Abstract 3998: Discovery of a small molecule inhibitor for triple-negative breast cancer (TNBC)
Authors: Saran Uttara , Chandrasekaran Balaji , Tyagi Ashish , Singh Amandeep , Ankem Murali K. , Sharma Arun K. , Damodaran Chendil 
Published: June 2022

Introduction: Little progress has been made towards developing alternative therapeutic strategies for triple-negative breast cancer (TNBC). Aberrant Notch1 activation, particularly in breast cancer stem cells (BCSCs) in TNBC tumors, enhances this cancer's aggressivity and resistance and consequently contributes to the poor overall survival of patients with TNBC. In contrast, inhibition of Notch1 has been shown to suppress TNBC growth. Thus, Notch1 presents as an attractive target for inhibiting TNBC growth. This study aims to overcome the limitations of current Notch1 inhibitors and identify a more specific inhibitor.

Methods: Small molecule screening was performed using structure-activity relationship (SAR) studies, and molecular docking and isothermal calorimetric (ITC) assays were used to determine the target sites of our rationally designed Notch1 inhibitor on the Notch1 receptor. The therapeutic potential of the identified small molecule Notch1 inhibitor was determined on BCSC and TNBC (ALDH+, BCSC, ALDH-, MDA-MB-231) cell lines using molecular studies and in vivo assays.

Results: We identified ASR490, a small molecule that binds to the NRR (the activation switch of the Notch receptor) of Notch1. Molecular docking studies indicated a strong interaction between ASR490 and the NRR domain with binding energy of -52.55 kcal/mol. Residue-wise interaction analysis estimated three hydrogen-bond interactions between ASR490 and NRR residues Asn-1483, Glu-1673, and Gly-1664 mediated by water molecules. The finding was further confirmed using biophysical assays (thermal shift assay and isothermal calorimetric analysis) using recombinant Notch1 protein. In vitro results demonstrated that ASR490 significantly inhibited TNBC growth (p &lt;0.001) at nM concentrations, with an IC50 of 760 nM at 24h. Further, the inhibitory effect was confirmed by TNBC’s colony-forming abilities. Western blot analysis confirmed that ASR490 downregulated the expressions of NICD (the active form of Notch1) and its downstream effectors Hey-1 and HES1 by ubiquitinating NRR of Notch1. ASR490 was found to be non-toxic to healthy cells. Moreover, ASR490 exhibited a maximum tolerated dose of more than 100 mg/kg body weight in mice and abrogated TNBC tumor growth in vivo xenograft models.

Conclusion: Our results demonstrate ASR490 as a potential therapeutic agent that abolishes Notch1 signaling in TNBC and BCSCs by specifically targeting Notch1 NRR. Further investigation is required for the clinical translational potential of this agent.

Citation Format: Uttara Saran, Balaji Chandrasekaran, Ashish Tyagi, Amandeep Singh, Murali K. Ankem, Arun K. Sharma, Chendil Damodaran. Discovery of a small molecule inhibitor for triple-negative breast cancer (TNBC) [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 3998.