Abstract 1115: PT-112 induces potent mitochondrial stress and immunogenic cell death in human prostate cancer cell lines
Authors: Soler-Agesta Ruth , Ames Tyler D. , Price Matthew , Jimeno José , Yim Christina Y. , Moreno-Loshuertos Raquel , Anel Alberto 
Published: June 2022

Abstract
Purpose: To investigate PT-112 in a human prostate cancer (PC) cell panel and assess differential sensitivity, cell death mechanism, induction of mitochondrial stress, and release of damage-associated molecular patterns (DAMPs).
Background: PT-112 is a novel pyrophosphate-platinum conjugate with clinical activity in advanced solid tumors including lung, thymoma and castration-resistant PC, and in multiple myeloma. PT-112’s cancer cell death was shown previously to be independent of DNA damage. In vitro and in vivo mouse experiments have shown that PT-112 causes mitochondrial reactive oxygen species (mtROS) accumulation, DAMP release, immunogenic cell death (ICD), and T cell infiltration.
Methods: Sensitivity to PT-112 was assessed in human PC cell lines (LNCap, LNCap-C4, LNCap-C4-2, DU-145, 22Rv1, VCap and PC-3) and the non-tumorigenic prostate cell line RWPE-1. We analyzed parameters involved in the cell death process such as apoptotic and necroptotic markers, mitochondrial membrane potential, and mtROS and autophagy by flow cytometry. We also evaluated PT-112’s induction of ICD markers calreticulin (CRT) cell surface exposure and ATP secretion. Finally, a possible link between HIF-1alpha expression and PT-112 sensitivity was investigated.
Results: PT-112 caused growth inhibition and cancer cell death without affecting healthy RWPE-1 cells. The pan-caspase inhibitor Z-VAD-fmk significantly reduced cell death, with more mild effects seen with the RIPK1/2 inhibitor necrostatin-1 in certain cell lines. PT-112-induced cell death was accompanied by a prominent increase of mtROS and decrease in mitochondrial membrane potential, as well as by DAMP emission (ATP release and CRT exposure). PT-112 activated markers of autophagy, and there was a positive relationship between HIF-1alpha expression and the sensitivity to PT-112 in this panel.
Conclusions: PT-112 was broadly active in the PC cell lines tested, while sparing benign prostate cells, indicative of PT-112 cancer cell selectivity and of activity that crosses the varied malignant prostate phenotypes, including androgen receptor positive and negative cell lines. Cell death was primarily apoptotic, as shown by the inhibitory effects of Z-VAD-fmk. Consistent with prior work reported in glycolytic murine cells, in this PC cell panel PT-112 induced mtROS accumulation and mitochondrial membrane depolarization, as well as DAMP release, demonstrating that these may be fundamental and linked responses of cancer cells to PT-112. The apparent induction of autophagy by PT-112 may be a cellular defense mechanism to drug-induced stress. The association between PT-112 sensitivity and HIF-alpha expression should be studied further, as validation of this finding could have clinical applications. Future studies will explore relationships across mitochondrial stress, ICD and HIF-1alpha in PT-112-treated cancer cells
Citation Format: Ruth Soler-Agesta, Tyler D. Ames, Matthew Price, José Jimeno, Christina Y. Yim, Raquel Moreno-Loshuertos, Alberto Anel. PT-112 induces potent mitochondrial stress and immunogenic cell death in human prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1115.