Abstract B162: NXP900, a novel YES1/SRC kinase inhibitor demonstrates inhibition of YAP1 nuclear localization and potent single agent anti-tumor activity in esophageal squamous cancer models
Authors: Carragher Neil O , Dash Sweta , Nyswaner Katherine , Harvey Mungo J B , King Ben , Woods Allison , Unciti-Broceta Asier , Poradosu Enrique , Brognard John 
Published: December 2023

Abstract
Background: NXP900 (eCF506) is a novel potent and selective SRC family kinase (SFK) inhibitor, (IC50 of 0.47 nM against YES1). NXP900 locks its target into its native “closed” conformation (type 1.5 inhibitor), thereby inhibiting both kinase activity and complex formation with protein partners (Temps et al. Cancer Res. 2021, 81, 5438-5450). In contrast, multi-kinase inhibitors, including dasatinib and bosutinib, block SRC in the active “open” conformation (type 1 inhibitors) promoting the association of SFK and signaling partners via allosteric facilitation (Higuchi et al. Cell Rep 2021, 34, 108876). Crosstalk between YES1 and the Hippo pathway suggests that NXP900 may have therapeutic potential in cancers with Hippo pathway alterations. YES1 and Hippo pathway alterations are prevalent in several squamous cancers including esophageal (ESCC), lung, head and neck, and cervical.
Methods: Animal strain: CD1 Nude mice (Charles River). Human ESCC cancer cell lines: KYSE70, OE21, KYSE410, KYSE30 and OE19 cells (Sigma), KYSE140 (DSMZ), TE5, and TE14 (Riken). Xenograft tumors were generated by subcutaneous implantation on the right lower flank of the thigh at a cell density of 2x106 to 1x107 cells/mouse. Experiment groups: Vehicle, NXP900 (40 mg/kg); treatment: QD for 28 days, oral gavage. Colony formation assay: 500 (KYSE70, OE21, KYSE410, KYSE30) or 1500 cells (OE19, TE5, TE14) were seeded in 24 well plates in triplicates and treated with NXP900 for 14 days followed by staining with crystal violet. KYSE70 and KYSE140 cells were treated with NXP900 for 24 hours before fixation and labelling with anti-YAP, phalloidin and hoechst. Images were analyzed using IN Carta® software. Expression and mutation status of hippo pathway modulators was determined by analysis of Dependency Map (DepMap) data.
Results: NXP900 strongly inhibited cell proliferation of KYSE70, KYSE410, KYSE30, OE21, OE19, TE5 and TE14 in a colony formation assay. NXP900 significantly inhibited the activating phosphorylation of SRC in KYSE70 cells at 10 nM. Potent dose-dependent reduction in YAP1 nuclear localization was observed in ESCC lines. Oral administration of NXP900 in a KYSE70 xenograft mouse model with YES1 gene amplification, resulted in an average decrease of tumor volume of 71% while in the vehicle control group tumor volume increased by 472% (p ≤ 0.001), demonstrating significant tumor regression. Body weight measurements indicate good tolerability. Bioinformatics analysis demonstrate elevated expression and mutation of several hippo pathway modulators in ESCC cells responding to NXP900 in this study.
Conclusions: NXP900 is a novel potent and selective YES1/SRC kinase inhibitor with subnanomolar IC50 against YES1. NXP900 potently inhibits YAP1 nuclear localization and cell proliferation in a panel of ESCC cell lines and induces tumor regressions in a KYSE70 xenograft model providing substantial proof of concept for targeting solid tumors with YES1 and Hippo pathway alterations. An IND for NXP900 has been cleared by the FDA and the start of the phase 1 study is pending.
Citation Format: Neil O Carragher, Sweta Dash, Katherine Nyswaner, Mungo J B Harvey, Ben King, Allison Woods, Asier Unciti-Broceta, Enrique Poradosu, John Brognard. NXP900, a novel YES1/SRC kinase inhibitor demonstrates inhibition of YAP1 nuclear localization and potent single agent anti-tumor activity in esophageal squamous cancer models [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 B162.