Triple-negative breast cancer (TNBC), defined by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), is the most aggressive and lethal form of breast cancer. The treatment of chemo-resistant TNBC tumors is particularly challenging due to the overexpression of efflux transporters, increased DNA repair, and the occurrence of genetic mutations that decrease the likelihood of apoptosis. Lack of clear targets and development of drug resistance allows TNBC to advance to metastatic stage thus resulting in poor prognosis and survival outcome. Recently, we discovered thieno-pyrimidin-hydrazinyl (TPH) class of small molecules that causes necroptotic (i.e. programmed necrosis) cell death in TNBC cells. We found that the lead compound TPH104 has an IC50 value of 160-400 nm and has ~400-fold selectivity towards different TNBC cell lines compared to normal breast cells. TPH104 selectively induced receptor interacting protein kinase 1 (RIPK1) - mediated necroptosis, while promoting autophagy thus resulting in significant inhibition of TNBC cell proliferation. We characterized eighteen novel TPH analogs for their cytotoxic potential and structure activity relation (SAR) and learned that the subtle changes in TPH pharmacophore could enhance or decrease efficacy against TNBC cells. We observed that 2-OH group is required to exert cytotoxic potency. Two new hit analogs of TPH104, TPH104c and TPH104m were found to activate necroptotic cell death markers (RIP, MLKL) with IC50 values similar to TPH104 in TNBC cells. Both analogs decreased the TNBC cell proliferation, and rate of colony formation compared to controls. Mechanistically, similar to TPH104, these analogs did not produce significant apoptosis induction i.e. no loss of mitochondrial membrane potential and arrested the TNBC cells in S-phase of cell cycle. Interestingly, unlike TPH104, TPH104c and TPH104m activate executioner caspases 3 and 7 only at extremely high concentration. TPH104 and its analogs reversed resistance mediated by ABCB1 and ABCG2 transporters through collateral sensitivity. TPH analogs in combination with doxorubicin and paclitaxel synergistically inhibited TNBC and TNBC/resistant (R) cells and reduced the dose-reduction index. Further understanding SAR of TPH analogs and the biology of non-apoptotic, necroptosis cell death by this new class of small molecules will help define a multimodal, non-apoptotic approach to overcome chemoresistance in TNBC patients. This will lead to the development of more selective and efficacious compounds that kills TNBC cells through unique cell death mechanism.
Citation Format: Saloni Malla, Diwakar Bastihalli Tukaramrao, Divya L. Dayanidhi, Pia Vogel, Shikha Kumari, Amit K. Tiwari. Necroptosis inducing thienopyridine analogs overcomes chemoresistance in TNBC [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 4119.