Medulloblastoma uses GABA transaminase to survive in the cerebrospinal fluid microenvironment and promote leptomeningeal dissemination.

Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum. Although abnormal GABAergic receptor activation has been described in MB, studies have not yet elucidated the contribution of receptor-independent GABA metabolism to MB pathogenesis. We find primary MB tumors globally display decreased expression of GABA transaminase (ABAT), the protein responsible for GABA metabolism, compared with normal cerebellum. However, less aggressive WNT and SHH subtypes express higher ABAT levels compared with metastatic G3 and G4 tumors. We show that elevated ABAT expression results in increased GABA catabolism, decreased tumor cell proliferation, and induction of metabolic and histone characteristics mirroring GABAergic neurons. Our studies suggest ABAT expression fluctuates depending on metabolite changes in the tumor microenvironment, with nutrient-poor conditions upregulating ABAT expression. We find metastatic MB cells require ABAT to maintain viability in the metabolite-scarce cerebrospinal fluid by using GABA as an energy source substitute, thereby facilitating leptomeningeal metastasis formation.

TORC1/2 kinase inhibition depletes glutathione and synergizes with carboplatin to suppress the growth of MYC-driven medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. An in silico analysis of these cells identified mTOR inhibitors as potential therapeutic agents. We hypothesized that the orally bioavailable TORC1/2 kinase inhibitor TAK228 would have activity against MYC-driven medulloblastoma. TAK228 inhibited mTORC1/2, decreased cell growth and caused apoptosis in high-MYC medulloblastoma cell lines. Comprehensive metabolic profiling of medulloblastoma orthotopic xenografts showed upregulation of glutathione compared to matched normal brain. TAK228 suppressed glutathione production. Because glutathione is required to detoxify platinum-containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin in medulloblastoma. TAK228 synergized with carboplatin to inhibit cell growth and induce apoptosis and extended survival in orthotopic xenografts of high-MYC medulloblastoma. Brain-penetrant TORC1/2 inhibitors and carboplatin may be an effective combination therapy for high-risk medulloblastoma.

Inhibition of enhancer of zest homologue 2 is a potential therapeutic target for high-MYC medulloblastoma.

MYC amplification is common in Group 3 medulloblastoma and is associated with poor survival. Group 3 and Group 4 medulloblastomas are also known to have elevated levels of histone H3-lysine 27-tri-methylation (H3K27me3), at least in part due to high expression of the H3K27 methyltransferase enhancer of zest homologue 2 (EZH2), which can be regulated by MYC. We therefore examined whether MYC expression is associated with elevated EZH2 and H3K27me3 in medulloblastoma, and if high-MYC medulloblastomas are particularly sensitive to pharmacological EZH2 blockade. Western blot analysis of low (DAOY, UW228, CB SV40) and high (DAOY-MYC, UW228-MYC, CB-MYC, D425) MYC cell lines showed that higher levels of EZH2 and H3K27me3 were associated with elevated MYC. In fixed medulloblastoma samples examined using immunohistochemistry, most MYC positive tumors also had high H3K27me3, but many MYC negative ones did as well, and the correlation was not statistically significant. All high MYC lines tested were sensitive to the EZH2 inhibitor EPZ6438. Many low MYC lines also grew more slowly in the presence of EPZ6438, although DAOY-MYC cells responded more strongly than parent DAOY cultures with lower MYC levels. We find that higher MYC levels are associated with increased EZH2, and pharmacological blockade of EZH2 is a potential therapeutic strategy for aggressive medulloblastoma with elevated MYC.

RITA downregulates Hedgehog-GLI in medulloblastoma and rhabdomyosarcoma via JNK-dependent but p53-independent mechanism.

Overactivation of the Hedgehog (HH) signaling pathway is implicated in many cancers. In this study, we demonstrate that the small molecule RITA, a p53 activator, effectively downregulates HH signaling in human medulloblastoma and rhabdomyosarcoma cells irrespective of p53. This is mediated by a ROS-independent activation of the MAP kinase JNK. We also show that in vitro RITA sensitized cells to the GLI antagonist GANT61, as co-administration of the two drugs had more pronounced effects on cell proliferation and apoptosis. In vivo administration of RITA or GANT61 suppressed rhabdomyosarcoma xenograft growth in nude mice; however, co-administration did not further enhance tumor suppression, even though cell proliferation was decreased. RITA was more potent than GANT61 in downregulating HH target gene expression; surprisingly, this suppressive effect was almost completely eliminated when the two drugs were administered together. Notably, RNA-seq demonstrated a broader response of pathways involved in cancer cell growth in the combination treatment, providing a plausible interpretation for tumor reduction in the absence of HH signaling downregulation.

Targeting Upstream Kinases of STAT3 in Human Medulloblastoma Cells.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. Despite improvement in overall survival rate, it still lacks an effective targeted treatment strategy. The Janus family of cytoplasmic tyrosine kinases (JAKs) and Src kinases, upstream protein kinases of signal transducer and activator of transcription 3 (STAT3), play important roles in medulloblastoma pathogenesis and therefore represent potential therapeutic targets. METHODS: In this report, we examined the inhibitory efficacy of the JAK1/2 inhibitor, ruxolitinib, the JAK3 inhibitor, tofacitinib and two Src inhibitors, KX2-391 and dasatinib. RESULTS: These small molecule drugs significantly reduce cell viability and inhibit cell migration and colony formation in human medulloblastoma cells in vitro. Src inhibitors have more potent efficacy than JAK inhibitors in inhibiting medulloblastoma cell migration ability. The Src inhibitors can inhibit both phosphorylation of STAT3 and Src while JAK inhibitors reduce JAK/STAT3 phosphorylation. We also investigated the combined effect of the Src inhibitor, dasatinib with cisplatin. The results show that dasatinib exerts synergistic effects with cisplatin in human medulloblastoma cells through the inhibition of STAT3 and Src. CONCLUSION: Our results suggest that the small molecule inhibitors of STAT3 upstream kinases, ruxolitinib, tofacitinib, KX2-391, and dasatinib could be novel and attractive candidate drugs for the treatment of human medulloblastoma.

Pyruvate Kinase Inhibits Proliferation during Postnatal Cerebellar Neurogenesis and Suppresses Medulloblastoma Formation.

Aerobic glycolysis supports proliferation through unresolved mechanisms. We have previously shown that aerobic glycolysis is required for the regulated proliferation of cerebellar granule neuron progenitors (CGNP) and for the growth of CGNP-derived medulloblastoma. Blocking the initiation of glycolysis via deletion of hexokinase-2 (Hk2) disrupts CGNP proliferation and restricts medulloblastoma growth. Here, we assessed whether disrupting pyruvate kinase-M (Pkm), an enzyme that acts in the terminal steps of glycolysis, would alter CGNP metabolism, proliferation, and tumorigenesis. We observed a dichotomous pattern of PKM expression, in which postmitotic neurons throughout the brain expressed the constitutively active PKM1 isoform, while neural progenitors and medulloblastomas exclusively expressed the less active PKM2. Isoform-specific Pkm2 deletion in CGNPs blocked all Pkm expression. Pkm2-deleted CGNPs showed reduced lactate production and increased SHH-driven proliferation. 13C-flux analysis showed that Pkm2 deletion reduced the flow of glucose carbons into lactate and glutamate without markedly increasing glucose-to-ribose flux. Pkm2 deletion accelerated tumor formation in medulloblastoma-prone ND2:SmoA1 mice, indicating the disrupting PKM releases CGNPs from a tumor-suppressive effect. These findings show that distal and proximal disruptions of glycolysis have opposite effects on proliferation, and that efforts to block the oncogenic effect of aerobic glycolysis must target reactions upstream of PKM. Cancer Res; 77(12); 3217-30. ©2017 AACR.

PLK1-associated microRNAs are correlated with pediatric medulloblastoma prognosis.

PURPOSE: Medulloblastoma (MB) is the most common malignant tumor of the central nervous system (CNS) in children. Despite its relative good survival rates, treatment can cause long time sequels and may impair patients' lifespan and quality, making the search for new treatment options still necessary. Polo like kinases (PLKs) constitute a five-member serine/threonine kinases family (PLK 1-5) that regulates different stages during cell cycle. Abnormal PLKs expression has been observed in several cancer types, including MB. As gene regulators, miRNAs have also been described with variable expression in cancer. METHODS: We evaluated gene expression profiles of all PLK family members and related miRNAs (miR-100, miR-126, miR-219, and miR-593*) in MB cell lines and tumor samples. RESULTS: RT-qPCR analysis revealed increased levels of PLK1-4 in all cell lines and in most MB samples, while PLK5 was found underexpressed. In parallel, miR-100 was also found upregulated while miR-129, miR-216, and miR-593* were decreased in MB cell lines. Variable miRNAs expression patterns were observed in MB samples. However, a correlation between miR-100 and PLK4 expression was observed, and associations between miR-100, miR-126, and miR-219 expression and overall and event free survival were also evinced in our cohort. Moreover, despite the lack of association with clinico-pathological features, when comparing primary tumors to those relapsed, we found a consistent decrease on PLK2, miR-219, and miR-598* and an increase on miR-100 and miR-126. CONCLUSION: Specific dysregulation on PLKs and associated miRNAs may be important in MB and can be used to predict prognosis. Although miRNAs sequences are fundamental to predict its target, the cell type may also be consider once that mRNA repertoire can define different roles for specific miRNA in a given cell.

Dual role of the integrated stress response in medulloblastoma tumorigenesis.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development. Our previous studies showed that the ISR is activated in medulloblastoma in both human patients and animal models, and that the decreased ISR via PERK heterozygous deficiency attenuates medulloblastoma formation in Patched1 heterozygous deficient (Ptch1+/-) mice by enhancing apoptosis of pre-malignant granule cell precursors (GCPs) during cell transformation. We showed here that GADD34 heterozygous mutation moderately enhanced the ISR and noticeably increased the incidence of medulloblastoma in adult Ptch1+/- mice. Surprisingly, GADD34 homozygous mutation strongly enhanced the ISR, but significantly decreased the incidence of medulloblastoma in adult Ptch1+/- mice. Intriguingly, GADD34 homozygous mutation significantly enhanced pre-malignant GCP apoptosis in cerebellar hyperplastic lesions and reduced the lesion numbers in young Ptch1+/- mice. Nevertheless, neither GADD34 heterozygous mutation nor GADD34 homozygous mutation had a significant effect on medulloblastoma cells in adult Ptch1+/- mice. Collectively, these data imply the dual role of the ISR, promoting and inhibiting, in medulloblastoma tumorigenesis by regulating apoptosis of pre-malignant GCPs during the course of malignant transformation.

High expression of DNA methyltransferases in primary human medulloblastoma.

Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

Checkpoint kinase 1 expression is an adverse prognostic marker and therapeutic target in MYC-driven medulloblastoma.

Checkpoint kinase 1 (CHK1) is an integral component of the cell cycle as well as the DNA Damage Response (DDR) pathway. Previous work has demonstrated the effectiveness of inhibiting CHK1 with small-molecule inhibitors, but the role of CHK1 mediated DDR in medulloblastoma is unknown. CHK1, both at the mRNA and protein level, is highly expressed in medulloblastoma and elevated CHK1 expression in Group3 medulloblastoma is an adverse prognostic marker. CHK1 inhibition with the small-molecule drug AZD7762, results in decreased cell growth, increased DNA damage and cell apoptosis. Furthermore, AZD7762 acts in synergy with cisplatin in reducing cell proliferation in medulloblastoma. Similar phenotypic changes were observed with another CHK1 inhibitor, PF477736, as well as genetic knockdown using siRNA against CHK1. Treatments with small-molecule inhibitors of CHK1 profoundly modulated the expression of both upstream and downstream target proteins within the CHK1 signaling pathways. This suggests the presence of a feedback loop in activating CHK1. Overall, our results demonstrate that small-molecule inhibition of CHK1 in combination with, cisplatin, is more advantageous than either treatment alone, especially for Group 3 medulloblastoma, and therefore this combined therapeutic approach serves as an avenue for further investigation.

PERK Activation Promotes Medulloblastoma Tumorigenesis by Attenuating Premalignant Granule Cell Precursor Apoptosis.

Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation.

Telomere dysfunction and chromothripsis.

Chromothripsis is a recently discovered form of genomic instability, characterized by tens to hundreds of clustered DNA rearrangements resulting from a single dramatic event. Telomere dysfunction has been suggested to play a role in the initiation of this phenomenon, which occurs in a large number of tumor entities. Here, we show that telomere attrition can indeed lead to catastrophic genomic events, and that telomere patterns differ between cells analyzed before and after such genomic catastrophes. Telomere length and telomere stabilization mechanisms diverge between samples with and without chromothripsis in a given tumor subtype. Longitudinal analyses of the evolution of chromothriptic patterns identify either stable patterns between matched primary and relapsed tumors, or loss of the chromothriptic clone in the relapsed specimen. The absence of additional chromothriptic events occurring between the initial tumor and the relapsed tumor sample points to telomere stabilization after the initial chromothriptic event which prevents further shattering of the genome.

PP2A inhibition with LB100 enhances cisplatin cytotoxicity and overcomes cisplatin resistance in medulloblastoma cells.

The protein phosphatase 2A (PP2A) inhibitor, LB100, has been shown in pre-clinical studies to be an effective chemo- and radio-sensitizer for treatment of various cancers. We investigated effects associated with LB100 treatment alone and in combination with cisplatin for medulloblastoma (MB) in vitro and in vivo in an intracranial xenograft model. We demonstrated that LB100 had a potent effect on MB cells. By itself, LB100 inhibited proliferation and induced significant apoptosis in a range of pediatric MB cell lines. It also attenuated MB cell migration, a pre-requirement for invasion. When used in combination, LB100 enhanced cisplatin-mediated cytotoxic effects. Cell viability in the presence of 1 uM cisplatin alone was 61% (DAOY), 100% (D341), and 58% (D283), but decreased with the addition of 2 µM of LB100 to 26% (DAOY), 67% (D341), and 27% (D283), (p < 0.005). LB100 suppressed phosphorylation of the STAT3 protein and several STAT3 downstream targets. Also, LB100 directly increased cisplatin uptake and overcame cisplatin-resistance in vitro. Finally, LB100 exhibited potent in vivo anti-neoplastic activity in combination with cisplatin in an intracranial xenograft model.

Characterization of anaplastic lymphoma kinase-positive medulloblastomas.

Medulloblastomas are the most common pediatric malignant primary brain tumor. To our knowledge, there are no known critical and druggable tyrosine kinases in medulloblastomas, precluding the use of established tyrosine kinase inhibitors that have shown efficacy in other tumor types. We studied the expression of anaplastic lymphoma kinase (ALK), a well-characterized tyrosine kinase and drug target, in a cohort of medulloblastomas by immunohistochemistry, and identified three ALK-positive cases. Mutational analyses did not reveal a definite underlying genetic mechanism for the ALK expression, although one of the cases showed increased ALK copy number. Our findings have clinical implications and warrant further pharmacological and functional studies, as well as evaluation in larger patient cohorts, to fully characterize the value of ALK as a prognostic and predictive therapeutic marker in medulloblastomas.

IDH1 R132H mutation in a pilocytic astrocytoma: a case report.

We present the case of a 72-year old female with a right cerebellar pilocytic astrocytoma WHO grade I with an Isocitrate dehydrogenase 1 (IDH1) R132H mutation. The patient is recurrence-free 6 years after the initial diagnosis. Only one single case with strikingly similar clinicopathological features has been reported before. Otherwise, IDH1/2 mutations are not seen in pilocytic astrocytomas. The clinical implications of these findings are discussed.

Immunohistochemical expression of sulfhydryl oxidase (QSOX1) in pediatric medulloblastomas.

BACKGROUND: Medulloblastoma is a malignant, invasive embryonal tumor of the cerebellum and accounts for 20% of intracranial tumors in children. QSOX1, whose functions include formation of disulphide bridges, which are needed for correct protein folding and stability, formation of the extracellular matrix, regulation of the redox status and cell cycle control, appears to be involved in apoptosis in pathological states such as cancer. Thus, the aim of this study was to investigate the immunohistochemical expression of QSOX1 in medulloblastomas and nonneoplastic cerebellum. METHODS: Histology blocks of pediatric medulloblastomas were separated and two representative areas of the tumors and non-neoplastic cerebellum samples were used to construct tissue microarrays (TMAs) that were stained with an anti-QSOX1 antibody, and the slides were read using image analysis software. RESULTS: QSOX1 immunoexpression was observed in the non-neoplastic cerebellum samples and the medulloblastoma samples. There was no statistically significant relationship between QSOX1 immunopositivity in the medulloblastoma samples and the clinical and pathological variables. CONCLUSIONS: Although QSOX1 did not prove useful for stratifying patients into risk groups, tumor cells and the fibrillar extracellular matrix were positive for this marker, indicating that this enzyme may be involved in the pathogenesis of medulloblastoma. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1822040654139436.

Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased ß1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

DHFR-mediated effects of methotrexate in medulloblastoma and osteosarcoma cells: the same outcome of treatment with different doses in sensitive cell lines.

Although methotrexate (MTX) is the most well-known antifolate included in many standard therapeutic regimens, substantial toxicity limits its wider use, particularly in pediatric oncology. Our study focused on a detailed analysis of MTX effects in cell lines derived from two types of pediatric solid tumors: medulloblastoma and osteosarcoma. The main aim of this study was to analyze the effects of treatment with MTX at concentrations comparable to MTX plasma levels in patients treated with high-dose or low-dose MTX. The results showed that treatment with MTX significantly decreased proliferation activity, inhibited the cell cycle at S-phase and induced apoptosis in Daoy and Saos-2 reference cell lines, which were found to be MTX-sensitive. Furthermore, no difference in these effects was observed following treatment with various doses of MTX ranging from 1 to 40 µM. These findings suggest the possibility of achieving the same outcome with the application of low-dose MTX, an extremely important result, particularly for clinical practice. Another important aspect of treatment with high-dose MTX in clinical practice is the administration of leucovorin (LV) as an antidote to reduce MTX toxicity in normal cells. For this reason, the combined application of MTX and LV was also included in our experiments; however, this application of MTX together with LV did not elicit any detectable effect. The expression analysis of genes involved in the mechanisms of resistance to MTX was a final component of our study, and the results helped us to elucidate the mechanisms of the various responses to MTX among the cell lines included in our study.

The PI3K inhibitor GDC-0941 displays promising in vitro and in vivo efficacy for targeted medulloblastoma therapy.

Deregulation of the Phosphoinositide 3-kinase (PI3K)/AKT signalling network is a hallmark of oncogenesis. Also medulloblastoma, the most common malignant brain tumor in children, is characterized by high levels of AKT phosphorylation and activated PI3K signalling in medulloblastoma is associated with enhanced cellular motility, survival and chemoresistency underscoring its role of as a potential therapeutic target. Here we demonstrate that GDC-0941, a highly specific PI3K inhibitor with good clinical tolerability and promising anti-neoplastic activity in adult cancer, also displays anti-proliferative and pro-apoptotic effects in pediatric human medulloblastoma cell lines. Loss in cell viability is accompanied by reduced phosphorylation of AKT, a downstream target of PI3K. Furthermore, we show that GDC-0941 attenuates the migratory capacity of medulloblastoma cells and targets subpopulations expressing the stem cell marker CD133. GDC-0941 also synergizes with the standard medulloblastoma chemotherapeutic etoposide. In an orthotopic xenograft model of the most aggressive human medulloblastoma variant we document that oral adminstration of GDC-0941 impairs tumor growth and significantly prolongs survival. These findings provide a rational to further investigate GDC-0941 alone and in combination with standard chemotherapeutics for medulloblastoma treatment.