Abstract 659: TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro
Authors: Giarrizzo Michael , LaComb Joseph F. , Patel Hetvi R. , Bialkowska Agnieszka B. , Reddy Rohan , Graves Lee M. , Iwanowicz Edwin J. 
Published: March 2024

Introduction: Ferroptosis is an emerging potential target for the treatment of colorectal cancer (CRC) and has been shown to sensitize cancer cells to combinatory chemotherapy. Our studies show that the TR-107 compound, an agonist of ClpP - the proteolytic subunit of mitochondrial maintenance complex - can synergize with Erastin, an inducer of ferroptosis cell death. TR-107 potently disrupts mitochondrial proteostasis and attenuates oxidative phosphorylation, resulting in excessive oxidative stress. Here, we show that Erastin synergizes with TR-107 activity by downregulating the cofactor of GPX4, a major intracellular antioxidant defense resulting in CRC cell apoptosis.

Methods: We first evaluated the efficacy of TR-107 against eight CRC cell lines belonging to four established consensus molecular subtypes. Cells were treated with control (DMSO) or TR-107 (10 nM, 50 nM, 1 µM) for 24, 48, and 72 hours. We determined IC50 values with Cell Titer-Glo, cell proliferation with the Beckman Coulter cell counter, cell cycle with flow cytometry, OXPHOS activity with Seahorse Real-Time ATP Rate assay, mitochondrial protein levels with western blot and proteomics, and differential gene expression using RNA sequencing and RT-PCR. Subsequently, we assessed the efficacy of TR-107 and Erastin co-treatment. Cells were treated with DMSO, TR-107 (10, 50 nM), Erastin (1, 10 µM), or combinations of the abovementioned concentrations. Cell viability was measured 72 hours post-treatment with Cell Titer-Glo. Synergy scores (HSA) were calculated with Synergyfinder on RStudio. Ferroptosis-related protein expression 24 hours after co-treatment was assessed with western blot and densitometry.

Results: TR-107 IC50 values ranged from 2-70 nM, producing a dose- and time-dependent inhibitory effect in cell proliferation. Western blot showed that 50 nM TR-107 abolished the expression of critical mitochondrial proteins ClpX, TFAM, and TUFM. Seahorse data showed a significant reduction in OXPHOS activity. Multi-omics revealed downregulation of respiratory chain complex subunits, mtDNA transcription/translation factors, and upregulation of mitophagy and ferroptosis-related pathways in treated cells. Co-treatment with Erastin produced synergistic HSA scores in 63% (5/8) of assessed CRC cell lines. RKO and NCI-H508 were highly sensitive to Erastin treatment, suggesting that lower Erastin concentrations may synergize with TR-107 and thus reduce dose toxicity. Western blot analysis 24 hours post-treatment showed a loss of GPX4 protein levels in co-treated cells compared to 50 nM TR-107 treatment. This suggests a rapid downregulation of intracellular antioxidant defense that may induce ferroptosis sooner and more effectively than single-agent treatment.

Conclusion: In vitro results suggest that co-treating with TR-107 and Erastin produces a significant, synergistic reduction in CRC cell viability.

Citation Format: Michael Giarrizzo, Joseph F. LaComb, Hetvi R. Patel, Agnieszka B. Bialkowska, Rohan Reddy, Lee M. Graves, Edwin J. Iwanowicz. TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 659.