Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high propensity for metastasis and recurrence, accounting for one-fourth of all breast cancer deaths. Despite being chemo-sensitive to anthracyclines and taxanes, TNBCs have poor prognosis due to development of resistance often due to the overexpression of efflux transporters, increased DNA repair, and occurrence of genetic mutations that decrease the likelihood of apoptosis. Recently, we discovered a novel class of thieno-pyrimidin-hydrazinyl (TPH) compounds that in the nanomolar range, selectively elicited a unique, caspase-independent cell death in TNBC cells. The lead compound, TPH104, selectively activates receptor interacting protein kinase 1 (RIPK1), thus inducing a necroptosis like cell death. Since necroptosis is known to release endogenous immunogenic markers, we wanted to study whether TPH104 can promote immunogenic cell death (ICD) in TNBC cells and activate cells in the tumor immune milieu. ICD is characterized by presence of damage-associated molecular patterns (DAMPs) such as cell surface exposure of calreticulin (CRT), secretion of ATP, and release of high-mobility group box protein B1 (HMGB1) from dying tumor cells.
Methods: Human TNBC cell line MDA-MB-231 cells were treated with TPH104 and cell survival was examined by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), clonogenic and live-cell imaging assays. Western blot analysis was used to assess death signal transducers. DAMPs were examined by ELISA, luminescence-based assay systems and immunofluorescence. Activation of mouse bone marrow derived dendritic cells (BMDCs) in response to conditioned media (CM) harvested after treatment of MDA-MB-231 cells with TPH104 (TPH104-CM) and/or mitoxantrone (MX, positive control) for 48h was evaluated using flow cytometry analysis of maturation markers (CD86, MHCII) expressed on CD11C+ BMDCs. Inflammatory gene expression was evaluated by q-PCR.
Results: The results demonstrated that TPH104 reduced cell survival and induced death of MDA-MB-231 cells in a dose-dependent pattern. TPH104 induced ICD, as evidenced by increased release of ATP, as well as surface exposure of CRT. Flow cytometric analysis revealed TPH104-CM treatment increased CD11C+CD86+MHC+ BMDCs as robustly as MX-CM. Further, TPH104 posed no toxicity to mice in a single dose toxicity study.
Conclusion: TPH104 induced selective ICD in MDA-MB-231 cells. TPH104 similar to MX primed cells to ICD and increased extracellular ATP levels and surface exposure of CRT. TPH104 promoted maturation of CD11C+ BMDCs and increased inflammatory gene expression. Our study demonstrated TPH104 as an ICD-inducer and immunomodulator in TNBC.
Citation Format: Diwakar Bastihalli Tukaramrao, Siddharth Saraiya, Ross A. Hanely, Saloni Malla, Amit K. Tiwari. Novel thienopyrimidine analog TPH104 induces immunogenic cell death in TNBC cells [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 2406.