Abstract 5393: Association of heat shock proteins as chaperone for STING: A potential link in a key immune activation mechanism revealed by the novel anti-cancer agent PV-10

Abstract

Abstract Introduction: The activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor system located in the endoplasmic reticulum, has been shown to augment antitumor immunity through the induction of pro-inflammatory cytokines. Currently, a number of STING agonists have been developed to treat refractory malignancies. Our previous studies have identified PV-10 (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) as a novel therapeutic agent with potent activity following intra-tumoral injection. Here we describe a previously unidentified mechanism by which PV-10 may facilitate sustained immune activation and therapeutic antitumor activity. Methods: The well-established acute monocytic leukemia (AML) cell line THP-1 was used as a model to study STING activation in vitro. Cells were treated with PV-10 and the induction of STING was evaluated by Western blot analysis using cGAMP as a positive control. Proteins that associate with STING in the presence of PV-10 were purified by immunoprecipitation and analyzed by mass spectrometry (LC-MS/MS, Mascot database). The culture supernatants from PV-10 treated cells were probed for a panel of 42 immune cytokines using the Bio-Plex multiplex bead-based assay system. Results: We show that the exposure of THP-1 cells to PV-10 leads to the appearance of a new 70-KD STING dimer band detected by specific antibodies. Compared to cGAMP controls no induction of PDL-1 was noted. Mass spectrometric analysis of immuno-precipitates of STING in these cells showed the presence of Heat Shock Proteins (HSP) 60, 70 and 90 as well as Polyadenylate Binding Protein 1 (PABP1) to the dimerized STING complex. The chemokine assays showed specific upregulation of a distinct set of pro-inflammatory and cytotoxic T-cell recruitment cytokines. A peak in the induction of MCP-3 and IFN gamma was seen at 24 hours (2 fold) and an approximately 10-fold increase in IL-6, IL-8 and IP-10 was seen in the 24 hours following exposure to PV-10. A significant increase in MCP-1 levels was also noted. Discussion: The compound PV-10 has been shown to induce tumor necrosis following intra-tumoral injection. Our present data show a possible mechanism of this agent through STING dimerization and HSP association leading to a sustained pro-inflammatory and immune response. We provide experimental data, for the first time, for a role of HSPs in STING-mediated immune activation pathways and the description of an agent that may play a role in effective single-agent immunotherapy or drug combination therapy approaches in future clinical studies. Citation Format: Satbir Thakur, Chunfen Zhang, Laurent Brechenmacher, Luis Murgia-Favela, Aru Narendran. Association of heat shock proteins as chaperone for STING: A potential link in a key immune activation mechanism revealed by the novel anti-cancer agent PV-10 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5393.