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5-AZA Upregulates SOCS3 and PTPN6/SHP1, Inhibiting STAT3 and Potentiating the Effects of AG490 against Primary
Michele Di Crosta1, Andrea Arena1, Rossella Benedetti1
1Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy.
Abstract:
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 t 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.
Insights
The DNA demethylating agent 5-Azacytidine (5-AZA) effectively reduces survival in primary effusion lymphoma (PEL) cells by upregulating STAT3 inhibitors. Combining 5-AZA with a STAT3 inhibitor potentiates its cytotoxic effect against PEL.
Area of Science:
- Epigenetics and Cancer Biology
- Molecular Oncology
- Hematologic Malignancies
Background:
- Epigenetic modifications, including DNA methylation and histone modifications, play a crucial role in carcinogenesis by altering oncogene and oncosuppressor gene expression.
- Aberrant methylation can activate pro-survival pathways like JAK/STAT, which are critical for the survival of certain cancers, including primary effusion lymphoma (PEL).
- Constitutive activation of Signal transducer and activator of transcription 3 (STAT3) is a key driver in PEL pathogenesis.
Purpose of the Study:
- To investigate the effects of DNA demethylating agent 5-Azacytidine (5-AZA) and histone demethylating agent DS-3201 on PEL cells.
- To evaluate the combination therapy of these agents with a STAT3 inhibitor (AG490) on PEL cell viability and molecular pathways.
- To elucidate the mechanisms by which epigenetic modulators affect STAT3 activation and survival in PEL.
Main Methods:
- Treatment of PEL cells with 5-AZA, DS-3201, and AG490 (STAT3 inhibitor), alone and in combination.
- Assessment of cell viability using trypan blue assay and FACS analysis.
- Western blot analysis for molecular changes and Luminex assay for cytokine release.
Main Results:
- 5-Azacytidine (5-AZA) upregulated STAT3 inhibitory molecules Suppressor of Cytokine Signaling 3 (SOCS3) and PTPN6/SHP-1, leading to reduced STAT3 activation and downstream pro-survival targets in PEL cells.
- 5-AZA treatment impaired PEL cell survival, and its combination with AG490 significantly potentiated the cytotoxic effect.
- DS-3201 (histone demethylating agent) showed minimal impact on STAT3 phosphorylation or PEL cell survival, alone or combined with AG490.
Conclusions:
- 5-Azacytidine (5-AZA) demonstrates therapeutic potential in PEL by targeting the JAK/STAT pathway through upregulation of endogenous STAT3 inhibitors.
- Combination therapy of 5-AZA with a STAT3 inhibitor (AG490) offers a promising strategy to enhance treatment efficacy in PEL.
- The findings highlight the differential roles of DNA and histone demethylating agents in modulating STAT3 signaling in PEL.
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