Pineoblastoma segregates into molecular sub-groups with distinct clinico-pathologic features: a Rare Brain Tumor Consortium registry study.

Pineoblastomas (PBs) are rare, aggressive pediatric brain tumors of the pineal gland with modest overall survival despite intensive therapy. We sought to define the clinical and molecular spectra of PB to inform new treatment approaches for this orphan cancer. Tumor, blood, and clinical data from 91 patients with PB or supratentorial primitive neuroectodermal tumor (sPNETs/CNS-PNETs), and 2 pineal parenchymal tumors of intermediate differentiation (PPTIDs) were collected from 29 centres in the Rare Brain Tumor Consortium. We used global DNA methylation profiling to define a core group of PB from 72/93 cases, which were delineated into five molecular sub-groups. Copy number, whole exome and targeted sequencing, and miRNA expression analyses were used to evaluate the clinico-pathologic significance of each sub-group. Tumors designated as group 1 and 2 almost exclusively exhibited deleterious homozygous loss-of-function alterations in miRNA biogenesis genes (DICER1, DROSHA, and DGCR8) in 62 and 100% of group 1 and 2 tumors, respectively. Recurrent alterations of the oncogenic MYC-miR-17/92-RB1 pathway were observed in the RB and MYC sub-group, respectively, characterized by RB1 loss with gain of miR-17/92, and recurrent gain or amplification of MYC. PB sub-groups exhibited distinct clinical features: group 1-3 arose in older children (median ages 5.2-14.0 years) and had intermediate to excellent survival (5-year OS of 68.0-100%), while Group RB and MYC PB patients were much younger (median age 1.3-1.4 years) with dismal survival (5-year OS 37.5% and 28.6%, respectively). We identified age < 3 years at diagnosis, metastatic disease, omission of upfront radiation, and chr 16q loss as significant negative prognostic factors across all PBs. Our findings demonstrate that PB exhibits substantial molecular heterogeneity with sub-group-associated clinical phenotypes and survival. In addition to revealing novel biology and therapeutics, molecular sub-grouping of PB can be exploited to reduce treatment intensity for patients with favorable biology tumors.

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.

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.

Molecular subgroups of atypical teratoid rhabdoid tumours in children: an integrated genomic and clinicopathological analysis.

BACKGROUND: Rhabdoid brain tumours, also called atypical teratoid rhabdoid tumours, are lethal childhood cancers with characteristic genetic alterations of SMARCB1/hSNF5. Lack of biological understanding of the substantial clinical heterogeneity of these tumours restricts therapeutic advances. We integrated genomic and clinicopathological analyses of a cohort of patients with atypical teratoid rhabdoid tumours to find out the molecular basis for clinical heterogeneity in these tumours. METHODS: We obtained 259 rhabdoid tumours from 37 international institutions and assessed transcriptional profiles in 43 primary tumours and copy number profiles in 38 primary tumours to discover molecular subgroups of atypical teratoid rhabdoid tumours. We used gene and pathway enrichment analyses to discover group-specific molecular markers and did immunohistochemical analyses on 125 primary tumours to evaluate clinicopathological significance of molecular subgroup and ASCL1-NOTCH signalling. FINDINGS: Transcriptional analyses identified two atypical teratoid rhabdoid tumour subgroups with differential enrichment of genetic pathways, and distinct clinicopathological and survival features. Expression of ASCL1, a regulator of NOTCH signalling, correlated with supratentorial location (p=0·004) and superior 5-year overall survival (35%, 95% CI 13-57, and 20%, 6-34, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·033) in 70 patients who received multimodal treatment. ASCL1 expression also correlated with superior 5-year overall survival (34%, 7-61, and 9%, 0-21, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·001) in 39 patients who received only chemotherapy without radiation. Cox hazard ratios for overall survival in patients with differential ASCL1 enrichment treated with chemotherapy with or without radiation were 2·02 (95% CI 1·04-3·85; p=0·038) and 3·98 (1·71-9·26; p=0·001). Integrated analyses of molecular subgroupings with clinical prognostic factors showed three distinct clinical risk groups of tumours with different therapeutic outcomes. INTERPRETATION: An integration of clinical risk factors and tumour molecular groups can be used to identify patients who are likely to have improved long-term radiation-free survival and might help therapeutic stratification of patients with atypical teratoid rhabdoid tumours. FUNDING: C17 Research Network, Genome Canada, b.r.a.i.n.child, Mitchell Duckman, Tal Doron and Suri Boon foundations.

Characterization of distinct immunophenotypes across pediatric brain tumor types.

Despite increasing evidence that antitumor immune control exists in the pediatric brain, these findings have yet to be exploited successfully in the clinic. A barrier to development of immunotherapeutic strategies in pediatric brain tumors is that the immunophenotype of these tumors' microenvironment has not been defined. To address this, the current study used multicolor FACS of disaggregated tumor to systematically characterize the frequency and phenotype of infiltrating immune cells in the most common pediatric brain tumor types. The initial study cohort consisted of 7 pilocytic astrocytoma (PA), 19 ependymoma (EPN), 5 glioblastoma (GBM), 6 medulloblastoma (MED), and 5 nontumor brain (NT) control samples obtained from epilepsy surgery. Immune cell types analyzed included both myeloid and T cell lineages and respective markers of activated or suppressed functional phenotypes. Immune parameters that distinguished each of the tumor types were identified. PA and EPN demonstrated significantly higher infiltrating myeloid and lymphoid cells compared with GBM, MED, or NT. Additionally, PA and EPN conveyed a comparatively activated/classically activated myeloid cell-skewed functional phenotype denoted in particular by HLA-DR and CD64 expression. In contrast, GBM and MED contained progressively fewer infiltrating leukocytes and more muted functional phenotypes similar to that of NT. These findings were recapitulated using whole tumor expression of corresponding immune marker genes in a large gene expression microarray cohort of pediatric brain tumors. The results of this cross-tumor comparative analysis demonstrate that different pediatric brain tumor types exhibit distinct immunophenotypes, implying that specific immunotherapeutic approaches may be most effective for each tumor type.

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.

Molecular sub-group-specific immunophenotypic changes are associated with outcome in recurrent posterior fossa ependymoma.

Better understanding of ependymoma (EPN) biology at relapse is needed to improve therapy at this critical event. Convincing data exist defining transcriptionally distinct posterior fossa (PF) sub-groups A and B at diagnosis. The clinical and biological consequence of these sub-groups at recurrence has not yet been defined. Genome and transcriptome microarray profiles and clinical variables of matched primary and first recurrent PF EPN pairs were used to identify biologically distinct patterns of progression between EPN sub-groups at recurrence. Key findings were validated by histology and immune function assays. Transcriptomic profiles were partially conserved at recurrence. However, 4 of 14 paired samples changed sub-groups at recurrence, and significant sub-group-specific transcriptomic changes between primary and recurrent tumors were identified, which were predominantly immune-related. Further examination revealed that Group A primary tumors harbor an immune gene signature and cellular functionality consistent with an immunosuppressive phenotype associated with tissue remodeling and wound healing. Conversely, Group B tumors develop an adaptive, antigen-specific immune response signature and increased T-cell infiltration at recurrence. Clinical distinctions between sub-groups become more apparent after first recurrence. Group A tumors were more often sub-totally resected and had a significantly shorter time to subsequent progression and worse overall survival. Minimal tumor-specific genomic changes were observed for either PF Groups A or B at recurrence. Molecular sub-groups of PF EPN convey distinct immunobiologic signatures at diagnosis and recurrence, providing potential biologic rationale to their disparate clinical outcomes. Immunotherapeutic approaches may be warranted, particularly in Group A PF EPN.

Increased immune gene expression and immune cell infiltration in high-grade astrocytoma distinguish long-term from short-term survivors.

Survival in the majority of high-grade astrocytoma (HGA) patients is very poor, with only a rare population of long-term survivors. A better understanding of the biological factors associated with long-term survival in HGA would aid development of more effective therapy and survival prediction. Factors associated with long-term survival have not been extensively studied using unbiased genome-wide expression analyses. In the current study, gene expression microarray profiles of HGA from long-term survivors were interrogated for discovery of survival-associated biological factors. Ontology analyses revealed that increased expression of immune function-related genes was the predominant biological factor that positively correlated with longer survival. A notable T cell signature was present within this prognostic immune gene set. Using immune cell-specific gene classifiers, both T cell-associated and myeloid linage-associated genes were shown to be enriched in HGA from long-term versus short-term survivors. Association of immune function and cell-specific genes with survival was confirmed independently in a larger publicly available glioblastoma gene expression microarray data set. Histology was used to validate the results of microarray analyses in a larger cohort of long-term survivors of HGA. Multivariate analyses demonstrated that increased immune cell infiltration was a significant independent variable contributing to longer survival, as was Karnofsky/Lansky performance score. These data provide evidence of a prognostic anti-tumor adaptive immune response and rationale for future development of immunotherapy in HGA.

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.

Pediatric rhabdoid tumors of kidney and brain show many differences in gene expression but share dysregulation of cell cycle and epigenetic effector genes.

BACKGROUND: Rhabdoid tumors (RTs) are aggressive tumors of early childhood that occur most often in brain (AT/RTs) or kidney (KRTs). Regardless of location, they are characterized by loss of functional SMARCB1 protein, a component of the SWI/SNF chromatin remodeling complex. The aim of this study was to determine genes and biological process dysregulated in common to both AT/RTs and KRTs. PROCEDURE: Gene expression for AT/RTs was compared to that of other brain tumors and normal brain using microarray data from our lab. Similar analysis was performed for KRTs and other kidney tumors and normal kidney using data from GEO. Dysregulated genes common to both analyses were analyzed for functional significance. RESULTS: Unsupervised hierarchical clustering of RTs identified three major subsets: two comprised of AT/RTs, and one of KRTs. Compared to other tumors, 1,187, 663, and 539 genes were dysregulated in each subset, respectively. Only 14 dysregulated genes were common to all three subsets. Compared to normal tissue, 5,209, 4,275, and 2,841 genes were dysregulated in each subset, with an overlap of 610 dysregulated genes. Among these genes, processes associated with cell proliferation, MYC activation, and epigenetic dysregulation were common to all three RT subsets. CONCLUSIONS: The low overlap of dysregulated genes in AT/RTs and KRTs suggests that factors in addition to SMARCB1 loss play a role in determining subsequent gene expression. Drugs which target cell cycle or epigenetic genes may be useful in all RTs. Additionally, targeted therapies tailored to specific RT subset molecular profiles should be considered.

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.

Pediatric brainstem gangliogliomas show overexpression of neuropeptide prepronociceptin (PNOC) by microarray and immunohistochemistry.

BACKGROUND: Gangliogliomas (GGs) primary to brainstem are rare, with the overwhelming majority of GGs occurring in supratentorial, especially temporal lobe, locations. A less favorable prognosis exists for brainstem GGs, despite their usually identical WHO grade I status. Few large clinical series, and limited biological information, exists on these tumors, especially gene expression. PROCEDURE: Seven pediatric brainstem GGs, all with classic histological features, seen at our institution since 2000 were identified. Frozen section material was available for gene expression microarray profiling from five of seven brainstem GGs and compared with that from three non-brainstem pediatric GGs. RESULTS: Significant upregulation of a number of genes was identified, most of which were involved in pathways of neural signaling, embryonic development, and pattern specification in pediatric brainstem GGs compared to non-brainstem. The single largest upregulated gene was a 256-fold increase in the expression of the neuropeptide prepronociceptin (PNOC); the protein product of this gene has been implicated in neuronal growth. Overexpression was validated by Western blot and by immunohistochemistry (IHC). Strong IHC expression of PNOC was seen in neoplastic neurons of 7/7 brainstem GGs, but was significantly weaker in non-brainstem GGs, and completely negative in normal pediatric autopsy brainstem controls. CONCLUSIONS: PNOC IHC was often superior to IHC for NeuN, synaptophysin, or neurofilament for highlighting neoplastic neurons.

Survey of MicroRNA expression in pediatric brain tumors.

BACKGROUND: A better understanding of pediatric brain tumor biology is needed to assist in the development of less toxic therapies and to provide better markers for disease stratification. MicroRNAs (miRNA) may play a significant role in brain tumor biology. The present study provides an initial survey of miRNA expression in pediatric central nervous system (CNS) malignancies including atypical teratoid/rhabdoid tumor, ependymoma, glioblastoma, medulloblastoma, and pilocytic astrocytoma. PROCEDURE: MicroRNA expression in pediatric brain tumors and normal tissue controls was examined by microarray. Three aberrantly expressed miRNAs were further studied in a larger cohort by quantitative real-time PCR (qRT-PCR). RESULTS: MicroRNA-129, miR-142-5p, and miR-25 were differentially expressed in every pediatric brain tumor type compared to normal tissue controls as measured by microarray. When further examined by qRT-PCR, these miRNAs demonstrated differential expression that significantly correlated with the microarray findings. Distinctive miRNA expression profiles were also observed in the different pediatric brain tumor types. CONCLUSIONS: MicroRNAs are differentially expressed between pediatric CNS neoplasms and normal tissue suggesting that they may play a significant role in oncogenesis. A greater understanding of aberrant miRNA expression in pediatric brain tumors may aid in the development of novel therapies. The characterization of tumor-specific miRNA signatures may aid in the discovery of biomarkers with diagnostic or prognostic utility.

Immune gene and cell enrichment is associated with a good prognosis in ependymoma.

Approximately 50% of children with ependymoma will suffer from tumor recurrences that will ultimately lead to death. Development of more effective therapies and patient stratification in ependymoma mandates better prognostication. In this study, tumor gene expression microarray profiles from pediatric ependymoma clinical samples were subject to ontological analyses to identify outcome-associated biological factors. Histology was subsequently used to evaluate the results of ontological analyses. Ontology analyses revealed that genes associated with nonrecurrent ependymoma were predominantly immune function-related. Additionally, increased expression of immune-related genes was correlated with longer time to progression in recurrent ependymoma. Of those genes associated with both the nonrecurrent phenotype and that positively correlated with time to progression, 95% were associated with immune function. Histological analysis of a subset of these immune function genes revealed that their expression was restricted to a subpopulation of tumor-infiltrating cells. Analysis of tumor-infiltrating immune cells showed increased infiltration of CD4(+) T cells in the nonrecurrent ependymomas. No genomic sequences for SV40, BK, JC, or Merkel polyomaviruses were found in nonrecurrent ependymoma. This study reveals that up-regulation of immune function genes is the predominant ontology associated with a good prognosis in ependymoma and it provides preliminary evidence of a beneficial host proinflammatory and/or Ag-specific immune response.

Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma.

Cyclin D1 (CCND1) is a well-known regulator of cell-cycle progression. It is overexpressed in several types of cancer including breast, lung, squamous, neuroblastoma, and lymphomas. The most well-known mechanism of overexpression is the t(11;14)(q13;q32) translocation found in mantle cell lymphoma (MCL). It has previously been shown that truncated CCND1 mRNA in MCL correlates with poor prognosis. We hypothesized that truncations of the CCND1 mRNA alter its ability to be down-regulated by microRNAs in MCL. MicroRNAs are a new class of abundant small RNAs that play important regulatory roles at the posttranscriptional level by binding to the 3' untranslated region (UTR) of mRNAs blocking either their translation or initiating their degradation. In this study, we have identified the truncation in CCND1 mRNA in MCL cell lines. We also found that truncated CCND1 mRNA leads to increased CCND1 protein expression and increased S-phase cell fraction. Furthermore, we demonstrated that this truncation alters miR-16-1 binding sites, and through the use of reporter constructs, we were able to show that miR-16-1 regulates CCND1 mRNA expression. This study introduces the role of miR-16-1 in the regulation of CCND1 in MCL.

Inhibition of Aurora Kinase A enhances chemosensitivity of medulloblastoma cell lines.

BACKGROUND: Medulloblastoma comprises approximately 20% of all primary pediatric brain tumors. Despite recent advances, the survival rate for high-risk patients and the morbidity associated with these treatments remains suboptimal. To improve outcomes and decrease morbidity, more targeted therapy is required. One possible target is the Aurora Kinase family. The objective of this study was to evaluate the impact of Aurora Kinase A inhibition in medulloblastoma cell lines. PROCEDURE: Cell proliferation was measured using an MTS assay after adding an Aurora Kinase inhibitor (C1368) at different concentrations. Cell cycle analysis was carried out by Flow Cytometry using propidium iodide (PI). RNAi experiments were performed using siRNA oligonucleotides. Luciferase experiments were carried out using the Cignal Finder 10 Pathway Reporter Arrays. RESULTS: Inhibition of Aurora Kinase A induces cell death in medulloblastoma cells and lowers the IC(50) of other chemotherapeutic agents (etoposide and cisplatin) used in medulloblastoma treatment. Cell arrest at G2/M phase was significantly increased in medulloblastoma cell lines treated with C1368 Sigma at IC(30) or transfected siRNA. Inhibition of Aurora Kinase A resulted in decreased activity of pro-proliferative signaling pathways including Wnt, Myc, and RB as measured by luciferase reporter assays. CONCLUSIONS: These data indicate that inhibition of Aurora Kinase A inhibits cell growth in medulloblastoma through inhibition of pro-proliferative signaling pathways Wnt, Myc, and RB. Additionally, combining Aurora Kinase A inhibition with other chemotherapeutic agents significantly lowers their IC(50), which make it a promising small molecule target for medulloblastoma therapy.

Senescence Induced by BMI1 Inhibition Is a Therapeutic Vulnerability in H3K27M-Mutant DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor of childhood characterized by histone mutations at lysine 27, which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. Using a combined RNAi and chemical screen targeting epigenomic regulators, we identify the polycomb repressive complex 1 (PRC1) component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy is enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 decreases cell self-renewal and attenuates tumor growth due to induction of senescence. Prolonged BMI1 inhibition induces a senescence-associated secretory phenotype, which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to enhance tumor cell killing in vivo.

Exosomal microRNA profiling to identify hypoxia-related biomarkers in prostate cancer.

Hypoxia and expression of hypoxia-related biomarkers are associated with disease progression and treatment failure in prostate cancer (PCa). We have reported that exosomes (nanovesicles of 30-150 nm in diameter) secreted by human PCa cells under hypoxia promote invasiveness and stemness in naïve PCa cells. Here, we identified the unique microRNAs (miRNAs) loaded in exosomes secreted by PCa cells under hypoxia. Using TaqMan® array microRNA cards, we analyzed the miRNA profile in exosomes secreted by human PCa LNCaP cells under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions. We identified 292 miRNAs loaded in both ExoHypoxic and ExoNormoxic. The top 11 miRNAs with significantly higher level in ExoHypoxic compared to ExoNormoxic were miR-517a, miR-204, miR-885, miR-143, miR-335, miR-127, miR-542, miR-433, miR-451, miR-92a and miR-181a; and top nine miRNA with significantly lower expression level in ExoHypoxic compared to ExoNormoxic were miR-521, miR-27a, miR-324, miR-579, miR-502, miR-222, miR-135b, miR-146a and miR-491. Importantly, the two differentially expressed miRNAs miR-885 (increased expression) and miR-521 (decreased expression) showed similar expression pattern in exosomes isolated from the serum of PCa patients compared to healthy individuals. Additionally, miR-204 and miR-222 displayed correlated expression patterns in prostate tumors (Pearson R = 0.66, p < 0.0001) by The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) genomic dataset analysis. Overall, the present study identified unique miRNAs with differential expression in exosomes secreted from hypoxic PCa cells and suggests their potential usefulness as a biomarker of hypoxia in PCa patients.

Targeting Polo-like kinase 1 in SMARCB1 deleted atypical teratoid rhabdoid tumor.

Atypical teratoid rhabdoid tumor (ATRT) is an aggressive and malignant pediatric brain tumor. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and essential for mitosis. Overexpression of PLK1 promotes chromosome instability and aneuploidy by overriding the G2-M DNA damage and spindle checkpoints. Recent studies suggest that targeting PLK1 by small molecule inhibitors is a promising approach to tumor therapy. We investigated the effect of PLK1 inhibition in ATRT. Gene expression analysis showed that PLK1 was overexpressed in ATRT patient samples and tumor cell lines. Genetic inhibition of PLK1 with shRNA potently suppressed ATRT cell growth in vitro. Treatment with the PLK1 inhibitor BI 6727 (Volasertib) significantly decreased cell growth, inhibited clonogenic potential, and induced apoptosis. BI6727 treatment led to G2-M phase arrest, consistent with PLK1's role as a critical regulator of mitosis. Moreover, inhibition of PLK1 by BI6727 suppressed the tumor-sphere formation of ATRT cells. Treatment also significantly decreased levels of the DNA damage proteins Ku80 and RAD51 and increased γ-H2AX expression, indicating that BI 6727 can induce DNA damage. Importantly, BI6727 significantly enhanced radiation sensitivity of ATRT cells. In vivo, BI6727 slowed growth of ATRT tumors and prolonged survival in a xenograft model. PLK1 inhibition is a compelling new therapeutic approach for treating ATRT, and the use of BI6727 should be evaluated in clinical studies.