Peter Atadja

Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation

Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.

Epigenetics in cancer: targeting chromatin modifications.

Posttranslational modifications to histones affect chromatin structure and function resulting in altered gene expression and changes in cell behavior. Aberrant gene expression and altered epigenomic patterns are major features of cancer. Epigenetic changes including histone acetylation, histone methylation, and DNA methylation are now thought to play important roles in the onset and progression of cancer in numerous tumor types. Indeed dysregulated epigenetic modifications, especially in early neoplastic development, may be just as significant as genetic mutations in driving cancer development and growth. The reversal of aberrant epigenetic changes has therefore emerged as a potential strategy for the treatment of cancer. A number of compounds targeting enzymes that regulate histone acetylation, histone methylation, and DNA methylation have been developed as epigenetic therapies, with some demonstrating efficacy in hematological malignancies and solid tumors. This review highlights the roles of epigenetic modifications to histones and DNA in tumorigenesis and emerging epigenetic therapies being developed for the treatment of cancer. [Mol Cancer Ther 2009;8(6):1409–20]

Development of the pan-DAC inhibitor panobinostat (LBH589): Successes and challenges

The histone deacetylase (HDAC) inhibitors are emerging as a highly useful class of anticancer agents that inhibit the enzyme HDAC involved in the deacetylation of histone and non-histone cellular proteins. The HDAC inhibitor, panobinostat (LBH589, Novartis Pharmaceuticals), achieves potent inhibition of all HDAC enzymes implicated in cancer and has demonstrated potent anti-tumor activity in preclinical models and promising clinical efficacy in cancer patients. In this review we discuss the successes and challenges surrounding the development of panobinostat, focusing on its proposed mechanism of action, preclinical anti-tumor activity, and early clinical efficacy in hematologic and solid tumors.

Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34(+) bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.

Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: the Hydroxamic Acid Derivative LBH589

Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase (HDAC) inhibitors have been shown to have activity against various tumor cell types by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589. Materials and Methods: To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion assays in vitro, and Matrigel plug assay in vivo. To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo. To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we did Western blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done to evaluate new blood vessel formation in vivo. Results: LBH589 induced acetylation of histone H3 and α-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated with induction of G2-M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth. Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.

Histone Deacetylase Inhibitor Panobinostat Induces Clinical Responses with Associated Alterations in Gene Expression Profiles in Cutaneous T-Cell Lymphoma

Purpose: Histone deacetylase inhibitors can alter gene expression and mediate diverse antitumor activities. Herein, we report the safety and activity of the histone deacetylase inhibitor panobinostat (LBH589) in cutaneous T-cell lymphoma (CTCL) and identify genes commonly regulated by panobinostat. Experimental Design: Panobinostat was administered orally to patients with CTCL on Monday, Wednesday, and Friday of each week on a 28-day cycle. A dose of 30 mg was considered excessively toxic, and subsequent patients were treated at the expanded maximum tolerated dose of 20 mg. Biopsies from six patients taken 0, 4, 8, and 24 h after administration were subjected to microarray gene expression profiling and real-time quantitative PCR of selected genes. Results: Patients attained a complete response (n = 2), attained a partial response (n = 4), achieved stable disease with ongoing improvement (n = 1), and progressed on treatment (n = 2). Microarray data showed distinct gene expression response profiles over time following panobinostat treatment, with the majority of genes being repressed. Twenty-three genes were commonly regulated by panobinostat in all patients tested. Conclusions: Panobinostat is well tolerated and induces clinical responses in CTCL patients. Microarray analyses of tumor samples indicate that panobinostat induces rapid changes in gene expression, and surprisingly more genes are repressed than are activated. A unique set of genes that can mediate biological responses such as apoptosis, immune regulation, and angiogenesis were commonly regulated in response to panobinostat. These genes are potential molecular biomarkers for panobinostat activity and are strong candidates for the future assessment of their functional role(s) in mediating the antitumor responses of panobinostat.

Class II Histone Deacetylases Are Associated with VHL-Independent Regulation of Hypoxia-Inducible Factor 1α

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1 alpha protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1 alpha inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1 alpha protein via a VHL-independent mechanism and reduction of HIF-1 alpha transcriptional activity. HIF-1 alpha inhibition by LAQ824 was associated with HIF-1 alpha acetylation and polyubiquitination. HIF-1 alpha immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1 alpha. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1 alpha at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1 alpha only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1 alpha protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1 alpha protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1 alpha stability and provide a rationale for targeting HIF-1 alpha with HDAC inhibitors against class II isozymes.

The Histone Deacetylase Inhibitor LBH589 Is a Potent Antimyeloma Agent that Overcomes Drug Resistance

Multiple myeloma represents an incurable disease, for which development of new therapies is required. Here, we report the effect on myeloma cells of LBH589, a new hydroxamic acid-derived histone deacetylase inhibitor. LBH589 was a potent antimyeloma agent (IC(50) < 40 nmol/L) on both cell lines and fresh cells from multiple myeloma patients, including cells resistant to conventional chemotherapeutic agents. In addition, LBH589 potentiated the action of drugs, such as bortezomib, dexamethasone, or melphalan. Using gene array, quantitative PCR, and Western analyses, we observed that LBH589 affected a large number of genes involved in cell cycle and cell death pathways. LBH589 blocked cell cycle progression, and this was accompanied by p21, p53, and p57 up-regulation. LBH589 induced cell death through an increase in the mitochondrial outer membrane permeability. LBH589 favored apoptosome formation by inducing cytochrome c release, Apaf-1 up-regulation, and caspase-9 cleavage. In addition, LBH589 stimulated a caspase-independent pathway through the release of AIF from the mitochondria. LBH589 down-regulated Bcl-2 and particularly Bcl-X. Moreover, overexpression of Bcl-X in multiple myeloma cells prevented LBH589-induced cell death. All these data indicate that LBH589 could be a useful drug for the treatment of multiple myeloma patients.

The histone deacetylase inhibitor NVP-LAQ824 Inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584

Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. Vascular endothelial growth factor (VEGF) and VEGF receptors represent critical molecular targets for antiangiogenesis therapy. In this study, we investigated the biological effect of the histone deacetylase inhibitor NVP-LAQ824 in combination with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on tumor growth and angiogenesis. We report that treatment with NVP-LAQ824 affected tumor and endothelial cells and was associated with increased histone acetylation, p21 up-regulation, and growth inhibition. In addition, NVP-LAQ824 treatment inhibited the expression of angiogenesis-related genes such as angiopoietin-2, Tie-2, and survivin in endothelial cells and down-regulated hypoxia-inducible factor 1-α and VEGF expression in tumor cells. Combination treatment with NVP-LAQ824 and PTK787/ZK222584 was more effective than single agents in inhibiting in vitro and in vivo VEGF-induced angiogenesis. Endothelial cell proliferation, tube formation, and invasion into the Matrigel plugs were reduced. In mouse models with established subcutaneous prostate (PC3) and orthotopic breast tumors (MDA-MB321), this combination treatment induced 80 to 85% inhibition of tumor growth without overt toxicity. These results suggest that the combination of histone deacetylase inhibitors and VEGF receptor inhibitors may target multiple pathways in tumor progression and angiogenesis and represents a novel therapeutic approach in cancer treatment.

Cotreatment with Histone Deacetylase Inhibitor LAQ824 Enhances Apo-2L/Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Induced Death Inducing Signaling Complex Activity and Apoptosis of Human Acute Leukemia Cells

Present studies demonstrate that treatment with the histone deacetylases inhibitor LAQ824, a cinnamic acid hydroxamate, increased the acetylation of histones H3 and H4, as well as induced p21WAF1 in the human T-cell acute leukemia Jurkat, B lymphoblast SKW 6.4, and acute myelogenous leukemia HL-60 cells. This was associated with increased accumulation of the cells in the G1 phase of the cell cycle, as well as accompanied by the processing and activity of caspase-9 and -3, and apoptosis. Exposure to LAQ824 increased the mRNA and protein expressions of the death receptors DR5 and/or DR4, but reduced the mRNA and protein levels of cellular FLICE-inhibitory protein (c-FLIP). As compared with treatment with Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or LAQ824 alone, pretreatment with LAQ824 increased the assembly of Fas-associated death domain and caspase-8, but not of c-FLIP, into the Apo-2L/TRAIL-induced death-inducing signaling complex. This increased the processing of caspase-8 and Bcl-2 interacting domain (BID), augmented cytosolic accumulation of the prodeath molecules cytochrome-c, Smac and Omi, as well as led to increased activity of caspase-3 and apoptosis. Treatment with LAQ824 also down-regulated the levels of Bcl-2, Bcl-xL, XIAP, and survivin. Partial inhibition of apoptosis due to LAQ824 or Apo-2L/TRAIL exerted by Bcl-2 overexpression was reversed by cotreatment with LAQ824 and Apo-2L/TRAIL. Significantly, cotreatment with LAQ824 increased Apo-2L/TRAIL-induced apoptosis of primary acute myelogenous leukemia blast samples isolated from 10 patients with acute myelogenous leukemia. Taken together, these findings indicate that LAQ824 may have promising activity in augmenting Apo-2L/TRAIL-induced death-inducing signaling complex and apoptosis of human acute leukemia cells.