(W1130-03-20) Discovery of Mitochondria Targeted Non-apoptotic Cell Death Inducers against Resistant Triple-Negative Breast Cancer

Purpose: More than 90% of patients with advanced triple-negative breast cancer (TNBC) develop multi-drug resistance (MDR) to first-line therapies such as taxanes and anthracyclines typically due to overexpression of the ATP-binding efflux transporters, metastasis, and reduced sensitivity to apoptosis regulated cell death resulting in early relapse and shorter survival times. Almost all the therapies available currently kills TNBC cells through apoptosis. We hypothesize that new opportunities to treat chemoresistance in TNBC patients could result from the identification of compounds that does not depend upon classical apoptotic pathways. Mitochondria has been in center for regulating several non-apoptotic cell deaths (NACD). We discovered a novel class of structurally constrained thieno-pyrimidin-4-yl-hydrazinylidene (TPH) small molecules that selectively induces a unique caspase-independent NACD by targeting Drp1, a mitochondrial fission protein that have been correlated with increased migration, invasion, metastasis and resistance in TNBC patients. The purpose of this study was to define the structural requirement necessary to induce NACD, identify the most potent Drp1 inhibitor that is drug like, and define the mechanism of action as how targeting Drp1 can suppress aggressive phenotypes of MDR TNBC.

Methods: We designed, synthesized, characterized, and screened a panel of 34 TPH compounds against a panel of TNBC cells (BT-20, MDA-MB-231, MDA-MB-468) to determine their structure-activity relationships. Drug likeness study was performed using ACD Percepta suite analysis. A variety of cell cytotoxicity assays including MTT, SRB, live-cell and CellTiter-Blue were used to determine cytotoxicity and selectivity of the most optimal lead, TPH104m, against TNBC and normal mammary epithelial cells (MCF-10A, HMEC). By staining for Annexin V/PI, detecting nuclear staining with Hoechst 33342, measuring caspase-3/7 activity, and inhibiting pan-caspase activity with Z-VAD-FMK, we assessed the induction of cell death by TPH104m and paclitaxel. An analysis of TPH104m's binding to Drp1 was performed. In addition, we examined the mechanism by which TPH104m induced cell death in TNBC cells by evaluating its effect on mitochondria. The mitochondrial membrane potential (MMP) was disrupted using TMRE staining, and cytochrome c localization was confirmed by immunofluorescence. A Western blot was used to profile mitochondrial fission and fusion proteins (DRP1, p-DRP1, MFF, FIS1, MFN1/2, OPA1). By incubating TNBC cells with or without TPH104m, we performed genomics and proteomics analyses and validated our findings. CRISPR/Cas9-mediated Drp1 knockout cell models were used to validate the functional relevance of DRP1 targeting. To evaluate metabolic reprogramming, Seahorse Mito Stress Test and ATP Real-Time Rate Assay were used to assess cellular ATP levels, mitochondrial respiration (OCR), and glycolytic function (ECAR).

Results: TPH104m, a drug-like compound, was identified as the most optimal hit that induced caspase-independent NACD in TNBC cells. TPH104m demonstrated potent, selective cytotoxicity against TNBC cells (IC₅₀: 0.18–0.47 µM) with minimal effects on normal mammary epithelial cells. Additionally, we defined the SAR of TPH compounds against TNBC cells. The treated TNBC cells showed morphological and phenotypic features consistent with NACD, including swelling and rupture without nuclear fragmentation. Z-VAD-FMK pretreatment did not rescue cell death and lack of caspase-3/7 activity assays confirmed caspase-independent death. A significant depolarization of mitochondrial membranes was observed without competing cytochrome c release or reactive oxygen species (ROS) production. Post-treatment with TPH104m, Western blots and immunofluorescence analyses revealed substantial downregulation of Drp1 and p-Drp1. CRISPR/Cas9-mediated Drp1 knockout reduced cellular sensitivity to TPH104m, establishing mitochondrial fission dysregulation as a critical mediator of cytotoxicity. TPH104m inhibits mitochondrial fission in TNBC without causing mitochondrial dysfunction in normal cells. A significant decrease in mitochondrial OCR and ATP production was observed in TNBC cells incubated with TPH104m, as well as elevated mitochondrial ROS and DNA copy number depletion. Treated TNBC cells with TPH10exhibited a metabolic shift characterized by loss of oxidative phosphorylation capacity and reduced bioenergetic health index (BHI). This shift away from functional mitochondrial respiration resulted in an ATP crisis, impaired survival, and non-apoptotic cell death without activation of caspase pathways.

Conclusion: We have identified a lead Drp1 specific, mitochondria targeted NACD inducers, TPH104m, against resistant TNBC cells. TPH104m offers a new therapeutic approach capable of overcoming apoptotic resistance by directly disrupting Drp1 expression and mitochondrial fission function. In addition to inhibiting Drp1, TPH104m induces mitochondrial dysfunction characterized by loss of mitochondrial membrane potential, ATP depletion, and collapse of bioenergetic capacity. For cancers that are apoptosis-resistant, our study highlights the potential of mitochondria- and Drp1 fission-targeted therapies to simultaneously impair survival signaling and metabolic adaptability, offering a next-generation strategy for effective treatment.

References: Malla, S.; Nyinawabera, A.; Neupane, R.; Pathak, R.; Lee, D.; Abou-Dahech, M.; Kumari, S.; Sinha, S.; Tang, Y.; Ray, A.; et al. Novel Thienopyrimidine-Hydrazinyl Compounds Induce DRP1-Mediated Non-Apoptotic Cell Death in Triple-Negative Breast Cancer Cells. Cancers 2024, 16, 2621. https://doi.org/10.3390/cancers16152621

Acknowledgements:

Funding: Winthrop P. Rockefeller Cancer Institute Funds (FD208-CC103501) and Arkansas Bioscience Institute Funds (GR020025/FD224) to A.K.T. Conflict of Interest: The authors declare no conflict of interest.


Figure 1: TPH104m induces non-apoptotic cell death in TNBC cells by targeting mitochondrial dynamics. TPH104m treatment leads to mitochondrial outer membrane permeabilization, increased mitochondrial reactive oxygen species (mROS), mitochondrial DNA depletion (mtDNA↓), ATP loss, and inhibition of DRP1-mediated fission. These mitochondrial disruptions result in loss of mitochondrial membrane potential and collapse of bioenergetic function, driving caspase-independent, non-apoptotic cell death.


Figure 2. The effect of TPH104c on apoptotic and anti-apoptotic proteins in BT-20 cells. (a) Representative images featuring morphological changes in BT-20 cells (20X magnification) after incubation with vehicle (media without the TPH compounds or paclitaxel (PTX)), 0.1, 0.3 or 1 μM of TPH104m, at 0, 24, 48 or 72h post-incubation. (b) Representative images of BT-20 cells incubated with vehicle (0 uM, 2 or 5 μM of TPH104m or PTX (1 μM,) a positive control) and stained with Hoescht 33342 dye. TPH104c did not produce condensed or fragmented nuclei, compared to cells incubated with PTX. Scale bar = 25 μM. (c) Western blot images for the apoptotic molecules, cleaved caspase-3, caspase-3, cleaved caspase-7, caspase-7, cleaved caspase-9, caspase-9, cleaved caspase-8, caspase-8, Bax, Bak, Bcl-2, cytochrome c, cleaved PARP and PARP, following incubation with Vehicle (0 uM), 0.5, 1, 2 or 5 μM of TPH104m. All of the proteins were normalized to β-actin levels. Histograms summarizing the levels of each protein is also shown. All data are presented as the means ± SEM of four independent studies; Clvd = cleaved; Csp = caspase; Cyt C = cytochrome C. (d) Caspase-Glo 3/ 7 assay results presented as a bar graph and as a curve, showing that the incubation of BT-20 cells with TPH104m for 24 hours, decreased the levels of caspase 3/7 in a concentration-dependent manner. In contrast, PTX (1 μM) increased the levels of caspase 3 and 7 (n = 2). (e) The IC50 values, using the MTT assay, for TPH104c in BT-20 cells that were preincubated with zVADfmk and then incubated with varying concentrations of TPH104c for 72 hours. The data represent the mean ± SD of three independent experiments performed in triplicate. Data are representative of two independent experiments performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


Figure 3. TPH104m impairs mitochondrial bioenergetics and induce mitochondrial dysfunction in TNBC BT-20 cells. BT-20 cells were treated with 2 or 5 μM of TPH104m for 24 h and subjected to Seahorse Mitostress test assay. (a) OCR and (b) ECAR of BT-20 cells treated with TPH104c. (c) Basal Respiration (d) ATP production, (e) Proton leak, (f) Maximal respiration (g) Spare Respiratory Capacity (h) non-mitochondrial oxygen consumption and (i) Bioenergetic Health Index (BHI) of BT-20 cells treated with 2 or 5 μM of TPH104m. Data analyzed using one-way ANOVA followed by Dunnett’s post-hoc test with n = 3 for each group performed in triplicate. (j) Effect of TPH104m on cellular ATP levels. ATP levels were measured using CellTiter-Glo assay. Data analyzed using two-way ANOVA followed by Dunnett’s post-hoc test with n = 2 for each group performed in triplicate All data were expressed as SEM. * P < 0.05 vs untreated control, ** P < 0.01, *** P < 0.001, **** P < 0.0001.