Roberto Rosato

RAC3 down-regulation sensitizes human chronic myeloid leukemia cells to TRAIL-induced apoptosis

The nuclear receptor coactivator RAC3 plays important roles in many biological processes and tumorigenesis. We found that RAC3 is over‐expressed in human chronic myeloid leukemia cells K562, which are normally resistant to TRAIL‐induced apoptosis. RAC3 down‐regulation by siRNA rendered these cells sensitive to TRAIL‐induced cell death. In addition to the up‐regulation of TRAIL receptors, the process involves Bid, caspases and PARP activation, loss of mitochondrial membrane potential, and release of AIF, cytochrome c and Smac/DIABLO to the cytoplasm. We conclude that RAC3 is required for TRAIL resistance and that this anti‐apoptotic function is independent of its role in hormone receptor signaling.

Resveratrol sensitizes acute myelogenous leukemia cells to histone deacetylase inhibitors through reactive oxygen species-mediated activation of the extrinsic apoptotic pathway

Histone deacetylase inhibitors (HDACIs) activate the prosurvival nuclear factor-κB (NF-κB) pathway by hyperacetylating RelA/p65, whereas the chemopreventive agent resveratrol inhibits NF-κB by activating the class III histone deacetylase Sirt1. Interactions between resveratrol and pan-HDACIs (vorinostat and panobinostat) were examined in human acute myelogenous leukemia (AML) cells. Pharmacologically achievable resveratrol concentrations (25–50 μM) synergistically potentiated HDACI lethality in AML cell lines and primary AML blasts. Resveratrol antagonized RelA acetylation and NF-κB activation in HDACI-treated cells. However, short hairpin RNA Sirt1 knockdown failed to modify HDACI sensitivity, which suggests that factors other than or in addition to Sirt1 activation contribute to resveratrol/HDACI interactions. These interactions were associated with death receptor 5 (DR5) up-regulation and caspase-8 activation, whereas cells expressing dominant-negative caspase-8 were substantially protected from resveratrol/HDACI treatment, which suggests a significant functional role for the extrinsic apoptotic pathway in lethality. Exposure to resveratrol with HDACI induced sustained reactive oxygen species (ROS) generation, which was accompanied by increased levels of DNA double-strand breaks, as reflected in γH2A.X and comet assays. The free radical scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride blocked ROS generation, DR5 up-regulation, caspase-8 activation, DNA damage, and apoptosis, which indicates a primary role for oxidative injury in lethality. Analyses of cell-cycle progression and 5-ethynyl-2′-deoxyuridine incorporation through flow cytometry revealed that resveratrol induced S-phase accumulation; this effect was abrogated by HDACI coadministration, which suggests that cells undergoing DNA synthesis may be particularly vulnerable to HDACI lethality. Collectively, these findings indicate that resveratrol interacts synergistically with HDACIs in AML cells through multiple ROS-dependent actions, including death receptor up-regulation, extrinsic apoptotic pathway activation, and DNA damage induction. They also raise the possibility that S-phase cells may be particularly susceptible to these actions.

LBH-589 (panobinostat) potentiates fludarabine anti-leukemic activity through a JNK- and XIAP-dependent mechanism.

Effects of the HDAC inhibitor LBH-589 (panobinostat) on fludarabine lethality toward acute myeloid leukemia (AML) cells were examined in vitro and in vivo. LBH-589 pretreatment sensitized U937, HL-60, and primary leukemia cells to fludarabine while blocking NF-κB activation accompanied by XIAP down-regulation and JNK activation. Pharmacologic or genetic JNK inhibition significantly attenuated LBH-589/fludarabine lethality, whereas XIAP over-expression diminished JNK activation and apoptosis. Combined in vivo treatment abrogated leukemia growth in a U937 xenograft murine model and substantially increased animal survival. These studies highlight the interplay between NF-κB activation, XIAP down-regulation, and JNK activation in anti-leukemic synergism between fludarabine and LBH-589.

Histone Deacetylase Inhibitors and Anticancer Activity

Structural alterations of DNA and their corresponding genetic defects are recognized as some of the fundamental characteristics of cancer, whereas gene mutations, deletions, and chromosomal alterations appear to play a critical role in the etiology of the disease. These epigenetic modifications have become the target of novel experimental therapeutic approaches representing a component of a more generalized concept in which the ultimate goal is to address the specific molecular alteration responsible for transformation. The notion of molecularly targeted therapies have radically changed the way cancer therapeutics is currently envisioned [1]. These compounds, in contradistinction to conventional cytotoxic drugs, which exert a more generally disruptive effect on key cellular structures or processes (e.g., DNA replication, formation of the mitotic spindle, etc.), are specifically designed to target pathways directly implicated in neoplastic transformation. There has been substantial progress in the development of drugs targeting epigenetic regulatory processes, and one of the areas that has become the subject of considerable attention is the development of HDAC inhibitors (HDACIs). The present chapter focuses on the analysis of this group of compounds as anticancer therapeutic agents. 2 Histone Deacetylases: HDAC Inhibitors

Abstract 4706: Resveratrol potentiates the lethality of HDAC inhibitors in acute myelogenous leukemia cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnPrevious studies from our group and others have shown that interference with NF-κB RelA acetylation/activation significantly potentiates the activity of histone deacetylase inhibitors (HDACIs) in human leukemia and other transformed cells (Dai et al., Mol Cell Biol 25; 5429,2005; Dai et al., J Biol Chem 286:34036, 2011). The natural polyphenol resveratrol (RSV) is an activator of the class III HDAC SIRT1. Recently, preclinical evidence of RSV activity in leukemia has been described. In addition, clinical trials have established that RSV is very well tolerated in humans, with plasma concentrations > 250 μM achievable. Based upon these considerations, we hypothesized that a SIRT1 agonist, by deacetylating RelA, might enhance the antileukemic activity of clinically relevant HDACIs. To test this possibility, the impact of RSV on HDACI antileukemic activity was investigated. Co-administration (24 hr) of RSV (50 μM) with LBH589 (15 nM) or vorinostat (1.5 μM) synergistically induced mitochondrial injury (cytochrome c and AIF release), caspase activation, and apoptosis in AML cell lines (e.g., U937 cells). Of note, caspase 8 cleavage/activation was particularly pronounced after co-treatment, suggesting a potentially important functional role for the extrinsic apoptotic pathway in RSV/HDACI interactions. Similar interactions were also observed in AML cell lines bearing FLT3-ITD (e.g., MV4-11) and in primary AML blasts. These events were associated with deacetylation of RelA accompanied by inactivation of NF-κB, increased ROS formation, a marked increase in DNA damage (reflected by αH2A.X formation, an indicator of double strand DNA breaks), and abrogation of RSV-mediated S phase arrest. Genetic knockdown of SIRT1 by shRNA resulted in increased RelA acetylation and NF-κB activity, consistent with SIRT1 deacetylase actions. However, SIRT1 knockdown increased rather than decreased HDACI lethality, suggesting the contribution of NF-κB independent RSV actions to cell death. On the other hand, cells expressing dominant-negative caspase 8 were substantially protected from HDACI and HDACI/RSV lethality. Finally, co-administration of the antioxidant TBAP sharply reduced ROS generation, diminished caspase 8 activation and γH2AX formation, and prevented cell death in cells exposed to HDACI/RSV. Together, these findings indicate that clinically relevant concentrations of RSV significantly increase HDACI antileukemic activity through a process involving induction of oxidative injury (ROS), promotion of DNA damage, engagement of the extrinsic caspase cascade, potentiation of mitochondrial injury, and interruption of cell cycle regulation, culminating in apoptosis. They also imply that factors other than RelA deacetylation by RSV are primarily responsible for the enhanced antileukemic activity of this regimen.nnCitation 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 4706. doi:1538-7445.AM2012-4706