Molecular sequelae of histone deacetylase inhibition in human malignant B cells

Histone acetylation modulates gene expression, cellular differentiation, and survival and is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nucleosomal histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of suberoylanilide hydroxamic acid (SAHA), the prototype of a series of hydroxamic acid-based HDAC inhibitors, in cell lines and patient cells from B-cell malignancies, including multiple myeloma (MM) and related disorders. SAHA induced apoptosis in all tumor cells tested, with increased p21 and p53 protein levels and dephosphorylation of Rb. We also detected cleavage of Bid, suggesting a role for Bcl-2 family members in regulation of SAHA-induced cell death. Transfection of Bcl-2 cDNA into MM.1S cells completely abrogated SAHA-induced apoptosis, confirming its protective role. SAHA did not induce cleavage of caspase-8, -9, or -3 in MM.1S cells during the early phase of apoptosis, and the pan-caspase inhibitor ZVAD-FMK did not protect against SAHA. Conversely, poly(ADP)ribose polymerase (PARP) was cleaved in a pattern indicative of calpain activation, and the calpain inhibitor calpeptin abrogated SAHA-induced cell death. Importantly, SAHA sensitized MM.1S cells to death receptor-mediated apoptosis and inhibited the secretion of interleukin 6 (IL-6) induced in bone marrow stromal cells (BMSCs) by binding of MM cells, suggesting that it can overcome cell adhesion-mediated drug resistance. Our studies delineate the mechanisms whereby HDAC inhibitors mediate anti-MM activity and overcome drug resistance in the BM milieu and provide the framework for clinical evaluation of SAHA, which is bioavailable, well tolerated, and bioactive after oral administration, to improve patient outcome.     

Role of autophagy in histone deacetylase inhibitor-induced apoptotic and nonapoptotic cell death

Autophagy is a cellular catabolic pathway by which long-lived proteins and damaged organelles are targeted for degradation. Activation of autophagy enhances cellular tolerance to various stresses. Recent studies indicate that a class of anticancer agents, histone deacetylase (HDAC) inhibitors, can induce autophagy. One of the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), is currently being used for treating cutaneous T-cell lymphoma and under clinical trials for multiple other cancer types, including glioblastoma. Here, we show that SAHA increases the expression of the autophagic factor LC3, and inhibits the nutrient-sensing kinase mammalian target of rapamycin (mTOR). The inactivation of mTOR results in the dephosphorylation, and thus activation, of the autophagic protein kinase ULK1, which is essential for autophagy activation during SAHA treatment. Furthermore, we show that the inhibition of autophagy by RNAi in glioblastoma cells results in an increase in SAHA-induced apoptosis. Importantly, when apoptosis is pharmacologically blocked, SAHA-induced nonapoptotic cell death can also be potentiated by autophagy inhibition. Overall, our findings indicate that SAHA activates autophagy via inhibiting mTOR and up-regulating LC3 expression; autophagy functions as a prosurvival mechanism to mitigate SAHA-induced apoptotic and nonapoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of SAHA.     

Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors

This study examines the basis of resistance and sensitivity of normal and transformed cells to histone deacetylase inhibitor (HDACi)-induced cell death, specifically the role of caspases and thioredoxin (Trx). An important attribute of HDACis is that they induce cancer cell death at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy. The mechanism underlying this selectivity has not been understood. In this study we found that the HDACi suberoylanilide hydroxamic acid (SAHA) and MS-275, a benzamide, cause an accumulation of reactive oxygen species (ROS) and caspase activation in transformed but not normal cells. Inhibition of caspases does not block HDACi-induced cell death. These studies provide a possible mechanism that can explain why normal but not certain transformed cells are resistant to HDACi-induced cell death. The HDACi causes an increase in the level of Trx, a major reducing protein for many targets, in normal cells but not in transformed cells. The SAHA-induced increase in Trx activity in normal cells is associated with no increase in ROS accumulation. Transfection of transformed cells with Trx small interfering RNA caused a marked decrease in the level of Trx protein with an increase in ROS, a decrease in cell proliferation, and an increase in sensitivity to SAHA-induced cell death. Thus, Trx, independent of the caspase apoptotic pathway, is an important determinant of resistance of cells to HDACi-induced cell death.     

Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance

Novel therapeutic strategies are needed to address the emerging problem of imatinib resistance. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) is being evaluated for imatinib-resistant chronic myelogenous leukemia (CML) and has multiple cellular effects, including the induction of autophagy and apoptosis. Considering that autophagy may promote cancer cell survival, we hypothesized that disrupting autophagy would augment the anticancer activity of SAHA. Here we report that drugs that disrupt the autophagy pathway dramatically augment the antineoplastic effects of SAHA in CML cell lines and primary CML cells expressing wild-type and imatinib-resistant mutant forms of Bcr-Abl, including T315I. This regimen has selectivity for malignant cells and its efficacy was not diminished by impairing p53 function, another contributing factor in imatinib resistance. Disrupting autophagy by chloroquine treatment enhances SAHA-induced superoxide generation, triggers relocalization and marked increases in the lysosomal protease cathepsin D, and reduces the expression of the cathepsin-D substrate thioredoxin. Finally, knockdown of cathepsin D diminishes the potency of this combination, demonstrating its role as a mediator of this therapeutic response. Our data suggest that, when combined with HDAC inhibitors, agents that disrupt autophagy are a promising new strategy to treat imatinib-refractory patients who fail conventional therapy.     

Ricolinostat (ACY‐1215) induced inhibition of aggresome formation accelerates carfilzomib‐induced multiple myeloma cell death

Proteasome inhibition induces the accumulation of aggregated misfolded/ubiquitinated proteins in the aggresome; conversely, histone deacetylase 6 (HDAC6) inhibition blocks aggresome formation. Although this rationale has been the basis of proteasome inhibitor (PI) and HDAC6 inhibitor combination studies, the role of disruption of aggresome formation by HDAC6 inhibition has not yet been studied in multiple myeloma (MM). The present study aimed to evaluate the impact of carfilzomib (CFZ) in combination with a selective HDAC6 inhibitor (ricolinostat) in MM cells with respect to the aggresome‐proteolysis pathway. We observed that combination treatment of CFZ with ricolinostat triggered synergistic anti‐MM effects, even in bortezomib‐resistant cells. Immunofluorescent staining showed that CFZ increased the accumulation of ubiquitinated proteins and protein aggregates in the cytoplasm, as well as the engulfment of aggregated ubiquitinated proteins by autophagosomes, which was blocked by ricolinostat. Electron microscopy imaging showed increased autophagy triggered by CFZ, which was inhibited by the addition of ACY‐1215. Finally, an in vivo mouse xenograft study confirmed a decrease in tumour volume, associated with apoptosis, following treatment with CFZ in combination with ricolinostat. Our results suggest that ricolinostat inhibits aggresome formation, caused by CFZ‐induced inhibition of the proteasome pathway, resulting in enhanced apoptosis in MM cells.     

Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1

Histone deacetylase (HDAC) inhibitors (HDACi) cause cancer cell growth arrest and/or apoptosis in vivo and in vitro. The HDACi suberoylanilide hydroxamic acid (SAHA) is in phase I/II clinical trials showing significant anticancer activity. Despite wide distribution of HDACs in chromatin, SAHA alters the expression of few genes in transformed cells. p21(WAF1) is one of the most commonly induced. SAHA does not alter the expression of p27(KIPI), an actively transcribed gene, or globin, a silent gene, in ARP-1 cells. Here we studied SAHA-induced changes in the p21(WAF1) promoter of ARP-1 cells to better understand the mechanism of HDACi gene activation. Within 1 h, SAHA caused modifications in acetylation and methylation of core histones and increased DNase I sensitivity and restriction enzyme accessibility in the p21(WAF1) promoter. These changes did not occur in the p27(KIPI) or epsilon-globin gene-related histones. The HDACi caused a marked decrease in HDAC1 and Myc and an increase in RNA polymerase II in proteins bound to the p21(WAF1) promoter. Thus, this study identifies effects of SAHA on p21(WAF1)-associated proteins that explain, at least in part, the selective effect of HDACi in altering gene expression.     

The proteasome inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl+ cells sensitive and resistant to STI571

Interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors (HDIs) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Coexposure of cells (24-48 hours) to minimally toxic concentrations of bortezomib + either suberoylanilide hydroxamic acid (SAHA) or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspases-3 and -8 cleavage and poly(adenosine diphosphate-ribose) polymerase (PARP) degradation. These events were accompanied by down-regulation of the Raf-1/mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) pathway as well as diminished expression of Bcr/Abl and cyclin D1, cleavage of p21CIP1 and phosphorylation of the retinoblastoma protein (pRb), and induction of the stress-related kinases Jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezomib/SAHA-mediated lethality. Coadministration of bortezomib and SAHA resulted in increased reactive oxygen species (ROS) generation and diminished nuclear factor kappa B (NF-kappa B) activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezomib/SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (imatinib mesylate)-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with STI571-resistant disease, as well as in Bcr/Abl- leukemia cells (eg, HL-60, U937, Jurkat). Together, these findings raise the possibility that combined proteasome/histone deacetylase inhibition may represent a novel strategy in leukemia, including apoptosis-resistant Bcr/Abl+ hematologic malignancies.     

Rheb controls misfolded protein metabolism by inhibiting aggresome formation and autophagy

Perinuclear aggresome formation is a key mechanism to dispose of misfolded proteins that exceed the degradative capacity of ubiquitin–proteasome and autophagy–lysosome systems. Functional blockade of either degradative system leads to an enhanced aggresome formation. The tuberous sclerosis complex–Ras homologue enriched in brain–mammalian target of rapamycin (TSC–Rheb–mTOR) pathway is known to play a central role in modulating protein synthesis and autophagy. However, in spite of the constitutive activation of mTOR and the abrogated autophagy activity in TSC1- or TSC2-deficient cells, the TSC mutant cells are defective in aggresome formation and undergo apoptosis upon misfolded protein accumulation both in vitro and in vivo. High Rheb activity in TSC mutant cells inhibits aggresome formation and sensitizes cell death in response to misfolded proteins. Surprisingly, this previously unrecognized function of Rheb is independent of TOR complex 1. Active Rheb disrupts the interaction between dynein and misfolded protein cargos, and therefore blocks aggresome formation by inhibiting dynein-dependent transportation of misfolded proteins. This study reveals a function of Rheb in controlling misfolded protein metabolism by modulating aggresome formation.     

Therapeutic potential of a synthetic lethal interaction between the MYC proto-oncogene and inhibition of aurora-B kinase

The Myc protein and proteins that participate in mitosis represent attractive targets for cancer therapy. However, their potential is presently compromised by the threat of side effects and by a lack of pharmacological inhibitors of Myc. Here we report that a circumscribed exposure to the aurora kinase inhibitor, VX-680, selectively kills cells that overexpress Myc. This synthetic lethal interaction is attributable to inhibition of aurora-B kinase, with consequent disabling of the chromosomal passenger protein complex (CPPC) and ensuing DNA replication in the absence of cell division; executed by sequential apoptosis and autophagy; not reliant on the tumor suppressor protein p53; and effective against mouse models for B-cell and T-cell lymphomas initiated by transgenes of MYC. Our findings cast light on how inhibitors of aurora-B kinase may kill tumor cells, implicate Myc in the induction of a lethal form of autophagy, indicate that expression of Myc be a useful biomarker for sensitivity of tumor cells to inhibition of the CPPC, dramatize the virtue of bimodal killing by a single therapeutic agent, and suggest a therapeutic strategy for killing tumor cells that overexpress Myc while sparing normal cells.     

Synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with histone deacetylase inhibitors,melphalan, or doxorubicin in multiple myeloma

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP‐AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM‐2, RPMI‐8226, U‐266, LP‐1, MM1.S, and primary myeloma cells were exposed to NVP‐AUY922 and one of the combination partners histone deacetylase inhibitor NVP‐LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP‐AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP‐AUY922 with SAHA, NVP‐LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI‐8226 cell line and relative resistance of some primary myeloma cells against NVP‐AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with melphalan, doxorubicin, NVP‐LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.     

The novel, orally bioavailable HSP90 inhibitor NVP-HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases

Heat shock protein 90 (HSP90) binds and stabilizes numerous proteins and kinases essential for myeloma cell survival and proliferation. We and others have recently demonstrated that inhibition of HSP90 by small molecular mass inhibitors induces cell death in multiple myeloma (MM). However, some of the HSP90 inhibitors involved in early clinical trials have shown limited antitumor activity and unfavorable toxicity profiles. Here, we analyzed the effects of the novel, orally bioavailable HSP90 inhibitor NVP-HSP990 on MM cell proliferation and survival. The inhibitor led to a significant reduction in myeloma cell viability and induced G2 cell cycle arrest, degradation of caspase-8 and caspase-3, and induction of apoptosis. Inhibition of the HSP90 ATPase activity was accompanied by the degradation of MM phospho-Akt and phospho-ERK1/2 and upregulation of Hsp70. Exposure of MM cells to a combination of NVP-HSP990 and either melphalan or histone deacetylase (HDAC) inhibitors caused synergistic inhibition of viability, increased induction of apoptosis, and was able to overcome the primary resistance of the cell line RPMI-8226 to HSP90 inhibition. Combined incubation with melphalan and NVP-HSP990 led to synergistically increased cleavage of caspase-2, caspase-9, and caspase-3. These data demonstrate promising activity for NVP-HSP990 as single agent or combination treatment in MM and provide a rationale for clinical trials.     

Edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts

Multiple myeloma (MM) is an incurable B-cell malignancy, requiring new therapeutic strategies. We have found that synthetic alkyl-lysophospholipids (ALPs) edelfosine and perifosine induced apoptosis in MM cell lines and patient MM cells, whereas normal B and T lymphocytes were spared. ALPs induced recruitment of Fas/CD95 death receptor, Fas-associated death domain-containing protein, and procaspase-8 into lipid rafts, leading to the formation of the death-inducing signaling complex (DISC) and apoptosis. TNF-related apoptosis-inducing ligand receptor-1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5, as well as Bid, were also recruited into lipid rafts, linking death receptor and mitochondrial signaling pathways. ALPs induced mitochondrial cytochrome c release. Bcl-X(L) overexpression prevented cytochrome c release and apoptosis. A Fas/CD95-deficient MM subline expressing DR4 and DR5 was resistant to edelfosine. Fas/CD95 retrovirus transduction bestowed edelfosine sensitivity in these cells. A Fas/CD95 mutant lacking part of the intracellular domain was ineffective. Lipid raft disruption prevented ALP-induced Fas/CD95 clustering, DISC formation, and apoptosis. ALP-induced apoptosis was Fas/CD95 ligand (FasL/CD95L) independent. ALP-induced recruitment of death receptors in lipid rafts potentiated MM cell killing by FasL/CD95L and TRAIL. These data uncover a novel lipid raft-mediated therapy in MM involving concentration of death receptors in membrane rafts, with Fas/CD95 playing a major role in ALP-mediated apoptosis.     

Epigenetic modulation of gene expression of human leukemia cell lines - induction of cell death and senescence

Histone deacetylase inhibitors (HDACi) are emerging new class of anticancer agents that act by inhibiting cell growth, inducing cell cycle arrest and apoptosis of various cancer cells. However, in some conditions, apoptosis can be blocked and non apoptotic cell death and irreversible growth arrest, namely senescence, can be activated as potential tumor-suppressor mechanism. Here we evaluated the dosage effects of HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) in a series of human leukaemia cell lines. We investigated, what concentration of SAHA and VPA can optimally induce apoptosis, growth inhibition or stress-induced premature senescence. We have found that SAHA inhibited proliferation and induced apoptosis in concentration 1000x lower than VPA. The senescence phenotype was preferentially induced by lower dosage of HDACi and required longer incubation time (5 days) while apoptosis was induced by higher dosage and appeared already after 24h. The optimal doses for the induction of cell death are 2,5-5 μM of SAHA and 2,5-5 mM of VPA. These doses of HDACi induce both apoptosis and senescence of studied leukemia cell lines.     

Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma (MM) cells in vitro irrespective of refractoriness to dexamethasone and chemotherapy. Because clinical trials with this anticancer agent are expected shortly, we investigated the signaling pathway of TRAIL-induced apoptosis in MM. We detected rapid cleavage of caspases-8, -9, -3, and -6, as well as the caspase substrates poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45 (DFF45), but not caspase-10, upon TRAIL treatment in sensitive MM cells, pointing to caspase-8 as the apical caspase of TRAIL signaling in MM cells. These phenomena were not observed or were significantly delayed in TRAIL-resistant MM cells, suggesting that resistance may arise from inhibition at the level of caspase-8 activation. Higher levels of expression for various apoptosis inhibitors, including FLICE-inhibitory protein (FLIP), and lower procaspase-8 levels were present in TRAIL-resistant cells and sensitivity was restored by the protein synthesis inhibitor cycloheximide (CHX) and the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), which both lowered FLIP and cellular inhibitor of apoptosis protein-2 (cIAP-2) protein levels. Forced expression of procaspase-8 or FLIP antisense oligonucleotides also sensitized TRAIL-resistant cells to TRAIL. Moreover, the cell permeable nuclear factor (NF)-kappaB inhibitor SN50, which sensitizes TRAIL-resistant cells to TRAIL, also inhibited cIAP2 protein expression. Finally, CHX, BIM, and SN50 facilitated the cleavage and activation of procaspase-8 in TRAIL-resistant cells, confirming that inhibition of TRAIL-induced apoptosis occurs at this level and that these agents sensitize MM cells by relieving this block. Our data set a framework for the clinical use of approaches that sensitize MM cells to TRAIL by agents that inhibit FLIP and cIAP-2 expression or augment caspase-8 activity.     

The histone deacetylase inhibitor SAHA arrests cancer cell growth,up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin

Suberoylanilide hydroxamic acid (SAHA) is a potent inhibitor of histone deacetylases (HDACs) that causes growth arrest, differentiation, and/or apoptosis of many tumor types in vitro and in vivo. SAHA is in clinical trials for the treatment of cancer. HDAC inhibitors induce the expression of less than 2% of genes in cultured cells. In this study we show that SAHA induces the expression of vitamin D-up-regulated protein 1/thioredoxin-binding protein-2 (TBP-2) in transformed cells. As the expression of TBP-2 mRNA is increased, the expression of a second gene, thioredoxin, is decreased. In transient transfection assays, HDAC inhibitors induce TBP-2 promoter constructs, and this induction requires an NF-Y binding site. We report here that TBP-2 expression is reduced in human primary breast and colon tumors compared with adjacent tissue. These results support a model in which the expression of a subset of genes (i.e., including TBP-2) is repressed in transformed cells, leading to a block in differentiation, and culture of transformed cells with SAHA causes re-expression of these genes, leading to induction of growth arrest, differentiation, and/or apoptosis.