The HDAC Inhibitor Domatinostat Promotes Cell-Cycle Arrest, Induces Apoptosis, and Increases Immunogenicity of Merkel Cell Carcinoma Cells

Abstract

Merkel cell carcinoma (MCC) is a rare, highly aggressive skin cancer for which immune modulation by immune checkpoint inhibitors shows remarkable response rates. However, primary or secondary resistance to immunotherapy prevents benefits in a significant proportion of patients. For MCC, one immune escape mechanism is insufficient for recognition by T cells owing to the downregulation of major histocompatibility complex I surface expression. Histone deacetylase inhibitors have been demonstrated to epigenetically reverse the low major histocompatibility complex I expression caused by the downregulation of the antigen-processing machinery. Domatinostat, an orally available small-molecule inhibitor targeting histone deacetylase class I, is currently in clinical evaluation to overcome resistance to immunotherapy. In this study, we present preclinical data on domatinostat's efficacy and mode of action in MCC. Single-cell RNA sequencing revealed a distinct gene expression signature of antigen processing and presentation, cell-cycle arrest, and execution phase of apoptosis on treatment. Accordingly, functional assays showed that domatinostat induced G2M arrest and apoptosis. In the surviving cells, antigen-processing machinery component gene transcription and translation were upregulated, consequently resulting in increased major histocompatibility complex I surface expression. Altogether, domatinostat not only exerts direct antitumoral effects but also restores HLA class I surface expression on MCC cells, therefore, restoring surviving MCC cells' susceptibility to recognition and elimination by cognate cytotoxic T cells.

Figure 1:. Domatinostat induces global transcriptional changes in MCC cells.

WaGa cells were either treated with domatinostat (2.5 μM, 24 hours) or solvent control before being subjected to single-cell RNA-seq. (a) Uniform Manifold Approximation and Projection (UMAP) of single-cell gene expression in WaGa cells annotated by treatment. (b) Comparison of average single-cell gene expression of domatinostat-treated and control cells. Significantly deregulated genes (adjusted p-value < 0.1) are labeled blue. (c) Metascape analysis of significantly enriched pathway terms in differentially expressed genes.

Figure 2:. Domatinostat promotes G2M cell cycle arrest.

(a) UMAP visualization of single cell gene expression in WaGa cells after domatinostat treatment (2.5 μM, 24 hours) and solvent controls. Cells were annotated by cell cycle phase and treatment condition. Quantification of cells in the different cell cycle phases were given as absolute numbers and as fractions. (b) Pre-ranked Gene Set Enrichment analysis (GSEA) of the hallmark G2M checkpoint gene set for differentially expressed genes sorted by log2-fold change between domatinostat and solvent treated control cells (NES=1,91; FDR<0.001). (c) Cell cycle analysis by BrdU incorporation and 7-AAD staining of WaGa cells treated 48 hrs. with domatinostat (2.5 μM) and solvent control or nocodazole (100 nM). (d) Quantification of cell cycle profiles in indicated cell lines, represented as mean + / −SD and presented as stacked bars (P values: ****, P < 0.0001). Experiments depicted in c and d were repeated twice.

Figure 3:. Domatinostat treatment promotes apoptosis in MCC cells.

(a) Viability of MCC cells and primary fibroblasts following a 24 hours treatment with indicated concentrations of domatinostat depicted as fold change as compared to solvent control. (b-d) In the NucView 488/MitoView 633 Apoptosis Assay, healthy cells with an intact mitochondrial membrane potential (ΔΨm) are stained with MitoView 633 (red), while late apoptotic cells (active caspase 3/7) are stained with NucView 488 (green). MCC cell lines and primary fibroblasts were treated with solvent control, 2.5 μM domatinostat or 100 nM nocodazole for 24 hours at 37°C. Visualization by flow cytometry (b) or fluorescence microscopy (d). Quantification of flow cytometry analysis is given in (c) as mean + / − SD and presented as stacked bars (whiskers: min to max) (P values: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). All experiments were repeated at least twice.

Figure 4:. Domatinostat induces expression of APM component gene expression and MHC class I surface expression by MCC cells.

(a) WaGa cells were treated with the indicated concentrations of domatinostat for 4, 8 and 24 hours at 37 °C. Gene expression fold change to solvent controls of LMP2 and TAP2 detected by RT-PCR are depicted. (b) TAP1, TAP2, LMP2 and LMP7 mRNA expression in MCC cells and primary fibroblasts treated for 24 hours at 37 °C with 2.5 μM domatinostat was measured by RT-qPCR. Only living cells were included in the experiments. Gene expression fold change is depicted as mean + / − SD (**, P < 0.01; ***, P <0.001; ****, P < 0.0001). (c) TAP1, TAP2, LMP2, LMP7, HLA-A and β2-microglobulin (B2M) protein expression in WaGa and MKL-1 cells treated with 2.5 μM domatinostat for 24 hours at 37 °C; β-tubulin was used as a loading control. Viable cells were enriched by Ficoll purification for preparation of whole cell lysates. (d) Dose dependent up-regulation of HLA class I (HLA-ABC) cell surface expression. Cells were treated with the indicated concentrations of domatinostat for 24 hours at 37 °C. (e) Quantification of HLA-I expression showing mean fluorescence intensity (FI, left) and % of HLA-I positive cells (right). All experiments were at least repeated twice.