Abstract 5825: Mechanisms of intrinsic and acquired resistance to antibodies targeting IGF-1R in pediatric sarcoma cell lines
Authors: Shackleford Terry J. , Hariharan Seethalakshmi , Phelps Doris , Bid Hemant , Zhong Hiahong , Houghton Peter 
Published: July 2017

Abstract
Background: Pediatric bone and soft tissue sarcomas are heavily dependent on the Type 1 insulin-like growth factor receptor (IGF-1R) signaling axis, which promotes tumorigenic properties such as proliferation, survival, angiogenesis and metastasis. While a substantial proportion of patients demonstrate intrinsic resistance to monoclonal antibodies targeting IGF-1R, a strong and sustained response is seen in a limited number of patients, and disease stabilization in about 30% of patients. Understanding these resistance mechanisms may identify combinations of targeted therapies that optimize therapeutic efficiency of IGF-1R-targeted antibodies. Methods: For intrinsic resistance, we used a panel of Ewing sarcoma (ES; n=10) and rhabdomyosarcoma (RMS; n=10) cell lines that, with the exception of Rh41, are all intrinsically resistant to TZ-1, a monoclonal antibody that targets IGF-1R. For acquired resistance, we developed a TZ-1 resistant RMS cell line, Rh41/TZ-1. We utilized Receptor Tyrosine Kinase (RTK) Arrays to analyze the phosphorylation status of 49 RTKs. The cell lines were treated with TZ-1 and changes in RTK expression were assessed by quantitative PCR. Results: RMS cells intrinsically resistant to TZ-1 treatment expressed multiple activated RTKs including EGFR family of receptors, IGF-1R, IR, PDGFR, FGFR4, HGFR4, RYK, ALK, DDR1, AXL, DTK. Interestingly, ES cells (n=5) had either a complex pattern of activated RTK expression similar to RMS cells or a relatively less complex set (n=5) of activated RTKs (IGF-1R, IR, HGFR, ERBB4, AXL, DTK). TZ-1 treatment of these cell lines with intrinsic resistance demonstrated decreased phosphorylation of IGF-1R, but had little effect on other RTKs. In contrast TZ-1 treatment of Rh41 cells rapidly induced activation of several RTKs including: AXL, PDGFRB, FGFR4 and ALK. Increased expression of these receptors was confirmed by qPCR. In contrast, Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed activated RTKs (PDGFRB, FGFR4, AXL, DTK, VEGFR1). In Rh41 cells treated with TZ-1, the BRD4 inhibitor, JQ1 (500 nM, 24 Hr), blocked the induction of IGF-1R, IRB, AXL, PDGFR-A/-B, VEGFR1 and ALK. However, it had less effect on the induction of FGFR 2/3/4 receptors. Further, combined treatment of TZ-1 and JQ1 reduced cellular proliferation of Rh41 cells more than TZ-1 treatment alone. Conclusions: We demonstrate that intrinsic resistance to the IGF-1R targeted antibody TZ-1 is characterized by constitutive activation of multiple RTKs. In response to TZ-1 treatment in a sensitive cell line there is rapid induction of multiple RTKs indicating a dynamic response to inhibition of IGF-1R. Our data suggests that BRD4 is involved in induction of these signaling molecules and that combined inhibition of BRD4 along with the IGF-1R targeted therapy can overcome this resistance and warrants further investigation. Support: US Public Health Service Grant CA165995.
Citation Format: Terry J. Shackleford, Seethalakshmi Hariharan, Doris Phelps, Hemant Bid, Hiahong Zhong, Peter Houghton. Mechanisms of intrinsic and acquired resistance to antibodies targeting IGF-1R in pediatric sarcoma cell lines [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 5825. doi:10.1158/1538-7445.AM2017-5825