Abstract 2994: Inhibition of the NRP2b:GSK3β binding interaction with peptides and macrocycles exerts anticancer effects in lung cancer

Introduction: Neuropilin-2b (NRP2b) expression is associated with pro-tumorigenic properties of NSCLCs. We previously reported that NRP2b promotes metastasis and drug resistance, while the canonical NRP2a isoform is inhibitory. Our recent work indicates that the NRP2b cytoplasmic domain recruits GSK3β to phosphorylate and promote degradation of co-recruited PTEN, thereby enhancing AKT activity leading to enhanced survival, migration and drug resistance. We identified a 15-amino acid motif near the NRP2b C-terminus required for interaction with GSK3β. Herein, we describe the anticancer effect of peptides and drug-like macrocycles designed to disrupt this interaction. Methods: We used the cytoplasmic sequence of NRP2b for decoy peptides designed to disrupt the interaction with GSK3β. Control peptides were synthesized with alanine replacements for three amino acids suggested as crucial for GSK3β recruitment. All peptides were N-terminally myristoylated to promote association with and flipping to the inner leaflet of the plasma membrane. A library of over 42,000 drug-like macrocycles was screened in silico for compounds predicted to fit and compete with the NRP2b:GSK3β binding interface. Decoy peptides and macrocycles were assessed for anti-NRP2b activity using assays for migration and drug-tolerant persister cells. Transwell migration assays were performed using a Neuro Probe Reusable Multiwell Chemotaxis Chamber. Persister cell assays were performed with EGFR mutant PC9 and HCC827 cells in the presence of EGFR inhibitors (gefitinib or osimertinib), followed by drug withdrawal and colony formation assays. Results: As previously reported, knockdown of NRP2b inhibited, while knockdown of NRP2a enhanced, migration of lung cancer cell lines. Importantly, emergence of drug-tolerant persister cells was similarly inhibited by knockdown of NRP2b and enhanced by knockdown of NRP2a. Wild type, but not control, peptides abrogated the pro-tumorigenic effects of NRP2a knockdown on both migration and persister cell formation with IC50s of ~250 nM. The most effective macrocycles, M3 and M7, inhibited NRP2b-dependent migration with IC50s of 1-3 µM. Both macrocycles also inhibited generation of persister cell colonies from shNRP2a-PC-9 cells. However, only M3 was effective at blocking persisters from shNRP2a-HCC827 cells. Conclusions: NRP2b:GSK3β interaction is a therapeutic target in NSCLC affecting migration/invasion and persister colony formation associated with the emergence of resistance to EGFR inhibitors. This work justifies further development of these peptides and drug-like macrocycles as therapeutic modalities in lung cancer. Citation Format: Cecile Nasarre, Yuri K. Peterson, Patrick Nasarre, Anastasios Dimou, Kent E. Armeson, Harry A. Drabkin, Nancy Demore, Chadrick E. Denlinger, Robert M. Gemmill. Inhibition of the NRP2b:GSK3β binding interaction with peptides and macrocycles exerts anticancer effects in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2994.