Abstract 3661: GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells

Ewing sarcoma (EWS) is characterized by TET-ETS fusions, most commonly the EWSR1-FLI1 fusion. GLI1, a transcription factor in the Hedgehog (HH) signal transduction pathway and target of canonical HH signaling, is a direct transcriptional target of EWSR1-FLI1. A role for the HH signaling pathway and GLI1 in acquisition of a multidrug resistance phenotype has been previously observed in several human cancers. The role of the HH signal transduction pathway and GLI1 in the biology of EWS remains incompletely understood. We hypothesize that the majority of EWS cell lines express GLI1 based on non-canonical up-regulation by EWSR1-FLI1 and that further up-regulation of GLI1 may be associated with the development of drug resistance. Here, we show that EWS cell lines (CHLA-9, CHLA-10, CHLA-258, TC-32 and TC-71) express canonical HH pathway components (PTCH1, SMO, GLI1 and GLI3) and that cells lines established at the time of recurrence have higher GLI1 expression (CHLA-258 and TC-71) than those established at the time of diagnosis (CHLA-9, CHLA-10 and TC-32). Exposure to Sonic HH ligand in order to activate canonical HH signaling did not affect cell viability (MTT assay), proliferation (BrdU assay), or apoptosis (caspase 3/7 assay). However, inhibition of GLI1 activity with GANT61 reduced GLI1 expression (qRT PCR), cell viability (MTT assay) and cell proliferation (BrdU assay) but increased apoptosis (caspase 3/7 assay) and sensitivity of the cells to vincristine (VCR) (MTT assay), suggesting GLI1 plays roles in each of these processes. We then established VCR-resistant EWS cell lines (TC-71 [45-fold increase in the IC-50] and TC-32 [27-fold increase in the IC-50]) by exposing cells to serially increasing concentrations of VCR. We used an 86-gene cancer drug resistance PCR array (Qiagen) to characterize gene expression differences between untreated vs. recurrent EWS cell lines and between parental vs. VCR-resistant EWS cell lines. Among the changes in gene expression, we showed higher GLI1 expression in the recurrent EWS cell lines (CHLA-258 cells [4.8-fold] and TC-71 [3.4-fold]) vs. untreated CHLA-9 cells, and in VCR-resistant TC-71 cells (2.2-fold) vs. parental TC-71 cells. Down-regulation of GLI1 by GANT61 or GLI1 siRNA enhanced sensitivity of the cells to VCR. Our results suggest that GLI1 expression contributes to proliferation, survival and drug-resistance of EWS cell lines. Strategies to inhibit GLI1 may have therapeutic benefit in EWS. Citation Format: Joon Won Yoon, Marilyn Lamm, Philip Iannaccone, David Walterhouse. GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3661.