Abstract A086: NST-628 is a novel molecular glue that inhibits signaling and pathway reactivation in oncogenic RAS-MAPK cancers
Authors: Quade Bradley , Ryan Meagan , Swalm Brooke , Cohen Steven , Fang Zhong , Ozen Aysegul , Huang Xin , Dar Arvin C , Han Yongxin , Hoeflich Klaus P , Hagel Margit , Hale Michael 
Published: December 2023

Abstract
Alterations in the RAS-MAPK pathway are identified in nearly 30% of cancers and are associated with poor patient prognosis. Although available therapies target various nodes of the RAS-MAPK pathway, clinical anti-tumor activity is limited by paradoxical bypass mechanisms, feedback signaling and pathway reactivation, and genetic resistance mechanisms. NST-628 aims to overcome the limitations of traditional RAS-MAPK pathway inhibitors by stabilizing all RAF-MEK complexes in a conformation that blocks MEK phosphorylation, to sequester these nodes from dynamic signaling processes that promote RAF-mediated bypass signaling. In comparison with clinically approved RAS-MAPK pathway inhibitors, only NST-628 robustly and durably decreases pathway reactivation, as measured by MEK phosphorylation, in KRAS-G13D tumor models in vitro and in vivo. Immunoprecipitation of endogenous ternary complexes demonstrates that NST-628 promotes potent stabilization of CRAF-MEK, BRAF-MEK, and ARAF-MEK complexes with complete shutdown of MEK, ERK, and RSK phosphorylation. Moreover, NST-628 treatment completely prevents RAF paralog heterodimerization in RAS-driven cells. Dose-dependent panRAF-MEK complex stabilization was confirmed using recombinant pre-formed CRAF-MEK, BRAF-MEK, and ARAF-MEK complexes, and MEK and RAF paralog affinity increases in the presence of NST-628 were quantified via surface plasmon resonance (SPR). In this assay, NST-628 significantly increased the affinity of MEK with both inactive monomeric and active dimeric RAF complexes. In parallel, we structurally characterized NST-628 bound to CRAF-MEK, BRAF-MEK, ARAF-MEK complexes using x-ray crystallography and cryogenic electron microscopy (cryo-EM). NST-628 binds the interfacial allosteric pocket along the MEK activation loop, thereby preventing RAF-mediated activation of MEK. We also demonstrate RAF paralog-specific pocket features that NST-628 can engage, helping us rationalize the large increase in affinity observed for MEK and CRAF in the presence of NST-628. The increased stabilization of CRAF-MEK complex by NST-628 may contribute to the decreased pathway reactivation in specific biomarker-driven tumors. Through our robust biophysical and cellular characterization coupled with novel structural insights, we have defined the unique mechanism of action for NST-628 and provide insight into its best-in-class potential in RAS-MAPK activated cancers.
Citation Format: Bradley Quade, Meagan Ryan, Brooke Swalm, Steven Cohen, Zhong Fang, Aysegul Ozen, Xin Huang, Arvin C Dar, Yongxin Han, Klaus P Hoeflich, Margit Hagel, Michael Hale. NST-628 is a novel molecular glue that inhibits signaling and pathway reactivation in oncogenic RAS-MAPK cancers [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 A086.