Targeting inflammatory macrophages rebuilds therapeutic efficacy of DOT1L inhibition in hepatocellular carcinoma

Abstract

Epigenetic reprogramming is a promising therapeutic strategy for aggressive cancers, but its limitations in vivo remain unclear. Here, we showed, in detailed studies of data regarding 410 patients with human hepatocellular carcinoma (HCC), that increased histone methyltransferase DOT1L triggered epithelial-mesenchymal transition-mediated metastasis and served as a therapeutic target for human HCC. Unexpectedly, although targeting DOT1L in vitro abrogated the invasive potential of hepatoma cells, abrogation of DOT1L signals hardly affected the metastasis of hepatoma in vivo. Macrophages, which constitute the major cellular component of the stroma, abrogated the anti-metastatic effect of DOT1L targeting. Mechanistically, NF-κB signal elicited by macrophage inflammatory response operated via a non-epigenetic machinery to eliminate the therapeutic efficacy of DOT1L targeting. Importantly, therapeutic strategy combining DOT1L-targeted therapy with macrophage depletion or NF-κB inhibition in vivo effectively and successfully elicited cancer regression. Moreover, we found that the densities of macrophages in HCC determined malignant cell DOT1L-associated clinical outcome of the patients. Our results provide insight into the crosstalk between epigenetic reprogramming and cancer microenvironments and suggest that strategies to influence the functional activities of inflammatory cells may benefit epigenetic reprogramming therapy.

Keywords: DOT1L; NF-κB; epigenetic reprogramming; hepatocellular carcinoma; immune microenvironment; inflammation; macrophages; metastasis; therapeutic strategy.

Graphical abstract

Figure 1

DOT1L serves as a prognostic factor and potential therapeutic target for human HCC (A) DOT1L distribution in 91 HCC tissues was analyzed by immunohistochemistry. Scale bar represents 100 μm. (B) DOT1L, H3K79me2, and H3K79me3 expression in HCC tissues was measured by immunoblotting (n = 5). (C and D) Association of DOT1L⁺ tumor cells with patients’ recurrence in human HCC. 91 patients were divided into two groups according to the median density of DOT1L⁺ cells in the tumor (C, median, 67): DOT1L^low (n = 45) and DOT1L^high (n = 46). Univariate and multivariate regression analyses of factors associated with recurrence were performed (D). (E–G) DOT1L-knockdown Huh-7 and Hepa1-6 cells were generated by transducing with pLKO-puro lentiviral particles containing shNC or shDOT1L sequences; migration of the cells (E, scale bar represents 100 μm), proteins of EMT genes (F and G, scale bar represents 20 μm), DOT1L, and H3K79me2 expression (F) were determined (n = 5). (H and I) Huh-7 and Hepa1-6 cells were left untreated or were treated with DMSO or an inhibitor against DOT1L (DOT1Lⁱ); migration of the cells (H), proteins of EMT genes, DOT1L, and H3K79me2 expression (I) were determined (n = 6). (J and K) Huh-7 cells were left untreated or were treated with DMSO or an inhibitor against DOT1L (DOT1Lⁱ); proliferation of cells under normal condition or serum starvation (2% FBS) was measured (J, n = 4). Proteins of survival genes in serum-starvation cells were determined by immunoblotting (K). Results represent three independent experiments. Data represent mean ± SEM. ∗∗∗p < 0.001, Student’s t test for (A) and (E), one-way ANOVA followed by Bonferroni’s correction for (H).

Figure 2

In vivo immune microenvironment dismantles therapeutic efficacy of DOT1L targeting (A–C) Wild-type (shNC) or DOT1L-knockdown (shDOT1L) Hepa1-6 cells were inoculated in liver of the mice for 22 days as described (A). Lung metastasis (B, scale bar represents 250 μm) and proteins of EMT genes, DOT1L, and H3K79me2 (C) in mice tumor tissues were analyzed (n = 6). (D) shNC or shDOT1L Hepa1-6 cells were left untreated or were treated with conditioned medium from mice tumor-infiltrating leukocytes (TIL-CM); migration of cells was determined (n = 5, scale bar represents 100 μm). (E) Effect of malignant DOT1L-knockdown on the infiltration of B cells (B220⁺), T helper cells (CD4⁺), cytotoxic T cells (CD8⁺), neutrophils (Gr1⁺), macrophages (F4/80⁺), and NK cells (NKp46⁺) in mouse hepatoma (n = 6, scale bar represents 100 μm). (F) Human HCC samples were divided into two groups according to the staining results of malignant DOT1L. B cell, T cell, neutrophil (TAN), macrophage (TAM), NK cell, and DC densities in HCC tissues were counted (n = 26). (G) 91 HCC patients were divided into four groups according to the median densities of CD68⁺ macrophages and DOT1L⁺ cells, or CD3⁺ T cells and DOT1L⁺ cells in tumors, respectively. Recurrence of the patients was calculated. (H) Gene set enrichment analysis (GSEA) of EMT signature (GSEA: M1373) in DOT1L^high versus DOT1L^low counterparts within HCC patients with low or high macrophage infiltration. Results represent three independent experiments. Data represent mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Bonferroni’s correction for (D).

Figure 3

Tumor macrophages abrogate the anti-metastatic effect of DOT1L targeting (A–D) shNC (A, C, and D) or shDOT1L (A–D) Huh-7 cells were left untreated or were treated with conditioned medium from tumor macrophages (TAM-CM), B cells, T cells, NK cells, or neutrophils as indicated; migration of the cells was determined (A and B, n = 6, scale bar represents 100 μm). Proteins of EMT genes were analyzed by immunoblotting (C) and immunofluorescence (D, scale bar represents 20 μm), respectively. (E and F) Huh-7 cells were left untreated or were treated with DMSO or an inhibitor against DOT1L in the absence or presence of TAM-CM; migration of the cells (E) and proteins of EMT genes (F) were determined (n = 5). (G) shNC or shDOT1L Huh-7 cells were left untreated or were treated with TAM-CM, DOT1L, H3K79me2, and H3K79me3 expression was determined (n = 5). (H) Huh-7 cells were left untreated or were treated with DMSO or an inhibitor against DOT1L in the absence or presence of TAM-CM, DOT1L, H3K79me2, and H3K79me3 expression was also determined (n = 5). Results represent four independent experiments. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Bonferroni’s correction for (A), (B), and (E).

Figure 4

Activated macrophages are more potent in eliminating the effect of DOT1L targeting (A) shDOT1L Huh-7 cells were left untreated or were treated with conditioned medium from tumor macrophages (TAM-CM) or paired blood monocytes (BMo-CM); migration of the cells was determined (n = 5). (B) Top 10 biological processes (Gene Ontology terms) enriched in 103 genes significantly upregulated in tumor macrophages compared with blood monocytes in HCC samples (GEO: GSE140228) (FC > 2; p < 0.05). (C) FACS analysis of HLA-DR, CD80, and CD86 expression on monocytes/macrophages from HCC tumor and paired blood sample (n = 5). (D and E) shDOT1L Huh-7 cells were left untreated or were treated with conditioned medium from HLA-DR⁻ or HLA-DR⁺ tumor macrophages; migration of the cells (D) and proteins of EMT genes (E) were determined (n = 5). (F and G) shDOT1L Huh-7 cells were cocultured with blood monocytes in the absence or presence of control peptide (Cpep) or hyaluronan-specific blocking peptide (Pep-1); migration (F) and proteins of EMT genes (G) of shDOT1L Huh-7 cells were determined (n = 5). (H and I) shDOT1L Huh-7 cells were left untreated or were treated with conditioned medium from untreated (BMo-CM) or hyaluronan-treated (HA-Mo-CM) blood monocytes; migration of the cells (H) and proteins of EMT genes (I) were determined (n = 5). Results represent four independent experiments. Data represent mean ± SEM. ∗∗∗p < 0.001, one-way ANOVA followed by Bonferroni’s correction for (A), (D), (F), and (H).

Figure 5

Tumor macrophages abrogate DOT1L inhibition efficacy in an NF-κB dependent manner (A) shNC or shDOT1L Huh-7 cells were left untreated or were incubated with conditioned medium from tumor macrophages (TAM-CM) for 15 min. Activation of indicated pathways was analyzed by immunoblotting (n = 4). (B and C) Effects of signaling pathway inhibitors on migration (B) and EMT genes expression (C) of shNC and shDOT1L Huh-7 cells in the presence of TAM-CM (n = 5). (D) Cytokine expression in tumor macrophages and blood monocytes in HCC samples (GSE140228) was compared (n = 4). (E) Cytokine production by monocytes/macrophages from HCC blood and tumor was examined by ELISA (n = 5). (F and G) Effects of cytokine neutralizing on p65 nuclear translocation (F) and cell migration (G) of shNC and shDOT1L (F and G) Huh-7 cells in the presence of TAM-CM (n = 5, scale bar represents 20 μm). (H and I) shNC and shDOT1L Huh-7 cells were left untreated or were incubated with TNF-α or IL-1β. Migration of cells (H) and EMT marker expression (I) were analyzed (n = 5, scale bar represents 20 μm). Results represent four independent experiments. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Bonferroni’s correction for (B), (G), and (H); Student’s t test for (E).

Figure 6

Suppressing macrophage-elicited NF-κB activation augments epigenetic reprogramming efficacy of DOT1L targeting (A and B) shNC and shDOT1L Hepa1-6 cells were inoculated in mice liver for 10 days. Thereafter, mice were injected with isotype Ab or αCSF1R Ab as described (A). Lung metastasis was analyzed (B, n = 5, scale bar represents 250 μm). (C and D) Mice bearing Hepa1-6 hepatoma were injected with PBS, DOT1L inhibitor, αCSF1R Ab, or DOT1L inhibitor plus αCSF1R Ab as described (C). Lung metastasis was analyzed (D, n = 5). (E and F) Mice bearing shNC or shDOT1L Hepa1-6 hepatoma were injected with PBS or NF-κB inhibitor as described (E). Lung metastasis was analyzed (F, n = 5). (G and H) Mice bearing Hepa1-6 hepatoma were injected with PBS, DOT1L inhibitor, NF-κB inhibitor, or DOT1L inhibitor plus NF-κB inhibitor as described (G). Lung metastasis was analyzed (H, n = 5). (I) Patients were divided into two groups according to the median value of NF-κB pathway score in 291 HCC samples from TCGA dataset. Associations of tumor DOT1L expression with patients’ recurrence in NF-κB score high and NF-κB score low HCC samples were analyzed. p value was calculated by chi-square test. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Bonferroni’s correction for (B), (D), (F), and (H).