Abstract P5-16-17: Targeting homologous recombination-proficient triple negative cancer cells with a novel RAD51 inhibitor
Authors: Neumann Carola Anke , Skoko John J , Freeman Bruce A , Schopfer Francisco , Woodcock Steven Robert , Chang Fei , Hong Lisa , Braden Dennis Carl , Uvalle Crystal 
Published: March 2022

Out of all breast cancers (BC), ~20% are triple negative breast cancers (TNBC) devoid of the three receptors that define breast cancer treatment strategies such as estrogen receptor (ER), progesterone receptor (PR) and ERBB2 (also known as HER2). Unlike other BC, TNBC disproportionally affects younger women and those of African origins and recurs early within 5 years after diagnosis in ~ 40% of the patients. Standard care for TNBC patients includes surgery, ionizing radiation (IR) treatment and chemotherapy. Successful targeting of the poly (ADP-ribose) polymerase (PARP) has revolutionized therapy for cancer patients carrying gmBRCA1/2. However, in TNBC patients optimal clinical usage of PARP inhibitors (PARPi) is stifled by a) only 15-20% of TNBC patients being positive for gmBRCA1/2 and currently receiving PARPi therapy, b) most PARPi-treated gmBRCA1/2 patients developing resistance due to restoration of HR-mediated DNA DSB repair and, c) PARPi treatment-associated toxicities that require dose adaptations or drug discontinuation. Our novel therapeutic approach addresses this clinical unmet need by further advancing a small molecule inhibitor of HR that includes the 80% TNBC patients wild type for BRCA1/2 genes, re-sensitizes patients resistant to PARPi due to restoration of HR-mediated DNA DSB repair and lowers PARPi doses by conferring greater sensitivity to PARPi actions through synthetic lethality approaches.We first defined OA-NO2 ((E)-9/10-nitro-octadec-9-enoic acid), an endogenous electrophilic fatty acid nitroalkene as an RAD51 inhibitor in TNBC. RAD51 is essential in HR-mediated DNA DSB repair, is a highly coveted drug target in cancer and is a revealing functional biomarker for PARPi sensitivity. OA-NO2 reacts with protein thiols via reverse Michael addition reaction and has broad spectrum anticancer activities. OA-NO2 inhibits RAD51 function by selectively alkylating RAD51 on Cys319, a residue essential in RAD51- guided HR. In combination with the PARPi talazoparib, OA-NO2 synergizes the killing of TNBC cells in vitro and in vivo. Two reasons motivated the further development of OA-NO2 as RAD51 inhibitor: 1) X-ray crystal structure modeling predicted superior fitting with the RAD51 C-terminus if the nitroalkene substituent is closer to the carboxylate terminus of the nitro-fatty acid, and 2) OA-NO2 exhibits off target effects by inhibiting c-GAS-STING signaling and activating Nrf2 activity, thus, possibly offsetting anti-cancer activities. To optimize nitroalkene-mediated RAD51 inhibition, a library of &gt;50 compounds was built that expands upon 5 chemical classes. The library was evaluated for structure activity relationships (SAR) and a new lead nitroalkene emerged, CP-23. This electrophilic small molecule kills as a monotherapy TNBC and not benign breast epithelial cells and unlike OA-NO2, has no impact on STING and Nrf2 signaling, or the cell cycle. CP-23 inhibits RAD51 DNA binding, shows potent inhibition of RAD51 nuclear foci formation after ionizing radiation (IR) treatment and HR in a GFP-reporter cell line, and demonstrated drug synergism with PARP inhibition and IR. At present, in vivo tumor growth inhibition, expansion of PARPi-CP-23 synthetic lethality studies and pilot PK/toxicology evaluation are underway.

Citation Format: Carola Anke Neumann, John J Skoko, Bruce A Freeman, Francisco Schopfer, Steven Robert Woodcock, Fei Chang, Lisa Hong, Dennis Carl Braden, Crystal Uvalle. Targeting homologous recombination-proficient triple negative cancer cells with a novel RAD51 inhibitor [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-17.