Histone deacetylase inhibitors activate NF-kappaB in human leukemia cells through an ATM/NEMO-related pathway.

Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-kappaB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (gamma-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-kappaB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-kappaB activation (e.g. in I kappaB alpha super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-kappaB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-kappaB activation, blocked TNFalpha- but not HDACI-mediated NF-kappaB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-kappaB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, gamma-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-kappaB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-kappaB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.

Mechanism and functional role of XIAP and Mcl-1 down-regulation in flavopiridol/vorinostat antileukemic interactions.

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-kappaB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat-mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat-mediated transcriptional repression of XIAP, Mcl-1-enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials.

Phase I study of vorinostat in combination with bortezomib for relapsed and refractory multiple myeloma.

PURPOSE: Vorinostat, a histone deacetylase inhibitor, enhances cell death by the proteasome inhibitor bortezomib in vitro. We sought to test the combination clinically. EXPERIMENTAL DESIGN: A phase I trial evaluated sequential dose escalation of bortezomib at 1 to 1.3 mg/m2 i.v. on days 1, 4, 8, and 11 and vorinostat at 100 to 500 mg orally daily for 8 days of each 21-day cycle in relapsed/refractory multiple myeloma patients. Vorinostat pharmacokinetics and dynamics were assessed. RESULTS: Twenty-three patients were treated. Patients had received a median of 7 prior regimens (range, 3-13), including autologous transplantation in 20, thalidomide in all 23, lenalidomide in 17, and bortezomib in 19, 9 of whom were bortezomib-refractory. Two patients receiving 500 mg vorinostat had prolonged QT interval and fatigue as dose-limiting toxicities. The most common grade >3 toxicities were myelo-suppression (n = 13), fatigue (n = 11), and diarrhea (n = 5). There were no drug-related deaths. Overall response rate was 42%, including three partial responses among nine bortezomib refractory patients. Vorinostat pharmacokinetics were nonlinear. Serum Cmax reached a plateau above 400 mg. Pharmacodynamic changes in CD-138+ bone marrow cells before and on day 11 showed no correlation between protein levels of NF-kappaB, IkappaB, acetylated tubulin, and p21CIP1 and clinical response. CONCLUSIONS: The maximum tolerated dose of vorinostat in our study was 400 mg daily for 8 days every 21 days, with bortezomib administered at a dose of 1.3 mg/m2 on days 1, 4, 8, and 11. The promising antimyeloma activity of the regimen in refractory patients merits further evaluation.

Chromatin-dependent regulation of the MMTV promoter by cAMP signaling is mediated through distinct pathways.

The nucleoprotein structure of the mouse mammary tumor virus (MMTV) promoter defines its response to cAMP signaling. A stably replicating MMTV template in highly organized chromatin is repressed in the presence of cAMP, whereas a transiently transfected template with a disorganized structure is activated. In this study, we investigate the nature of the cAMP-induced signal(s) by which these opposing responses occur to gain insight into their mechanism. We demonstrate that the transcriptional changes observed at both templates are mediated through cAMP-dependent protein kinase A (PKA). In addition, the MMTV promoter lacks a consensus cAMP response element (CRE) and neither template requires cAMP response element-binding protein (CREB) to elicit a response to cAMP signaling. However, the responses of the two templates differ mechanistically in that the CREB-binding protein p300 potentiates activation from the transient template in a manner dependent on its Cys/His-rich region 3, but does not appear to affect the repression of the replicating chromatin template. Chromatin immunoprecipitation assays show that cAMP treatment results in a decrease in acetylation of histone H4, and in multiple modifications of histone H3 at specific nucleosomes in the promoter region of the stable MMTV template. These findings suggest novel CREB-independent, chromatin-dependent pathways for transcriptional regulation by cAMP.