Abstract 981: SRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-kB/STAT3 complex to its promoter in malignant lymphoid cells

Abstract

Abstract SIRT1 is an NAD+-dependent class III histone deacetylase, which is not affected directly by HDAC inhibitors (HDACi). It remains controversial whether inhibition of SIRT1 or its activation is more efficacious in anticancer therapy. We have previously reported that the antitumor activity of the HDAC inhibitor LBH589 in pre-B acute leukemia cells is partly mediated by upregulation of the growth arrest and DNA damage response gene GADD45G, associated with histone hyperacetylation at the promoter level. In the present study we explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with LBH589 in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis (10-100 uM and 1-20 uM, respectively), concomitant with deacetylation of STAT3 and NF-kB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA damage response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA binding capacity of both NF-kB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with LBH589 enhanced the anti-growth and anti-survival effects mediated by either compound alone. Q-PCR confirmed that the LBH589 plus SRT2183 combination leads to significantly higher expression of GADD45G than the single agents. Enhanced upregulation of GADD45G was validated also for the combination of LBH589 with either SRT501 or resveratrol. LBH589 plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-kB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-kB. Moreover, the binding of NF-kB/STAT3 complexes to the GADD45G promoter is inhibited following LBH589, SRT501 or resveratrol treatment. In addition, the combination of LBH589 with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. In summary, SRT501 and SRT2183 show growth inhibitory and pro-apoptotic activity in malignant lymphoid cells alone and further enhanced activity in combination with LBH589. At least one of the mechanisms of action, which explains the combination effect, is mediated through enhanced upregulation of GADD45G as a result of the repressed binding of NF-kB/STAT3 complex to the GADD45G promoter together with hyperacetylation of the promoter. Thus, this study validates for the first time STAT3 as a corepressor of GADD45G and provides in vitro proof-of-concept that the combination of HDACi with SIRT1 activators is a potential new strategy for therapy of lymphoid malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 981. doi:1538-7445.AM2012-981