Single-cell transcriptional analysis of human endothelial colony-forming cells from patients with low VWF levels.

von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.

Cellular Therapy for Children with Central Nervous System Tumors: Mining and Mapping the Correlative Data.

PURPOSE OF REVIEW: Correlative studies should leverage clinical trial frameworks to conduct biospecimen analyses that provide insight into the bioactivity of the intervention and facilitate iteration toward future trials that further improve patient outcomes. In pediatric cellular immunotherapy trials, correlative studies enable deeper understanding of T cell mobilization, durability of immune activation, patterns of toxicity, and early detection of treatment response. Here, we review the correlative science in adoptive cell therapy (ACT) for childhood central nervous system (CNS) tumors, with a focus on existing chimeric antigen receptor (CAR) and T cell receptor (TCR)-expressing T cell therapies. RECENT FINDINGS: We highlight long-standing and more recently understood challenges for effective alignment of correlative data and offer practical considerations for current and future approaches to multi-omic analysis of serial tumor, serum, and cerebrospinal fluid (CSF) biospecimens. We highlight the preliminary success in collecting serial cytokine and proteomics from patients with CNS tumors on ACT clinical trials.

The effects of ephrinB2 signaling on proliferation and invasion in glioblastoma multiforme.

The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane-bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti-invasive but pro-proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti-proliferative and anti-invasive effects, which correlated with decrease in PCNA, p-ERK, vimentin, Snail, Fak, and increase in the E-cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.

Targeting MYC-driven replication stress in medulloblastoma with AZD1775 and gemcitabine.

PURPOSE: MYC-driven medulloblastomas are highly aggressive childhood tumors with dismal outcomes and a lack of new treatment paradigms. We identified that targeting replication stress through WEE1 inhibition to suppress the S-phase replication checkpoint, combined with the attenuation of nucleotide synthesis with gemcitabine, is an effective strategy to induce apoptosis in MYC-driven medulloblastoma that could be rapidly translated into early phase clinical trials in children. Attenuation of replication stress is a key component of MYC-driven oncogenesis. Previous studies revealed a vulnerability in MYC medulloblastoma through WEE1 inhibition. Here, we focused on elucidating combinations of agents to synergize with WEE1 inhibition and drive replication stress toward cell death. METHODS: We first analyzed WEE1 expression in patient tissues by immunohistochemistry. Next, we used high-throughput drug screens to identify agents that would synergize with WEE1 inhibition. Synergy was confirmed by in vitro live cell imaging, ex vivo slice culture models, and in vivo studies using orthotopic and flank xenograft models. RESULTS: WEE1 expression was significantly higher in Group 3 and 4 medulloblastoma patients. The WEE1 inhibitor AZD1775 synergized with inhibitors of nucleotide synthesis, including gemcitabine. AZD1775 with gemcitabine suppressed proliferation and induced apoptosis. Ex vivo modeling demonstrated efficacy in Group 3 medulloblastoma patients, and in vivo modeling confirmed that combining AZD1775 and gemcitabine effectively suppressed tumor growth. CONCLUSION: Our results identified a potent new synergistic treatment combination for MYC-driven medulloblastoma that warrants exploration in early phase clinical trials.

Inhibition of MYC attenuates tumor cell self-renewal and promotes senescence in SMARCB1-deficient Group 2 atypical teratoid rhabdoid tumors to suppress tumor growth in vivo.

Loss of SMARCB1 is the hallmark genetic event that characterizes rhabdoid tumors in children. Rhabdoid tumors of the brain (ATRT) occur in young children and are particularly challenging with poor long-term survival. SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. Recent studies demonstrate that ATRT consists of 3 genomic subgroups with a subset of poor outcome tumors expressing high BMP and MYC pathway activation. Here we show that MYC occupies distinct promoter loci in ATRT compared to embryonic stem (ES) cells. Furthermore, using human ATRT cell lines, patient-derived cell culture, ex vivo patient-derived tumor, and orthotopic xenograft models, we show that MYC inhibition is a molecular vulnerability in SMARCB1-deleted tumors and that such inhibition effectively suppresses BMP and pluripotency-associated genomic programs, attenuates tumor cell self-renewal, promotes senescence, and inhibits ATRT tumor growth in vivo. Transgenic expression of Omomyc (a bona-fide MYC dominant negative) or chemical inhibition of MYC transcriptomic programs with the BET inhibitor JQ1 phenocopy genetic depletion of MYC, effectively restricting ATRT tumor growth and opening a promising therapeutic avenue for rhabdoid tumors in children.

Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma.

BACKGROUND: Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro. METHODS: The expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting. RESULTS: We observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others. CONCLUSIONS: Overall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models.

Histone deacetylation contributes to low extracellular superoxide dismutase expression in human idiopathic pulmonary arterial hypertension.

Epigenetic mechanisms, including DNA methylation and histone acetylation, regulate gene expression in idiopathic pulmonary arterial hypertension (IPAH). These mechanisms can modulate expression of extracellular superoxide dismutase (SOD3 or EC-SOD), a key vascular antioxidant enzyme, and loss of vascular SOD3 worsens outcomes in animal models of pulmonary arterial hypertension. We hypothesized that SOD3 gene expression is decreased in patients with IPAH due to aberrant DNA methylation and/or histone deacetylation. We used lung tissue and pulmonary artery smooth muscle cells (PASMC) from subjects with IPAH at transplantation and from failed donors (FD). Lung SOD3 mRNA expression and activity was decreased in IPAH vs. FD. In contrast, mitochondrial SOD (Mn-SOD or SOD2) protein expression was unchanged and intracellular SOD activity was unchanged. Using bisulfite sequencing in genomic lung or PASMC DNA, we found the methylation status of the SOD3 promoter was similar between FD and IPAH. Furthermore, treatment with 5-aza-2'-deoxycytidine did not increase PASMC SOD3 mRNA, suggesting DNA methylation was not responsible for PASMC SOD3 expression. Though total histone deacetylase (HDAC) activity, histone acetyltransferase (HAT) activity, acetylated histones, and acetylated SP1 were similar between IPAH and FD, treatment with two selective class I HDAC inhibitors increased SOD3 only in IPAH PASMC. Class I HDAC3 siRNA also increased SOD3 expression. Trichostatin A, a pan-HDAC inhibitor, decreased proliferation in IPAH, but not in FD PASMC. These data indicate that histone deacetylation, specifically via class I HDAC3, decreases SOD3 expression in PASMC and HDAC inhibitors may protect IPAH in part by increasing PASMC SOD3 expression.

Polo-like Kinase 1 as a potential therapeutic target in Diffuse Intrinsic Pontine Glioma.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive, fatal, childhood tumors that arise in the brainstem. DIPGs have no effective treatment, and their location and diffuse nature render them inoperable. Radiation therapy remains the only standard of care for this devastating disease. New therapeutic targets are needed to develop novel therapy for DIPG. METHODS: We examined the expression of PLK1 mRNA in DIPG tumor samples through microarray analysis and found it to be up regulated versus normal pons. Using the DIPG tumor cells, we inhibited PLK1 using a clinically relevant specific inhibitor BI 6727 and evaluated the effects on, proliferation, apoptosis, induction of DNA damage and radio sensitization of the DIPG tumor cells. RESULTS: Treatment of DIPG cell lines with BI 6727, a new generation, highly selective inhibitor of PLK1, resulted in decreased cell proliferation and a marked increase in cellular apoptosis. Cell cycle analysis showed a significant arrest in G2-M phase and a substantial increase in cell death. Treatment also resulted in an increased γH2AX expression, indicating induction of DNA damage. PLK1 inhibition resulted in radiosensitization of DIPG cells. CONCLUSION: These findings suggest that targeting PLK1 with small-molecule inhibitors, in combination with radiation therapy, will hold a novel strategy in the treatment of DIPG that warrants further investigation.

MicroRNA 218 acts as a tumor suppressor by targeting multiple cancer phenotype-associated genes in medulloblastoma.

Aberrant expression of microRNAs has been implicated in many cancers. We recently demonstrated differential expression of several microRNAs in medulloblastoma. In this study, the regulation and function of microRNA 218 (miR-218), which is significantly underexpressed in medulloblastoma, was evaluated. Re-expression of miR-218 resulted in a significant decrease in medulloblastoma cell growth, cell colony formation, cell migration, invasion, and tumor sphere size. We used C17.2 neural stem cells as a model to show that increased miR-218 expression results in increased cell differentiation and also decreased malignant transformation when transfected with the oncogene REST. These results suggest that miR-218 acts as a tumor suppressor in medulloblastoma. MicroRNAs function by down-regulating translation of target mRNAs. Targets are determined by imperfect base pairing of the microRNA to the 3'-UTR of the mRNA. To comprehensively identify actual miR-218 targets, medulloblastoma cells overexpressing miR-218 and control cells were subjected to high throughput sequencing of RNA isolated by cross-linking immunoprecipitation, a technique that identifies the mRNAs bound to the RNA-induced silencing complex component protein Argonaute 2. High throughput sequencing of mRNAs identified 618 genes as targets of miR-218 and included both previously validated targets and many targets not predicted computationally. Additional work further confirmed CDK6, RICTOR, and CTSB (cathepsin B) as targets of miR-218 and examined the functional role of one of these targets, CDK6, in medulloblastoma.

Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma.

BACKGROUND: Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. METHODS: To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor. RESULTS: Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent. CONCLUSIONS: Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma.

Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma.

MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening across MB cell lines, we identified the Mediator-associated kinase CDK8 as the top dependence for MYC-driven MB. Loss of CDK8 markedly reduces MYC expression and impedes MB growth. Mechanistically, we demonstrate that CDK8 depletion suppresses ribosome biogenesis and mRNA translation. CDK8 regulates occupancy of phospho-Polymerase II at specific chromatin loci facilitating an epigenetic alteration that promotes transcriptional regulation of ribosome biogenesis. Additionally, CDK8-mediated phosphorylation of 4EBP1 plays a crucial role in initiating eIF4E-dependent translation. Targeting CDK8 effectively suppresses cancer stem and progenitor cells, characterized by increased ribosome biogenesis activity. We also report the synergistic inhibition of CDK8 and mTOR in vivo and in vitro . Overall, our findings establish a connection between transcription and translation regulation, suggesting a promising therapeutic approach targets multiple points in the protein synthesis network for MYC-driven MB.

Inhibition of cyclin-dependent kinase 6 suppresses cell proliferation and enhances radiation sensitivity in medulloblastoma cells.

Medulloblastoma accounts for 20 % of all primary pediatric intracranial tumors. Current treatment cures 50-80 % of patients but is associated with significant long-term morbidity and thus new therapeutic targets are needed. One such target is cyclin-dependent kinase 6 (CDK6), a serine/threonine kinase that plays a vital role in cell cycle progression and differentiation. CDK6 is overexpressed in medulloblastoma patients and is associated with an adverse prognosis. To investigate the role of CDK6 in medulloblastoma, we assayed the effect of CDK6 inhibition on proliferation by depleting expression with RNA interference (RNAi) or by inhibiting kinase function with a small molecule inhibitor, PD0332991. Cell proliferation was assessed by colony focus assay or by the xCELLigence system. We then investigated the impact of CDK6 inhibition on differentiation of murine neural stem cells by immunofluorescence of relevant markers. Finally we evaluated the effects of PD0332991 treatment on medulloblastoma cell cycle and radiosensitivity using colony focus assays. Gene expression analysis revealed that CDK6 mRNA expression is higher than normal cerebellum in fifteen out of sixteen medulloblastoma patient samples. Inhibition of CDK6 by RNAi significantly decreased medulloblastoma cell proliferation and colony forming potential. Interestingly, CDK6 inhibition by RNAi increased differentiation in murine neural stem cells. PD0332991 treatment significantly decreased medulloblastoma cell proliferation and led to a G0/G1 cell cycle arrest. Furthermore, PD0332991 pretreatment sensitized medulloblastoma cells to ionizing radiation. Our findings suggest that targeting CDK6 with small molecule inhibitors may prove beneficial in the treatment of medulloblastoma, especially when combined with radiation.

Targeting B7­H3 through EZH2 inhibition in MYC­positive Group 3 medulloblastoma.

The most aggressive subtype of medulloblastoma (MB), Group 3, is characterized by MYC amplifications. However, targeting MYC has proven unsuccessful, and there remains a lack of therapeutic targets for treating MB. Studies have shown that the B7 homolog 3 (B7­H3) promotes cell proliferation and tumor cell invasion in a variety of cancers. Similarly, it was recently revealed that B7­H3 promotes angiogenesis in Group 3 MB and likely facilitates MB metastasis through exosome biogenesis. While therapies targeting B7­H3 remain in the early stages of development, targeting upstream regulators of B7­H3 expression may be more effective for halting MB progression. Notably, MYC and the enhancer of zeste homolog 2 (EZH2) are known to regulate B7­H3 expression, and a previous study by the authors suggested that B7­H3 amplifications present in MB are likely the result of EZH2­MYC mediated activities. In the present study, it was reported that overexpression of EZH2 is associated with lower overall survival in Group 3 MB patients. It was also revealed that inhibition of EZH2 significantly reduces B7­H3 and MYC transcript levels and upregulates miR­29a, indicating that EZH2 post­transcriptionally regulates B7­H3 expression in Group 3 MB cells. Pharmacological inhibition of EZH2 using EPZ005687 attenuated MB cell viability and reduced the expression of B7­H3. Similarly, pharmacological inhibition and knockdown of EZH2 led to the downregulation of MYC, B7­H3, and H3K27me3. Further, EZH2 silencing induced apoptosis and reduced colony­forming ability in MB cells, whereas EZH2 inhibition in MYC­amplified C17.2 neural stem cells induced G2/M phase arrest while downregulating B7­H3 expression. Collectively, the current study positions EZH2 as a viable target for the future development of MB treatments and that targeting EZH2 in combination with B7­H3 immunotherapy may be an effective treatment for halting MB progression.

The PARP inhibitor Rucaparib synergizes with radiation to attenuate atypical teratoid rhabdoid tumor growth.

Background: Atypical teratoid rhabdoid tumors (ATRT) are highly aggressive pediatric brain tumors. The available treatments rely on toxic chemotherapy and radiotherapy, which themselves can cause poor outcomes in young patients. Poly (ADP-ribose) polymerases (PARP), multifunctional enzymes which play an important role in DNA damage repair and genome stability have emerged as a new target in cancer therapy. An FDA-approved drug screen revealed that Rucaparib, a PARP inhibitor, is important for ATRT cell growth. This study aims to investigate the effect of Rucaparib treatment in ATRT. Methods: This study utilized cell viability, colony formation, flow cytometry, western blot, immunofluorescence, and immunohistochemistry assays to investigate Rucaparib's effectiveness in BT16 and MAF737 ATRT cell lines. In vivo, intracranial orthotopic xenograft model of ATRT was used. BT16 cell line was transduced with a luciferase-expressing vector and injected into the cerebellum of athymic nude mice. Animals were treated with Rucaparib by oral gavaging and irradiated with 2 Gy of radiation for 3 consecutive days. Tumor growth was monitored using In Vivo Imaging System. Results: Rucaparib treatment decreased ATRT cell growth, inhibited clonogenic potential of ATRT cells, induced cell cycle arrest and apoptosis, and led to DNA damage accumulation as shown by increased expression of γH2AX. In vivo, Rucaparib treatment decreased tumor growth, sensitized ATRT cells to radiation and significantly increased mice survival. Conclusion: We demonstrated that Rucaparib has potential to be a new therapeutic strategy for ATRT as seen by its ability to decrease ATRT tumor growth both in vitro and in vivo.

Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

Targeting the enhancer of zeste homologue 2 in medulloblastoma.

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors.

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

BACKGROUND: Medulloblastoma 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. METHODS: We 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. RESULTS: Analysis 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 initiating cells. CONCLUSIONS: Our 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.

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.