The HDAC inhibitor domatinostat induces type I interferon α in Merkel cell carcinoma by HES1 repression

Abstract

Background: Class I selective histone deacetylase inhibitors (HDACi) have been previously demonstrated to not only increase major histocompatibility complex class I surface expression in Merkel cell carcinoma (MCC) cells by restoring the antigen processing and presentation machinery, but also exert anti-tumoral effect by inducing apoptosis. Both phenomena could be due to induction of type I interferons (IFN), as has been described for HDACi. However, the mechanism of IFN induction under HDACi is not fully understood because the expression of IFNs is regulated by both activating and inhibitory signaling pathways. Our own preliminary observations suggest that this may be caused by suppression of HES1.

Methods: The effect of the class I selective HDACi domatinostat and IFNα on cell viability and the apoptosis of MCPyV-positive (WaGa, MKL-1) and -negative (UM-MCC 34) MCC cell lines, as well as, primary fibroblasts were assessed by colorimetric methods or measuring mitochondrial membrane potential and intracellular caspase-3/7, respectively. Next, the impact of domatinostat on IFNA and HES1 mRNA expression was measured by RT-qPCR; intracellular IFNα production was detected by flow cytometry. To confirm that the expression of IFNα induced by HDACi was due to the suppression of HES1, it was silenced by RNA interference and then mRNA expression of IFNA and IFN-stimulated genes was assessed.

Results: Our studies show that the previously reported reduction in viability of MCC cell lines after inhibition of HDAC by domatinostat is accompanied by an increase in IFNα expression, both of mRNA and at the protein level. We confirmed that treatment of MCC cells with external IFNα inhibited their proliferation and induced apoptosis. Re-analysis of existing single-cell RNA sequencing data indicated that induction of IFNα by domatinostat occurs through repression of HES1, a transcriptional inhibitor of IFNA; this was confirmed by RT-qPCR. Finally, siRNA-mediated silencing of HES1 in the MCC cell line WaGa not only increased mRNA expression of IFNA and IFN-stimulated genes but also decreased cell viability.

Conclusion: Our results demonstrate that the direct anti-tumor effect of HDACi domatinostat on MCC cells is at least in part mediated via decreased HES1 expression allowing the induction of IFNα, which in turn causes apoptosis.

Keywords: HES1; Histone deacetylase inhibitor; Interferon-stimulated genes; Interferons; Merkel cell carcinoma.

Fig. 1

Induction of IFNα expression and apoptosis in MCC cells by domatinostat. a Viability of MCC cells (WaGa, MKL-1 and UM-MCC34) and primary fibroblasts treated with 2.5 µM and 5 µM domatinostat for 24 h at 37 °C was measured using CellTiter 96 AQueous One Solution Cell Viability Assay (Promega, Mannheim, Germany), absorbance was measured at 490 nm using a Spectramax microplate reader (Molecular Devices, San Jose, CA). b IFNA mRNA expression, in MCC cell lines and primary fibroblasts treated with 2.5 µM domatinostat for 24 h at 37 °C, was quantified by RT-qPCR using SYBR green assay. Relative quantification normalized to RPLPO, calculated by the 2^−ΔΔCt method, is depicted as mean + SD (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). All the experiments were repeated at least twice. c Intracellular IFNα expression in WaGa cells after 6 h of treatment with domatinostat or solvent control in the presence of brefeldin A was quantified by flow cytometry. The values on the dot plots indicate the percentage of cells stained positive for IFNα. The experiment was repeated three times. d Viability of MCC cells and primary fibroblasts treated with the indicated concentrations of IFNα 2a for 7 days at 37 °C under was measured with CellTiter 96 AQueous One Solution Cell Viability Assay, absorbance was measured at 490 nm using a Spectramax microplate reader. e In the NucView 488/MitoView 633 Apoptosis Assay, healthy cells with an intact mitochondrial membrane potential are stained with MitoView633 (ΔΨm), while late apoptotic cells are stained with NucView488 (active caspase-3/7). MCC cells and primary fibroblasts were treated with 50,000 ng/ml IFNα 2a for 7 days at 37 °C. Visualized by flow cytometry and quantification is given as mean + SD and presented as stacked bars. All the experiments were repeated at least twice

Fig. 2

Domatinostat reduces the expression of transcription factor HES1. a Uniform Manifold Approximation and Projection (UMAP) of scRNAseq data from WaGa cells treated with domatinostat (blue) or not (red); normalized data were regressed for cell cycle effect. b In the same UMAP visualization as a, cells were annotated according to log-normalized HES1 expression. c HES1 mRNA expression in the MCC cell lines- WaGa, MKL-1 and UM-MCC34, as well as, primary fibroblasts treated for 24 h at 37 °C with 2.5 µM domatinostat were quantified by RT-qPCR. d mRNA expression of interferon-stimulated genes IFNAR1, IFNAR2, JAK2, and IRF3 in WaGa cells treated for 24 h at 37 °C with 2.5 µM domatinostat were quantified by RT-qPCR. Relative quantification normalized to RPLPO calculated by the 2^−ΔΔCt method is depicted as mean + SD (****, p < 0.0001). All the experiments were repeated at least twice

Fig. 3

HES1 repression and induction of IFNα in MCC cells. a HES1 knockdown was performed in WaGa cells using dicer-substrate RNAi (TriFECTa kit, Integrated DNA Technologies, Leuven, Belgium) and Lipofectamine™ RNAiMAX (Thermofischer, Dreieich, Germany) with HES1 specific siRNA (HES1 k.d.) or scrambled control siRNA (control). HES1 mRNA expression was measured 36 h post-transfection by RT-qPCR using SYBR. b mRNA expression of IFNA and IFNB and interferon-stimulated genes, IFNAR1, IFNAR2, JAK2, and IRF3 was quantified in WaGa cells 36 h after HES1 knockdown or scrambled control by RT-qPCR. Relative quantification normalized to RPLPO, calculated by the 2^−ΔΔCt method, is depicted as mean + SD (*, P < 0.05; **, P < 0.01***, p < 0.001). All the experiments were repeated at least twice