Abstract 3560: Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development
Authors: Venkataramany Akila S. , Khurshid Safiya , Miller Anthony R. , Wijeratne Saranga , Wang Pin-Yi , Cripe Timothy P. , Chandler Dawn S. 
Published: April 2023

Introduction: Ewing sarcoma (ES) is the second most common pediatric bone cancer, and patients with metastases have five-year survival rates of &lt;30%. Both local and metastatic ES tumors are characterized by the EWS-FLI1 chromosomal translocation, resulting in an aberrant transcription factor that increases ES cell proliferation and metastasis. EWS-FLI1 activity and ES transformation are reliant upon increased IGF-1 signaling, so initial therapies have centered on IGF-1R inhibition. However, compensatory upregulation of insulin receptor A (IR-A), an oncogenic spliced isoform of IR, provides a viable resistance mechanism to support ES growth and progression. IR has two spliced isoforms formed by the inclusion or exclusion of exon 11 (IR-B and IR-A respectively), and IR-A is differentially expressed in cancers due to its high affinity binding to IGF-II. We hypothesize that the pathogenesis and metastatic potential of ES is driven in part by alternative splicing of IR and other key genes. Furthermore, modulation of this splicing via antisense oligonucleotides (ASOs) aided by adeno-associated viral (AAV) delivery may be a novel therapeutic avenue for ES.

Results: We performed long-read mRNA sequencing to identify differentially spliced isoforms between the matched ES cell lines CHLA-9 and CHLA-10 (derived from a localized and metastatic ES tumor respectively). Our analysis revealed global differences in alternative splicing between CHLA-9 and CHLA-10 and identified gene isoforms in the insulin receptor/IGF signaling pathway (IGFBP3) that are unique to CHLA-10. Additionally, we detected both previously reported and novel isoforms for CD99, RHOA, RACK1, splicing factors (PTBP1), and eukaryotic initiation factors, among other genes.

To further understand IR alternative splicing, our labs have developed an IR ASO targeting the CUG-BP1 regulatory site upstream of exon 11, and we have previously validated its efficacy in HeLa cells transfected with an IR minigene and in pediatric sarcoma cell lines. In ES cell lines, we observed significant IR-A upregulation in a panel of ES cell lines and patient-derived xenografts derived from local and metastatic tumors, highlighting the opportunity for intervention with our ASO. To facilitate delivery, we have explored the use of AAV vectors, a clinically approved option for gene therapy, and shown successful transduction of AAV-8 over a time-course in ES cells.

Conclusions: Our results indicate that alternative splicing is differential between local and metastatic ES and produces isoforms unique to the metastatic setting. Additionally, the oncogenic IR-A isoform and other spliced isoforms are viable targets for ASO therapy aided by AAV delivery. By better understanding splicing in ES, we can identify targetable interactions at the transcriptional level and develop innovative therapeutics that can improve the clinical outcomes of ES patients.

Citation Format: Akila S. Venkataramany, Safiya Khurshid, Anthony R. Miller, Saranga Wijeratne, Pin-Yi Wang, Timothy P. Cripe, Dawn S. Chandler. Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3560.