Vorinostat Treatment Research Articles

The Mechanism of Action of the Histone Deacetylase Inhibitor Vorinostat Involves Interaction with the Insulin-Like Growth Factor Signaling Pathway

A correlation between components of the insulin-like growth factor (IGF) system and endometrial cancer risk has been shown in recent studies. The antitumor action of vorinostat, a histone deacetylase inhibitor, involves changes in the expression of specific genes via acetylation of histones and transcription factors. The aim of this study was to establish whether vorinostat can modify the expression of specific genes related to the IGF-I receptor (IGF-IR) signaling pathway and revert the transformed phenotype. Human endometrioid (Type I, Ishikawa) and uterine serous papillary (Type II, USPC-2) endometrial cancer cell lines were treated with vorinostat in the presence or absence of IGF-I. Vorinostat increased IGF-IR phosphorylation, produced acetylation of histone H3, up-regulated pTEN and p21 expression, and reduced p53 and cyclin D1 levels in Ishikawa cells. Vorinostat up-regulated IGF-IR and p21 expression, produced acetylation of histone H3, and down-regulated the expression of total AKT, pTEN and cyclin D1 in USPC-2 cells. Of interest, IGF-IR activation was associated with a major elevation in IGF-IR promoter activity. In addition, vorinostat treatment induced apoptosis in both cell lines and abolished the anti-apoptotic activity of IGF-I both in the absence or presence of a humanized monoclonal IGF-IR antibody, MK-0646. Finally, vorinostat treatment led to a significant decrease in proliferation and colony forming capability in both cell lines. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cells, thus suggesting that endometrial cancer may be therapeutically targeted by vorinostat.

Vorinostat induced cellular stress disrupts the p38 mitogen activated protein kinase and extracellular signal regulated kinase pathways leading to apoptosis in Waldenström macroglobulinemia cells

Histone deacetylases (HDACs) are aberrantly expressed, and inhibitors of HDACs induce apoptosis in lymphoplasmacytic cells (LPCs) in Waldenström macroglobulinemia (WM). The molecular profile by which these agents induce apoptosis in WM LPCs remains to be delineated. We examined the activity of the histone deacetylase inhibitor, vorinostat, and dissected its pro-apoptotic pathways in WM LPCs. Vorinostat induced apoptosis in WM cells through activating specific caspases at varying times. Inhibitors of apoptosis (IAPs) were down-regulated after vorinostat treatment. Cellular stress induced in vorinostat-treated WM cells was reflected by changes in the mitogen activated protein kinase (MAPK) pathways. Activated phospho-p38 MAPK was up-regulated at 12 h, while phospho-extracellular signal-regulated kinase (Erk) abruptly decreased at 24 h. Bortezomib did not augment vorinostat induced primary WM cell killing as reported in other B-cell disorders. These studies support that stress induced apoptosis in vorinostat-treated WM LPCs is mediated through disrupting the activity of the Erk and p38 MAPK pathways.

HDAC inhibitor vorinostat enhances the antitumor effect of gefitinib in squamous cell carcinoma of head and neck by modulating ErbB receptor expression and reverting EMT

Potentiation of epidermal growth factor receptor (EGFR) inhibitors is required in squamous cell carcinoma of head and neck (SCCHN) to improve their therapeutic index. We demonstrated that the histone deacetylase inhibitor vorinostat in combination with the EGFR tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation, migration, and invasion as well as induction of apoptosis in SCCHN cells, including cells resistant to gefitinib. We provided evidence suggesting that differential modulation of ErbB receptors together with reversion of epithelial-to-mesenchymal transition (EMT) by vorinostat represent mechanistic bases for the observed synergism. We demonstrated in epithelial CAL27 cells expressing EGFR, ErbB2, and ErbB3 that vorinostat downregulated the expression and signaling of all three receptors. In gefitinib-resistant KB and Hep-2 cells, both of which had undergone EMT and expressed very low levels of ErbB3, vorinostat reverted the mesenchymal phenotype by inducing both E-cadherin and ErbB3 and downregulating vimentin as well as EGFR and ErbB2. Both transcriptional and post-translational mechanisms were involved in the modulation of ErbB receptors by vorinostat. Attenuation of all ErbB transcripts in CAL27 cells as well as induction of ErbB3 transcript in Hep-2 and KB cells was seen upon vorinostat treatment. We showed that vorinostat induced ubiquitination of EGFR and ErbB2 and targeted them predominantly to lysosome-degradation in all cell lines, while the induction of ErbB3-ubiquitination in CAL27 cells led to proteasomes-degradation. Overall, this study suggests that the vorinostat/gefitinib combination represents a promising therapeutic strategy that warrants further clinical evaluation in SCCHN, including tumors intrinsically resistant to EGFR-inhibitors.

Histone deacetylase inhibition attenuates diabetes-associated kidney growth: potential role for epigenetic modification of the epidermal growth factor receptor

Clinical trials and experimental studies have highlighted the importance of epigenetic processes in the development of diabetic complications. One of the earliest features of diabetic nephropathy is renal enlargement. The epidermal growth factor (EGF) has a pivotal role in the development of diabetic nephromegaly and transactivation of its receptor has been implicated in the pathogenesis of later-stage disease. As EGF signaling is altered by the acetylation status of histone proteins, we measured the effects of the histone deacetylase (HDAC) inhibitor, vorinostat, in mediating renal enlargement in diabetes focusing on the EGF-EGF receptor (EGFR) axis. In cultured proximal tubule (normal rat kidney) cells, vorinostat treatment reduced EGFR protein and mRNA, and attenuated cellular proliferation. Within 72 h of diabetes induction with streptozotocin, urinary EGF excretion was increased approximately threefold and was unaffected by vorinostat, even though the kidneys of vorinostat-treated diabetic rats had reduced tubular epithelial cell proliferation. Daily treatment of diabetic rats with vorinostat for 4 weeks blunted renal growth and glomerular hypertrophy. Thus, early renal changes in diabetes are amenable to epigenetic intervention. Attenuating effects of HDAC inhibition, although multifactorial, are likely to be mediated in part through downregulation of the EGFR.

Vorinostat modulates cell cycle regulatory proteins in glioma cells and human glioma slice cultures

Chromatin modification through histone deacetylase inhibition has shown evidence of activity against malignancies. The mechanism of action of such agents are pleiotropic and potentially tumor specific. In this study, we studied the mechanisms of vorinostat-induced cellular effects in gliomas. The effects of vorinostat on proliferation, induction of apoptosis and cell cycle effects were studied in vitro (D54, U87 and U373 glioma cell lines). To gain additional insights into its effects on human gliomas, vorinostat-induced changes were examined ex vivo using a novel organotypic human glioma slice model. Vorinostat treatment resulted in increased p21 levels in all glioma cells tested in a p53 independent manner. In addition, cyclin B1 levels were transcriptionally downregulated and resulted in reduced kinase activity of the cyclin B1/cdk1 complex causing a G2 arrest. These effects were associated with a dose- and time-dependent inhibition of cellular proliferation and anchorage-independent growth in association with hyperacetylation of core histones and induction of apoptosis. Of particular significance, we demonstrate histone hyperacetylation and increased p21 levels in freshly resected human glioma specimens maintained as organotypic slice cultures and exposed to vorinostat similar to cell lines suggesting that human glioma can be targeted by this agent. Our data suggest that the effects of vorinostat are associated with modulation of cell cycle related proteins and activation of a G2 checkpoint along with induction of apoptosis. These effects are mediated by both transcriptional and post-translational mechanisms which provide potential options that can be exploited to develop new therapeutic approaches against gliomas.

Long-term administration of the histone deacetylase inhibitor vorinostat attenuates renal injury in experimental diabetes through an endothelial nitric oxide synthase-dependent mechanism.

Epigenetic changes in gene expression play a role in the development of diabetic complications, including nephropathy. Histone deacetylases (HDACs) are a group of enzymes that exert epigenetic effects by altering the acetylation status of histone and nonhistone proteins. In the current study, we investigated the action of the clinically available HDAC inhibitor vorinostat in a mouse model of diabetic nephropathy, with the following aims: to define its effect on the progression of renal injury and to explore its mechanism of action by focusing on its role in regulating the expression of endothelial nitric oxide synthase (eNOS). Control and streptozotocin-diabetic wild-type and eNOS(-/-) mice were treated with vorinostat by daily oral dosing for 18 weeks. Without affecting either blood glucose concentration or blood pressure, vorinostat decreased albuminuria, mesangial collagen IV deposition, and oxidative-nitrosative stress in streptozotocin-wild-type mice. These attenuating effects were associated with a >50% reduction in eNOS expression in mouse kidneys and in cultured human umbilical vein endothelial cells. Vorinostat treatment had no effect on albuminuria, glomerular collagen IV concentration, or mesangiolysis in diabetic mice genetically deficient in eNOS. These observations illustrate the therapeutic efficacy of long-term HDAC inhibition in diabetic nephropathy and emphasize the importance of the interplay between eNOS activity and oxidative stress in mediating these effects.

Abstract 342: Murine transgenic lung cancer models guide a proof of principle histone deacetylase inhibitor trial in lung cancer patients

Abstract Lung cancer is the most common cause of cancer mortality in the United States. New treatments for lung cancer are therefore needed. We previously engineered murine transgenic cyclin E models that recapitulated frequent features of lung cancers in patients. These included chromosome instability, hedgehog pathway and cyclin D1 activation as well as single or multiple pre-malignant and malignant (adenocarcinoma) lung lesions. Metastases were also observed. Cell lines (ED-1 and ED-2) were derived from these murine lung cancers. After tail vein injection of ED-1 or ED-2 cells into syngeneic FVB mice, neoplastic lung lesions form within 1-2 weeks. These models are useful tools to identify promising anti-neoplastic agents. Among a panel of drugs that caused growth inhibition of lung cancer cells and repression of cyclin D1 protein, the histone deacetylase inhibitor (Vorinostat) was found as most potent. Vorinostat treatment caused dose- (1-5μM) and time-dependent (2 and 4 days) growth inhibition and apoptosis induction in murine lung cancer cells. Effects were reversed by washouts of Vorinostat. Similar reversible Vorinostat effects on growth inhibition and apoptosis were observed in human lung cancer cell lines (HOP62, H522 and H23). Vorinostat treatments repressed cyclin D1 and cyclin E proteins, but increased p27 expression in ED-1 cells. Notably, Vorinostat treatments of cyclin E transgenic mice also substantially repressed cyclin D1 expression in pre-malignant and malignant lung tissues. Vorinostat treatment also significantly reduced lung tumor formation (p = 0.047) in FVB mice harboring transplanted syngeneic murine lung cancer cells. To translate studies into the clinic, an Institutional Review Board-approved proof of principle Vorinostat trial was launched for early stage non-small cell lung cancer cases undergoing surgical resection. Patients receive Vorinostat treatments pre-operatively. Post- versus pre-treatment biopsies will be scored for changes in immunohistochemical profiles of cyclin D1, cyclin E, p27, and ki-67. Trial accrual ends January 2011. Results will then be analyzed with findings reported at this meeting. Taken together, Vorinostat treatments repressed lung cancer cell growth by augmenting apoptosis and reducing cyclin D1 expression. Substantial anti-neoplastic effects of Vorinostat were seen in murine transgenic and transplantable lung cancer models. These studies underscore the value of clinically-relevant murine lung cancer models for guiding clinical trials in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2011-342

Abstract 2609: Tissue transglutaminase (TG2) promotes resistance to HDAC inhibitor (HDI) vorinostat in cancer cells

Abstract Vorinostat has shown preclinical and clinical effects in human cancers and it is the first HDI approved by FDA for cutaneous T-cell-lymphoma treatment. TG2 is a multifunctional enzyme that catalyze a Ca2+-dependent transamidating reaction resulting in covalent cross-links between proteins. TG2 may also act as a G-protein in transmembrane signalling, as well as a cell surface adhesion mediator. TG2 upregulation has been demonstrated in several cancers and its expression levels correlate with resistance to chemotherapy and metastatic potential. In the present study, we demonstrated that the antiproliferative effects of vorinostat is paralleled by the induction of both TG2 mRNA and protein expression in breast, colorectal and oral cancer cell lines. This effect was also shared by other pan-HDIs and by class I HDIs but not by the specific inhibitor of HDAC-6. Vorinostat-induced TG2 upregulation correlated with an increased transamidating activity. Confocal microscopy analysis confirmed TG2 induction as well as TG2 cytosolic aggregation upon vorinostat treatment. Compartment analysis of TG2 protein expression revealed that vorinostat induces cytosolic, membrane and cytoskeleton but not nuclear upregulation of TG2. Apparently this effect seems confined to tumor cells, since same results were not observed in ex vivo vorinostat-treated peripheral blood lymphocytes from healthy donors. Notably high basal levels of TG2 protein correlated with lower antiproliferative activity of vorinostat. In order to confirm that TG2 play a role in vorinostat antitumor effect, we silenced by specific shRNA TG2 expression in high TG2 expressing HT29 colorectal cancer cells, and overexpressed full length TG2 in low TG2 expressing MCF7 breast cancer cells. We demonstrated that in TG2 silenced cells vorinostat antiproliferative and proapoptotic effects were enhanced whereas in TG2 overexpressed cells they were impaired, demonstrating that TG2 is directly involved in the mechanism of antitumor effect exerted by vorinostat. Moreover cotreatment of tumor cells with two specific inhibitors of TG2 transamidating activity (KCC009 and monodansylcadaverine) potentiated the antitumor effect of vorinostat, suggesting that TG2 cross-linking activity is important for vorinostat antitumor effect. Vorinostat also induced TG2 acetylation, however this posttranslational modification has no relevant effect on protein half-life as demonstrated by cotreatment with cycloheximide. Finally, vorinostat-resistant MCF-7 cell line selected by stepwise increasing concentrations of vorinostat up to 12 μM, significantly overexpressed TG2 protein compared with parental control cell line. Overall this study demonstrated that TG2 overexpression is a common mechanism of intrinsic or acquired resistance to vorinostat and that inhibition of TG2 transamidating activity may potentiate vorinostat antitumor effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2609. doi:10.1158/1538-7445.AM2011-2609

Abstract 2136: Expression of fusion proteins in acute myeloid leukemia cells increases sensitivity to histone deacetylase inhibitors

Abstract Acute myeloid leukemias (AMLs) are often characterized by chromosomal translocations resulting in the expression of fusion proteins (FP). Some FP have been demonstrated to recruit histone deacetylases (HDACs) and repress the expression of DNA repair genes. This may result in an increased sensitivity to histone deacetylase inhibitors (HDI) due to their ability to also down-regulate DNA repair gene expression and induce DNA damage. We have tested the sensitivity of PLZF-RARα, PML-RARα and AML1-ETO inducible cell lines to HDI in order to characterize changes in the mechanisms of cell death. Treatment of U937 cells with vorinostat results in DNA damage as measured by the COMET assay, followed by cell death. To test the effect of FP expression, U937 cells stably transfected with PLZF-RARα, PML-RARα or AML1-ETO cDNA under the control of a tetracycline-off or a zinc-inducible system were treated with vorinostat and assayed for cell death in the presence/absence of FP. FP expression resulted in increased cell death and caspase-3/7 activation. This effect was found to be largely caspase driven as pre-treatment with the pan-caspase inhibitor Z-VAD-FMK resulted in protection against vorinostat-induced cell death. In addition, FP expressing cells were also exposed to the DNA-targeting agents Doxorubicin, Etoposide, Cisplatin and ionizing radiation. Again, FP expression resulted in increased cell death. In addition, we investigated the effect of FP expression on vorinostat sensitivity using a PLZF-RARα murine hematopoietic model. A retroviral expression system was used to overexpress PLZF-RARα in lineage-depleted (Lin-) murine hematopoietic progenitors, followed by vorinostat treatment in the absence/presence of PLZF-RARα. The increased sensitivity of FP expressing cells to HDI and DNA-targeting agents suggests a mechanism where the combination of an FP expression and HDI results in an increased accumulation of DNA damage, leading to enhanced cell death. These findings are significant as they point to FP expressing AMLs as a target group that may respond better to HDI-based therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2136. doi:10.1158/1538-7445.AM2011-2136

Abstract 1932: Bortezomib sensitizes malignant human glioma cells to histone deacetylase inhibitors depends on reactive oxygen species production, mitochondrial dysfunction, Noxa upregulation and Mcl-1 cleavage

Abstract Glioblastomas are invasive tumors with poor prognosis despite current therapies. Unfortunately, individual targeted therapies have failed to offer long-term survival benefit. Histone deacetylase inhibitors (HDACIs) represent a class of agents that can modulate gene expression to reduce tumor growth. Suberoylanalide hydroxamic acid (SAHA, vorinostat) and LBH-589 are inhibitors of several members of the HDAC family, and we and others have noted some antiglioma activity, although insufficient to warrant use as single-agent therapy. Proteasome inhibitors represent a promising class of anticancer agents, already in clinical use, as bortezomib (PS-341/Velcade) has been approved for the treatment of multiple myeloma with inactivation of NF-κB signaling pathway. We have recently demonstrated that bortezomib exhibited potent anti-glioma activity and dramatically sensitized highly resistant glioma cells to TRAIL cytotoxicity, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to HDAC inhibition in glioma cells. These studies showed that bortezomib enhanced vorinostat and LBH-589-induced apoptosis in malignant glioma cells. Although vorinostat or LBH-589 treatment alone did not induce apoptosis in glioma cells, efficacy was enhanced by addition of bortezomib, which promoted activation of proapoptotic mitochondrial injury, generation of ROS, and inhibition of the prosurvival NF-κB signaling pathway. Our results also revealed that cotreatment with bortezomib and vorinostat enhanced apoptosis by increasing Mcl-1, Noxa cleavage and other pro-apoptotic protein expression. The significantly enhanced antitumor activity that results from the combination of bortezomib and HDACIs offers promise as a novel treatment for glioma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2011-1932

Vorinostat Increases Expression of Functional Norepinephrine Transporter in Neuroblastoma In Vitro and In Vivo Model Systems

Histone deacetylase (HDAC) inhibition causes transcriptional activation or repression of several genes that in turn can influence the biodistribution of other chemotherapeutic agents. Here, we hypothesize that the combination of vorinostat, a HDAC inhibitor, with (131)I-meta-iodobenzylguanidine (MIBG) would lead to preferential accumulation of the latter in neuroblastoma (NB) tumors via increased expression of the human norepinephrine transporter (NET). In vitro and in vivo experiments examined the effect of vorinostat on the expression of NET, an uptake transporter for (131)I-MIBG. Human NB cell lines (Kelly and SH-SY-5Y) and NB1691-luc mouse xenografts were employed. The upregulated NET protein was characterized for its effect on (123)I-MIBG biodistribution. Preincubation of NB cell lines, Kelly, and SH-SY-5Y, with vorinostat caused dose-dependent increases in NET mRNA and protein levels. Accompanying this was a corresponding dose-dependent increase in MIBG uptake in NB cell lines. Four- and 2.5-fold increases were observed in Kelly and SH-SY-5Y cells, respectively, pretreated with vorinostat in comparison to untreated cells. Similarly, NB xenografts, created by intravenous tail vein injection of NB1691-luc, and harvested from nude mice livers treated with vorinostat (150 mg/kg i.p.) showed substantial increases in NET protein expression. Maximal effect of vorinostat pretreatment in NB xenografts on (123)I-MIBG biodistribution was observed in tumors that exhibited enhanced uptake in vorinostat-treated [0.062 ± 0.011 μCi/(mg tissue-dose injected)] vs. -untreated mice [0.022 ± 0.003 μCi/(mg tissue-dose injected); P < 0.05]. The results of our study provide preclinical evidence that vorinostat treatment can enhance NB therapy with (131)I-MIBG.

Combination of vorinostat and adenovirus-TRAIL exhibits a synergistic antitumor effect by increasing transduction and transcription of TRAIL in lung cancer cells

Soluble TRAIL and adenovirus (ad)-TRAIL exhibit a strong antitumor effect by inducing apoptosis. Vorinostat is the histone deacetylase (HDAC) inhibitor that induces cell death in cancer cell lines and regulates the expression of epigenetically silenced genes, such as Coxackie adenoviral receptor (CAR), the receptor for adenoviral entry. We propose a new strategy in which vorinostat will induce high expression of ad-TRAIL and a strong antitumor response, and investigated the mechanism involved. The effect of vorinostat on transcription and expression of TRAIL from ad-TRAIL-transduced lung cancer cells were confirmed by reverse transciption-PCR (RT-PCR), quantitative real time-PCR and western blot assay. Anti-tumor effects were measured after cotreatment of vorinostat and ad-TRAIL, and the drug interactions were analyzed. After combined treatment of vorinostat and ad-TRAIL, apoptosis and western blot assays for Akt, Bcl-2 and caspase were performed. Vorinostat increased the expression of CAR in lung cancer cell lines and increased the expression of luciferase (luc) from ad-luc-transduced cells and TRAIL from ad-TRAIL-transduced cells. RT-PCR and quantitative real time-PCR, after sequential vorinostat treatment, revealed that vorinostat may enhance TRAIL expression from ad-TRAIL by increasing transduction through enhanced CAR expression and increasing adenoviral transgene transcription. Combined vorinostat and ad-TRAIL treatment showed the synergistic anti-tumor effect in lung cancer cell lines. Combined vorinostat and ad-TRAIL induced stronger apoptosis induction, suppression of NF-κB activation and breakdown of the anti-apoptotic molecule Bcl-2. In conclusion, the vorinostat synergistically enhanced the anti-tumor effect of ad-TRAIL by (1) increasing adenoviral transduction through the increased expression of CAR and (2) increasing adenoviral transgene (TRAIL) transcription in lung cancer cell lines.

The Histone Deacetylase Inhibitor, Vorinostat, Reduces Tumor Growth at the Metastatic Bone Site and Associated Osteolysis, but Promotes Normal Bone Loss

Vorinostat, an oral histone deacetylase inhibitor with antitumor activity, is in clinical trials for hematologic and solid tumors that metastasize and compromise bone structure. Consequently, there is a requirement to establish the effects of vorinostat on tumor growth within bone. Breast (MDA-231) and prostate (PC3) cancer cells were injected into tibias of SCID/NCr mice and the effects of vorinostat on tumor growth and osteolytic disease were assessed by radiography, micro-computed tomography, and histologic and molecular analyses. Vorinostat-treated and control mice without tumors were also examined. Tumor growth in bone was reduced ∼33% by vorinostat with inhibited osteolysis in the first few weeks of the experiment. However, osteolysis became more severe in both the vehicle and vorinostat-treated groups. Vorinostat increased the expression of tumor-derived factors promoting bone resorption, including PTHrP, IL-8, and osteopontin. After 4 weeks of vorinostat therapy, the non-tumor-bearing contralateral femurs and limbs from vorinostat-treated tumor-free SCID mice showed significant bone loss (50% volume density of controls). Thus, our studies indicate that vorinostat effectively inhibits tumor growth in bone, but has a negative systemic effect reducing normal trabecular bone mass. Vorinostat treatment reduces tumor growth in bone and accompanying osteolytic disease as a result of decreased tumor burden in bone. However, vorinostat can promote osteopenia throughout the skeleton independent of tumor cell activity.

Efficacy of Vorinostat In a Murine Model of Polycythemia Vera

AbstractAbstract 629The JAK2V617F mutation is present in most patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET), and primary myelofibrosis (PMF). Inhibitors of histone deacetylases (HDAC), which regulate gene transcription, cell cycle progression and programmed cell death, are emerging as a new class of potential anticancer drugs. A number of HDAC inhibitors are in clinical trials for various neoplasms. Vorinostat (also known as SAHA or Zolinza) was the first FDA approved HDAC inhibitor for clinical use in treating cutaneous T cell lymphoma (CTCL). In this study, we have investigated the efficacy of vorinostat in hematopoietic cells expressing JAK2V617F and in a murine model of PV. We found that proliferation of Ba/F3-EpoR cells expressing JAK2V617F or human erythroleukemia (HEL) cells harboring JAK2V617F mutation was significantly reduced upon treatment with vorinostat at 0.5–1.0 μM. However, proliferation of wild type JAK2 expressing BCR-ABL-transformed K562 cells was only modestly affected by vorinostat. Vorinostat treatment also resulted in marked apoptosis in HEL cells but not in wild-type JAK2 expressing K562 or UT7 cells as determined by annexin V staining and flow cytometry. Moreover, vorinostat treatment (at 1.0 μM) completely inhibited the clonogenic growth of Ba/F3-EpoR-JAK2V617F and HEL cells in a colony assay. Clonogenic growth of K562 cells was only modestly affected by vorinostat at 1.0 μM. Biochemical analyzes revealed significant inhibition of phosphorylation of JAK2, STAT5, Akt, Erk1/2 in JAK2V617F-positive HEL cells but not in wild-type JAK2-expressing K562 cells. Expression of JAK2 and STAT5 was also significantly downregulated by vorinostat treatment in HEL cells but not in K562 cells. In addition, we observed a marked reduction in c-Myc and Pim-1, and cleavage of caspase 3 and PARP in HEL cells, but not in K562 cells, upon treatment with vorinostat. These results suggest that vorinostat selectively targets JAK2V617F mutated cells and inhibits the activation/expression of JAK2 and STAT5. We reported previously the generation of an inducible JAK2V617F knock-in mouse (Akada et al., Blood 2010; 115: 3589–3597). Expression of the JAK2V617F in the knock-in mice reproducibly produced all the features of human PV. We have utilized this JAK2V617F knock-in mouse model to test the in vivo effects of vorinostat. We found that vorinostat treatment (200 mg/kg/day by IP injection) for 2 weeks significantly reduced the increase in red blood cells (RBC), hemoglobin and hematocrit as well as spleen size in mice expressing JAK2V617F compared with vehicle-treatment. Furthermore, vorinostat markedly inhibited the clonogenic growth of primary erythroid progenitors expressing JAK2V617F without exhibiting any significant toxicity towards wild-type JAK2-expressing normal hematopoietic progenitors in mice. Thus, our in vitro and in vivo results suggest that vorinostat may have therapeutic potential for treatment of PV. Disclosures:No relevant conflicts of interest to declare.

Activity of the Histone Deacetylase Inhibitor, Vorinostat, Against BCR-ABL Positive Leukemia Cells with Random Mutagenesis and In Combination with Nilotinib

AbstractAbstract 4460The clinical use of imatinib, a specific BCR-ABL tyrosine kinase inhibitor (TKI) is effective in inducing a complete hematological and cytogenetic remission in a high percentage of chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (ALL) patients. However, imatinib does not efficiently kill leukemic stem cells and is limited by the emergence of resistance due to the point mutations in the BCR-ABL kinase domain. Histone acetyltransferases (HAT) and histone deacetylases (HDAC) control the acetylation of histones and intracellular proteins, and regulate the transcription and function of the proteins. HDAC inhibitor is a structurally diverse class of targeted anti-cancer agent. One of the pan-HDAC inhibitor, vorinostat (suberoylanilide hydroxamic acid: SAHA) is a small-molecule inhibitor of most human class I and class II HDAC, and is reported the efficacy of malignant cells including lymphomas and myeloid malignancies.Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitor and an HDAC inhibitor, vorinostat may help prevent CML relapse due to BCR-ABL point mutation and may improve their long-term outcome. In this study, we investigated the efficacy of vorinostat by using the Ph-positive leukemia cell line, K562 and Ba/F3 BCR-ABL cell in a random mutagenesis study for BCR-ABL mutation. We first performed a comprehensive drug combination experiment using vorinostat and BCR-ABL tyrosine kinase inhibitor, imatinib or nilotinib. The treatment of imatinib or nilotinib exhibits cell growth inhibition partially against Ba/F3 BCR-ABL cell in a random mutagenesis. We also found the BCR-ABL point mutation such as T315I or M344V after 2 weeks nilotinib treatment by direct sequence analysis. We show that vorinostat potently induced cell growth inhibition of K562 and Ba/F3 BCR-ABL cells in a random mutagenesis in a dose dependent manner. Combined treatment of Ba/F3 BCR-ABL cell in a random mutagenesis with vorinostat and nilotinib or imatinib caused significantly more cytotoxicity than each drug alone by colony assay. We investigated the intracellular signaling of vorinostat. Phosphorylation of BCR-ABL, Crk-L were reduced after vorinostat treatment for 24 hours in a dose dependent manner. Caspase 3 and poly (ADP-ribose) polymerase (PARP) activation were increased after vorinostat treatment. Vorinostat potently enhanced cell growth inhibition of Ba/F3 BCR-ABL point mutants (G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T and H396P) compared with Ba/F3 expressing Wt BCR-ABL cells. The protein level of BCR-ABL was reduced after vorinostat treatment. BCR-ABL degradations in BCR-ABL mutant cells were significantly enhanced compared with Ba/F3 Wt BCR-ABL cells. Although long term culture of Ba/F3 BCR-ABL cell in a random mutagenesis with 2μ M vorinostat significantly decreased cell growth, the cells were increased after removal of vorinostat. We found these cells were wild type BCR-ABL by direct sequence analysis. Data from this study suggested that administration of the vorinostat may mediate its effects on BCR-ABL positive cells included BCR-ABL point mutation and enhance cytotoxic effects of nilotinib or imatinib in BCR-ABL mutant cells, and provide information of potential therapeutic relevance. Disclosures:Ohyashiki:Nippon Shinyaku Co., Ltd.: Research Funding.

Synergistic Killing Effect between Vorinostat and Target of CD146 in Malignant Cells

Although histone deacetylase inhibitors (HDACi) are emerging as a new class of anticancer agents, one of the most significant concerns is that interactions with a wide array of substrates using these agents might initiate both therapeutic and undesired protective responses. Here, we sought to identify the potential protective reactions initiated by HDACi and determine whether targeting these reactions would enhance the antitumoral activity of HDACi. Gene expression profiles were analyzed by cDNA microarray in Molt-4 cells before and after treatment of vorinostat. Induction of CD146 by vorinostat was examined in a wide range of tumors and nonmalignant cells. AA98, an anti-CD146 monoclonal antibody, was used to target CD146 function. Synergistic antitumoral and antiangiogenic effects between AA98 and vorinostat were examined both in vitro and in vivo. The potential effect of combined AA98 and vorinostat treatment on the AKT pathway was determined by Western blotting. The induction of CD146 is a common phenomenon in vorinostat-treated cancer but not in nonmalignant cells. Targeting of CD146 with AA98 substantially enhanced vorinostat-induced killing via the suppression of activation of AKT pathways in cancer cells. Moreover, AA98 in combination with vorinostat significantly inhibited angiogenesis. In vivo, AA98 synergized with vorinostat to inhibit tumor growth and metastasis. The present study provided the first evidence that an undesired induction of CD146 could serve as a protective response to offset the antitumor efficacy of vorinostat. On the other hand, targeting CD146 in combination with vorinostat could be exploited as a novel strategy to more effectively kill cancer cells.

Sorafenib Activates CD95 and Promotes Autophagy and Cell Death via Src Family Kinases in Gastrointestinal Tumor Cells

Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95; the present studies have determined how sorafenib and vorinostat individually contribute to CD95 activation. Sorafenib (3-6 micromol/L) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib-induced (3 micromol/L) CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 micromol/L), or sorafenib (3 micromol/L) and vorinostat (500 nmol/L) treatment promoted higher levels of CD95 phosphorylation which correlated with DISC formation, receptor surface localization, and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to localize plasma membrane or induce autophagy. Knockdown/knockout of Src family kinases abolished sorafenib-induced CD95 tyrosine phosphorylation, DISC formation, and the induction of cell death and autophagy. Knockdown of platelet-ived growth factor receptor-beta enhanced Src Y416 and CD95 tyrosine phosphorylation, which correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased reactive oxygen species levels, and in a delayed NF kappa B-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95; however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by approximately 50%. Thus, sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation, whereas vorinostat contributes to CD95 activation via the initial facilitation of reactive oxygen species generation and subsequently of FAS-L expression.

Vorinostat and Sorafenib Increase CD95 Activation in Gastrointestinal Tumor Cells through a Ca2+-De novo Ceramide-PP2A-Reactive Oxygen Species–Dependent Signaling Pathway

The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study, we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat, but not the individual drugs, rapidly increased reactive oxygen species (ROS), Ca(2+), and ceramide levels in gastrointestinal tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation, and cell killing was also blocked by quenching of induced Ca(2+) levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS; however, combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca(2+). Quenching of ROS did not affect drug-induced ceramide/dihydro-ceramide levels, whereas quenching of Ca(2+) reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knockdown of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings show that induction of cytosolic Ca(2+) by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.

Efficacy of MK-0457 and in combination with vorinostat against Philadelphia chromosome positive acute lymphoblastic leukemia cells

Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division and Aurora kinases are overexpressed in a number of human cancers. In this study, we examined the intracellular signaling of Aurora kinases inhibitor, MK-0457 (VX-680), in BCR-ABL positive cell lines and in primary samples. MK-0457 induced apoptosis. Caspase 3 and poly (ADP-ribose) polymerase (PARP) were activated and heat shock proteins were reduced. A combination of MK-0457 and histone deacetylase inhibitor, vorinostat, increased apoptosis. Caspase 3 and PARP were activated and phosphorylation of BCR-ABL, Lyn, and Crk-L were reduced. BCR-ABL and Aurora A and B were reduced after vorinostat treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the extent of apoptosis in primary acute lymphoblastic leukemia cells with T315I mutation. Our study increases insight into how MK-0457 may mediate its effects on BCR-ABL positive leukemia cells with T315I mutation, and information of potential therapeutic relevance.

Abstract LB-41: Histone deacetylase (HDAC) inhibition induces a resistance mechanism via the insulin-like growth factor-1 receptor (IGF-1R) signaling pathway: Rational basis for cotargeting of IGF-1R and HDAC in non-small cell lung cancer

Abstract Background: Histone deacetylase (HDAC), which modulate chromatin structure and gene expression, has attracted attention as a promising therapeutic target for hematologic and solid tumors including non-small cell lung cancer (NSCLC). However, the mechanisms resulting in the resistance to HDAC inhibitors are not yet well-known. In the current study, we investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in resistance to HDAC inhibitor in NSCLC.]\ Material and Methods: We assessed response of a panel of 14 NSCLC cells to vorinostat-based treatment by MTT assay. Because cells growing in monolayer on standard tissue culture plates could have different response to drug treatment compared with cells growing in vivo in three-dimensional environment, we also tested the effects of vorinostat on the cells cultured as spheroids on poly-HEMA coated plate or in soft agar. The antitumor effects of vorinostat, either alone or in combination with a novel anti-IGF-1R antibody, MK0646 (Merck) were evaluated in vitro using representative vorinostat-resistant NSCLC cell lines. The antitumor potency of the combination was evaluated in vivo using xenograft tumor models established in nude mice. The mice were treated twice a week with vorinostat (50 mg/kg), MK0646 (15 mg/kg) or their combination by intraperitoneal (IP) injection. Western blot, real time quantitative PCR, and immunohistochemical analyses were performed to assess biochemical changes, especially those related with IGF-1R signaling components. Results: Treatment with vorinostat at doses ranging from 0.5 to 50 μM exhibited a range of sensitivities after 3-day treatment. IGF-1R expression remained unchanged or increased by vorinostat treatment in relatively resistant cells while relatively sensitive cells showed decreased expression of IGF-1R after the drug treatment. Addition of MK0646 enhanced cytotoxic activities of vorinostat in relatively resistant cells. In xenograft model, combined treatment of vorinostat and MK0646 significantly delayed the growth of H1299 and H226Br xenograft tumors compared with vehicle or single agent treatment. Conclusions: Our results suggested that (a) resistance to vorinostat is related to induction expression and subsequent activation of IGF-1R and (b) integration of IGF-IR-targeted agents is needed to the treatment regimens with vorinostat for patients with lung cancer. Results from this study provide a proof-of principle for clinical trials of MK0646 and vorinostat combination in patients with vorinostat-resistant NSCLC. Background: Histone deacetylase (HDAC), which modulate chromatin structure and gene expression, has attracted attention as a promising therapeutic target for hematologic and solid tumors including non-small cell lung cancer (NSCLC). However, the mechanisms resulting in the resistance to HDAC inhibitors are not yet well-known. In the current study, we investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in resistance to HDAC inhibitor in NSCLC. Materials and Methods: We assessed response of a panel of 14 NSCLC cells to vorinostat-based treatment by MTT assay. Because cells growing in monolayer on standard tissue culture plates could have different response to drug treatment compared with cells growing in vivo in three-dimensional environment, we also tested the effects of vorinostat on the cells cultured as spheroids on poly-HEMA coated plate or in soft agar. The antitumor effects of vorinostat, either alone or in combination with a novel anti-IGF-1R antibody, MK0646 (Merck) were evaluated in vitro using representative vorinostat-resistant NSCLC cell lines. The antitumor potency of the combination was evaluated in vivo using xenograft tumor models established in nude mice. The mice were treated twice a week with vorinostat (50 mg/kg), MK0646 (15 mg/kg) or their combination by intraperitoneal (IP) injection. Western blot, real time quantitative PCR, and immunohistochemical analyses were performed to assess biochemical changes, especially those related with IGF-1R signaling components. Results: Treatment with vorinostat at doses ranging from 0.5 to 50 μM exhibited a range of sensitivities after 3-day treatment. IGF-1R expression remained unchanged or increased by vorinostat treatment in relatively resistant cells while relatively sensitive cells showed decreased expression of IGF-1R after the drug treatment. Addition of MK0646 enhanced cytotoxic activities of vorinostat in relatively resistant cells. In xenograft model, combined treatment of vorinostat and MK0646 significantly delayed the growth of H1299 and H226Br xenograft tumors compared with vehicle or single agent treatment. Conclusions: Our results suggested that (a) resistance to vorinostat is related to induction expression and subsequent activation of IGF-1R and (b) integration of IGF-IR-targeted agents is needed to the treatment regimens with vorinostat for patients with lung cancer. Results from this study provide a proof-of principle for clinical trials of MK0646 and vorinostat combination in patients with vorinostat-resistant NSCLC. Supported by the National Institutes of Health Grants R01 CA109520 (to H-Y. Lee) and CA100816 (to H-Y. Lee) and a Study Agreement with Merck and Company. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-41.