Abstract 49: Modulating insulin receptor splicing as a potential therapeutic approach for rhabdomyosarcoma

Abstract

Abstract The insulin receptor (IN-R) is subject to alternative splicing to produce two isoforms: the full-length IN-R B isoform and an isoform lacking exon 11 known as IN-R A which is the predominant isoform expressed in sarcoma cells. This short exon encodes for 12 amino acids but the resulting receptor has high affinity for a growth hormone called insulin-like growth factor 2 (IGF2), which exquisitely responds to autocrine and paracrine signaling. Increased IN-R A levels observed in rhabdomyosarcoma (RMS) coupled with increased expression of IGF2, programs the tumor cell for enhanced growth and angiogenesis, two important factors in tumorigenesis. Furthermore, IGF2 can maintain angiogenesis through IN-R A when the cognate receptor for IGF2, the IGF-1 receptor, is blocked. Interestingly we have shown that hypoxia increases alternative splicing to produce more IN-R A. This is an interesting finding since adaptation to the hypoxic environment is a hallmark of the neoplastic phenotype. Therefore, we hypothesize that expression of splicing factors is altered under hypoxic conditions which leads to increased alternative splicing and generation of IN-R A, allowing the cancer cells a growth advantage which contributes to metastasis beyond the micrometastatic phase. To characterize sequence elements and splicing factors involved in the regulation of IN-R alternative splicing, we have developed a hypoxia-inducible splicing system, which recapitulates the splicing patterns observed in tumors. We have shown that sequence elements preceding exon 11 are critical to the increased alternative splicing we see under hypoxic conditions. As such, we have targeted these regions for antisense oligonucleotide (ASO) development to increase full-length splicing of IN-R, with the goal of restricting proliferative signaling by IGF2 activation of IN-R A. We performed an ASO walk to target regions important for exon inclusion or exclusion and have successfully targeted a region known to be a binding site for the splicing factor CUGBP1. RMS-derived cell lines almost exclusively express IN-R A but when treated with our lead ASO compound we see a dramatic decrease in alternative splicing resulting in increased levels of the full-length insulin receptor. We have also shown that RMS-derived cells treated with our splice modulating ASO exhibit reduced cell proliferation and migratory properties. With these promising data we postulate that modulation of IN-R splicing can be used in conjunction with already established anti-IGF-1 receptor therapies in the treatment of RMS. Citation Format: Brianne Sanford, Chelsea Brown, Hemant Bid, Thomas Beebe, Daniel Comiskey, Frank Rigo, Peter Houghton, Dawn Chandler. Modulating insulin receptor splicing as a potential therapeutic approach for rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 49. doi:10.1158/1538-7445.AM2017-49