Abstract 1286: ONC201 inhibits RET and IGFBP2 signaling through ATF4 mediated- Integrated stress response in medullary thyroid cancer
Authors: Bagheri-Yarmand Rozita , Li Ling , Tarapore Rohinton , Allen Joshua E. , Sherman Steven I. , Gagel Robert F. 
Published: August 2019

Abstract
Medullary thyroid cancer (MTC) is an aggressive tumor with frequent lymph node and distant metastasis at diagnosis. Activating mutations of the RET receptor tyrosine kinase occur frequently and prevent apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum stress. Prior studies have demonstrated shRNA depletion of ATF4 increases RET protein levels and cause resistance to tyrosine kinase inhibitor-mediated cell death. Conversely, forced expression of ATF4 in MTC cells promotes RET ubiquitination and degradation. Here, we report the anti-cancer efficacy of ONC201 in MTC models. ONC201 is a selective DRD2 antagonist that is in clinical trials for a range of advanced cancers. Downstream signaling studies have found that ONC201 induces ATF expression and activates the integrated stress response (ISR) in a variety of cancers. ONC201 decreased cell survival of MTC cell lines (TT and MZCRC1) harboring activating RET mutations and increased the percentage of sub-G1 DNA content. Moreover, ONC201 decreased, in a dose-dependent manner, the abundance of RET protein and induced the mRNA and protein levels of ATF4 and its target genes. In our attempt to uncover RET targets, we performed reverse phase protein array analysis using TT- shRNA-RET knockdown cells. Among the top 5 proteins regulated by RET in this screen, we found insulin-like growth factor-binding protein IGFBP2, to be downregulated in shRNA-RET cells. ELISA based cytokine array of MTC-conditioned media showed high levels of secreted IGFBP2. IGFBP2 have been reported to be an oncogene in many human cancers and we show that elevated IGFBP2 protein levels are associated with poor prognosis of primary MTC. The treatment of MTC cells with ONC201 or overexpression of ATF4 inhibited the secretion of IGFBP2 into the media and decreased IGFBP2 protein levels, suggesting that ONC201 targets IGF signaling through activation of ISR. To investigate the efficacy of ONC201 in vivo, established MTC xenografts (TT or MZCRC1) were treated with a weekly dose of 120 mg/kg orally for 8 weeks and tumor burden was assessed every week. We observed that ONC201 decreased tumor growth of TT and MZCRC1 xenografts by 80-90%. There was no evidence of toxicity, as indicated by weight loss in mice. Immunohistochemical and western blot analysis of tumors at the termination of the study demonstrated decreased IGFBP2 protein levels in mice treated with ONC201. These studies identify IGFBP2 as a novel target of the RET proto-oncogene and demonstrate that ONC201, through its effect on ATF4, reverses RET-mediated signaling. These results support the further investigation of ONC201 as a therapeutic agent for the treatment of MTC alone or in combination with tyrosine kinase inhibitors.
Citation Format: Rozita Bagheri-Yarmand, Ling Li, Rohinton Tarapore, Joshua E. Allen, Steven I. Sherman, Robert F. Gagel. ONC201 inhibits RET and IGFBP2 signaling through ATF4 mediated- Integrated stress response in medullary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1286.