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.

Pre-therapeutic efficacy of the CDK inhibitor dinaciclib in medulloblastoma cells.

Medulloblastoma (MB) is the most common aggressive paediatric brain tumour and, despite the recent progress in the treatments of MB patients, there is still an urgent need of complementary or alternative therapeutic options for MB infants. Cyclin Dependent Kinase inhibitors (CDKi) are at the front-line of novel targeted treatments for multiple cancers and the CDK4/6 specific inhibitor palbociclib has been pre-clinically identified as an effective option for MB cells. Herein, we identified the pan-CDKi dinaciclib as a promising alternative to palbociclib for the suppression of MB cells proliferation. We present evidence supporting dinaciclib's ability to inhibit MB cells in vitro proliferation at considerably lower doses than palbociclib. Sequencing data and pathway analysis suggested that dinaciclib is a potent cell death inducer in MB cells. We found that dinaciclib-triggered apoptosis is triggered by CDK9 inhibition and the resultant reduction in RNA pol II phosphorylation, which leads to the downregulation of the oncogenic marker MYC, and the anti-apoptotic protein MCL-1. Specifically, we demonstrated that MCL-1 is a key apoptotic mediator for MB cells and co-treatment of dinaciclib with BH3 mimetics boosts the therapeutic efficacy of dinaciclib. Together, these findings highlight the potential of multi-CDK inhibition by dinaciclib as an alternative option to CDK4/6 specific inhibition, frequently associated with drug resistance in patients.

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.

Involvement of Heparan Sulfate and Heparanase in Neural Development and Pathogenesis of Brain Tumors.

Brain tumors are aggressive and devastating diseases. The most common type of brain tumor, glioblastoma (GBM), is incurable and has one of the worst five-year survival rates of all human cancers. GBMs are invasive and infiltrate healthy brain tissue, which is one main reason they remain fatal despite resection, since cells that have already migrated away lead to rapid regrowth of the tumor. Curative therapy for medulloblastoma (MB), the most common pediatric brain tumor, has improved, but the outcome is still poor for many patients, and treatment causes long-term complications. Recent advances in the classification of pediatric brain tumors reveal distinct subgroups, allowing more targeted therapy for the most aggressive forms, and sparing children with less malignant tumors the side-effects of massive treatment. Heparan sulfate proteoglycans (HSPGs), main components of the neurogenic niche, interact specifically with a large number of physiologically important molecules and vital roles for HS biosynthesis and degradation in neural stem cell differentiation have been presented. HSPGs are composed of a core protein with attached highly charged, sulfated disaccharide chains. The major enzyme that degrades HS is heparanase (HPSE), an important regulator of extracellular matrix (ECM) remodeling which has been suggested to promote the growth and invasion of other types of tumors. This is of clinical interest because GBM are highly invasive and children with metastatic MB at the time of diagnosis exhibit a worse outcome. Here we review the involvement of HS and HPSE in development of the nervous system and some of its most malignant brain tumors, glioblastoma and medulloblastoma.

Pharmacological mTOR targeting enhances the antineoplastic effects of selective PI3Kα inhibition in medulloblastoma.

Despite recent advances in the treatment of medulloblastoma, patients in high-risk categories still face very poor outcomes. Evidence indicates that a subpopulation of cancer stem cells contributes to therapy resistance and tumour relapse in these patients. To prevent resistance and relapse, the development of treatment strategies tailored to target subgroup specific signalling circuits in high-risk medulloblastomas might be similarly important as targeting the cancer stem cell population. We have previously demonstrated potent antineoplastic effects for the PI3Kα selective inhibitor alpelisib in medulloblastoma. Here, we performed studies aimed to enhance the anti-medulloblastoma effects of alpelisib by simultaneous catalytic targeting of the mTOR kinase. Pharmacological mTOR inhibition potently enhanced the suppressive effects of alpelisib on cancer cell proliferation, colony formation and apoptosis and additionally blocked sphere-forming ability of medulloblastoma stem-like cancer cells in vitro. We identified the HH effector GLI1 as a target for dual PI3Kα and mTOR inhibition in SHH-type medulloblastoma and confirmed these results in HH-driven Ewing sarcoma cells. Importantly, pharmacologic mTOR inhibition greatly enhanced the inhibitory effects of alpelisib on medulloblastoma tumour growth in vivo. In summary, these findings highlight a key role for PI3K/mTOR signalling in GLI1 regulation in HH-driven cancers and suggest that combined PI3Kα/mTOR inhibition may be particularly interesting for the development of effective treatment strategies in high-risk medulloblastomas.

Casein kinase 2 inhibition sensitizes medulloblastoma to temozolomide.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Since surviving patients experience severe neurocognitive disabilities, better and more effective treatments are needed to enhance their quality of life. Casein kinase 2 (CK2) is known to regulate cell growth and survival in multiple cancers; however, the role of CK2 in MB is currently being studied. In this study, we verified the importance of CK2 in MB tumorigenesis and discovered that inhibition of CK2 using the small molecule inhibitor, CX-4945, can sensitize MB cells to a well-known and tolerated chemotherapeutic, temozolomide (TMZ). To study the role of CK2 in MB we modulated CK2 expression in multiple MB cells. Exogenous expression of CK2 enhanced cell growth and tumor growth in mice, while depletion or inhibition of CK2 expression decreased MB tumorigenesis. Treatment with CX-4945 reduced MB growth and increased apoptosis. We conducted a high-throughput screen where 4000 small molecule compounds were analyzed to identify compounds that increased the anti-tumorigenic properties of CX-4945. TMZ was found to work synergistically with CX-4945 to decrease cell survival and increase apoptosis in MB cells. O-6-methylguanine-DNA methyltransferase (MGMT) activity is directly correlated to TMZ sensitivity. We found that loss of CK2 activity reduced ß-catenin expression, a known MGMT regulator, which in turn led to a decrease in MGMT expression and an increased sensitivity to TMZ. Our findings show that CK2 is important for MB maintenance and that treatment with CX-4945 can sensitize MB cells to TMZ treatment.

Human Sialic acid O-acetyl esterase (SIAE) - mediated changes in sensitivity to etoposide in a medulloblastoma cell line.

Medulloblastoma (MB), the most common malignant paediatric brain tumour occurs in the cerebellum. Advances in molecular genomics have led to the identification of defined subgroups which are associated with distinct clinical prognoses. Despite this classification, standard therapies for all subgroups often leave children with life-long neurological deficits. New therapeutic approaches are therefore urgently needed to reduce current treatment toxicity and increase survival for patients. GD3 is a well-studied ganglioside which is known to have roles in the development of the cerebellum. Post-partum GD3 is not highly expressed in the brain. In some cancers however GD3 is highly expressed. In MB cells GD3 is largely acetylated to GD3A. GD3 is pro-apoptotic but GD3A can protect cells from apoptosis. Presence of these gangliosides has previously been shown to correlate with resistance to chemotherapy. Here we show that the GD3 acetylation pathway is dysregulated in MB and as a proof-of-principle we show that increased GD3 expression sensitises an MB cell line to etoposide.

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.

Itch/ß-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis.

Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/ß-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis.

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.

Polo-Like Kinase 1 Pharmacological Inhibition as Monotherapy or in Combination: Comparative Effects of Polo-Like Kinase 1 Inhibition in Medulloblastoma Cells.

BACKGROUND: Medulloblastoma (MB) is one of most frequent malignant tumors that affect children. Despite the relatively good survival rate, long time sequels still represent a challenge for MB. Therefore, in an attempt to reduce treatment aftereffects, new therapeutic targets are constantly being explored. Polo like kinase 1 (PLK1) is a master cell cycle regulator that is increased in proliferative cells, while its depletion has been repeatedly proposed as an oncological therapeutic strategy. OBJECTIVES: Here, we evaluated and compared the effects of PLK1 inhibition alone and in combination with currently used radio- and chemotherapy in MB cells. METHODS: UW402, UW473, ONS-76 and DAOY MB cell lines were treated with BI 2536, BI 6727, GW843682X, and GSK461364 PLK1 inhibitors and cell proliferation, apoptosis, clonogenicity, cell invasion, adhesion and cell cycle distribution were evaluated. In addition, the combinatorial effect with gamma irradiation or etoposide, cisplatin and temozolomide was evaluated. RESULTS: We show that PLK1 inhibition causes a significant decrease on cell proliferation, clonogenic capacity, cell invasion and adhesion, with modest differences between inhibitors. Yet, the four drugs cause G2/M arrest followed by increased cell death. PLK1 inhibition proved to be efficient to sensitize MB cells to radiation irrespective of the inhibitor, even though it showed thrifty results when combined with chemotherapy. CONCLUSIONS: We proved that all PLK1 inhibitors have anti-mitotic effects on MB cells, supporting the idea of using them as radiosensitizers. Taken together, our results strengthen the potential of using PLK1 as a therapeutic target to improve treatment strategy for this tumor.

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.

Polo-like Kinase Inhibitor Volasertib Exhibits Antitumor Activity and Synergy with Vincristine in Pediatric Malignancies.

BACKGROUND: Polo-like kinase 1 (PLK1) controls the main cell-cycle checkpoints, suggesting utility of its inhibition for cancer treatment, including of highly proliferative pediatric cancer. This preclinical study explored the selective PLK1 inhibitor volasertib (BI 6727) alone and combined with chemotherapy in pediatric malignancies. MATERIALS AND METHODS: Inhibition of proliferation was explored in vitro using dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assay. Mice bearing human xenografts were treated with weekly intravenous injections of volasertib. RESULTS: Volasertib inhibited proliferation in all 40 cell lines tested, with a mean half-maximal growth inhibitory concentration of 313 nmol/l (range: 4-5000 nmol/l). Volasertib was highly active against RMS-1 alveolar rhabdomyosarcoma xenografts, resulting in 100% tumor regression. Activity was associated with complete and prolonged G2/M arrest and subsequent apoptotic cell death. Volasertib showed synergistic activity with vincristine but antagonistic effects with etoposide. CONCLUSION: These findings support the further exploration of volasertib for pediatric malignancies, particularly alveolar rhabdomyosarcoma, and its combination with mitotic spindle poison.