*2.3.1. Molecular basis of Group 3 medulloblastomas*

Another approach to developing combination drug therapies has been to identify additional signaling pathways that impact SHH-driven medulloblastoma. Research has demonstrated

**•** p53: Tumor suppressor p53 is highly mutated in pediatric medulloblastomas and is a significant factor in determining prognosis [6]. A cohort study found that 5-year survival rates differed between 41 and 82%, respectively, for SHH medulloblastoma cases with and without p53 mutations [59]. In mice, the incidence of medulloblastoma increases to nearly 100% with p53 loss [60]. Therefore, regulators of p53 activity might serve as highly attrac‐ tive drug candidates for combination therapy with Smo inhibitors. For example, driving down levels of MDM2, a negative regulator of p53, has been shown to decrease expres‐

**•** cAMP: In general, researchers have discovered that the levels of second messenger cAMP are inversely correlated with tumor grade and growth. Ablation of the G protein Gαs is sufficient to initiate SHH medulloblastoma, and mice harboring the *GNAS* mutation

**•** TGF-β: Expression analysis of Ptch1 heterozygous and Smo/Smo mouse medulloblastoma tumors of varying clinical severities found a correlation between TGF-β expression levels and medulloblastoma progression. In general, it was found that activation of the TGF-β pathway correlated with better prognosis with patients [63]. For instance, positive nuclear staining of SMAD3, a downstream component of TGF-β signaling, was associated with longer patient survival [63]. Therefore, regulation of the TGF-β signaling pathway in

**•** Basic FGF: Overall, basic FGF (bFGF) signaling appears to have an inhibitory role on SHHinduced proliferation. The addition of bGFG to tumor cultures has been shown to limit tumor formation and proliferation and to inhibit expression of the transcriptional prod‐

While these intersecting pathways contain possible targets, determining the exact mecha‐ nism by which they impact SHH medulloblastoma is the limiting step to uncovering the best

While Wnt and SHH medulloblastomas have been identified by mutations within these pathways, more comprehensive biological pathways have not been delineated for Group 3 and Group 4 medulloblastomas. Hence, these have been so named untilthe underlying biology

Conventional diagnosis of Group 3 medulloblastomas is accomplished through transcription‐ al profiling [3]. Group 3 medulloblastoma is associated with increased MYC expression and enrichment for photoreceptor pathway-associated genes; these genes are overexpressed in Group 3 [3]. In addition, Group 3 can be divided into subtype based on MYC expression. In Group 3α subtype, all patients contain MYC amplification and this is associated with poor

demonstrate decreased tumor proliferation when cAMP levels are elevated [62].

conjunction with SHH signaling may be another venue of combination therapy.

ucts of SHH signaling, namely *Gli1, Nmyc*, and *cyclin D1* [64].

that these pathways play a role in medulloblastoma development:

sion of Gli1 and Gli2 [61].

392 Neurooncology - Newer Developments

candidates to target.

is further elucidated.

**2.3. Group 3 medulloblastomas**

While many details about the molecular makeup of Group 3 medulloblastomas remain unresolved, recent literature therapeutically targeting Group 3 medulloblastoma may reveal clues to the molecular pathways driving this subgroup. The folate synthesis inhibitor peme‐ trexed and nucleoside analog gemcitabine demonstrated a synergistic effect in increasing the survival of mice bearing MYC-overexpressing tumors [65]. The same drug combination had little effect on mice medulloblastomas of the SHH subgroup [65]. These observations are supported by gene set enrichment analysis showing that Group 3 medulloblastomas are enriched in the folate and purine metabolism pathways compared to Group 4 and SHH medulloblastoma [65].

The antihelmintic drug, mebendazole, has been shown to inhibit angiogenesis in medullo‐ blastoma [66]. While it acts as a microtubule synthesis inhibitor in worms, studies with medulloblastoma models suggest that it can inhibit vascular endothelial growth factor receptor 2 (VEGFR2) [66]. Targeting class I histone deacetylase 2 has also been shown to impact Group 3 medulloblastoma tumor cell viability [67].

The International Cancer Genome Consortium (ICGC) PedBrain Tumor Project published in 2014 the analyses of deep sequencing of Group 3 and Group 4 tumors. This study uncovered novel information about the biology between this subgroup. Tetraploidy was a common event for both Group 3 and Group 4 tumors, respectively, and tetraploid tumors displayed signs of genomic instability [68]. With Group 3, the most frequently mutated gene was SMARCA4 [68]. Together, both *in vitro* drug assays and genome-wide mining of Wnt medulloblastomas introduce molecular pathways for further exploration in uncovering Group 3 medulloblasto‐ ma biology and which may reveal possible drug targets.

#### **2.4. Group 4 medulloblastomas**

Group 4 is the most prevalent medulloblastoma subgroup, accounting for about 34% of all medulloblastomas [6]. A high frequency (66%) of isochromosome 17q is associated with Group 4 medulloblastomas [6]. Strikingly, 80% of women with Group 4 medulloblastoma also have X chromosome loss [6]. Group 4 medulloblastomas have a prognosis comparable to SHH group medulloblastomas [6].

#### *2.4.1. Molecular basis of Group 4 medulloblastomas*

The ICGC PedBrain Project found that KDM64, a histone 3 lysine 27 demethylase, was mutated in 10% of Group 4 tumors [68]. These mutations reveal the genetic and molecular pathways that go awry in Group 3 and Group 4 tumors. For example, the ICGC PedBrain uncovered an association between TBR1 and Group 4 medulloblastomas [68]. TBR1 is a T-box transcrip‐ tion factor shown to play a role in brain development. Of particular interest is the gene CTDNEP1, found mutated in 10% of Group 4 tumors and which is located on 17q [68]. CTDNEP1 encodes a nuclear membrane phosphatase and in mammals is shown to play a role in nuclear membrane biogenesis and in lipid activation. As 66% of Group 4 medulloblasto‐ mas contain 17q, mutations found on this isochromosome are particularly important for study.

#### **2.5. Future clinical and basic science directions for medulloblastoma**

Clearly, with respect to Group 3 and Group 4 medulloblastomas, further studies about the molecular basis for these subgroups are needed. These two subgroups pose great clinical challenges: Group 4 is the most prevalent group, while Group 3 has the poorest diagnosis. Yet a dearth of knowledge about the molecular basis behind each group limits drug targeting. The growing body of studies which include genome-wide mining for enrichments within each subgroup along with *in vitro* studies for Group 3 and Group 4 may soon intersect to reveal a broader picture of the molecular pathways behind these subgroups.

Currently, there are a number of clinical trials evaluating the safety and efficacy of Wnttargeted therapies in patients with other malignancies that overexpress the Wnt pathway; however, none of these are being tested in medulloblastomas. The efficacy of these agents in treating Wnt medulloblastoma remains to be assessed. Additionally, in light of the high survival rates of standard risk patients with Wnt medulloblastoma, additional studies would be helpful to identify optimal treatment regimens that will maintain these high survival rates while minimizing treatment side effects. With respect to SHH-driven medulloblastoma, identification of noveltargets especially for combination drug therapy will address the concern for drug resistance and limited efficacy of current treatments. For example, the identification and assessment of novel Gli inhibitors for SHH-mediated cancers should be evaluated in the context of medulloblastoma. In addition, the effects of the crosstalk of intersecting pathways on medulloblastoma tumorigenesis should be further studied.
