Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma
Authors: Workman Paul 
Published: December 2023

Abstract
My presentation will describe our work on the discovery, nonclinical antitumor properties, mechanism of action (MOA), identification of initial target patient population, and early clinical development of NXP-800. We discovered this agent in our academic drug discovery centre at ICR following our initial identification of the ‘bisamide’ hit CCT245232 from a diverse ~200,000-compound library using an innovative, imaging-based (ArrayScan™) phenotypic screen for inhibitors of the HSF1-mediated activation of the heat shock response in U2OS human osteosarcoma cells. Subsequent multiparameter medicinal chemistry optimisation of the bisamide series, containing the N,N′-4-methyl-1,3-phenylenediamide core, focused on establishing the cell-based SAR for both inhibition of cell proliferation and linked HSF-1 mediated HSP72 induction in the sensitive ARID1A-mutant, platinum-resistant SK-OV-3 human ovarian carcinoma cell line. This generated our orally bioavailable, in vivo-active lead compound and chemical tool CCT251236 – exhibiting oral antitumor efficacy in the SK-OV-3 human ovarian cancer xenograft model in nude mice (Cheeseman et al J Med Chem 60 180-201 2017). Final multiparameter optimization yielded the clinical development candidate CCT361814/NXP800 (Pasqua et al J Med Chem 66 5907-36 2023). With the screening hit bisamide compound already active in the single to low double digit nanomolar range, the early focus was on improving kinetic solubility while retaining the desired antiproliferative and linked HSF1 pathway inhibitory activity together with simultaneous optimisation of PK/PD properties to maximize oral antitumour efficacy; finally requiring modification to the fluorobisamide NXP800 to eliminate PGP-mediated efflux and achieve optimal PK/PD, Pharmacologic Audit Trail, antitumor efficacy and tolerability. In view of these properties, its clean safety panel profile, clear therapeutic index and acceptable dose-to-human prediction, NXP800 was progressed into clinical development.
Evaluation in a mini-panel of human cancer cell lines and tumor xenografts revealed high sensitivity to NXP800 in ARIDIA-deficient human ovarian cancer models, confirmed in the large Sanger cancer cell line panel and in isogenic pair systems. Activity exceeded that of cisplatin in both platinum-sensitive and platinum-resistant ARID1A-mutant ovarian cancer xenograft models. By RNA-Seq we identified two sets of consistent, major gene expression profile changes in human cancer cell lines exposed to NXP800, namely: 1) the expected changes in HSF1-regulated genes, together with 2) activation of the integrated stress response (ISR), including ATF4-regulated gene expression. This latter did not indicate a global stress response to NXP800 as we saw no activation of the unfolded protein response (UPR). Physiologically, ISR activation is mediated by phosphorylation of EIF2α which is tightly regulated by four stress-controlled kinases, GCN2, HRI, PKR and PERK. Using each of systematic siRNA knockdown or pharmacologic inhibition by two small-molecule tool compounds from different chemotypes, we discovered that GCN2 alone was necessary and sufficient for ISR activation and cell growth inhibition by NXP800 in SK-OV-3 ovarian cancer cells. Also, orthogonal siRNA depletion or pharmacologic inhibition of GCN2 both markedly reduced the antiproliferative activity of NXP800 in these cells. Global phospho-proteome analysis demonstrated defined changes in response to NXP800 which were reversed by co-treatment with a GCN2 inhibitor. Other follow-on studies at the protein level demonstrated that pharmacological concentrations of NXP800 induced GCN2-dependent phosphorylation of EIF2α, inhibition of global cap-dependent protein translation, and selective translation of ATF4 – a transcription factor that activates downstream genes such as CHAC1 and CHOP in the ISR – both in ARID1A-mutant human ovarian cells in vitro and in corresponding tumor xenograft models in vivo. Using siRNA silencing, we showed that prevention of ATF4 induction substantially reduced the antiproliferative response of SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. Controls used in these experiments included a closely matched, inactive regioisomer.
Furthermore, we showed that ISR induction by NXP800 inhibited HSF1 activation, in SK-OV-3 cells, confirming the mechanistic link between ISR activation and inhibition of HSF1-mediated transcription. We further showed that GCN2 activation was not due to the canonical mechanism of amino acid starvation.
In summary, we used an HSF1 pathway-focused phenotypic screen to discover the mechanistically novel, first-in-class drug NXP800, which acts potently on ARID1A-mutant, human ovarian cancer cells to stimulate GCN2 and thereby activate the ISR pathway, leading to ATF4 induction and inhibition of HSF1-mediated gene transcription in ARID1A-mutant, platinum-resistant human ovarian cancer cells. Through this mechanism, NXP800 shows highly promising activity in models of this cancer type, including potent inhibition of proliferation in vitro and substantial regression of tumor xenografts in vivo. Studies are currently underway to determine precisely how NXP800 stimulates GCN2 activity and the role of ARID1A deficiency in its pharmacological effects. Recent ChIP-Seq studies are informing on the molecular interactions between ARID1A mutation and the expression of ISR/ATF4 and HSF1-regulated genes. With Nuvectis Pharma, the Phase 1a dose escalation study has been completed and a multicentre Phase 1b expansion cohort study in ARID1A-mutated, platinum-resistant ovarian cancer – a high unmet medical need – has been initiated (NCT05226507) in collaboration with the GOG Foundation and the European Network of Gynaecological Oncological Trial Group (ENGOT). FDA has issued a Fast Track designation to NXP800 in this setting. In addition, NXP800 shows therapeutic potential in non-clinical models of endometrial, gastro-intestinal and bile duct cancer, with FDA Orphan Drug status granted in the last of these indications.
Citation Format: Paul Workman. From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA014.