Leigh Ellis

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]

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.

Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma.

Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Eμ-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Eμ-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.

Targeting tumor angiogenesis with histone deacetylase inhibitors

Solid tumor malignancies including breast, lung and prostate carcinomas are considered to be angiogenesis dependent. Tumor angiogenesis is often mediated by hypoxia secondary to tumor growth or by increased oncogenic signaling. Both mechanisms result in increased hypoxia-inducible factor-1 alpha (HIF-1alpha) signaling and its transcriptional target vascular endothelial growth factor (VEGF). Critical to HIF-1alpha signaling are post translational modifications including acetylation mediated by histone acetyltransferases (HATS) and deacetylation by histone deacetylases (HDACs). More recently, HDACs were shown to be up-regulated in response to hypoxia mediating increased HIF-1alpha signaling. HDAC inhibitors represent a new class of anti-cancer therapeutics which show great promise at inhibiting angiogenesis in pre-clinical animal models and early phase clinical trials. This review will discuss the role of HIF-1alpha and VEGF influence on tumor angiogenesis and how HDACs play a critical role in HIF-1alpha transcriptional activity. Furthermore it will also be discussed how targeting HDACs via their inhibition create new avenues in treating solid malignancies by increasing the activity of established and novel therapeutic applications.

Reversible Epithelial to Mesenchymal Transition and Acquired Resistance to Sunitinib in Patients with Renal Cell Carcinoma: Evidence from a Xenograft Study

Tyrosine kinase inhibitors (TKI) targeting angiogenesis via inhibition of the vascular endothelial growth factor pathway have changed the medical management of metastatic renal cell carcinoma. Although treatment with TKIs has shown clinical benefit, these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. To address this question, we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response to sunitinib and eventually progressed with therapy. Tumor pieces were grafted s.c. in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density, and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor-derived cell line was established and assessed in vitro for potential direct antitumor effects of sunitinib. To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg, sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histologic examination of the original skin metastasis revealed evidence of epithelial to mesenchymal transition, whereas the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. In conclusion, the histologic examination in this xenograft study suggests that reversible epithelial to mesenchymal transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. Mol Cancer Ther; 9(6); 1525–35. ©2010 AACR.

SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer

Evading cancer drugs by identity fraud Prostate cancer growth is fueled by male hormones called androgens. Drugs targeting the androgen receptor (AR) are initially efficacious, but most tumors eventually become resistant (see the Perspective by Kelly and Balk). Mu et al. found that prostate cancer cells escaped the effects of androgen deprivation therapy through a change in lineage identity. Functional loss of the tumor suppressors TP53 and RB1 promoted a shift from AR-dependent luminal epithelial cells to AR-independent basal-like cells. In related work, Ku et al. found that prostate cancer metastasis, lineage switching, and drug resistance were driven by the combined loss of the same tumor suppressors and were accompanied by increased expression of the epigenetic regulator Ezh2. Ezh2 inhibitors reversed the lineage switch and restored sensitivity to androgen deprivation therapy in experimental models. Science, this issue p. 84, p. 78; see also p. 29 Prostate cancer cells escape androgen deprivation therapy by morphing into a cell type that does not require androgens. Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. We use in vitro and in vivo human prostate cancer models to show that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR)–dependent luminal epithelial cells to AR-independent basal-like cells. This lineage plasticity is enabled by the loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2, and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching.

Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance

Evading cancer drugs by identity fraud Prostate cancer growth is fueled by male hormones called androgens. Drugs targeting the androgen receptor (AR) are initially efficacious, but most tumors eventually become resistant (see the Perspective by Kelly and Balk). Mu et al. found that prostate cancer cells escaped the effects of androgen deprivation therapy through a change in lineage identity. Functional loss of the tumor suppressors TP53 and RB1 promoted a shift from AR-dependent luminal epithelial cells to AR-independent basal-like cells. In related work, Ku et al. found that prostate cancer metastasis, lineage switching, and drug resistance were driven by the combined loss of the same tumor suppressors and were accompanied by increased expression of the epigenetic regulator Ezh2. Ezh2 inhibitors reversed the lineage switch and restored sensitivity to androgen deprivation therapy in experimental models. Science, this issue p. 84, p. 78; see also p. 29 Prostate cancer cells escape androgen deprivation therapy by morphing into a cell type that does not require androgens. Prostate cancer relapsing from antiandrogen therapies can exhibit variant histology with altered lineage marker expression, suggesting that lineage plasticity facilitates therapeutic resistance. The mechanisms underlying prostate cancer lineage plasticity are incompletely understood. Studying mouse models, we demonstrate that Rb1 loss facilitates lineage plasticity and metastasis of prostate adenocarcinoma initiated by Pten mutation. Additional loss of Trp53 causes resistance to antiandrogen therapy. Gene expression profiling indicates that mouse tumors resemble human prostate cancer neuroendocrine variants; both mouse and human tumors exhibit increased expression of epigenetic reprogramming factors such as Ezh2 and Sox2. Clinically relevant Ezh2 inhibitors restore androgen receptor expression and sensitivity to antiandrogen therapy. These findings uncover genetic mutations that enable prostate cancer progression; identify mouse models for studying prostate cancer lineage plasticity; and suggest an epigenetic approach for extending clinical responses to antiandrogen therapy.

The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy

LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Emu-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-X(L) protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. Emu-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response.

Class I histone deacetylase inhibitor entinostat suppresses regulatory T cells and enhances immunotherapies in renal and prostate cancer models.

Background Immunosuppressive factors such as regulatory T cells (Tregs) limit the efficacy of immunotherapies. Histone deacetylase (HDAC) inhibitors have been reported to have antitumor activity in different malignancies and immunomodulatory effects. Herein, we report the Tregs-targeting and immune-promoting effect of a class I specific HDAC inhibitor, entinostat, in combination with either IL-2 in a murine renal cell carcinoma (RENCA) model or a survivin-based vaccine therapy (SurVaxM) in a castration resistant prostate cancer (CR Myc-CaP) model. Methods and Results RENCA or CR Myc-CaP tumors were implanted orthotopically or subcutaneously, respectively. Inoculated mice were randomized into four treatment groups: vehicle, entinostat, cytokine or vaccine, and combination. Tregs in the blood were assessed by FACS analysis. Real time quantitative PCR and Western blot analysis of isolated T cell subpopulations from spleen were performed to determine Foxp3 gene and protein expression. The suppressive function of Tregs was tested by T cell proliferation assay. Low dose (5 mg/kg) entinostat reduced Foxp3 levels in Tregs and this was associated with enhanced tumor growth inhibition in combination with either IL-2 or a SurVaxM vaccine. Entinostat down-regulated Foxp3 expression transcriptionally and blocked Tregs suppressive function without affecting T effector cells (Teffs). In vitro low dose entinostat (0.5 µM) induced STAT3 acetylation and a specific inhibitor of STAT3 partially rescued entinostat-induced down-regulation of Foxp3, suggesting that STAT3 signaling is involved in Foxp3 down-regulation by entinostat. Conclusions These results demonstrate a novel immunomodulatory effect of class I HDAC inhibition and provide a rationale for the clinical testing of entinostat to enhance cancer immunotherapy.

Histone Deacetylase Inhibitors: Advancing Therapeutic Strategies in Hematological and Solid Malignancies.

Advancement in the understanding of cancer development in recent years has identified epigenetic abnormalities as a common factor in both tumorigenesis and refractory disease. One such event is the dysregulation of histone deacetylases (HDACs) in both hematological and solid tumors, and has consequently resulted in the development of HDAC inhibitors (HDACI) to overcome this. HDACI exhibit pleiotropic biological effects including inhibition of angiogenesis and the induction of autophagy and apoptosis. Although HDACI exhibit modest results as single agents in preclinical and clinical data, they often fall short, and therefore HDACI are most promising in combinational strategies with either standard treatments or with other experimental chemotherapies and targeted therapies. This review will discuss the induction of autophagy and apoptosis and the inhibition of angiogenesis by HDACI, and also pre-clinical and clinical combination strategies using these agents.