Di Crosta Michele, Arena Andrea, Benedetti Rossella, Gilardini Montani Maria Saveria, Cirone Mara
Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy.
Curr Issues Mol Biol. 2024 Mar 14;46(3):2468-2479. doi: 10.3390/cimb46030156.
Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism software (version 9) and analyzed by Student's test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine-protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells.
表观遗传修饰，包括原癌基因和抑癌基因启动子处发生的异常DNA甲基化以及组蛋白修饰，可导致癌症发生。由组蛋白甲基转移酶介导的异常甲基化与组蛋白一起，可影响参与调控如JAK/STAT等促生存途径的蛋白质的甲基化，并促使其激活。在本研究中，我们分别使用DNA或组蛋白去甲基化剂5-氮杂胞苷（5-AZA）或DS-3201（瓦列莫司他）来处理原发性渗出性淋巴瘤（PEL）细胞，单独使用或与信号转导和转录激活因子3（STAT3）抑制剂AG490联合使用。通过台盼蓝测定法和流式细胞术分析来研究细胞活力。通过蛋白质免疫印迹分析来研究5-AZA和/或AG490处理诱导的分子变化，而通过Luminex评估这些药物处理的PEL细胞的细胞因子释放。使用Graphpad Prism软件（版本9）进行统计分析，并通过学生检验或非参数单因素方差分析进行分析。本研究获得的结果表明，5-AZA上调了抑制STAT3酪氨酸磷酸化的分子，即细胞因子信号转导抑制因子3（SOCS3）和非受体型酪氨酸蛋白磷酸酶（PTPN）6/含Src同源区2结构域的磷酸酶-1（SHP-1），减少了STAT3的激活，并下调了PEL细胞中几个STAT3促生存靶点。由于这种淋巴瘤高度依赖于STAT3的组成性激活，5-AZA损害了PEL细胞的存活，并且当与AG490（JAK2/STAT3抑制剂）联合使用时，增强了其细胞毒性作用。与5-AZA不同，DS-3201对EZH
