Abstract 3346: A20/TNFAIP3, a novel target of histone deacetylase inhibitor-induced NF-κB activation, functionally disables the extrinsic apoptotic pathway in human leukemia cells.
Authors: Zhou Liang , Chen Shuang , Lin Hui , Pei Xin-Yan , Aust Mandy M. , Rahmani Mohamed , Rosato Roberto , Dai Yun , Grant Steven 
Published: August 2014

Abstract
The zinc finger protein A20/TNFAIP3 is a novel regulator of the pro-survival NF-κB signaling pathway. TNFα induces A20 via an NF-κB-dependent process to inhibit NF-κB, thus acting as a negative feedback regulator. Unlike the classic inhibitor IκBα, which sequesters NF-κB in the cytoplasm, A20 inactivates NF-κB by altering ubiquitination and proteasomal degradation of the receptor-interacting protein (RIP) in the TNF-R1-recruiting complex containing the FAS-associated death domain (FADD). Recently, we and others have found that histone deacetylase inhibitors (HDACIs) trigger sustained NF-κB activation, representing a compensatory cytoprotective response blunting the anti-cancer activity of these agents. Here, we identify, for the first time, A20 as a downstream target of HDACI-mediated NF-κB activation, which plays an important role in antagonizing HDACI lethality by preventing activation of the extrinsic apoptotic pathway. First, like TNFα and PMA, HDACIs (e.g., vorinostat and LBH-589) robustly increased A20 protein expression in various human leukemia cells (e.g., U937, MV4-11, and MOLM-13). Second, qPCR demonstrated increased mRNA levels after HDACI exposure. A20 expression occurred 16 h after HDACI exposure, similar to induction of p21, an NF-κB-dependent gene implicated in HDACI-induced differentiation. Moreover, A20 induction occurred (16 h) later than IκBα S32/S36 and RelA S536 phosphorylation (&lt; 4h), suggesting an NF-κB activation-dependent process. Indeed, the IKK inhibitor Bay 11-7092 clearly diminished HDACI-induced A20 expression and transfection with an IκBα-super repressor (IκBα-SR) almost completely blocked this event. Third, prevention of A20 induction by shRNA dramatically increased (4 - 5-fold) sustained NF-κB activation by TNFα. In contrast, A20 shRNA failed to increase NF-κB activation by HDACI s, but did prolong this response from 8 h to 28 h. Fourth, disruption of A20 induction by shRNA sensitized leukemic cells to HDACIs, manifested by sharply diminished viability and increased apoptosis. The increased HDACI sensitivity of cells expressing A20 shRNA was associated with marked activation of the extrinsic pathway, reflected by increased caspase 8 cleavage and enzyme activity using IETD-pNA as substrate. Conversely, doxycycline-induced A20 expression significantly blocked HDACI-mediated caspase 8 and PARP cleavage in a tet-inducible system. Last, dominant-negative caspase 8 largely diminished HDACI lethality. Together, these findings indicate that HDACIs induce A20 expression through an NF-κB-dependent mechanism, and that A20 induction plays a significant functional role in limiting HDACI anti-leukemia activity by preventing extrinsic apoptotic pathway activation. They also raise the possibility that A20 may represent a therapeutic target for improving HDACI activity.
Citation Format: Liang Zhou, Shuang Chen, Hui Lin, Xin-Yan Pei, Mandy M. Aust, Mohamed Rahmani, Roberto Rosato, Yun Dai, Steven Grant. A20/TNFAIP3, a novel target of histone deacetylase inhibitor-induced NF-κB activation, functionally disables the extrinsic apoptotic pathway in human leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3346. doi:10.1158/1538-7445.AM2013-3346