The DNA damage response (DDR) represents a complex network of multiple signaling pathways involving cell cycle checkpoints, DNA repair, transcriptional programs, and apoptosis, through which cells maintain genomic integrity following various endogenous (metabolic) or environmental stresses. In cancer treatment, the DDR occurs in response to various genotoxic insults by diverse cytotoxic agents and radiation, representing an important mechanism limiting chemotherapeutic and radiotherapeutic efficacy. This has prompted the development of agents targeting DDR signaling pathways, particularly checkpoint kinase 1 (Chk1), which contributes to all currently defined cell cycle checkpoints, including G1/S, intra-S-phase, G2/M, and the mitotic spindle checkpoint. Although numerous agents have been developed with the primary goal of enhancing the activity of DNA-damaging agents or radiation, the therapeutic outcome of this strategy remains to be determined. Recently, new insights into DDR signaling pathways support the notion that Chk1 represents a core component central to the entire DDR, including direct involvement in DNA repair and apoptotic events in addition to checkpoint regulation. Together, these new insights into the role of Chk1 in the DDR machinery could provide an opportunity for novel approaches to the development of Chk1 inhibitor strategies. Clin Cancer Res; 16(2); 376–83

Purpose: The purpose of this study was to examine interactions between the proteasome inhibitor bortezomib (Velcade) and the histone deacetylase (HDAC) inhibitors sodium butyrate and suberoylanilide hydroxamic acid in human multiple myeloma (MM) cells that are sensitive and resistant to conventional agents. Experimental Design: MM cells were exposed to bortezomib for 6 h before the addition of HDAC inhibitors (total, 26 h), after which reactive oxygen species (ROS), mitochondrial dysfunction, signaling and cell cycle pathways, and apoptosis were monitored. The functional role of ROS generation was assessed using the free radical scavenger N-acetyl-l-cysteine. Results: Preincubation with a subtoxic concentration of bortezomib markedly sensitized U266 and MM.1S cells to sodium butyrate- and suberoylanilide hydroxamic acid-induced mitochondrial dysfunction; caspase 9, 8, and 3 activation; and poly(ADP-ribose) polymerase degradation; resulting in synergistic apoptosis induction. These events were associated with nuclear factor κB inactivation, c-Jun NH₂-terminal kinase activation, p53 induction, and caspase-dependent cleavage of p21^CIP1, p27^KIP1, and Bcl-2, as well as Mcl-1, X-linked inhibitor of apoptosis, and cyclin D1 down-regulation. The bortezomib/HDAC inhibitor regimen markedly induced ROS generation; moreover, apoptosis and c-Jun NH₂-terminal kinase activation were attenuated by N-acetyl-l-cysteine. Dexamethasone- or doxorubicin-resistant MM cells failed to exhibit cross-resistance to the bortezomib/HDAC inhibitor regimen, nor did exogenous interleukin 6 or insulin-like growth factor I block apoptosis induced by this drug combination. Finally, bortezomib/HDAC inhibitors induced pronounced lethality in primary CD138⁺ bone marrow cells from MM patients, but not in the CD138⁻ cell population. Conclusions: Sequential exposure to bortezomib in conjunction with clinically relevant HDAC inhibitors potently induces mitochondrial dysfunction and apoptosis in human MM cells through a ROS-dependent mechanism, suggesting that a strategy combining these agents warrants further investigation in MM.

NF-κB activation is reciprocally regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and deacetylases (HDACs). Here we demonstrate that in leukemia cells, NF-κB activation by the HDAC inhibitors (HDACIs) MS-275 and suberoylanilide hydroxamic acid was associated with hyperacetylation and nuclear translocation of RelA/p65. The latter events, as well as the association of RelA/p65 with IκBα, were strikingly diminished by either coadministration of the IκBα phosphorylation inhibitor Bay 11-7082 (Bay) or transfection with an IκBα superrepressor. Inhibition of NF-κB by pharmacological inhibitors or genetic strategies markedly potentiated apoptosis induced by HDACIs, and this was accompanied by enhanced reactive oxygen species (ROS) generation, downregulation of Mn-superoxide dismutase and XIAP, and c-Jun N-terminal kinase 1 (JNK1) activation. Conversely, N-acetyl l-cysteine blocked apoptosis induced by Bay/HDACIs by abrogating ROS generation. Inhibition of JNK1 activation attenuated Bay/HDACI lethality without affecting NF-κB inactivation and ROS generation. Finally, XIAP overexpression dramatically protected cells against the Bay/HDACI regimen but failed to prevent ROS production and JNK1 activation. Together, these data suggest that HDACIs promote the accumulation of acetylated RelA/p65 in the nucleus, leading to NF-κB activation. Moreover, interference with these events by either pharmacological or genetic means leads to a dramatic increase in HDACI-mediated lethality through enhanced oxidative damage, downregulation of NF-κB-dependent antiapoptotic proteins, and stress-related JNK1 activation.

Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1alpha markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1alpha or XBP1, disruption of PERK activity, or inhibition of eIF2alpha phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.