Abstract 1387: Novel thienopyrimidine-hydrazinyl analog, TPH104m inhibits mitochondrial respiration and induces caspase-independent cell death in triple-negative breast cancer cells
Authors: Malla Saloni , Terrero David , Karki Sushma , Abou-Dahech Mariam Sami , Kumari Shikha , Tiwari Amit K. 
Published: April 2023

Abstract
A triple negative breast cancer (TNBC) is an aggressive and difficult-to-treat breast cancer subtype. Neoadjuvant chemotherapy using apoptosis-inducing taxanes and/or anthracyclines has been shown to produce early therapeutic responses. Unfortunately, drug resistance often results from mutations in apoptotic pathways, which can lead to metastasis and poor prognosis. There are emerging studies suggesting that TNBC tumors display altered mitochondrial dynamics and have overexpressed dynamin related protein 1 (Drp1), which drives mitochondrial fission. Drp1 deregulation also influences metabolic changes and mitochondrial energetics, which support TNBC tumor growth. Therefore, bypassing apoptosis signaling cascade, suppressing Drp1 and reversing aberrant mitochondrial dynamics may be an effective therapy for TNBC. We report here for the first time the identification of novel thienopyrimidine-hydrazinyl analog of parent TPH104, namely, TPH104m, which inhibits Drp1 phosphorylation and induces a non-apoptotic TNBC cell death. TPH104m significantly inhibited TNBC proliferation at nanomolar concentrations, and was more selective for TNBC than normal human mammary epithelial cells. TPH104m induced a unique type of non-apoptotic cell death, where cells swelled without shrinkage, nuclear fragmentation or apoptotic bleb formation typical of apoptosis. Moreover, TPH104m did not induce the cleavage of initator (caspase-8, -9) and executioner caspases (caspase-3, -7), and zVAD.fmk, a pan-caspase inhibitor, did not rescue TNBC cell death when incubated with TPH104m. Furthermore, TPH104m did not significantly increase reactive oxygen species (ROS) production in BT-20 cells compared to Paclitaxel treated cells. Although mitochondrial membrane potential (MMP) was significantly reduced, cytochrome C release was not increased, but instead decreased, explaining why MMP loss did not lead to apoptosis. TPH104m remarkably downregulated the phosphorylated and total forms of Drp1 protein in TNBC cells. A docking-based reverse screening approach and Surface Plasmon Resonance (SPR) data indicated that TPH104m binds to Drp1 more strongly than Mdivi-1, a weak non-specific Drp1 inhibitor. A complete and partial Drp1 KO cell line was created in SUM-159 TNBC cells to investigate the role of Drp1 in TPH104m-induced non-apoptotic cell death. Genetically deleting Drp1 from TNBC cells reduced TPH104m's cytotoxic potential suggesting Drp1's role in TPH104m's anticancer mechanism. Moreover, mito-stress assay showed that TPH104m severely impaired oxygen consumption rate, resulting in decreased ATP production and inhibition of mitochondrial respiration. Development of alternative treatments for TNBC will be facilitated by a deeper understanding of mitochondrial role in non-apoptotic cell death.
Citation Format: Saloni Malla, David Terrero, Sushma Karki, Mariam Sami Abou-Dahech, Shikha Kumari, Amit K. Tiwari. Novel thienopyrimidine-hydrazinyl analog, TPH104m inhibits mitochondrial respiration and induces caspase-independent cell death in triple-negative breast cancer cells [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 1387.