279 might be therapeutically effective in ES patients.

Fig. 1.. YK-4-279 synergizes with VCR, significantly increasing apoptosis and leading to improved survival of ES-xenografted mice.

(A) Cell viability assessed by WST-1 staining in A4573 cells treated with different concentrations of VCR and YK-4-279. Dose-response curves through nonlinear regression analysis are shown. (B and C) Apoptosis assessed by (B) caspase-3 activity and (C) flow cytometry for annexin V (AV)/propidium iodide (PI) staining in A4573 cells treated with YK-4-279 (3 μM), VCR (10 nM), both, or DMSO. (D and E) Change in tumor volume (D) and percent survival (E) assessed in A4573 xenograft mice intraperitoneally injected with YK-4-279 (YK; 10, 50, 100, and 150 mg/kg), VCR (1 mg/kg), or DMSO. Figure key shows number (n) of mice per group. (F and G) TUNEL staining (white arrows) (F) and quantification (G) to assess apoptosis in A4573 xenografts from mice intraperitoneally injected with YK-4-279 (150 mg/kg), VCR (1 mg/kg), or DMSO for 3 days. Magnification, ×800; (scale bars, 50 μm). Data are means ± SEM of greater than or equal to seven xenografts. All other data are means ± SEM of greater than or equal to three independent experiments. *P < 0.05, **P < 0.01, ***P = 0.001, and ****P < 0.0001 by unpaired, two-tailed t test (B, D, and G) or log-rank test (E). hpf, high-power field.

Fig. 2.. YK-4-279 leads to a G2-M cell cycle arrest, which is enhanced upon combination with VCR.

(A and B) Abnormal chromatin condensation (ACC; white arrows) assessed by hematoxylin and eosin (H&E) staining (A) and quantified (B) in A4573 xenografts from mice after treatment as described for Fig. 1F. Magnification for large images, ×400 for large images (scale bars, 100 μm); magnification for inset, ×800 (scale bars, 50 μm). Data are means ± SEM of greater than or equal to seven different xenografts. (C) Cell cycle analysis by fluorescence-activated cell sorting (FACS) in TC32 cells treated with YK-4-279 (3 μM), VCR (30 nM), both, or DMSO. (D) Western blot analysis for p-H3Thr¹¹ in lysates from A4573 cells after treatment with YK-4-279 (3 μM) as indicated. Blots are representative of greater than or equal to three independent experiments. (E and F) Cell cycle analysis by immunohistochemical staining for p-H3Ser¹⁰ in A4573 xenograft tissue from mice. Treatment and quantification were as described for Fig. 1 (F and G). Magnification, ×800 (scale bars, 50 mm). Data are means ± SEM of greater than or equal to three independent experiments. *P < 0.05, **P < 0.01, ***P = 0.001, and ****P < 0.0001 by unpaired, two-tailed t test.

Fig. 3.. Cyclin B1 expression increases after YK-4-279 and VCR treatments, in part, through decreased EWS-FLI1-driven UBE2C.

(A) Cyclin B1 abundance assessed by immunofluorescence staining in TC32 cells after 20 hours of treatment with YK-4-279 (3 μM) or DMSO. Magnification for large images, ×400 (scale bars, 100 μm); magnification for inset, ×800 (scale bars, 50 μm). (B) Western blot analysis for cyclin B1, UBE2C, CDK1, and p-CDK1 (Tyr¹⁵) on lysates from A4573 cells treated with YK-4-279 (3 μM), VCR (10 nM), both, or DMSO. (C and D) Immunohistochemical staining for cyclin B1 (white arrows) of A4573 xenograft tissue from mice; treatment, magnification, and analysis were as described for Fig. 2 (E and F). **P < 0.01 and ***P < 0.001 by unpaired, two-tailed t test. (E) RNA-seq analysis for the expression of UBE2C in TC32 cells after transfection with either EWS-FLI1 shRNA or empty vector (EV), or after treatment with YK-4-279 (3 μM) for 12 hours; the depth of all exon reads is represented in fragments per kilobase and permillion RNA-seq fragments of the sample (FPKM). (F and H) Western blot analysis for UBE2C, cyclin B1, and p-H3Ser¹⁰ on A4573 cell lysates after overexpression of DDK-tagged UBE2C (OE) (F) or shRNA depletion of cyclin B1 (CCNB1) (H), or after treatment with YK-4-279 (3 μM) for 24 hours. Blots are representative. (G) Cell viability assays in A4573 cells after transfection with either DDK-tagged UBE2C vector (OE) or control (EV), and after treatment with different concentrations of YK-4-279 for 72 hours; analysis was as described for Fig. 1A. (I) Cell cycle analysis by FACS in A4573 cells after transfection and treatment as in (H); percentagesof cells in G₂-M are indicated. Data are means ± SEM of greater than or equal to three independent experiments.

Fig. 4.. YK-4-279 potentiates spindle perturbation of VCR and induces chromosomal alignment defects.

(A) Morphological analysis of spindle, chromatin, and centrosome formation assessed by immunofluorescence staining for β-tubulin, DNA, and γ-tubulin in TC32 cells after 8 hours of treatment with YK-4-279 (1 μM), VCR (30 nM), both, or DMSO. Magnification, ×800 (scale bars, 50 μm). (B) Western blot analysis for β-tubulin on lysates from A4573 cells treated with YK-4-279 (3 μM), VCR (10 nM), both, or DMSO. (C) Representative cells with tridentate chromosome formation assessed by confocal microscopy after immunofluorescence staining from (A). White arrows mark residual chromosomes at spindle poles (top) and prominent astral microtubules (middle). Magnification, ×1200 (scale bars, 25 μm). (D) RNA-seq analysis for the expression of CENPE, KIF22, and KIF2C in TC32 cells with wild-type (WT) EWS-FLI1 expression, shRNA reduction of EWS-FLI1 (ΔEF), or treated 12 hours with YK-4-279 (3 μM). The depth of all exon reads is represented in FPKM. (E) Apoptosis assessed by caspase-3 activity in A4573 cells after single and combinatorial treatments with YK-4-279 (3 μM) and VCR (10 nM) nonsequentially (No) or sequentially (Yes; 4 hours of pretreatment with either drug). *P < 0.05 by unpaired, two-tailed t test.

Fig. 5.. YK-4-279 induces alternative proapoptotic splicing of MCL1 that is confined to ES cell lines and leads to altered protein ratios while reducing Mcl-1L.

(A) RNA-seq analysis and de novo isoform reconstruction from TC32 cells with wild-type (WT) EWS-FLI1 expression, after shRNA reduction of EWS-FLI1 (ΔEF), or after treatment with YK-4-279 (3 μM for 12 hours). Reconstructed transcripts were coded as TCONS and displayed with the adjacent presumed protein isoform. Annotation of TCONS to RefSeq mRNA reference ID is shown in table S4. On the basis of the RefSeq database, the MCL1 protein-coding region of each transcript is indicated by dashed lines. The aligned reads map to the gene transcript of each condition (WT, ΔEF, or YK). (B) Absolute isoform expression based on RNA-seq reads from each sample (WT, ΔEF, or YK). (C) Validation of MCL1 splicing by qRT-PCR using specific primer pairs [black arrows in (A)], from exons 1 and 3 in TC32 and HEK293 cells after 18 hours of treatment as indicated (YK, 3 μM; YK1, 1 μM; YK3, 3 μM; VCR, 30 nM). Blots are representative. (D) Annotated is fold change of MCL1S expression with respect to DMSO after densitometric quantification of short, two-exon comprising PCR product bands and normalization to 18S ribosomal RNA in TC32 and HEK293 cells after treatment as described for (C). Data are means ± SEM of greater than or equal to three independent experiments. *P < 0.05 by unpaired, two-tailed t test. (E) Western blot analysis for Mcl-1 isoforms on A4573 cell lysates after treatment with YK-4-279 (3 μM), VCR (10 nM), both, or DMSO. Blots are representative. (F) Change in ratio of Mcl-1S/Mcl-1L assessed by densitometric protein quantification after normalization to actin based on Western blot analysis as described for (E). Data are means ± SEM of greater than or equal to three independent experiments after treatment as described for (E).

Fig. 6.. YK-4-279 reverses EWS-FLI1–induced antiapoptotic alternative splicing of BCL2, leading to expression of proapoptotic Bcl-2 beta protein isoform after single and combinatorial treatments.

(A and B) RNA-seq analysis for BCL2 acquired and presented as described for data in Fig. 5 (A and B). (C) Validation of BCL2 splicing by qRT-PCR using specific primer pairs [black arrows in (A)] from exons 1 and 2 in TC32 and HEK293 cells after treatment as described for Fig. 5C. Blots are representative. (D) Change in the ratio of BCL2 beta to BCL2 alpha assessed by densitometric RNA quantification after normalization to 18S. Data are means ± SEM of greater than or equal to three independent experiments after treatment as described for Fig. 5C. (E) Western blot analysis for Bcl-2 alpha on TC32 cell lysates after treatment with YK-4-279 (3 μM), VCR (10 nM), both, or DMSO. Blots are representative. (F) Apoptosis assessed by caspase-3 activity in TC32 and NB1643 cells treated with ABT-737. *P < 0.05, ***P < 0.001, and ****P < 0.0001 by unpaired, two-tailed t test.

Fig. 7.. Proposed model of synergistic cytotoxicity between YK-4-279 and VCR in ES.

VCR and YK-4-279 induce a cell cycle arrest at the G₂-M transition, a presumed VCR-sensitive stage for microtubule depolymerization. YK-4-279 advances a potent G₂-M arrest that is sustained by persistent amounts of cyclin B1 after reduced expression of UBE2C. VCR and YK-4-279 induce spindle and centrosome perturbation in ES cells including decreased expression of microtubule associated proteins (MAPs). YK-4-279 then flips the final switch to apoptosis by altering the ratios of MCL1 and BCL2 transcripts and corresponding protein isoforms.