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Background Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. Methods Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. Results Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. Conclusion Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Medulloblastoma is the most common type of pediatric malignant primary brain tumor, and about one-third of patients die due to disease recurrence and most survivors suffer from long-term side effects. MB is clinically, genetically, and epigenetically heterogeneous and subdivided into at least four molecular subgroups: WNT, SHH, Group 3, and Group 4. We evaluated common differentially expressed genes between a Brazilian RNA-seq GSE181293 dataset and microarray GSE85217 dataset cohort of pediatric MB samples using bioinformatics methodology in order to identify hub genes of the molecular subgroups based on PPI network construction, survival and functional analysis. The main finding was the identification of five hub genes from the WNT subgroup that are tumor suppressors, and whose lower expression is related to a worse prognosis for MB patients. Furthermore, the common genes correlated with the five tumor suppressors participate in important pathways and processes for tumor initiation and progression, as well as development and differentiation, and some of them control cell stemness and pluripotency. These genes have not yet been studied within the context of MB, representing new important elements for investigation in the search for therapeutic targets, prognostic markers or for understanding of MB biology.

The Group 3 Medulloblastoma (Grp3-MB) is an aggressive molecular subtype with a high incidence of metastasis and deaths. In this study, were used an RNA sequencing data (RNA-Seq) from a Brazilian cohort of MBs to identify hub genes associated with the metastatic risk. Data validation were performed by using multiple large datasets from MBs (GSE85217, GSE37418, and EGAS00001001953). DESeq2 package in R software was used to identify the differentially expressed genes (DEGs) in our RNA-Seq data. The DEGs data were accessed to construct the modules/graphs of co-expression and to identify hub genes through Cytoscape platform. The coregulated genes were enriched by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and the protein-protein interaction (PPI) network was visualized by Cytoscape. The Kaplan-Meier plotter and ROC curves were used to validate the diagnostic and prognostic values of specific biomarkers identified through this model. We identified that inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) as a downregulated hub gene, with a high diagnostic accuracy to Grp3-MBs and associated with tumor metastasis. In addition, we identified genes significantly correlated with ITPR1 that were associated with metastasis in Grp3-MB (ATP1A2, MTTL7A, and RGL1) and worst overall survival in MBs (ANTXR1 and RGL1). Our findings suggest that the ITPR1 hub gene is potentially involved in the metastatic process for Grp3-MB. Our data also provide evidence of targets that may serve as prognostic predictors and/or regulators for the metastatic process that maybe explored for further research of individualized therapy to Grp3-MBs.

Groups (Grp) 3 and 4 are aggressive molecular subgroups of medulloblastoma (MB), with high rates of leptomeningeal dissemination. To date, there is still a paucity of biomarkers for these subtypes of MBs. The RNA-binding protein Musashi-1 (MSI1) is a neural stem cell marker, characterized as a gene translation regulator and associated with high oncogenicity in several human cancers. In this study, we investigated the clinical significance and biological functions of MSI1 in Grp3/Grp4-MBs. First, we assessed the expression profile of MSI1 in 59 primary MB samples (15-WNT, 18-SHH, 9-Grp3, 17-Grp4 subgroups) by qRT-PCR. MSI1 mRNA expression levels were also validated in an additional public dataset of MBs (GSE85217). The ROC curve was used to validate the diagnostic standards of MSI1 expression. Cell cycle, cell viability, and apoptosis were evaluated in D283 Med cell-line (Grp3/Grp4/MBs) after shRNA-mediated knockdown of MSI1 plus cisplatin treatment. We identified an overexpression of MSI1 with a high accuracy to discriminate Grp3/Grp4-MBs from non- Grp3/Grp4-MBs. In addition, MSI1 knockdown promoted cell cycle interruption in the G1/S transition and, consequently, decreased the number of cells in the G2/M phase, repressed cell proliferation and sensitized D283 Med cells to cisplatin treatment by enhancing cell apoptosis. The results of the present study are the first to demonstrate that MSI1 may play a role as biomarker for Grp3/Grp4-MBs. In addition, MSI1 knockdown combined with cisplatin may offer a potential strategy to be further explored in Grp3/Grp4-MBs.

We evaluated the potential effects of ATO in different pediatric SHH-MB cell lines (ONS-76: TP53-wild type; DAOY and UW402: TP53-mutated). MB cell lines molecular subgroup was confirmed and TP53 mutations were validated. Cell viability, clonogenicity and apoptosis were evaluated after ATO treatment at different concentrations (1–16 µM) alone or combined with irradiation doses (0.5, 1, 2 and 4 Gy). Rad51 and Ku86 proteins were evaluated by WB. ATO treatment reduced cell viability for all SHH-MB cell lines. Significant decrease of clonogenic capacity and higher apoptosis rates were also observed after ATO exposure, being cell death more pronounced (>70%) for the SHH-MB TP53-mutated. Combined treatment of ATO with irradiation also reduced colonies formation in UW402 tumor cells, which was independent of DNA damage repair proteins Rad51 and Ku86. In silico analyses suggested that a set of genes from cell cycle and p53 pathways are differentially expressed in SHH tumor subtypes, suggesting that cell lines may respond to therapies according to the gene expression profiles. Herein, we showed ATO cytotoxicity in pediatric SHH cell lines, with marked radiosensitizing effect for the MB-SHH TP53-mutated cells. These results highlight the potential of ATO, alone or in combination with radiotherapy, supporting further clinical investigations.

Glioblastoma is the most common aggressive primary brain tumor. Standard care includes maximal safe surgical resection, radiation, and chemotherapy with temozolomide. However, the impact of this therapeutic approach on patient survival is disappointing and poor outcomes are frequently observed. Therefore, new therapeutic targets are needed to treat this potentially deadly tumor. Aurora kinases are one of today's most sought-after classes of therapeutic targets to glioblastoma therapy. They are a family of proteins composed of three members: Aurora-A, Aurora-B, and Aurora-C that play different roles in the cell division through regulation of chromosome segregation. Deregulation of these genes has been reported in glioblastoma and a progressive number of studies have shown that inhibition of these proteins could be a promising strategy for the treatment of this tumor. This review discusses the preclinical and early clinical findings on the potential use of the Aurora kinases as new targets for the treatment of glioblastoma. KEY MESSAGES: GBM is a very aggressive tumor with limited therapeutic options. Aurora kinases are a family of serine/threonine kinases implicated in GBM pathology. Aurora kinases are critical for glioblastoma cell growth, apoptosis, and chemoresistance. Inhibition of Aurora kinases has a synergistic or sensitizing effect with chemotherapy drugs, radiotherapy, or with other targeted molecules in GBM. Several Aurora kinase inhibitors are currently in clinical trials.

Patients diagnosed with acute lymphoblastic leukemia (ALL) bearing t(4;11)/MLL-AF4 have aggressive clinical features, poor prognosis and there’s an urgent need for new therapies to improve outcomes. Panobinostat (LBH589) has been identified as a potential therapeutic agent for ALL with t(4;11) and studies suggest that the antineoplastic effects is associated with reduced MLL-AF4 fusion protein and reduced expression of HOX genes. Here, we evaluated the in vitro effects of the combination of LBH589 with methotrexate (MTX) or 6-mercaptopurine (6MP) by cell proliferation assays and Calcusyn software in ALL cell line (RS4;11); the in vivo effects of LBH589 in xenotransplanted NOD-scid IL2Rgammanull mice meansuring human lymphoblasts by flow cytometry; and the expression of HOX genes by qPCR after treatment in an adult model of ALL with t(4;11). LBH589 combination with MTX or 6MP did not promote synergistic effects in RS4;11 cell line. LBH589 treatment leads to increased overall survival and reduction of blasts in xenotransplanted mice but caused no significant changes in HOXA7, HOXA9, HOXA10, and MEIS1 expression. The LBH589, alone, showed promising antineoplastic effects in vivo and may represent a potential agent for chemotherapy in ALL patients with t(4;11).

Patients diagnosed with acute lymphoblastic leukemia (ALL) bearing t(4;11)/MLL-AF4 have aggressive clinical features, poor prognosis and there is an urgent need for new therapies to improve outcomes. Panobinostat (LBH589) has been identified as a potential therapeutic agent for ALL with t(4;11) and studies suggest that the antineoplastic effects are associated with reduced MLL-AF4 fusion protein and reduced expression of HOX genes. Here, we evaluated the in vitro effects of the combination of LBH589 with methotrexate (MTX) or 6-mercaptopurine (6MP) by cell proliferation assays and Calcusyn software in ALL cell line (RS4;11); the in vivo effects of LBH589 in xenotransplanted NOD-scid IL2Rgamma

Background: Medulloblastoma, a genetically heterogeneous tumor, is the most frequent malignant brain tumor in children. Although several studies have been carried out, the molecular mechanism underlying medulloblastoma tumorigenesis is not completely known. microRNA (miRNA) expression profiles have been associated with development, progression, and prognosis of human cancers, including medulloblastoma. However, the role of miRNAs in pediatric medulloblastoma has been poorly explored.Methods: Global miRNA expression in 24 microdissected medulloblastoma specimens (19 pediatric and 5 adult specimens) was evaluated by microarray assay. miR-512-3p, the most differentially expressed miRNA in these two groups, was analyzed by qRT-PCR in a cohort of 51 consecutive pediatric medulloblastoma samples and 7 pediatric non-neoplastic cerebellum control samples, and its clinical significance was assessed. Further in silico miRNA prediction of target genes was performed with bioinformatics tools.Results: Compared to the controls, miR-512-3p was significantly downregulated in the pediatric medulloblastoma samples. Higher miR-512-3p was associated with incomplete degree of resection, high risk group classification, and poor overall survival. In silico analysis in an independent cohort of medulloblastoma identified that some of the miR-512-3p target genes (SMAD9, SSX2IP, MAPK10, PTCH1, CCDC6, and BMPR2) were statistically correlated with overall survival, metastasis, and death.Conclusions: For the first time, our results have shown that miR-512-3p is significantly associated with poor clinical outcome in pediatric medulloblastoma, suggesting that miR-512-3p is a potential biomarker of prognosis.

The Group 3 Medulloblastoma ( Grp3-MB ) is an aggressive molecular subtype with a high incidence of metastasis and deaths. In this study, were used an RNA sequencing data ( RNA-Seq ) from a Brazinian cohort of MBs to identify hub genes associated with the metastatic risk. Data validation were performed by using multiple large datasets from MBs (GSE85217, GSE37418, EGAS00001001953). DESeq2 package in R software was used to identify the differentially expressed genes ( DEGs ) in our RNA-Seq data. The DEGs data were accessed to construct the modules/graphs of coexpression and to identify hub genes through Cytoscape platform. The co-regulated genes were enriched by the Kyoto Encyclopedia of Genes and Genomes ( KEGG ) pathway and the Protein-protein interaction ( PPI ) network was visualized by Cytoscape. The Kaplan–Meier plotter and ROC curves were used to validate the diagnostic and prognostic values of specific biomarkers identified through this model. We identified that Inositol 1,4,5-triphosphate receptor type 1 ( ITPR1 ) as a downregulated hub gene, with a high diagnostic accuracy to Grp3-MBs and associated with tumor metastasis. In addition, we identified genes significantly correlated with ITPR1 that were associated with metastasis in Grp3-MB ( ATP1A2 , MTTL7A and RGL1) , and worst overall survival in MBs ( ANTXR1 and RGL1 ). Our findings suggest that the ITPR1 hub gene is potentially involved in the metastatic process for Grp3-MB. Our data also provide evidence of targets that may serve as prognostic predictors and/or regulators for the metastatic process that maybe explored for further research of individualized therapy to Grp3-MBs.

RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.

Medulloblastoma (MB) is the most frequent malignant brain tumor in children and it is subgrouped into 4 entities (SHH, WNT, Group 3, and Group 4). Molecular pathways involved in these different subgroups still are evolving and can be of clinical relevance to therapy. The YAP1-CTGF axis is known to regulate cell proliferation, differentiation, and cell death; however, its role in MB is poorly explored. We aimed to investigate the role of YAP1 gene in the MB SHH cell line DAOY and evaluate cell proliferation, doubling time and 3D spheroids invasion and its consequence on CTGF regulation. We assessed CTGF expression from 22 children with MB. Lastly, we validated our findings through in silico analysis in large cohorts dataset of patients. We observed an increased invasion rate of DAOY cells and CTGF downregulation under YAP1 knockdown (p

Glioblastoma (GBM) is the most common malignant primary brain tumor affecting adults. In pediatric patients, GBM exhibits genetic variations distinct from those identified in the adult GBM phenotype. This tumor exhibits complex genetic changes leading to malignant progression and resistance to standard therapies including radiotherapy and temozolomide treatment. The GDF15 gene codes for a growth factor whose expression is altered in the presence of inflammations and malignancies. GDF15 is associated with a poor prognosis and with radio- and chemoresistance in a variety of tumors. The aim of this study was to compare the response to GDF15 knockdown in adult (U343) and pediatric (KNS42) GBM cell line models. The expression of the GDF15 gene was investigated by qRT-PCR and overexpression was identified in both GBM cell lines. The KNS42 and U343 cell lines were submitted to lentiviral transduction with shRNA of GDF15 and validated at the protein level. To understand the difference between cell lines, RNAseq was performed after GDF15 knockdown. The data obtained demonstrated that the pathways were differentially expressed in adult GBM and pediatric GBM cell lines. This was confirmed by functional assays perfomed after independent treatments (radiotherapy and TMZ). These results demonstrated that GBM cell lines had distinct responses to GDF15 knockdown, a fact that can be explained by the different molecular profile of pediatric and adult GBM.

Simple Summary Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Currently, MB is assigned in four molecular subgroups (SHH, WNT, Group 3, and Group 4) and subtyped in 12 variants. The alpha subtype of the SHH subgroup bears TP53 mutation and is considered very high risk by the World Health Organization (WHO). In the current study, we have investigated the role of YAP1 expression in SHH MBs. Herein, we show: (1) SHH MB patients genotypically profiled as resistant to SMOi and the aggressive alpha subtype overexpress YAP1; (2) SHH-like cell lines bearing TP53 mutation show improved responsiveness to SMOi upon YAP1 depletion; (3) Sonidegib (smoothened inhibitor) and Verteporfin (YAP1 inhibitor) synergize at specific doses; (4) distinct cell populations in the single-cell RNA-seq patient setting express YAP1 and SMO. Abstract Advances in genomics have led to the identification of twelve relevant molecular subtypes within medulloblastoma (MB). The alpha subtype of Sonic hedgehog-driven MB is resistant to therapy (including smoothened inhibitors) due to activation of genes from the non-canonical SHH pathway, such as MYCN, YAP1, or TP53. Using retrospective cohort microarray data, we found that YAP1 is overexpressed in SHH alpha MB and patients profiled as resistant to SMO inhibitors compared to good responders. Here, we performed YAP1 depletion via CRISPR/Cas9 in two in vitro models of SHH-like MB cells and found that this protein is involved in responsiveness to the SMO inhibitor regarding proliferation, apoptosis, and colony formation. Further, considering the synergic combination of YAP1 depletion with SMO inhibition, we assessed single-cell RNA-seq data from five patients and found that SMO and YAP1 are enriched within cells of SHH MB. Importantly, our data suggest that YAP1 is not only a reliable biomarker for cellular response to SMOi but may indicate prospective testing of combination therapy using YAP1 and SMO inhibitors in preclinical models of SHH MB.

TP53 p.R337H germline mutation is highly prevalent in the Southern region of Brazil. We sought to investigate TP53 p.R337H mutation in pediatric tumor samples from a population settled in a geographic area of high prevalence for this variant. Mutation assessment and genetic counseling for carriers/relatives were provided. 6/57 tumor samples were heterozygous for TP53 p.R337H. As expected, a high frequency was observed within adrenocortical tumors (3/3) and choroid plexus carcinomas (2/2). Interestingly, the TP53 R337H mutation was found in one case of pediatric rhabdomyosarcoma with Li-Fraumeni pedigree. Our finding expands the spectrum of childhood cancer associated with this germline mutation.

Medulloblastoma (MB) is the most frequent malignant brain tumor in children and it is subgrouped into 4 entities (SHH, WNT, Group 3, and Group 4). Molecular pathways involved in these different subgroups still are evolving and can be of clinical relevance to therapy. The YAP1-CTGF axis is known to regulate cell proliferation, differentiation, and cell death; however, its role in MB is poorly explored. We aimed to investigate the role of YAP1 gene in the MB SHH cell line DAOY and evaluate cell proliferation, doubling time and 3D spheroids invasion and its consequence on CTGF regulation. We assessed CTGF expression from 22 children with MB. Lastly, we validated our findings through in silico analysis in large cohorts dataset of patients. We observed an increased invasion rate of DAOY cells and CTGF downregulation under YAP1 knockdown (p 0.0001). Additionally CTGF is overexpressed in MB with extensive nodularity subtype and an indicative of higher survival rates in pediatric MB (p 0.05). Interestingly, no difference of CTGF expression was observed between molecular subgroups. These results provide new evidence ofCTGF as a potential prognostic marker for MB, corroborating to the role of YAP1 in restricting MB cell.

Next-generation sequencing platforms are routinely used for molecular assignment due to their high impact for risk stratification and prognosis in medulloblastomas. Yet, low and middle-income countries still lack an accurate cost-effective platform to perform this allocation. TaqMan Low Density array (TLDA) assay was performed using a set of 20 genes in 92 medulloblastoma samples. The same methodology was assessed in silico using microarray data for 763 medulloblastoma samples from the GSE85217 study, which performed MB classification by a robust integrative method (Transcriptional, Methylation and cytogenetic profile). Furthermore, we validated in 11 MBs samples our proposed method by Methylation Array 450 K to assess methylation profile along with 390 MB samples (GSE109381) and copy number variations. TLDA with only 20 genes accurately assigned MB samples into WNT, SHH, Group 3 and Group 4 using Pearson distance with the average-linkage algorithm and showed concordance with molecular assignment provided by Methylation Array 450 k. Similarly, we tested this simplified set of gene signatures in 763 MB samples and we were able to recapitulate molecular assignment with an accuracy of 99.1% (SHH), 94.29% (WNT), 92.36% (Group 3) and 95.40% (Group 4), against 97.31, 97.14, 88.89 and 97.24% (respectively) with the Ward.D2 algorithm. t-SNE analysis revealed a high level of concordance (k = 4) with minor overlapping features between Group 3 and Group 4. Finally, we condensed the number of genes to 6 without significantly losing accuracy in classifying samples into SHH, WNT and non-SHH/non-WNT subgroups. Additionally, we found a relatively high frequency of WNT subgroup in our cohort, which requires further epidemiological studies. TLDA is a rapid, simple and cost-effective assay for classifying MB in low/middle income countries. A simplified method using six genes and restricting the final stratification into SHH, WNT and non-SHH/non-WNT appears to be a very interesting approach for rapid clinical decision-making. Electronic supplementary material The online version of this article (10.1186/s40478-019-0681-y) contains supplementary material, which is available to authorized users.

High-grade gliomas harboring H3 G34R/V mutations exclusively occur in the cerebral hemispheres of adolescents and young adults, suggesting a distinct neurodevelopmental origin. Combining multimodal bulk and single-cell genomics with unbiased genome-scale CRISPR/Cas9 approaches, we here describe a GABAergic interneuron progenitor lineage as the most likely context from which these H3 G34R/V mutations drive gliomagenesis, conferring unique and tumor-selective gene targets essential for glioma cell survival, as validated genetically and pharmacologically. Phenotypically, we demonstrate that while H3 G34R/V glioma cells harbor the neurotransmitter GABA, they are developmentally stalled, and do not induce the neuronal hyperexcitability described in other glioma subtypes. These findings offer a striking counter-example to the prevailing view of glioma origins in glial precursor cells, resulting in distinct cellular, microenvironmental, and therapeutic consequences.

Background: Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Despite improvement in current treatment, children still suffer from long-term sequelae resulting from standard care therapy. World Health Organization considers MB as 4 distinct entities: SHH, WNT, Group 3, and Group 4. The most aggressive variant of SHH MB is the bearer of TP53 mutation and it is resistant to standard chemo/radiation therapy. Therefore, novel treatments modalities are urgently needed. In this study we aimed to evaluate the biologic relevance of YAP1 in SHH MB human cell lines bearers of TP53 mutation and its potential as a new therapeutic target. Methods: We generated YAP1 CRISPR/CAS9 knockout single-cell clones of human cell lines DAOY and UW228 assigned as “MB SHH-like” and bearers of TP53 mutation. We picked sonidegib as a standard-care chemotherapy for in vitro SHH MB model and assessed the apoptosis/necrosis using Annexin V/Propidium-Iodide staining to identify positive cells through flow cytometry. Proliferation and colony formation were assessed by cell counting Kit 8 proliferation assay and GIEMSA staining, respectively. Additionally, we used verteporfin, a YAP1 inhibitor, and evaluated its effect on cell proliferation. Findings: Using single-cell cloning, we successfully generated DAOY and UW228 cell lines with depleted YAP1 protein. YAP1 knockout cell lines DAOY and UW228 showed improved response to sonidegib regarding i) decrease in proliferation (p Conclusion: Although YAP1 depletion showed association with better response to sonidegib, a smoothened inhibitor, more experiments are needed to evaluate if the in vitro findings might shed new light on a potential synergistic combination between sonidegib and verteporfin and provide a good rationale for targeted therapy. Citation Format: Gustavo Alencastro Veiga Cruzeiro, Taciani de Almeida Magalhães, Graziella de Sousa Ribeiro, Pablo Ferreira das Chagas, Ricardo Bonfim Silva, Sampurna Chatterje, Carlos Alberto Scrideli, Luiz Gonzaga Tone. YAP1 knockout in vitro models of medulloblastoma showed improved response to sonidegib [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A22.

Introduction: Recently we found that RELA ependymomas (EPN) demonstrate inappropriate activation of the Hedgehog (Hh) pathway. However, how this activation is modulated remains to be investigated. Primary cilia are essential to positive and negative modulation of Hh signaling and changes in ciliation seem to be linked to Hh drug resistance in pediatric cancers. In contrast, targeting cilia integrity by molecules that prevents ciliary disassembly, such as Aurora-A (AURKA), has been shown to overcome this resistance. However, the role of the Hh pathway and the prevalence and function of cilia in pediatric RELA ependymoma (EPN) subgroup have not been examined. Thus, we explored the effect of Hh inhibitor, sonidegib, alone or in combination with Alisertib, AURKA inhibitor, in EPN RELA cell line. Methods: Hh pathway activation of RELA cell line (BXD-1425) was evaluated by immunostaining using ARL13B, a marker specific for ciliary membranes, proto-oncogene Smoothened (SMO) and Hh effector, GLI, antibodies in untreated cells or after stimulation with SMO agonist (SAG). Western blot was performed for protein expression of GLI1, GLI2, and full-length (FL) and repressor form (R) of Hh transcriptional program GLI3. Cell proliferation was assessed by CCK8 assay. Cell apoptosis was detected with Annexin V-FITC by flow cytometry. Results: We showed that EPN RELA cell line is frequently ciliated. Fluorescence intensity showed SMO and GLI recruitment to cilia in both BXD untreated cells and after stimulation with SAG, implicating in activation of Hh signals. Surprisingly, sonidegib treatment reduced ciliary formation in BXD cell line. Cilia loss was followed by increase of GLI recruitment to cilia and GLI2 expression, potentially by reduction of GLI3R. Our results indicate that impaired GLI processing probably by cilia disruption could explain persistent activation of downstream Hh pathway. To test this hypothesis, we targeted the cilia integrity through the combination of sonidegib with alisertib, to see if the cilia maintenance might affect Hh function and overcome sonidegib resistance. The combination prevented the primary cilia disruption followed by upregulation of GLI3R and downregulation of GLI recruitment to cilia, GLI2, and GLI3FL expression. Our data point towards to a synergistic combination that enhances sonidegib efficiency by primary cilia preservation, reducing cell proliferation and increasing apoptosis BXD cells compared to drugs alone treatment. Conclusion: Our findings suggest a synergistic combination of sonidegib with alisertib that enhances the inhibition of Hh major effectors such as GLIs (GLI2 and GLI3FL) and induces GLI3R expression. Our data suggest that combination of these agents may represent a novel approach for treatment of ST-EPN-RELA tumors to overcome chemoresistance and these mechanisms are potentially mediated by primary cilia, a key regulator for Hh activity. Citation Format: Taciani de Almeida Magalhães, Gustavo Alencastro Veiga Cruzeiro, Kleiton Silva Borges, Cherry Liu Yulu, Graziella Ribeiro de Souza, Keteryne Rodrigues da Silva, Carlos Alberto Scridelli, Adrian Salic, Luiz Gonzaga Tone. Alisertib acts synergistically with sonidegib by modulating primary cilia assembly in a pediatric RELA ependymoma cell line [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A30.

Background: Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. Objective: Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. Method: qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. Results: A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. Conclusion: HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

Medulloblastoma (MB) is an embryonal tumour that originates from genetic deregulation of cerebellar developmental pathways and is classified into 4 molecular subgroups: SHH, WNT, group 3, and group 4. Hydroxymethylation levels progressively increases during cerebellum development suggesting a possibility of deregulation in MB pathogenesis. The aim of this study was to investigate global hydroxymethylation levels and changes in TET and IDH gene expression in MB samples compared to control cerebellum samples. The methods utilized were qRT-PCR for gene expression, dot-blot and immunohistochemistry for global hydroxymethylation levels and sequencing for the investigation of IDH mutations. Our results show that global hydroxymethylation level was decreased in MB, and low 5hmC level was associated with the presence of metastasis. TET1 expression levels were decreased in the WNT subgroup, while TET3 expression levels were decreased in the SHH subgroup. Reduced TET3 expression levels were associated with the presence of events such as relapse and death. Higher expression of IDH1 was observed in MB group 3 samples, whereas no mutations were detected in exon 4 of IDH1 and IDH2. These findings suggest that reduction of global hydroxymethylation levels, an epigenetic event, may be important for MB development and/or maintenance, representing a possible target in this tumour and indicating a possible interaction of TET and IDH genes with the developmental pathways specifically activated in the MB subgroups. These genes could be specific targets and markers for each subgroup.

O objetivo deste trabalho foi identificar, por meio da análise de mapeamento associativo, os marcadores moleculares relacionados à produtividade do arroz de terras altas e aos seus caracteres componentes. Foram usadas 113 linhagens e cultivares de arroz de terras altas, da Coleção Nuclear de Arroz da Embrapa, com reduzido vínculo genético entre si. Os seguintes caracteres componentes da produtividade foram avaliados: número de panículas por metro, número de grãos por panícula e peso de 100 grãos. Dos 115 marcadores utilizados, 25 (21,7%) associaram-se significativamente a um ou mais caracteres. Entre os 29 SSR ("simple sequence repeats") colocalizados em QTL ("quantitative trait loci") de produtividade de arroz, 12 foram associados aos caracteres avaliados e considerados como candidatos para uso na seleção assistida por marcadores. Os marcadores NP914540, Q6ZGD1 e Q69JE3, associados ao número de grãos por panícula, ainda não foram anotados no arroz e podem constituir o ponto de partida para estudos de genômica funcional. Entre os marcadores derivados de sequências transcritas, NP914526 e NP914533 destacam-se por pertencer a rotas metabólicas relacionadas ao aumento do potencial produtivo de arroz. The objective of this work was to identify, through analysis of associative mapping, the molecular markers related to upland rice yield and its component traits. One hundred thirteen lines and cultivars of upland rice, with reduced admixture, from the Rice Core Collection of Embrapa, were used. The following yield component traits were evaluated: number of panicles per meter, number of grains per panicle, and weight of 100 grains. Out of the 115 used markers, 25 (21.7%) were significantly associated with one or more traits. Among the 29 SSR (simple sequence repeats) co-located in yield QTL (quantitative trait loci) in rice, 12 were associated with the evaluated traits and considered candidates for use in marker-assisted selection. The markers NP914540, Q6ZGD1, and Q69JE3, associated with the number of grains per panicle, are not yet annotated in rice and should constitute the starting point for functional genomics studies. Among the markers derived from transcribed sequences, NP914526 and NP914533 stand out for belonging to metabolic pathways related to increased yield potential of rice.

A cellular hypoxic condition is a key event in several human cancers, but knowledge about its role in childhood acute lymphoblastic leukemia (ALL) is very limited. In the present study, the gene expression profile of hypoxia-related genes (HIF1A, CA9, VEGF and SCL2A1) was evaluated in bone marrow samples of 113 pediatric patients. HIF1A mRNA up-regulation was significantly associated with higher 5-year event-free survival rates in patients with B-ALL as well as in the overall ALL population in both univariate and multivariate analysis (p = 0.023 and p = 0.041, respectively). In gene expression analysis, low oxygen levels promoted HIF1A activation in a time-dependent manner, in both ALL cell lines. In vitro cytotoxic assays suggested an initial trend toward hypoxia-related resistance in the first 24 h, but evaluation at later time points (48-72 h) clearly showed that there was no relevant difference in drug response when comparing hypoxic and normal oxygen level conditions. Modulation of mRNA expression of several hypoxia-related genes was also observed after hypoxic exposure in a cell specific manner, suggesting that HIF1A mRNA expression could play a different role in specific subtypes of leukemia. Despite the remaining questions regarding hypoxia-mediated mechanisms, these findings could be helpful to provide new insights into the role of hypoxia in childhood ALL.

Chromosomal heterogeneity is a hallmark of most tumors and it can drive critical events as growth advantages, survival advantages, progression and karyotypic evolution. Medulloblastoma (MB) is the most common malignant central nervous system tumor in children. This work attempted to investigate chromosomal heterogeneity and instability profiles of two MB pediatric cell lines and their relationship with cell phenotype. We performed GTG-banding and cytokinesis-block micronucleus cytome assays, as well as morphological characterization, cell population doubling time, colony-forming efficiency, and chemo-sensitivity assays in two pediatric MB cell lines (UW402 and UW473). Both MB cells showed a high chromosomal heterogeneity. UW473 cells showed ~2 fold higher both clonal- and non-clonal chromosomal alterations than UW402 cells. Besides, UW473 showed two clonal-groups well-differentiated by ploidy level ( and ) and also presented a significantly higher number of chromosomal instability biomarkers. These results were associated with high morphological heterogeneity and survival advantages for UW473 and proliferation advantages for UW402 cells. Moreover, UW473 was significantly more sensitive to methotrexate, temozolomide and cisplatin while UW402 cells were more sensitive to doxorubicin. These data suggest that distinct different degrees of karyotypic heterogeneity and instability may affect neoplasic phenotype of MB cells. These findings bring new insights into cell and tumor biology.

Introduction: Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood and represents a heterogeneous disease regarding its biology and prognosis. Major improvements have been made in the ALL treatment, which achieved successful rates of approximately 80% of long-terms survival. Despite the significant percentage of success, the remaining 20% still presents treatment failure and the molecular mechanisms involved in the resistance process remains unclear. The SHOC2 gene encodes a leucine-rich-repeats (LRR) protein which acts as a positive modulator of the RAS-MAPK signaling cascade. Its gene expression profile was previously related to daunorubicin sensibility in children with ALL, suggesting the correlation of SHOC2 with good prognostic factors. Thus, the present study was undertaken to better understand the possible mechanisms which involve SHOC2 gene to the chemotherapy response process. Methods: Jurkat, ALL T-type cells, were cultured in standard RPMI medium supplemented with 10% of fetal calf serum (FCS) and transfected with siRNA using lipossomal transfection reagent to silence the gene SHOC2. Mock transfections were also performed. The silencing confirmation was done by real time quantitative PCR and Western blotting. Responsiveness of leukemia cells to vincristine, methotrexate and L-asparaginase was determined by the 4-day XTT in vitro drug resistance assay. The concentrations of L-asparaginase, methotrexate and vincristine were 2–12 IU/mL ASP, 0.25–2.0uM/mL MTX and 4.0–14.0 ng/mL VCR. SHOC2 effect on apoptosis was analyzed by Annexin V staining by flow cytometer. The t-test was conducted to determine statistical significance between two groups in triplicate of two independent experiments. The significance level was set at p ≤ 0.05. Results: When treated with vincristine and methotrexate, the survival rates of Jurkat cell lines were significantly lower in non-transfected cells than in transfected group (p Conclusions: this data suggests that the SHOC2 gene could play an important role in leukemic cell's sensibility to chemotherapy agents by modifying the cell proliferation and the apoptosis' processes. Thus, this gene could be considered as a target for developing new therapeutic interventions for ALL treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C158.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor and is majorly caused by the constitutive activation of mutant KRAS - found in more than 90% of PDAC cases. The undruggability of KRAS mutations has led to efforts of finding new therapeutic targets that focus on downstream molecules in the MAPK pathways. The regulation of these kinase activities is orchestrated by a negative feedback network played by a series of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), which in turn activate and inhibit the phosphatase function. DUSP6 is a dual-specificity phosphatase that regulates ERK1/2 phosphorylation and, therefore, RAS pathway activation. Data from different PDAC datasets revealed that DUSP6 is overexpressed in metastatic tumor samples compared to primary tumor samples and to non-tumoral pancreatic tissue. Overall survival analysis indicated that patients with high DUSP6 expression have a worse prognosis than patients with low DUSP6 expression, reaffirming its clinical relevance. Moreover, we observed that DUSP6 is overexpressed in the quasimesenchymal/stromal subtype, which was previously described to be correlated with the glycolytic phenotype and the worst prognosis among all the other described PDAC subtypes. Considering the aforementioned, we hypothesized that DUSP6 could play a role in metabolism reprogramming in PDAC and, therefore, induce a more aggressive phenotype, leading to metastasis development. To investigate DUSP6 role in metastasis development and progression we developed DUSP6 stable knockdown in PDAC cells lines and performed genotypic and phenotypic analysis to evaluate metastatic and metabolic behaviors. Surprisingly, we observed different phenotypes among the cell lines used, which we believe is derived from the different genetic backgrounds and metabolic subtypes involved. Overall, results indicate that DUSP6 play a role in the metastatic process in PDAC, modifying phenotypes that are closely related to the cells capacity to survive and thrive in an unfamiliar environment. Also, DUSP6 plays a role in the metabolic reprogramming of these cells, as we observe that its knockdown induces glycolysis in these cells under blockage of the mitochondrial respiration. Nevertheless, the mechanism behind these changes remains to be further investigated. O adenocarcinoma ductal pancreático (ADP) é um tumor altamente agressivo e majoritariamente causado pela ativação constitutiva de KRAS mutante - encontrado em mais de 90% dos casos de ADP. Os desafios em desenvolver um inibidor para o oncogene KRAS tem levado a esforços para encontrar novos alvos terapêuticos em moléculas a jusante das vias MAPK. A regulação da atividade dessas quinases é orquestrada por uma retroalimentação negativa que envolve uma série de proteínas tirosinas quinases e proteínas tirosina fosfatases que, por sua vez, ativam e inibem a função das fosfatases. DUSP6 é uma fosfatase de dupla especificidade que regula a fosforilação de ERK1/2 e, dessa forma, a ativação da via RAS. RNA-seq de diferentes bancos de dados demonstram que DUSP6 está hiperexpressa em amostras metastáticas em comparação a amostras de tumor primário e de tecido pancreático não-tumoral. Uma análise de sobrevida global indica que pacientes com alta expressão de DUSP6 apresentam pior prognóstico que os pacientes com baixa expressão de DUSP6, reafirmando a importância clínica desse alvo. Ainda, observamos que DUSP6 está hiperexpressa no subtipo quasimesenquimal/estromal, que foi previamente correlacionado com o fenótipo glicolítico e com o pior prognóstico entre todos os demais subtipos descritos. Considerando o descrito, a hipótese deste trabalho é de que DUSP6 pode desempenhar um papel na reprogramação metabólica do ADP, contribuindo assim para um fenótipo mais agressivo e, consequentemente, o desenvolvimento de metástase. Visando investigar o papel de DUSP6 no desenvolvimento e progressão tumoral, linhagens de ADP com inibição estável de DUSP6 foram utilizadas em ensaios funcionais in vitro para avaliar a capacidade de migração, invasão e o comportamento metabólico dessas células. Surpreendentemente, observamos diferentes fenótipos entre as linhagens celulares utilizadas, o que acreditamos estar relacionado ao fato de que tais linhagens possuem alterações genéticas distintas e subtipos metabólicos específicos. Em suma, os resultados obtidos indicam que DUSP6 pode impactar o processo metastático do ADP, por meio da modulação da capacidade migratória e invasiva das células. Ainda, DUSP6 pode controlar a reprogramação metabólica, uma vez que observamos que a inibição dessa fosfatase promove o aumento da glicólise diante do bloqueio da respiração mitocondrial. No entanto, os mecanismos que permeiam essas mudanças ainda precisam ser esclarecidos.