The anticancer thiosemicarbazone triapine exerts immune-enhancing activities via immunogenic cell death induction and FAS upregulation

Abstract

The anticancer thiosemicarbazone Triapine is currently in a phase III clinical trial in combination with radiation therapy and cisplatin. Noteworthy, while radiotherapy induces an immune-activating cell death, so called immunogenic cell death (ICD), cisplatin possesses immunomodulatory and ICD-enhancing functions. Interestingly, although there are several indications that suggest that Triapine could also enhance the immune recognition of cancer cells, no investigations in this direction have been reported so far. Indeed, immune cells (especially cytotoxic T-cells) were found to enhance the anticancer activity of Triapine. This effect might be based on endoplasmic reticulum (ER) stress induction, which on the one hand led to ICD of the cancer cells as indicated by ATP release, calreticulin exposure, high-mobility group box 1 secretion and in vivo vaccination experiments. On the other hand, the Triapine-induced ER stress resulted in FAS upregulation in cell culture as well as in vivo via NFκB signaling. This, in turn, rendered cancer cells more susceptible to FASL (predominantly expressed by lymphoid immune cells)-induced caspase 8-mediated apoptosis. Consequently, our study is the first to unveil the significant role of the (adaptive) immune system in the anticancer activity of Triapine, positioning it as a promising partner for combination with immunotherapy and other immunogenic agents.

Keywords: Adaptive immune system; Anticancer therapy; Cytotoxic t cells; FAS; Immunogenic cell death; Immunomodulation; NFκB; Thiosemicarbazones; Triapine.

Fig. 1

The role of the adaptive immune system in the anticancer activity of Triapine. (A) Top 20 upregulated GO-terms in a transcriptomic dataset (SW480, 1 µM Triapine, 15 h) compared to control. (B) Impact of immune status on the anticancer activity of Triapine in murine allograft models. Mean tumor volume (mm³) ± SEM is given. (C) Flow cytometry analysis on day seven of CT-26 tumor-infiltrating immune cells of female Balb/c mice. Mean percent cells of the (grand)parental gate ± SD is given. Effect of a CD8-blocking antibody on (D) tumor growth (mean tumor volume (mm³) ± SEM) of Triapine-treated CT-26 allografts and (E) overall survival. Pink lines indicate Triapine treatment. (F) Representative confocal microscopy images of CT-26 cells stained for CALR (green) and DAPI (blue) after 24 h Triapine treatment (10 µM, scale bar: 50 μm). (G) Flow cytometry analysis of CALR (24 h). (H) Representative fluorescence images and (I) associated quantification of CT-26 cells stained for HMGB1(green) and DAPI (blue) after 24 h Triapine treatment (10 µM; scale bar: 15 μm). Triapine induced (J) HMGB1 and (K) ATP release into the supernatant after 24 h treatment. Values given are the mean ± SD of at least three independent experiments, normalized to untreated control. (L) Percentage of tumor-free mice at the site of re-challenge site after vaccination with Triapine-treated cancer cells. The control group received freezed/thawed (F/T)-lysed cells. Significant differences were calculated by mixed-effects analysis corrected for multiple comparisons by Sidak, unpaired T-test, one-way, two-way ANOVA corrected for multiple comparisons by Sidak or Dunnett, or Log-rank and Mantel-Cox post-test (**** p < 0.0001, *** p < 0.001, **p < 0.01; *p < 0.05, n.s. = not significant)

Fig. 2

Triapine stimulates FAS upregulation via ER stress and NFκB. (A) Expression of receptor genes in SW480 cells (1 µM Triapine, 18 h). Values are mean log fold change compared to control. (B) Representative flow cytometry histograms and quantification (mean fluorescence intensities (MFI) ± SD) of FAS expression of Triapine-treated CT-26 cells. (C) Representative IHC images (scale bar: 100 μm) and (D) associated quantification of FAS expression (mean % ± SEM) in CT-26 tumors after treatment with Triapine. (E) Caspase 8 activity and (F) cell viability of HCT116 cells after 24 h Triapine preincubation, followed by 24 h FASL treatment. Values are mean ± SD from one representative experiment out of three, normalized to untreated control. (G) Impact of the ER-stress inhibitor 4-PhB on Triapine-induced NFκB activity was analyzed using NFκB reporter HEK293-Blue™ hTLR7 cells after 24 h. Values are mean ± SD of three independent experiments normalized to control and cell number. Impact of (H) 4-PhB or ± (I) Bay 11-7082 on Triapine-induced FAS expression in HCT116 cells with functional caspase 8 signaling after 24 h treatment. Values are MFI ± SD of three independent experiments, normalized to control. Significance was determined by one-way, two-way ANOVA with Bonferroni´s or Dunnett´s multiple comparisons test or unpaired T-test (**** p < 0.001, *** p < 0.001, ** p < 0.01, * p < 0.05). (J) Scheme summarizing the immune-enhancing activities of Triapine via induction of ICD and FAS upregulation.