DOT1L inhibition exerts the anti-tumor effect by activating interferon signaling in breast cancer cells

Abstract

Background: DOT1L, a histone H3 lysine 79 (H3K79) methyltransferase, is a potential therapeutic target in various malignancies. In the present study, we aimed to clarify the anti-tumor effect of DOT1L inhibition in breast cancer.

Methods: Estrogen receptor (ER)-positive/HER2-negative breast cancer cells (MCF7) and ER-negative/HER2-positive cells (SKBR3) were treated with a DOT1L inhibitor (SGC0946, EPZ-5676), after which colony formation assays, cell cycle assays, flow cytometry, gene expression microarray analysis, chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) were performed. Genetic ablation of STING was performed using the CRISPR/Cas9 system.

Results: Treatment with a DOT1L inhibitor suppressed proliferation and induced cell cycle arrest and apoptosis in both ER-positive/HER2-negative and ER-negative/HER2-positive cells. Transcriptome and epigenome analysis revealed that DOT1L inhibition activated transcription of a number of interferon (IFN)-related genes (IRGs) in breast cancer cells. We also found that DOT1L inhibition upregulated type I and type III IFNs as well as cell surface human leukocyte antigen (HLA) class I expression. Notably, DOT1L inhibition induced DNA damage and upregulated levels of cytoplasmic DNA in breast cancer cells. CRISPR/Cas9-mediated knockout of STING in breast cancer cells significantly suppressed the IFN signaling activated by DOT1L inhibition and attenuated the anti-tumor effect. Moreover, scATAC-seq analysis revealed that DOT1L inhibition suppressed expression of ERBB2 in HER2-positive breast cancer cells.

Conclusion: These findings suggest that the anti-breast cancer effect of DOT1L inhibition is mediated by multiple mechanisms, including activation of innate immune signaling.

Keywords: DNA damage; DOT1L; Immune response; Interferon; STING.

Fig. 1

The anti-tumor effects of DOT1L inhibitors in breast cancer cells. A Western blot analysis of H3K79me1, H3K79me2 and H3K79me3 in MCF7 and SKBR3 cells treated for 12 days with DMSO, SGC0946 (SGC) or EPZ-5676 (EPZ). Total histone H3 is shown as an endogenous control. B Colony formation assays in breast cancer cells treated with DMSO or the indicated DOT1L inhibitor. Representative results are shown on the left, and summarized results are on the right. (n = 3). C, D Cell cycle (C) and apoptosis (D) analyses in breast cancer cells treated for 9 days with DMSO or the indicated DOT1L inhibitor. Representative results are shown on the left, and summarized results are on the right. (n = 3). Error bars represent SDs. ***P < 0.001

Fig. 2

DOT1L inhibition activates interferon (IFN) signaling in breast cancer cells. A Gene expression microarray analysis in MCF7 (left) and SKBR3 (right) cells treated for 6 days with DMSO or SGC0946. Summarized results of GSEA using genes upregulated by SGC0946 are shown. NES, normalized enrichment score; FDR, false discovery rate. B Results of GSEA of the hallmark IFN-α response gene set in breast cancer cells treated for 6 days with the indicated DOT1L inhibitor. C Heatmaps showing the microarray data of the hallmark IFN-α response genes in breast cancer cells treated with DMSO, SGC0946 (SGC) or EPZ-5676 (EPZ) for indicated periods. D qRT-PCR analysis of the indicated IFN-related genes (IRGs) in breast cancer cells treated for 6 days with DMSO (D), SGC0946 (S) or EPZ-5676 (E). (n = 3). Error bars represent SDs. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

DOT1L inhibition upregulates HLA expression in breast cancer cells. A Heatmap showing microarray data of HLA genes in MCF7 cells treated with DMSO, SGC0946 (SGC) or EPZ-5676 (EPZ) for indicated periods. B Flow cytometry analysis showing cell surface expression of HLA class I and II molecules in MCF7 cell lines treated with indicated agents for 6 days

Fig. 4

DOT1L inhibition activates IFN-Stat1 signaling in breast cancer cells. A Heatmaps showing histone H3 lysine 4 trimethylation (H3K4me3) peaks detected by ChIP-seq analysis in MCF7 and SKBR3 cells treated for 6 days with DMSO, SGC0946 (SGC) or EPZ-5676 (EPZ). B Motif analysis of H3K4me3 peaks in (A) in breast cancer cells treated as indicated. C qRT-PCR analysis of the indicated IFN genes in MCF7 cells treated for 6 days and SKBR3 cells treated for 9 days with the indicated agents. D ELISA of IFN-λ in MCF7 cells treated for 6 days with the indicated agents. E Western blot analysis of total and phosphorylated Stat1 in MCF7 cells treated for 6 days and SKBR3 cells treated for 9 days with indicated agents. Error bars represent SDs. *P < 0.05, ***P < 0.001

Fig. 5

The STING pathway is involved in the DOT1L inhibition-induced IFN signaling in breast cancer cells. A Western blot analysis of histone H3 and γH2AX in the cytoplasm of MCF7 and SKBR3 cells treated for 6 days with DMSO, SGC0946 (SGC) or EPZ-5676 (EPZ). B qRT-PCR of CXCL10 in breast cancer cells treated for 6 days with the indicated agents. (n = 3). C Western blot analysis of total and phosphorylated Stat1 and STING in control and STING knockout (KO) MCF7 cells treated for 6 days with the indicated agents. D Results of RNA-seq analysis in control and STING KO MCF7 cells. Summarized results of GSEA of the indicated gene sets using genes downregulated in KO cells are shown. E Heatmap showing expression levels of the hallmark IFN-α response genes in control and STING KO MCF7 cells treated for 6 days with the indicated agents. F GSEA of the hallmark IFN-α response gene set in control and STING KO MCF7 cells treated for 6 days with the indicated agents. G qRT-PCR analysis of the indicated IFN genes and IRGs in control and STING KO MCF7 cells treated for 6 days with DMSO (D), SGC0946 (S) or EPZ-5676 (E). (n = 3). Error bars represent SDs. ***P < 0.001

Fig. 6

The STING pathway contributes to the anti-tumor effect of DOT1L inhibition in breast cancer cells. A Colony formation assays in control and STING knockout (KO) MCF7 cells treated with DMSO, SGC0946 or EPZ-5676. Representative results are shown on the left, and summarized results are on the right. (n = 3). B, C Cell cycle (B) and apoptosis (C) assays in control and STING KO MCF7 cells treated for 9 days with DMSO, SGC0946 (SGC) or EPZ-5676 (EPZ). Representative results are shown on the left, and summarized results are on the right. NS, not significant; ***P < 0.001

Fig. 7

Single-cell ATAC-seq (scATAC-seq) analysis in SKBR3 cells treated with a DOT1L inhibitor. A Uniform manifold approximation and projection (UMAP) of the scATAC-seq data from SKBR3 cells treated with DMSO, SGC0946 (SGC) or EPZ-5676 (EPZ). Results of unsupervised clustering are shown on the right. B Proportions of the respective clusters in cells treated as indicated. C Summarized results of a pseudotime analysis of motifs identified by scATAC-seq. D Results of a pseudotime trajectory analysis of IRF1 and STAT1 motifs. E Gene scores of representative IRGs. F Gene scores of ERBB2 gene. G Results of a qRT-PCR analysis of ERBB2 in SKBR3 cells treated with the indicated agents. (n = 3). H Results of ChIP-seq analysis showing levels of H3K4me3 at the ERBB2 gene in SKBR3 cells treated as indicated