Abstract 1597: Inhibition of the novel therapeutic target pregnancy associated plasma protein A (PAPP-A) in Ewing sarcoma enhances efficacy of IGF1R targeting in vivo
2017
Background: Despite intensive treatment regimens, Ewing sarcoma (EWS) patients with metastatic or relapsed disease still face a 5-year overall survival of less than 20%, indicating a clear need for novel targeted therapies. Utilizing ribosomal depleted RNA sequencing of 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal samples of variable tissues, we previously reported PAPP-A as one of the top 5 genes overexpressed in EWS (> than 4-fold) compared to normal tissue. Notably, PAPP-A showed substantial expression in tumors (median log2FPKM = 3.933, n=122) and minimal expression in normal tissue (median log2FPKM = -1.564, n=96). Pregnancy Associated Plasma Protein A (PAPP-A) is a secreted zinc metalloproteinase, that anchors to cell surface of heparan sulfate proteoglycans and enhances local IGF signaling. Through the release of IGFs from IGFBP-4, IGFBP-2 and IGFBP-5, free, bioactive IGF is being increased in close proximity to its receptors. In pregnancy PAPP-A is highly expressed by placental trophoblasts and represents a key regulator of fetal growth. The impact of IGF signaling in EWS is illustrated by the modest activity of IGF-1R inhibition as a single agent treatment in clinical trials, an approach that is limited by the rapid development of resistance. Methods: In an attempt to investigate the role of PAPP-A in Ewing sarcoma, we used CRISPR/Cas9 technology to target the PAPP-A locus in EWS cell lines and generated PAPP-A knockout clones (EW8, TC32) for further investigation in vitro and in vivo. Additionally, we tested inhibition of PAPP-A proteolytic function in a xenograft NSG mouse model, as a single agent treatment as well as in conjunction with IGF1R inhibition. Results: Knockout of PAPP-A in EWS cell lines utilizing CRISPR/Cas9 technology completely abrogated PAPP-A secretion and metalloproteinase activity in single cell clones, resulting in significantly increased complexed IGFBP-4 and diminished bioactive IGF-1, together with decreased cell growth in vitro. This phenotype could be rescued by recombinant, soluble PAPP-A. In vivo experiments showed that treatment with a monoclonal PAPP-A neutralizing antibody that inhibits the IGFBP-4 proteolytic activity of PAPP-A delays tumor growth of xenografted EWS tumor cells (EW8) in NSG mice. Furthermore, in conjunction with IGF1R inhibition (mAB h7C10), PAPP-A neutralization significantly decreased tumor growth and resulted in prolonged survival of mice (p=0.021).Conclusions: PAPP-A is a novel biologically relevant and highly tumor specific cell surface target in Ewing sarcoma. Our data suggest that neutralization of PAPP-A is a new therapeutic option that merits further evaluation for the treatment of Ewing9s Sarcoma. Citation Format: Sabine Heitzeneder, John F. Shern, Lee J. Helman, Javed Khan, Crystal L. Mackall. Inhibition of the novel therapeutic target pregnancy associated plasma protein A (PAPP-A) in Ewing sarcoma enhances efficacy of IGF1R targeting in vivo [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 1597. doi:10.1158/1538-7445.AM2017-1597
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