Abstract 2996: IT-139 targets GRP78 in stressed cancer cells
Authors: Sethuraman Jyothi , Costich Tara Lee , Vojkovsky Tomas , Crouse Rick , Cognet Valentin , Bakewell Suzanne 
Published: July 2016

IT-139, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is a first in class small molecule that has successfully completed a phase I clinical trial against solid refractory cancers. 11 of 41 patients achieved stable disease with manageable side effects and 8 patients showed tumor size reduction including a durable partial response. Preclinical studies suggest IT-139 down-regulates the chaperone GRP78, but the mechanism of action is not fully understood. GRP78 (also known as BiP and HSPA5) is the primary sensor and regulator of the endoplasmic reticulum (ER). In cancer cells it is preferably expressed on the cell surface, but also translocates to the mitochondria and cytosol where it regulates critical oncogenic signaling pathways, thus making GRP78 an excellent target for therapy. Here we show in vitro results of IT-139 treatment in a panel of cancer cell lines. IC50 levels measured at 72 hour varied ranging from 15 μM to 180 μM. Annexin V/PI staining by flow cytometry in HCT-116, HT-29, A375 and A549 cell lines at 24 hour show early and late apoptosis, which corresponds to their relative IC50s. UPR induction and overexpression of GRP78 has been reported to cause cell cycle arrest in G1. But in IT-139 treated cell lines HCT116, HT-29, A549 and LnCAP, cell cycle arrest occurred in G2, whereas in A375 cells IT-139 induced cell cycle arrest at G1. These data suggest an inhibition of GRP78 levels and that cell cycle arrest status is also dependent upon the cell line. Electron microscopic (EM) images of HCT-116 and HT-29 cells treated with IT-139 at 24 hours showed significant vacuolization, ER expansion and disorganization of intracellular organelles, strongly suggesting ER stress. However, there is an absence of double membrane vacuoles or autophagosome formation in response to the ER stress. Also, immunofluorescent analysis displayed no LC3-II punctate distribution, although protein immunoblots showed LC3 and p62 protein levels increasing in IT-139 treated cells. These data suggest an inhibition of autophagy. Additionally, EM images showed vacuolated mitochondria and distortion of cristae. In all cell lines treated with IT-139 even below their corresponding IC50 concentration, JC-1 staining showed an increased loss of mitochondrial membrane potential. These observations hypothetically indicate an inhibition of stress-induced GRP78. Quantitative real-time PCR showed IT-139 treatment in combination with thapsigargin exhibits a significant fold change decrease in the GRP78 mRNA expression. These results suggest that IT-139 downregulates GRP78 leading to an increase in ER stress, mitochondrial damage, decreased autophagy, increased apoptosis and cell death.

Citation Format: Jyothi Sethuraman, Tara Lee Costich, Tomas Vojkovsky, Rick Crouse, Valentin Cognet, Suzanne Bakewell. IT-139 targets GRP78 in stressed cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2996.