Development of an Ewing sarcoma cell line with resistance to EWS‑FLI1 inhibitor YK‑4‑279

Abstract

Despite Ewing sarcoma (ES) being the second most common pediatric malignancy of bone and soft tissue, few novel therapeutic approaches have been introduced over the past few decades. ES contains a pathognomonic chromosomal translocation that leads to a fusion protein between EWSR1 and an ets family member, most often FLI1. EWS‑FLI1 is the most common type of fusion protein and is a well‑vetted therapeutic target. A small molecule inhibitor of EWS‑FLI1, YK‑4‑279 (YK) was developed with the intention to serve as a targeted therapy option for patients with ES. The present study investigated resistance mechanisms by developing an ES cell line specifically resistant to YK. The ES cell line A4573 was treated with YK to create resistant cells by long term continuous exposure. The results revealed that resistance in A4573 was robust and sustainable, with a >27‑fold increase in IC50 lasting up to 16 weeks in the absence of the compound. Resistant ES cells were still sensitive to standard of care drugs, including doxorubicin, vincristine and etoposide, which may be valuable in future combination treatments in the clinic. Resistant ES cells revealed an increased expression of CD99. RNA sequencing and qPCR validation of resistant ES cells confirmed an increased expression of ANO1, BRSK2 and IGSF21, and a reduced expression of COL24A1, PRSS23 and RAB38 genes. A functional association between these genes and mechanism of resistance remains to be investigated. The present study created a cell line to investigate YK resistance.

Keywords: ewing sarcoma; YK-4-279; resistance; eWS-Fli1; cd99; ano1.

Figure 1.

Development of a YK resistant A4573-R (A4573-R) cell line. (A) A4573 cells developed a significant resistance to YK, exhibiting a near 30-fold increase in IC₅₀. (B) A4573-R cells maintained resistance to YK in the absence of the compound in media for 16 weeks. IC₅₀ values for 1 week, 4 weeks and 16 weeks are presented. YK, YK-4-279; R, resistant.

Figure 2.

A4573-R resistance to YK is specific. (A) Treatment with increasing concentrations of the p-glycoprotein pump inhibitor, verapamil, does not affect A4573-R's sensitivity to YK. IC₅₀ values for each condition are presented. Viability curves comparing sensitive and resistant cell lines with (B) doxorubicin, (C) vincristine, (D) etoposide and (E) imatinib are provided. A4573-R cells did not exhibit cross-resistance to any of these compounds. R, resistant; YK, YK-4-279.

Figure 3.

CD99 expression is increased in A4573-R cells. (A) Quantitative PCR analysis was performed for EF, CD99 and 18S ribosomal RNA in A4573 sensitive cells and resistant cells at multiple timepoints. CD99 RNA expression is significantly elevated compared with sensitive cells at all timepoints. *P<0.05 and ****P<0.0001 vs. sensitive. (B) Western blot anlaysis of CD99 and EF in A4573 sensitive and resistant cells. Expression levels were calculated using densitometry analysis relative to sensitive cell lines. CD99 expression is elevated in A4573-R cells. R, resistant; EF, EWS-FLI1.

Figure 4.

YK treatment of A4573 ×enografts results in an elevated expression of CD99 in vivo. (A) Western blot analysis for CD99 expression in tumors collected after 5 days of YK treatment at concentrations of 10, 50 and 100 mg/kg, or DMSO control treatment. (B) Tumor volume at endpoint. No significant difference was identified between the groups. (C) Densitometry quantification of western blotting relative to DMSO control tumor expression. Circles represent individual samples. CD99 expression was significantly elevated in all treatment groups. *P<0.05 and ***P<0.001 vs. DMSO. YK, YK-4-279.

Figure 5.

Quantitative PCR for upregulated genes identified through RNA sequencing in A4573 cells. Relative expression was evaluated at multiple time points in resistant cells compared with sensitive cells. (A) ANO1, BRSK2, and IGSF21 were upregulated. (B) COL24A1, PRSS23 and RAB38 were downregulated. *P<0.05, **P<0.01 and ***P<0.001 vs. sensitive. ANO1, anoctamin 1; BRSK2, BR serine/threonine kinase 2; IGSF21, immunoglobulin superfamily member 21; COL24A1, collagen type XXIV alpha 1 chain; PRSS23, serine protease 23; RAB38, Ras-related protein Rab-38.