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ment (Figure 4C, right panel). To search for more evidence of tumor cell apoptosis, a TUNEL assay was performed on mice tumor sections. Compared with the untreated group and the groups treated with single agents, the number of apoptotic tumor cells was increased following combined treatment (Figure 4D). In accordance with in vitro data, upregulation of H3K4me3 was more significant in the combination treatment group than in the single-agent and the untreated group (Figure 4E).
To determine KMT2D-H3K4me3 DNA binding tar- gets, ChIP-seq was performed using H3K4me3 antibody in KMT2D V5486M mutated Jurkat cells treated with chidamide (5 mm) alone or in combination with decitabine (5 mm) for 48 h. Presentation of the data in a
Venn diagram identified a significant non-overlapping portion of H3K4me3 binding promoters in the combina- tion group, excluding 663 promoters overlapping with the chidamide group and 17 with the decitabine group (Figure 5A,B). Consistent with previous studies, H3K4me3 peaks were found at gene promoters. The group of promoters, whose H3K4me3 levels were affected by combined chidamide and decitabine treatment, but not by either chidamide or decitabine treatment alone, was enriched with binding site motifs for PU.1, a transcription factor that activates gene expression during myeloid and B-cell lymphoid cell development15,16 (Figure 5C). Furthermore, RNA sequencing analysis indicated that, in comparison with the untreated group and the single-agent
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Figure 4. Effect of chidamide and decitabine in KMT2D-mutated and EP300-mutated T-lymphoma. (A) Combination index (CI) curve calculated by Compusyn soft- ware in KMT2D-mutated and EP300-mutated Jurkat cells treated with chidamide (CHID, 5 mm) and/or decitabine (DECI, 5 mm) for 48 h. (B) KMT2D-mutated Jurkat cell apoptosis and cell cycle determined by flow cytometry of cells treated with CHID and/or DECI for 48 h. *P<0.05, **P<0.01 compared with the untreated cells. (C) In vivo effect of the CHID and DECI combination in a murine T-lymphoma xenograft model. Tumor volume (left panel) and standardized uptake value (SUV) intensity of micro-positron emission tomograpy-computed tomography (right panel) of xenograft nude mice injected subcutaneously with KMT2D V5486-mutated Jurkat cells treated with CHID (12.5 mg/kg, twice weekly for 3 weeks), DECI (0.5 mg/kg, twice weekly for 3 weeks), either alone or in combination. **P<0.01 compared with the untreated group that received RPMI1640. (D) Apoptotic cells detected by the TUNEL assay (×400). Bar=20 mm. (E) Immunohistochemical assay of H3K4me3 in murine tumor samples treated with CHID and/or DECI. **P<0.01 compared with the untreated group. Bar=50 mm.
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haematologica | 2018; 103(4)