Abstract 1658: ADT-007 binds RAS and inhibits RAS signaling

Abstract

RAS is a critically important oncogenic protein that is mutated in approximately 1/3 of cancers resulting in aberrant activation of downstream signaling, which drives malignant transformation. Current molecular targeted therapeutics, and several in development, inhibit only specific mutant alleles (G12C, G12D). In addition, compounds which directly inhibit RAS via proteolytic degradation or inhibit RAS activation by SOS1 are in preclinical development and are referred to as Pan-KRAS inhibitors. Previously investigators have reported that the NSAID, sulindac, can selectively inhibit RAS mutant tumorigenesis by a cyclooxygenase (COX)-independent mechanism. Sulindac, and more potent analogs have also previously been reported to inhibit RAS-mediated transformation and directly bind RAS. Here we describe an ultra-potent non-COX inhibitory derivative of sulindac, ADT-007, which binds to and inhibits RAS nucleotide binding and RAS-effector association. ADT-007 binding to KRAS was evaluated by Micro-Tag cell target engagement and by Cellular Thermal Shift Assay (CETSA) which demonstrated a potency of target engagement (EC50) value in the subnanomolar range. Consistent with molecular docking studies, HSQC NMR spectroscopy using recombinant KRAS revealed that ADT-007 interacted with KRAS after Mg2+ chelation to obtain a nucleotide free (NF) state, resulting in chemical shift changes and signal attenuation of residues in the P-loop and nucleotide binding domain. Similarly, biochemical assays confirmed that ADT-007 prevented MANT-GTP binding to recombinant NF KRAS but did not compete with bound GTP. Functional assays also showed that KRAS binding to RAF-RBD(GST) was inhibited by ADT-007. The compound inhibited constitutive RAS activation (RAF-RBD pulldown) in serum starved MiaPaCa-2 pancreatic cancer cells harboring a KRAS-G12C mutation and demonstrated Pan-RAS inhibition in serum- or EGF-stimulated cells. Further, ADT-007 inhibited AKT phosphorylation and EGF-stimulated downstream ERK1/2 phosphorylation. Finally, ADT-007 demonstrated RAS-selective growth inhibition in isogenic pancreatic and colorectal cancer cell pairs (BxPC-3, HT29). Together, these experiments support further development of ADT-007 and related analogs for treatment of RAS-driven cancers. Citation Format: Adam B. Keeton, Xi Chen, Jacob Valiyaveettil, Chung-Hui Huang, Tyler E. Mattox, Khalda Fadlalla, Jeremy B. Foote, Donald J. Buchsbaum, Kristy L. Berry, Elmar Nurmemmedov, Ivan Babic, Vadim Gaponenko, Gregory Gorman, Lori Coward, Yulia Y. Maxuitenko, Forrest T. Smith, Gary A. Piazza. ADT-007 binds RAS and inhibits RAS signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1658.