Joanna Triscott

Recurrence patterns across medulloblastoma subgroups: an integrated clinical and molecular analysis

BACKGROUND Recurrent medulloblastoma is a therapeutic challenge because it is almost always fatal. Studies have confirmed that medulloblastoma consists of at least four distinct subgroups. We sought to delineate subgroup-specific differences in medulloblastoma recurrence patterns. METHODS We retrospectively identified a discovery cohort of all recurrent medulloblastomas at the Hospital for Sick Children (Toronto, ON, Canada) from 1994 to 2012 (cohort 1), and established molecular subgroups using a nanoString-based assay on formalin-fixed paraffin-embedded tissues or frozen tissue. The anatomical site of recurrence (local tumour bed or leptomeningeal metastasis), time to recurrence, and survival after recurrence were assessed in a subgroup-specific manner. Two independent, non-overlapping cohorts (cohort 2: samples from patients with recurrent medulloblastomas from 13 centres worldwide, obtained between 1991 and 2012; cohort 3: samples from patients with recurrent medulloblastoma obtained at the NN Burdenko Neurosurgical Institute [Moscow, Russia] between 1994 and 2011) were analysed to confirm and validate observations. When possible, molecular subgrouping was done on tissue obtained from both the initial surgery and at recurrence. RESULTS Cohort 1 consisted of 30 patients with recurrent medulloblastomas; nine with local recurrences, and 21 with metastatic recurrences. Cohort 2 consisted of 77 patients and cohort 3 of 96 patients with recurrent medulloblastoma. Subgroup affiliation remained stable at recurrence in all 34 cases with available matched primary and recurrent pairs (five pairs from cohort 1 and 29 pairs from cohort 2 [15 SHH, five group 3, 14 group 4]). This finding was validated in 17 pairs from cohort 3. When analysed in a subgroup-specific manner, local recurrences in cohort 1 were more frequent in SHH tumours (eight of nine [89%]) and metastatic recurrences were more common in group 3 and group 4 tumours (17 of 20 [85%] with one WNT, p=0·0014, local vs metastatic recurrence, SHH vs group 3 vs group 4). The subgroup-specific location of recurrence was confirmed in cohort 2 (p=0·0013 for local vs metastatic recurrence, SHH vs group 3 vs group 4,), and cohort 3 (p<0·0001). Treatment with craniospinal irradiation at diagnosis was not significantly associated with the anatomical pattern of recurrence. Survival after recurrence was significantly longer in patients with group 4 tumours in cohort 1 (p=0·013) than with other subgroups, which was confirmed in cohort 2 (p=0·0075), but not cohort 3 (p=0·70). INTERPRETATION Medulloblastoma does not change subgroup at the time of recurrence, reinforcing the stability of the four main medulloblastoma subgroups. Significant differences in the location and timing of recurrence across medulloblastoma subgroups have potential treatment ramifications. Specifically, intensified local (posterior fossa) therapy should be tested in the initial treatment of patients with SHH tumours. Refinement of therapy for patients with group 3 or group 4 tumours should focus on metastases.

YB-1 Bridges Neural Stem Cells and Brain Tumor–Initiating Cells via Its Roles in Differentiation and Cell Growth

The Y-box binding protein 1 (YB-1) is upregulated in many human malignancies including glioblastoma (GBM). It is also essential for normal brain development, suggesting that YB-1 is part of a neural stem cell (NSC) network. Here, we show that YB-1 was highly expressed in the subventricular zone (SVZ) of mouse fetal brain tissues but not in terminally differentiated primary astrocytes. Conversely, YB-1 knockout mice had reduced Sox-2, nestin, and musashi-1 expression in the SVZ. Although primary murine neurospheres were rich in YB-1, its expression was lost during glial differentiation. Glial tumors often express NSC markers and tend to loose the cellular control that governs differentiation; therefore, we addressed whether YB-1 served a similar role in cancer cells. YB-1, Sox-2, musashi-1, Bmi-1, and nestin are coordinately expressed in SF188 cells and 9/9 GBM patient-derived primary brain tumor-initiating cells (BTIC). Silencing YB-1 with siRNA attenuated the expression of these NSC markers, reduced neurosphere growth, and triggered differentiation via coordinate loss of GSK3-β. Furthermore, differentiation of BTIC with 1% serum or bone morphogenetic protein-4 suppressed YB-1 protein expression. Likewise, YB-1 expression was lost during differentiation of normal human NSCs. Consistent with these observations, YB-1 expression increased with tumor grade (n = 49 cases). YB-1 was also coexpressed with Bmi-1 (Spearmans 0.80, P > 0.001) and Sox-2 (Spearmans 0.66, P > 0.001) based on the analysis of 282 cases of high-grade gliomas. These proteins were highly expressed in 10/15 (67%) of GBM patients that subsequently relapsed. In conclusion, YB-1 correlatively expresses with NSC markers where it functions to promote cell growth and inhibit differentiation.

Personalizing the treatment of pediatric medulloblastoma: Polo-like kinase 1 as a molecular target in high-risk children.

Medulloblastoma is the most common malignant brain tumor in children. This disease is heterogeneous and is composed of four subtypes of medulloblastoma [WNT, Sonic Hedgehog (SHH), Group 3, and Group 4]. An immediate goal is to identify novel molecular targets for the most aggressive forms of medulloblastoma. Polo-like kinase 1 (PLK1) is an oncogenic kinase that controls cell cycle and proliferation, making it a strong candidate for medulloblastoma treatment. In this study, pediatric medulloblastomas were subtyped in two patient cohorts (discovery cohort, n = 63 patients; validation cohort, n = 57 patients) using NanoString nCounter analysis and PLK1 mRNA was assessed. We determined that the SHH and Group 3 subtypes were independently associated with poor outcomes in children as was PLK1 using Cox regression analyses. Furthermore, we screened a library of 129 compounds in clinical trials using a model of pediatric medulloblastoma and determined that PLK1 inhibitors were the most promising class of agents against the growth of medulloblastoma. In patient-derived primary medulloblastoma isolates, the PLK1 small-molecule inhibitor BI2536 suppressed the self-renewal of cells with high PLK1 but not low PLK1 expression. PLK1 inhibition prevented medulloblastoma cell proliferation, self-renewal, cell-cycle progression, and induced apoptosis. In contrast, the growth of normal neural stem cells was unaffected by BI2536. Finally, BI2536 extended survival in medulloblastoma-bearing mice with efficacy comparable with Headstart, a standard-of-care chemotherapy regimen. We conclude that patients with medulloblastoma expressing high levels of PLK1 are at elevated risk. These preclinical studies pave the way for improving the treatment of medulloblastoma through PLK1 inhibition.

Polo‐Like Kinase 1 Inhibition Kills Glioblastoma Multiforme Brain Tumor Cells in Part Through Loss of SOX2 and Delays Tumor Progression in Mice

Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer and given these new therapies are urgently needed. To identify molecular targets, we queried a microarray profiling 467 human GBMs and discovered that polo‐like kinase 1 (PLK1) was highly expressed in these tumors and that it clustered with the proliferative subtype. Patients with PLK1‐high tumors were more likely to die from their disease suggesting that current therapies are inactive against such tumors. This prompted us to examine its expression in brain tumor initiating cells (BTICs) given their association with treatment failure. BTICs isolated from patients expressed 110‐470 times more PLK1 than normal human astrocytes. Moreover, BTICs rely on PLK1 for survival because the PLK1 inhibitor BI2536 inhibited their growth in tumorsphere cultures. PLK1 inhibition suppressed growth, caused G2/M arrest, induced apoptosis, and reduced the expression of SOX2, a marker of neural stem cells, in SF188 cells. Consistent with SOX2 inhibition, the loss of PLK1 activity caused the cells to differentiate based on elevated levels of glial fibrillary acidic protein and changes in cellular morphology. We then knocked glial fibrillary acidic protein (GFAP) down SOX2 with siRNA and showed that it too inhibited cell growth and induced cell death. Likewise, in U251 cells, PLK1 inhibition suppressed cell growth, downregulated SOX2, and induced cell death. Furthermore, BI2536 delayed tumor growth of U251 cells in an orthotopic brain tumor model, demonstrating that the drug is active against GBM. In conclusion, PLK1 level is elevated in GBM and its inhibition restricts the growth of brain cancer cells. STEM CELLS2012;30:1064–1075

FoxG1 Interacts with Bmi1 to Regulate Self‐Renewal and Tumorigenicity of Medulloblastoma Stem Cells

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. Recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Groups 1 and 2 are characterized by relatively good clinical outcomes and activation of the Wnt and Shh pathways, respectively. In contrast, groups 3 and 4 (“non‐Shh/Wnt MBs”) are distinguished by metastatic disease, poor patient outcome, and lack a molecular pathway phenotype. Current gene expression platforms have not detected brain tumor‐initiating cell (BTIC) self‐renewal genes in groups 3 and 4 MBs as BTICs typically comprise a minority of tumor cells and may therefore go undetected on bulk tumor analyses. Since increasing BTIC frequency has been associated with increasing tumor aggressiveness and poor patient outcome, we investigated the subgroup‐specific gene expression profile of candidate stem cell genes within 251 primary human MBs from four nonoverlapping MB transcriptional databases (Amsterdam, Memphis, Toronto, Boston) and 74 NanoString‐subgrouped MBs (Vancouver). We assessed the functional relevance of two genes, FoxG1 and Bmi1, which were significantly enriched in non‐Shh/Wnt MBs and showed these genes to mediate MB stem cell self‐renewal and tumor initiation in mice. We also identified their transcriptional regulation through reciprocal promoter occupancy in CD15+ MB stem cells. Our work demonstrates the application of stem cell data gathered from genomic platforms to guide functional BTIC assays, which may then be used to develop novel BTIC self‐renewal mechanisms amenable to therapeutic targeting. STEM Cells2013;31:1266–1277

Concise Review: Bullseye: Targeting Cancer Stem Cells to Improve the Treatment of Gliomas by Repurposing Disulfiram

Cancer stem cells (CSCs) are thought to be at the root of cancer recurrence because they resist conventional therapies and subsequently reinitiate tumor cell growth. Thus, targeting CSCs could be the bullseye to successful cancer therapeutics in the future. Brain tumors are some of the most challenging types of cancer to treat and the median survival following the initial diagnosis is 12–18 months. Among the different types of brain tumors, glioblastoma (GBM) is considered the most aggressive and remains extremely difficult to treat. Despite surgery, radiation, and chemotherapy, most patients develop refractory disease. Temozolomide (TMZ) is a chemotherapy used to treat GBM however resistance develops in most patients. The underlying mechanisms for TMZ resistance (TMZ‐resistant) involve the expression of DNA repair gene O(6)‐methylguanine‐DNA methyltransferase. CSC genes such as Sox‐2, BMI‐1, and more recently Y‐box binding protein‐1 also play a role in resistance. In order to develop novel therapies for GBM, libraries of small interfering RNAs and off‐patent drugs have been screened. Over the past few years, several independent laboratories identified disulfiram (DSF) as an off‐patent drug that kills GBM CSCs. Reportedly DSF has several modes of action including its ability to inhibit aldehyde dehydrogenases, E3 ligase, polo‐like kinase 1, and NFkB. Due to the fact that GBM is a disease of heterogeneity, chemotherapy with multitargeting properties may be the way of the future. In broader terms, DSF kills CSCs from a range of different cancer types further supporting the idea of repurposing it for “target practice.” Stem Cells 2015;33:1042–1046

Overcoming resistance to Sonic Hedgehog inhibition by targeting p90 ribosomal S6 kinase in pediatric medulloblastoma.

Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However, resistance inevitably emerges against these therapies, particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets.

Abstract 811: VAL083, a novel N7 alkylating agent, surpasses temozolomide activity and inhibits cancer stem cells providing a new potential treatment option for glioblastoma multiforme

Glioblastoma (GBM) remains one of the most difficult tumors to treat in part because many new agents fail to cross the blood brain barrier (BBB) and secondly due to intrinsic drug resistance. Temozolomide (TMZ) is a front-line therapy for the treatment of GBM, however, it is often ineffective due to drug inactivation by O6-methylguanine-DNA methyltransferase (MGMT). Cancer stem cells (CSC) are a subpopulation of the tumor that resist therapy and give rise to relapse. Here we described VAL083 a novel alkylating agent that creates N7 methylation on DNA, which was initially intriguing because it crosses the BBB. We addressed how it compared to TMZ, whether it could be used to overcome MGMT-driven drug resistance and if it has activity against CSCs. Addressing these questions provides further preclinical support for VAL083, which is currently undergoing human clinical trials in the USA against refractory GBM. VAL083 inhibited U251 and SF188 cell growth in monolayer and as neurospheres better then TMZ and caused apoptosis after 72 hrs. In a 10-day colony formation assay, VAL083 (5uM) suppressed SF188 growth by ∼95%. T98G cells are classically TMZ resistant and express MGMT yet VAL083 inhibited their growth in monolayer after 72 hrs in a dose-dependent manner (IC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 811. doi:1538-7445.AM2012-811

Abstract 3552: Subgroup-specific pattern of recurrence in medulloblastoma.

Background: Medulloblastoma constitutes the most common malignant brain tumor of childhood. Although multimodel treatment strategies, encompassing surgery, chemotherapy, and radiation, results in up to 80% five-year overall survival, recurrent medulloblastoma is almost always uniformly fatal. Recent integrated genomic studies have shown that medulloblastoma comprises 4 clinical and biologically distinct variants. We sought to delineate subgroup-specific differences in recurrent medulloblastoma. Methods: We identified a discovery cohort of all recurrent medulloblastomas at the Hospital for Sick Children between 1994-2012, and subgrouped cases using nanoString. Clinical details were ascertained via retrospective chart review. Our findings were confirmed through analysis of an independent validation cohort of 85 recurrences. Primary and recurrent matched pairs were evaluated where possible. Results: Twenty-nine recurrent cases were identified, ten with a local recurrence within the tumor bed only, and 19 recurred with metastases. Notably, SHH tumors recurred more frequently in the tumor bed (8/11, 73%) whereas Group 3 and Group 4 developed with metastatic relapses more frequently (16/18, 89%; p Conclusions: Significant differences in the pattern of recurrence exist across medulloblastoma subgroups. Longer surveillance periods across the entire neuro-axis may be required for Group 4 patients even in the absence of local tumor bed or supratentorial recurrence. Intensified local therapy should be considered upon initial treatment for SHH patients. Citation Format: Vijay Ramaswamy, Marc Remke, Eric Bouffet, David Shih, Claudia Faria, Ulrich Schuller, Sri Gururangan, Roger McLendon, Nada Jabado, Adam Fontebasso, Sandra Dunn, Joanna Triscott, Cynthia Hawkins, Uri Tabori, Kari Codispoti, Roger Packer, Stefan M. Pfister, Andrey Korshunov, Michael D. Taylor. Subgroup-specific pattern of recurrence in medulloblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3552. doi:10.1158/1538-7445.AM2013-3552