Jeffrey Knipstein

Inhibition of EZH2 suppresses self-renewal and induces radiation sensitivity in atypical rhabdoid teratoid tumor cells.

INTRODUCTION Overexpression of the Polycomb repressive complex 2 (PRC2) subunit Enhancer of Zeste 2 (EZH2) occurs in several malignancies, including prostate cancer, breast cancer, medulloblastoma, and glioblastoma multiforme. Recent evidence suggests that EZH2 may also have a role in rhabdoid tumors. Atypical teratoid/rhabdoid tumor (ATRT) is a rare, high-grade embryonal brain tumor that occurs most commonly in young children and carries a very poor prognosis. ATRTs are characterized by absence of the chromatin remodeling protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes, we investigated the role of EZH2 in ATRT. METHODS Microarray analysis was used to evaluate expression of EZH2 in ATRT tumor samples. We used shRNA and a chemical inhibitor of EZH2 to examine the impact of EZH2 inhibition on cell growth, proliferation, and tumor cell self-renewal. RESULTS Here, we show that targeted disruption of EZH2 by RNAi or pharmacologic inhibition strongly impairs ATRT cell growth, suppresses tumor cell self-renewal, induces apoptosis, and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity, we found the cyclin D1-E2F axis to be repressed after EZH2 depletion in ATRT cells. CONCLUSIONS Our observations provide evidence that EZH2 disruption alters cell cycle progression and may be an important new therapeutic target, particularly in combination with radiation, in ATRT.

Polo-like kinase 1 (PLK1) inhibition suppresses cell growth and enhances radiation sensitivity in medulloblastoma cells

BackgroundMedulloblastoma is the most common malignant brain tumor in children and remains a therapeutic challenge due to its significant therapy-related morbidity. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and regulates critical steps in mitotic progression. Recent studies suggest that targeting PLK1 with small molecule inhibitors is a promising approach to tumor therapy.MethodsWe examined the expression of PLK1 mRNA in medulloblastoma tumor samples using microarray analysis. The impact of PLK1 on cell proliferation was evaluated by depleting expression with RNA interference (RNAi) or by inhibiting function with the small molecule inhibitor BI 2536. Colony formation studies were performed to examine the impact of BI 2536 on medulloblastoma cell radiosensitivity. In addition, the impact of depleting PLK1 mRNA on tumor-initiating cells was evaluated using tumor sphere assays.ResultsAnalysis of gene expression in two independent cohorts revealed that PLK1 mRNA is overexpressed in some, but not all, medulloblastoma patient samples when compared to normal cerebellum. Inhibition of PLK1 by RNAi significantly decreased medulloblastoma cell proliferation and clonogenic potential and increased cell apoptosis. Similarly, a low nanomolar concentration of BI 2536, a small molecule inhibitor of PLK1, potently inhibited cell growth, strongly suppressed the colony-forming ability, and increased cellular apoptosis of medulloblastoma cells. Furthermore, BI 2536 pretreatment sensitized medulloblastoma cells to ionizing radiation. Inhibition of PLK1 impaired tumor sphere formation of medulloblastoma cells and decreased the expression of SRY (sex determining region Y)-box 2 (SOX2) mRNA in tumor spheres indicating a possible role in targeting tumor inititiating cells.ConclusionsOur data suggest that targeting PLK1 with small molecule inhibitors, in combination with radiation therapy, is a novel strategy in the treatment of medulloblastoma that warrants further investigation.

Entinostat for treatment of solid tumors and hematologic malignancies

Introduction: A key feature of malignant cells is inappropriate gene suppression resulting in uncontrolled proliferation, continued cell cycling and a lack of differentiation. Histone deacetylase inhibitors (HDACi) are an emerging class of antineoplastic agents that counteract this effect and thus permit re-expression of silenced genes. Entinostat is an emerging HDACi that has shown promise in multiple preclinical studies. Additionally, Phase I and II clinical trials have begun to demonstrate its potential as a well-tolerated agent with anti-tumor activity. Areas covered: The pharmacokinetics, pharmacodynamics, mechanisms of action, safety and tolerability, and clinical trials of entinostat are reviewed. Sources for this review included all relevant, publicly available, entinostat-related peer-reviewed publications and meeting abstracts up to March 2011. Expert opinion: Entinostat is a well-tolerated HDACi that demonstrates promising therapeutic potential in both solid and hematologic malignancies. Its efficacy does not appear directly dose-related, and as such, more relevant biomarkers are needed to adequately assess its activity. Future clinical trials will likely focus on its use in combination with other agents that are able to exploit the epigenetic changes rendered by deacetylase inhibition.

Targeting Aurora Kinase A enhances radiation sensitivity of atypical teratoid rhabdoid tumor cells

Atypical teratoid/rhabdoid tumors (ATRT) are rare, highly malignant, embryonal CNS tumors with a poor prognosis. Therapy relies on highly toxic chemotherapy and radiotherapy. To improve outcomes and decrease morbidity, more targeted therapy is required. Gene expression analysis revealed elevated expression of multiple kinases in ATRT tissues. Aurora Kinase A was one of the candidate kinases. The objective of this study was to evaluate the impact of Aurora Kinase A inhibition in ATRT cell lines. Our analysis revealed that inhibition of Aurora Kinase A induces cell death in ATRT cells and the small molecule inhibitor MLN 8237 sensitizes these cells to radiation. Furthermore, inhibition of Aurora Kinase A resulted in decreased activity of pro-proliferative signaling pathways. These data indicate that inhibition of Aurora Kinase A is a promising small molecule target for ATRT therapy.

Histone deacetylase inhibition decreases proliferation and potentiates the effect of ionizing radiation in atypical teratoid/rhabdoid tumor cells

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant central nervous system neoplasm that primarily occurs in children less than 3 years of age. Because of poor outcomes with intense and toxic multimodality treatment, new therapies are urgently needed. Histone deacetylase inhibitors (HDIs) have been evaluated as novel agents for multiple malignancies and have been shown to function as radiosensitizers. They act as epigenetic modifiers and lead to re-expression of inappropriately repressed genes, proteins, and cellular functions. Because of the underlying chromatin remodeling gene mutation in ATRT, HDIs are ideal candidates for therapeutic evaluation. To evaluate the role of HDIs against ATRT in vitro, we assessed the effect of drug treatment on proliferation, apoptosis, and gene expression. In addition, we examined HDI pretreatment as a radiosensitization strategy for ATRT. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium with phenazine methosulfate electron coupling reagent (MTS) and clonogenic assays demonstrated that HDI treatment significantly reduces the proliferative capacity of BT-12 and BT-16 ATRT cells. In addition, the HDI SNDX-275 was able to induce apoptosis in both cell lines and induced p21(Waf1/Cip1) protein expression as measured by Western blot. Evaluation of differential gene expression by microarray and pathway analysis after HDI treatment demonstrated alterations of several key ATRT cellular functions. Finally, we showed that HDI pretreatment effectively potentiates the effect of ionizing radiation on ATRT cells as measured by clonogenic assay. Our findings suggest that the addition of HDIs to ATRT therapy may prove to be beneficial, especially when administered in combination with current treatment modalities, such as radiation.

Sweet syndrome in an infant with chronic granulomatous disease.

Sweet syndrome is characterized by painful, erythematous cutaneous lesions containing neutrophilic infiltrates. Although more commonly seen in adults, Sweet syndrome has also been recognized in several pediatric cases. Two previous cases of pediatric Sweet syndrome and 1 adult case have been described in chronic granulomatous disease (CGD) patients. We report the case of an infant with known CGD who was presented with methicillin-sensitive Staphylococcus aureus lymphadenitis and subsequently developed Sweet syndrome. CGD patients are prone to several disorders of inflammation. This case illustrates that Sweet syndrome may be part of the spectrum of inflammatory conditions to which CGD patients are predisposed.

Extraspinal sacrococcygeal ependymoma masquerading as sacrococcygeal teratoma in the pediatric patient

Ependymoma is a glial cell tumor that arises from the ependymal lining of the central nervous ventricular system [1]. These tumors are most commonly seen in children and account for 10% of all central nervous system tumors [1, 2]. There have been rare cases of extraspinal sacrococcygeal ependymomas reported in the literature. These tumors are thought to originate from a group of heterotopic ependymal cells called the coccygeal medullary vestige [3]. While these tumors are often initially misdiagnosed as pilonidal disease, the differential diagnosis also includes congenital tumors such as chordoma, teratoma, and dermoid cysts. This case describes the presentation of an extradural sacrococcygeal subcutaneous ependymoma misdiagnosed as a sacrococcygeal teratoma.