Abstract 4132: ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway

Abstract

Abstract Alternative splicing is a process contributing to structural transcript variation and proteome diversity but is often disrupted in cancer. Like most other transcripts, the insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. The expression of these isoforms is tightly regulated during development, however there is an aberrant increase in IN-RA expression in childhood cancers like Rhabdomyo- & Osteo-sarcoma. This increased IN-RA expression is deleterious to the cells because it encodes for a receptor which has high affinity for both insulin & IGF2 growth hormones and it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks like proliferation & angiogenesis. Rhabdosarcoma is the 3rd most frequent solid tumor and the most common soft-tissue sarcoma in children. Despite improvement in the treatment for this disease, there is substantial inadequacy of less toxic alternative therapies. We found that there is a significantly increased expression of IN-RA levels in a cohort of 40 Rhabdosarcoma (RMS) patients (embryonal & alveolar) as compared to the control samples. We confirmed this increased expression in multiple RMS cell lines. Furthermore, we went on to show that cellular stress such as hypoxia increases alternative splicing to produce more IN-RA isoform. In order to get a mechanistic insight into this phenomenon, we utilized this hypoxia-inducible splicing system and performed an antisense-oligonucleotide (ASO) screen to characterize sequence elements and splicing factors involved in the regulation of IN-R splicing. We found that sequence elements flanking exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. We then performed a refined ASO walk to target the regions important for exon inclusion or exclusion and identified a region known to be a binding site for the splicing-factor CUGBP1. RMS derived cell lines almost exclusively express IN-RA but when treated with our lead ASO compound that targets this region, they show a dramatic decrease in the IN-RA expression. More importantly, the ASO treated cells exhibit a significant reduction in cell proliferation and migration. Application of ASO compound attenuates the IGF signaling pathway measured by decreased pAKT staining. Additionally, when tested in RMS xenograft models, we see decreased extravasation of blood vessels in the grafts cells transfected with the ASO that blocks IN-RA expression compared to controls. Our current data shows promising insight into how we can impede the IGF2 pathway by reducing IN-RA expression and consequently mitigate tumor hallmarks like cell-proliferation, migration and angiogenesis. The goal is to use these ASO compounds as therapeutic interventions in conjunction with already established anti-IGF1 receptor therapies to treat pediatric Rhabdomyosarcoma. Citation Format: Safiya Khurshid, Matias Montes, Brianne Sanford, Chelsea Brown, Daniel Comiskey, Frank Rigo, Peter Houghton, Dawn Chandler. ASO screen uncovers splicing as a therapeutic vulnerability in theinsulin-like growth factor (IGF) signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4132.