Abstract LB234: Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800

Abstract

Abstract HSF1 is a stress-inducible transcription factor that regulates the eukaryotic heat shock response (HSR). HSF1 activation induces the expression of multiple proteins needed for cellular recovery from stress. HSF1 also plays a key role in tumorigenesis and regulates the expression of a cancer-specific gene signature which is unique to malignant cells and distinct from that activated during the HSR. We discovered NXP800, the first-in-class orally active HSF1 pathway inhibitor which is now undergoing Phase 1 clinical trial, based on a phenotypic pathway screen. Here we employed various techniques to investigate the mechanism of action of NXP800. Using RNAseq in a panel of human carcinoma cell lines, we identified overlapping gene expression changes in response to NXP800. These included genes regulated by HSF1 and interestingly also genes associated with activation of the integrated stress response (ISR). In contrast, we found no evidence for activation of the unfolded protein response. Consistent with the observed NXP800-induced phosphorylation of eIF2α which is a critical regulator of the ISR, NXP800 increased the protein expression of downstream ISR markers ATF4, CHOP and CHAC1, both in human tumor cells in vitro and in human tumor xenograft models in vivo. Induction of the ISR is controlled by four stress-activated protein kinases (PKs) that phosphorylate eIF2α. To further explore these kinases in the mechanism of action of NXP800 we used genetic knockdown by siRNA and inhibition by small-molecule tool compounds. Silencing each of the ISR-regulatory PKs revealed that GCN2 was required for ISR activation by NXP800. This was confirmed using two GCN2 inhibitors from different chemical series. Global phospho-proteome analysis showed that altered protein phosphorylation following NXP800 exposure was reversed upon co-treatment with a GCN2 inhibitor. We also demonstrated that activation of the ISR caused inhibition of HSF1 activation when stimulated with an HSF1 activator, thus confirming the link between ISR induction and inhibition of HSF1 activation. Activation of GCN2 and the ISR can occur in response to a variety of stimuli including amino acid deprivation. However, we did not detect a difference in the uptake of amino acids following exposure to NXP800, indicating that NXP800 does not directly impair amino acid uptake. Using an siRNA approach to determine if activation of the ISR components was contributing to growth inhibition following NXP800 exposure, we found that blocking the induction of ATF4 reduced the response of NXP800-sensitive SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. In summary, NXP800 acts on cancer cells to induce activation of the ISR pathway via GCN2, which then leads to inhibition of HSF1 activation. Further studies are underway to determine the precise molecular target of NXP800 and the mechanism of HSF1 pathway inhibition. Citation Format: Marissa V. Powers, Swee Y. Sharp, Eirini-Maria Lampraki, Toby Roe, Loredana Pellegrino, Maria Taskinen, Robert te Poele, Florence Raynaud, Suzanne Eccles, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB234.