Interactions between histone deacetylase inhibitors (HDACIs) and the novel proteasome inhibitor carfilzomib (CFZ) were investigated in GC- and activated B-cell–like diffuse large B-cell lymphoma (ABC-DLBCL) cells. Coadministration of subtoxic or minimally toxic concentrations of CFZ) with marginally lethal concentrations of HDACIs (vorinostat, SNDX-275, or SBHA) synergistically increased mitochondrial injury, caspase activation, and apoptosis in both GC- and ABC-DLBCL cells. These events were associated with Jun NH2-terminal kinase (JNK) and p38MAPK activation, abrogation of HDACI-mediated nuclear factor-κB activation, AKT inactivation, Ku70 acetylation, and induction of γH2A.X. Genetic or pharmacologic JNK inhibition significantly diminished CFZ/vorinostat lethality. CFZ/vorinostat induced pronounced lethality in 3 primary DLBCL specimens but minimally affected normal CD34+ hematopoietic cells. Bortezomib-resistant GC (SUDHL16) and ABC (OCI-LY10) cells exhibited partial cross-resistance to CFZ. However, CFZ/vorinostat dramatically induced resistant cell apoptosis, accompanied by increased JNK activation and γH2A.X expression. Finally, subeffective vorinostat doses markedly increased CFZ-mediated tumor growth suppression and apoptosis in a murine xenograft OCI-LY10 model. These findings indicate that HDACIs increase CFZ activity in GC- and ABC-DLBCL cells sensitive or resistant to bortezomib through a JNK-dependent mechanism in association with DNA damage and inhibition of nuclear factor-κB activation. Together, they support further investigation of strategies combining CFZ and HDACIs in DLBCL.

). The present findings document the intrinsic ability of sphingoid bases to induce apoptosis in HL-60 and U937 cells. Exposure to either sphingosine or sphinganine (0.001-10 M) for 6 h promoted apoptotic degradation of genomic DNA as indicated by (a) electrophoretic resolution of 50-kilobase pair DNA loop fragments and 0.2-1.2-kilobase pair DNA fragment ladders on agarose gels, and (b) spectrofluorophotometric determination of the formation and release of double-stranded fragments and corresponding loss of integrity of bulk DNA. DNA damage correlated directly with reduced cloning efficiency and was associated with the appearance of apoptotic cytoarchitectural traits. At sublethal concentrations ( 750 nM), however, sphingoid bases synergistically augmented the apoptotic capacity of ceramide (10 M), producing both a leftward shift in the ceramide concentration-response profile and a pronounced increase in the response to maximally effective levels of ceramide. Thus, sphingosine and sphinganine increased both the potency and efficacy of ceramide. The apoptotic capacity of bacterial sphingomyelinase (50 milliunits/ml) was similarly enhanced by either (a) acute co-exposure to highly selective pharmacological inhibitors of protein kinase C such as calphostin C and chelerythrine or (b) chronic pre-exposure to the nontumor-promoting protein kinase C activator bryostatin 1, which completely down-modulated total assayable protein kinase C activity. These findings demonstrate that inhibition of protein kinase C by physiological or pharmacological agents potentiates the lethal actions of ceramide in human leukemia cells, providing further support for the emerging concept of a cytoprotective function of the protein kinase C isoenzyme family in the regulation of leukemic cell survival.Recent investigation has examined the participation of sphingophospholipid-and glycerophospholipid-derived messengers in the regulation of leukemic cell survival. We (1, 2) and others (3) have demonstrated that increased intracellular availability of ceramide induces programmed cell death or apoptosis in the human myeloid leukemia cell lines HL-60 and U937. Ceramide interacts with at least two distinct intracellular target enzymes, ceramide-activated protein kinase (4 -6) and ceramide-activated protein phosphatase (7-9). A cytotoxic role for ceramide-activated protein phosphatase and ceramideactivated protein kinase in ceramide action has been inferred, although the relative contributions of these enzymes to the initiation of apoptosis is presently uncertain (10, 11). A contrasting cytoprotective function of diglyceride and, therefore, of one or more isoforms of protein kinase C (PKC) 1 is supported by several lines of evidence. Increased intracellular availability of diglyceride abrogates the initiation of apoptotic DNA damage by ceramide in both HL-60 and U937 cells (1, 2); this effect is mimicked by such diverse pharmacological PKC activators as the stage 1 tumor promoters phorbol dibutyrate (2) and phorbol myristate acetate (2, 3), the stag...

IntroductionCheckpoint kinases (ie, Chk1 and Chk2) represent key components of the DNA damage checkpoint machinery, which monitors DNA breaks caused by endogenous/metabolic or environmental genotoxic insults or by replication stress. 1,2 In response to DNA damage, cells activate checkpoint pathways, resulting in cell-cycle arrest, which permits the DNA repair machinery to rectify the damage. Depending on the nature of the DNA lesions and the context in which damage occurs, cells either survive and resume cell-cycle progression through a recovery mechanism when repair is successful or are eliminated by apoptosis if repair fails. Thus, checkpoints provide normal cells with critical surveillance machinery designed to promote genomic integrity and survival. Conversely, checkpoint dysfunction contributes to tumorigenesis by permitting cell proliferation in the face of genomic instability. 3,4 Moreover, checkpoints are activated by numerous chemotherapeutic agents and ionizing radiation. 5 This has prompted the development of anticancer strategies targeting checkpoint machinery. 5,6 Among the diverse checkpoint pathway components, Chk1 represents a particularly attractive target for several reasons, that is, (1) Chk1 is functionally associated with all known checkpoints (eg, the G2-M transition, G1, intra-S, 5 and, most recently, the mitotic spindle checkpoint 7 ); (2) Chk1 is essential for maintenance of genomic integrity, whereas the role of Chk2 is conditional 3 ; and (3) for multiple checkpoints, Chk2 function can be mimicked by Chk1, whereas Chk1 cannot be replaced by a functionally overlapping kinase such as Chk2. 3 Chk1 inhibition (eg, by the Chk1 inhibitor UCN-01) results in abrogation of checkpoints induced by DNA-damaging chemotherapy and radiation, leading to enhanced tumor cell killing. 8,9 Given these findings, a major emphasis has been placed on efforts to combine Chk1 inhibitors (eg, UCN-01 10 or CHIR-124 11 ) with diverse DNA-damaging agents. However, an alternative strategy is based on the concept that transformed cells may be ill-equipped to survive simultaneous interruption of both checkpoint machinery and prosurvival signaling. In this context, our group has reported that exposure of human leukemia and multiple myeloma (MM) cells to UCN-01 induces pronounced activation of MEK1/2 and ERK1/2, 12,13 key components of the Ras/Raf/MEK/ERK cascade that plays a critical role in proliferation and survival of malignant cells. 14 Significantly, disruption of ERK1/2 activation by pharmacologic MEK1/2 inhibitors, 12,13 farnesyltransferase inhibitors (FTIs; eg, L744832) 15,16 or HMG-CoA reductase inhibitors (ie, statins) 17 results in a dramatic increase in apoptosis of hematopoietic malignant cells. Together, these findings suggest that activation of Ras/MEK/ERK signaling cascade may represent a compensatory response to Chk1 inhibitor lethality, and that interruption of this response lowers the death threshold.Although the observation that MEK1/2 inhibitors or FTIs antagonize UCN-01-mediated ERK1/2 activati...

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a)

Purpose: The goal of this study was to characterize interactions between the proteasome inhibitor bortezomib and the histone deacetylase (HDAC) inhibitors (HDACI) romidepsin or belinostat in chronic lymphocytic leukemia (CLL) cells. Experimental Design: Primary and cultured (JVM-3 and MEC-2) CLL cells were exposed to agents alone or in combination, after which cell death was determined by 7-aminoactinomycin D staining/flow cytometry. Acetylation of target proteins, activation of caspase cascades, and expression of apoptosis-regulatory proteins were monitored by Western blot analysis. Nuclear factor-nB (NF-nB) activity was determined by luciferase reporter assay. Cells were transiently transfected with wild-type and acetylation site-mutated (inactive) RelA(p65) (e.g., K221R, K310R, or K281/221/310R) and assessed for HDACI sensitivity. Results: Combined exposure to very low concentrations of romidepsin or belinostat (i.e., low nanomolar and submicromolar, respectively) in combination with low nanomolar concentrations of bortezomib synergistically induced cell death in primary and cultured CLL cells. These events were likely associated with prevention of HDACI-mediated RelA acetylation and NF-nB activation by bortezomib, down-regulation of antiapoptotic proteins (i.e., Bcl-xL, Mcl-1, and XIAP), as well as up-regulation of the proapoptotic protein Bim, resulting in activation of caspase cascade. Finally, CLL cells transfected with inactive RelA displayed a significant increase in HDACI lethality. Conclusions: Coadministration of the clinically relevant HDACIs romidepsin or belinostat with bortezomib synergistically induces cell death in CLL cells, likely through mechanisms involving, among other factors, NF-nB inactivation and perturbation in the expression of proapoptotic and antiapoptotic proteins. A strategy combining HDAC with proteasome inhibition warrants further attention in CLL.