Abstract 1817: EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity

Abstract

Abstract Background. Novel therapeutic targets are needed to improve the outcome of individuals with cancer. EG-011 is a small molecule with in vitro and in vivo anti-tumor activity in lymphoma and acute leukemias, but, at least so far, no activity in solid tumor models (Gaudio et al AACR 2019). Since no information was available on its target, we have now performed experiments showing that EG-011 targets WASp. Methods. Target identification: kinase screens with DiscoverX KINOMEscan, ProQinase Wildtype-Profiler; thermal proteomic profiling. Target validation: pyrene actin polymerization assay. In vitro drug treatment of cell lines followed for changes in actin filaments (F-actin) by confocal imaging with Alexa Fluor 488 Phalloidin. Results. Extensive kinome screens excluded kinases as targets of EG-011. We then applied thermal proteomic profiling to identify new protein targets interacting with EG-011. Over 3,300 proteins from the soluble proteome from the EG-011 sensitive mantle cell lymphoma cell line REC1 were analyzed and 48 possible protein targets were initially identified. Among the proteins undergoing a thermal shift, WASp was among the most highly destabilized by EG-011. Due to the pattern of expression of WASp, compatible with the anti-tumor activity observed only in hematological cancers (Gaudio et al AACR 2019), we performed further experiments to confirm the possibility that EG-011 targets WASp. One of the main functions of WASp is the regulation of actin filaments formation. Pyrene actin polymerization assays demonstrated that EG-011 activated the auto-inhibited form of WASP with strong actin polymerization. Further confirmation was obtained using confocal imaging of cell lines exposed to DMSO or EG-011 (500 nM, 5 μM) and stained for F-actin. An increase in actine polymerization was seen in EG-011 sensitive (VL51) and not in resistant (Z138) cell lines at 4, 8 and 24h with both concentrations. Conclusions. These data demonstrate that EG-011 is the “first-in-class” activator of the auto-inhibited form of WASp with selective anti-tumor activity in lymphomas. Citation Format: Filippo Spriano, Laura Barnabei, Ana Maria Carrasco Del Amor, Meagan R. Tomasso, Chiara Tarantelli, Eugenia Riveiro, Natalina Pazzi, Shae B. Padrick, Susana Cristobal, Franco Cavalli, Eugenio Gaudio, Francesco Bertoni. EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1817.