Challenges and Recent Advances in Medulloblastoma Therapy

Feature Review

Challenges and Recent Advances in Medulloblastoma Therapy Vinod Kumar,1 Virender Kumar,1 Timothy McGuire,2 Donald W. Coulter,3 John G. Sharp,4 and Ram I. Mahato1,* Medulloblastoma (MB) is the most common childhood brain tumor, which occurs in the posterior fossa. MB tumors are highly heterogeneous and have diverse genetic make-ups, with differential microRNA (miRNA) expression profiles and variable prognoses. MB can be classified into four subgroups, each with different origins, pathogenesis, and potential therapeutic targets. miRNA and small-molecule targeted therapies have emerged as a potential new therapeutic paradigm in MB treatment. However, the development of chemoresistance due to surviving cancer stem cells and dysregulation of miRNAs remains a challenge. Combination therapies using multiple drugs and miRNAs could be effective approaches. In this review we discuss various MB subtypes, barriers, and novel therapeutic options which may be less toxic than current standard treatments. Advances in MB Therapy MB is the most common pediatric brain tumor and is a leading cause of cancer-related deaths in children. MB accounts for 15–20% of pediatric brain tumors with an overall cure rate that ranges from 40% to 90% depending on the molecular subtype. MB is also called cerebellar primitive neuroectodermal tumor (PNET) because it starts in a region of the brain at the base of the skull called the posterior fossa. Different subgroups of MB have different origin. Its peak incidence is in children between the ages of three to nine years, and about 10% of cases occur in infants [1]. Unlike most brain tumors, MB rapidly metastasizes to various locations via the cerebrospinal fluid (CSF). Typically, patients become listless, with repeated episodes of vomiting and morning headaches which may lead to a misdiagnosis of a gastrointestinal disease or migraine. Initially it was thought that MB originates from embryonal cells or immature cells at their earliest stage of development. It is highly likely that MB arises from embryonic/fetal cells that have abnormalities of pluripotency network genes (NANOG, OCT3/4, SOX, and others). The consequences are several different downstream abnormalities. However, it is now evident that the cell of origin depends on the subgroup. In MB the cerebellar stem cells stop dividing and differentiating into their normal progeny. Abnormal cell differentiation could be one of the reasons for high variability in the earlier histologic classification of MB. However, the most recent classification systems are based molecular pathway characteristics. MB is currently classified into four groups: the wingless (Wnt), sonic hedgehog (Shh), group 3, and group 4 subtypes, with MYC expression as a negative prognostic indicator. In the group 1 Wnt subtype the canonical Wnt signaling pathway is upregulated. The group 2 Shh subtype is characterized by activation of the Shh signaling cascade. The group 3 subtype is characterized by amplification of various proto-oncogenes including MYC (16.7%), PVT1 (11.9%), SMARCA4 (10.5%), and OTX2 (7.7%). This subgroup has the worst outcome among all MB subtypes [2]. The group

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Trends Medulloblastoma accounts for 15– 20% of all pediatric brain tumor and is a leading cause of cancer-related deaths in children. The latest classification categorizes medulloblastoma into four subtypes – Wnt, Shh, group 3, and group 4 – based on signaling pathways and whole-genome sequence analysis. Chemoresistance caused by stem cells, miRNA dysregulation, and the blood–brain barrier is the main obstacle in medulloblastoma therapy. Polymeric nanomedicines carrying small-molecule inhibitors and miRNA have potential to cross the blood–brain barrier and treat the disease effectively.

1 Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Department of Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE 68198, USA 3 Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE 68198, USA 4 Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, USA

*Correspondence: [email protected] (R.I. Mahato).

http://dx.doi.org/10.1016/j.tips.2017.09.002 © 2017 Elsevier Ltd. All rights reserved.

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4 MB subtype is characterized by molecular abnormalities associated with chromosome 17. This group is also characterized by frequent mutation in the KDM6A gene which regulates methylation of lysine 27 of histone H3 [3]. The current treatment for MB consists of surgery followed by craniospinal irradiation and chemotherapy. Because irradiating the central nervous system (CNS) can be damaging to the developing brain, radiation therapy is usually avoided in children under the age of three, but this can compromise disease control and survival. Therefore, there is a need for new treatments that can be tolerated in the younger population to treat therapy-resistant disease as well as to decrease potential side effects. Adjuvant chemotherapy and irradiation are used to treat any residual tumor after surgery and to reduce the risk of metastases through the CSF. To improve the current outlook for patients with MB, targeted therapies that are more effective in killing tumor cells and less toxic are being developed. The use of miRNA and small-molecule inhibitors of various pathways involving in MB prognosis has potential to treat the disease effectively and offer long-term benefits. These therapies must have activity against chemoresistant cells and should cross the blood–brain barrier (BBB). Therefore, in this review we discuss the pathophysiological characteristics of MB, the therapeutic potential of various uncategorized drugs and miRNAs, and their targeted delivery to MB.

Types of MB According to 2007 World Health Organization (WHO) classification of CNS tumors, MB can be classified histopathologically into five recognizable subtypes: classic, desmoplastic/nodular, MB with extensive nodularity (MBEN), anaplastic, and large-cell MB [4,5]. The latest classification of MB has four subtypes based on their extensive genomic and pathway analysis: Wnt group, Shh group, group 3, and group 4. Recently Cavalli et al. analyzed 763 primary MB samples and further classified MB into 12 subtypes: two Wnt, four Shh, three group 3, and three group 4 subgroups. Heterogeneity within subgroups accounts for previously unexplained variation [6]. Schwalbe et al. analyzed 428 primary MB samples and classified MB into seven groups: Wnt, group 4 low risk, group 4 high risk, group 3 low risk, group 3 high risk, Shh infant, and Shh child. These subgroups have distinct pathological and molecular features, and underpin current disease subclassification. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup [7]. These subgroups have different pathological and molecular features which result in substantial biological heterogeneity and different survival rates. Therefore, this molecular subclassification is important for treatment. Classic MB Classic MB is the most common histopathological subtype, and accounts for about 70–80% of total MB [5,8]. In classic MB the tumor tissue is typically composed of sheets of densely packed, small round cells with large dark nuclei: in other words, small round blue cell (neuroectodermal) tumors. It has a high nucleus to cytoplasm ratio, with hyperchromatic nuclei being visible after hematoxylin and eosin (H&E) staining [9]. Desmoplastic/Nodular (DN) MB The DN variant of MB is seen in 10–15% of cases [10,11]. It is predominantly observed in young children [8] where it accounts for
50% of infant cases (<3 years of age at diagnosis) and only 5% of cases aged 3–15 years. Scattered ovoid or round nodules separated by reticulin-rich desmoplastic internodular regions characterize this subtype of MB. Frequent desmoplasia is common in infant cases [11]. In internodular/desmoplastic regions the tumor cells are densely packed and highly pleomorphic compared to the nodular region [9]. The occurrence of excessive nodular density has been associated with improved prognosis [12].

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MB with Extensive Nodularity (MBEN) 2007 WHO classification of CNS tumors identified a new and different type of histological subgroup, MBEN, which resembles the DN disease variant. This subtype is characterized by markedly expanded lobular architecture and a comparatively reduced internodular component. This histological subtype of MB accounts for approximately 1–2% of total MB. Histologically, this subgroup contains uniform nuclei in round cells with high levels of neuronal differentiation and a low proliferative index [4]. This subtype is mostly found in infant patients ( < 3 years of age at diagnosis) [9]. Large Cell MB This subtype of MB comprises
4% of total MB. The main characteristic feature of this subtype is a pleomorphic nucleus in large cells, prominent nucleoli, and abundant cytoplasm. This subtype has a higher mitotic and apoptotic rate, often resulting in necrosis, and also has a very poor prognosis. Anaplastic MB This subtype comprises approximately 10–20% of total MB. It is characterized by cells with nuclear pleomorphism and molding of cells in which cells wrap around each other. Although areas of anaplasia are found in all MB histopathological subtypes, it is more prominent in this subtype. The large cell and anaplastic variants share a poor prognosis, and are typically grouped as a single large cell/anaplastic (LCA) category in MB studies.

Signaling Pathways Involved in MB As previously discussed, the most widely accepted classification of MB identifies four distinct molecular subtypes – Wnt, Shh, group 3, and group 4 – with MYC expression being a negative prognostic indicator in the last two categories. Wnt patients have the best prognosis while group 3 patients have the worst. This classification is based on whole-genome sequencing, DNA copy-number analysis, mRNA expression patterns, and somatic copy-number aberrations. These molecular subtypes have different genetic, epigenetic, and phenotypic profiles (Figure 1). The signaling pathways involved in these subtypes differ greatly from each other. In addition, the proper development of the cerebellum is controlled by the coordinated activities of multiple signal transduction pathways including Shh, Wnt, and Notch. Inappropriate activation and/or dysregulation of these pathways is the main cause of MB (Figure 2). The role of various signaling pathways in the development of the cerebellum and their involvement in MB is discussed below. Wnt The name Wnt is derived from the Drosophila melanogaster segment-polarity gene Wingless and its vertebrate homolog Integration site 1. The Wnt proteins constitute a large family of secreted glycoproteins that play a central role in embryogenesis, differentiation, cell motility, cell proliferation, and adult tissue homeostasis. The Wnt signaling pathway comprises a group of proteins that provide signals to cells through surface receptors. The pathway can be broadly divided into three parts: (i) the canonical Wnt pathway (b-catenin pathway), (ii) the noncanonical Wnt pathway (planar cell polarity pathway), and (iii) the Wnt/Ca2+ pathway. The Wnt pathway is activated when Wnt protein ligand binds to frizzled (Fz) family receptors which then transfer the signal inside the cell via disheveled protein (Dsh/Dvl). The b-catenin pathway then activates target genes in the nucleus; the planar cell polarity pathway involves Jun Nterminal kinase (JNK) and regulates the cytoskeleton, while Wnt/Ca2+ pathway regulates cellular Ca2+ levels [13,14]. A characteristic feature of Wnt pathway activation is an increase in the level of b-catenin protein in the cytoplasm. In the absence of Wnt signaling, b-catenin is phosphorylated by various kinases [15,16]. The molecular interaction of b-catenin with these kinases is facilitated by the proteins axin and adenomatous polyposis coli (APC) [17,18]. These

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Figure 1. Molecular Subtypes of Medulloblastoma (MB). MB is divided into four subtypes based on genomics and signaling pathway analysis: Wnt, Shh, group 3, and group 4. Each subgroup has different prognosis, metastasis, MYC status, and signaling pathways. The occurrence of different subtypes of MB also varies among infants, children, and adults. MB in infants is dominated by Shh and group 3 subtypes. MB in children is dominated by group 4 and 3 subtypes, whereas MB in adults is dominated by Shh and group 4 subtypes. This classification highlights the features of four MB subgroups including the molecular genetics and clinical outcome.

proteins together form the b-catenin degradation complex which facilitates phosphorylation of b-catenin by b-TrCP [19]. Wnt involvement in cancer was confirmed by its interactions with the tumor-suppressor APC protein. In mouse, mutation of Wnt causes severe defects in the developing midbrain, hindbrain, and spinal cord [20,21]. In human, mutation of the gene encoding b-catenin and loss of chromosome 6 are the characteristic features of Wnt-type MB [22]. Other common mutations observed in Wnt group of MB include TP53 and dead-box RNA helicases (DDX3X). Wnt tumors generally exhibit classic histology [23], are rarely metastatic, and are found equally in males and females [24]. This type of tumor is more common in older children and teenagers, and is rare in infants. Hedgehog (Hh) The Hh signaling pathway transmits information to embryonic cells for cell differentiation, proliferation, stem cell maintenance, and tissue polarity. The concentration of Hh signaling proteins varies in different parts of the embryo. Hh protein is an important regulator of embryonic development and plays a crucial role in adult tissue maintenance, renewal, and regeneration. There are three known mammalian homologs of hedgehog – desert hedgehog (Dhh); sonic hedgehog (Shh), and indian hedgehog (Ihh) – of which Shh is the best-studied. The Hh signaling cascade is initiated through binding of Hh ligand to the canonical receptor, patched 1 (PTCH1). In the absence of Hh, the transmembrane protein PTCH1 catalytically inhibits the activity of smoothened (SMO) protein. Binding of Hh to PTCH1 results in loss of PTCH1 and SMO activities. Activated SMO transduces the Hh signal via the activator and repressor form of Ci (cubitus interruptus)/GLI family of zinc-finger transcription factors. The GLI transcription factors exist in three isoforms: GLI1 and GLI2, which act as transcriptional

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Figure 2. Pathways Involved in Medulloblastoma (MB) and Their Inhibitors. Signaling pathways reported in MB include Wnt, Shh, Notch, p53, and PI3K. The regulation of these pathways is essential in normal cerebellum development and their dysregulation has been linked to MB tumorigenesis. These pathways involve various receptors (tyrosine kinases, cMET, ERBB2, IGF-R), transcription factors (GLI, b-catenin), and oncoprotein MYC. Various inhibitors specific to these pathways have been developed (GDC-0449, LGK-974, crizotinib, dibenzazepine). These signaling pathways are helpful in selecting therapeutic agents and treatment strategies.

activators, and GLI 3 which acts as a transcriptional repressor [25]. GLI1 activation is often considered as a readout of Hh pathway activation. The main mammalian Hh target genes include GLI1, PTCH1, Hh-interacting protein (HHIP), cyclin D (CCND), MYC, BMI1, BCL2, and VEGF [26,27]. GLI activation is regulated at several different levels via phosphorylation by inhibitors such as SuFu, Ren, protein kinase A (PKA), glycogen synthase kinase 3b (GSK3b), and by activators such as Dyrk1, RAS, and AKT [28]. The Hh pathway can also be activated by several SMO-independent pathways. Aberrant activation of the Hh signaling pathway can lead to cancer. The importance of Hh pathway activation in MB is now widely recognized, and Shh signaling pathway activation accounts for approximately 25% of MB [29–31]. Germline mutation of Hh pathway components leads to MB. Mutation in the Shh pathway varies in age-dependent manner. Typically, PTCH1 mutations occur among all age groups, whereas SUFU mutations are mainly found in infants, SMO mutations are mainly found in adults, and amplification of MYCN and GLI2 is found in children of >3 years of age. Shh-type MB tumors harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant [32]. The involvement of the Hh pathway in MB has been elucidated in various mouse models. Approximately 10–15% of Ptch+/ mice develop MB and express prominent

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levels of GLI1, consistent with activation of the Hh pathway [33]. Tumor incidence in this model can be increased by external beam irradiation of the brain [34]. Most MB mouse models represent the Shh group. Similar to Wnt group, the sex ratio in the Shh group of MB is 1:1, and metastasis is not common in this type of MB although CD31 positive microvessels are increased in such tumors and may be targets for anti-angiogenic therapies. The Shh group of MB is predominantly seen in infants and adults and rarely affects children. TP53 Gene The p53 tumor-suppressor protein encoded by TP53 becomes dysfunctional in almost 40% of MB tumors, and this is considered to be one of the leading causes of treatment failure [35]. A functional TP53 gene limits the survival of potentially pre-neoplastic cells by inducing cell-cycle arrest and apoptosis, whereas TP53 mutations facilitate MB development. MDM2 is the principal cellular antagonist of p53 and thus negatively regulates p53 activity through the induction of p53 protein degradation. In the majority of cases (90%), MDM2 serves as an E3 ubiquitin ligase for p53: it catalyzes polyubiquitination and subsequently induces proteasomal degradation to downregulate p53. Therefore, functional reactivation of p53 or inhibition of the MDM2 is a promising strategy for treatment. Nutlin-3 binds selectively to a p53 interaction domain of MDM2 and completely blocks the degradation of p53. Annette et al. showed antitumor activity of nutlin-3 against MB cells with wild-type p53, both in vitro and in vivo, leading to reduced MDM2 activity and reactivation of the p53 pathway [36]. Chemokines and Cytokines Chemokines are a group of proteins that bind to the G protein chemokine receptors [37,38]. Chemokines are cytokines which play an important role in tumor progression, metastasis, and cancer-related inflammation. Based on the pattern of cysteine residues, chemokines and their receptors are categorized into four groups: CXC, CC, CX3C, and C (C, cysteine; X, noncysteine amino acid) [39,40]. The chemokine CXCL12 and its receptor CXCR4 regulate the development of brain and cerebellum, and thus are potential chemotherapeutic targets for treating MB. A study by Sengupta et al. demonstrated that CXCR4 was present in Wnt- and Shh-type MB, but only Shh subtype tumors displayed relative CXCR4 overexpression [41]. CXCR4 signaling is activated in most proliferating cerebellar astrocytes. CXCL12 and CXCR4 are known to be overexpressed in various brain tumors, but CXCR4 levels are significantly higher in MB. Human cord blood-derived stem cells (hUCBSCs) can develop into MB via MMP2 expression, which is mediated by the SDF1/CXCR4 axis [42]. The CXCR4 antagonist AMD3100 inhibits the intracranial growth of MB, demonstrating that CXCR4 can be an effective target to treat both adult and pediatric brain tumors [43]. CXCL12 activity can also be blocked by the CXCR4 antagonist (plerixafor) or pertussis toxin. Schuller et al. reported A157C and C414T mutations in the CXCR4 transmembrane region in MB patients. These mutations could result in resistance to plerixafor in this subtype of patients. CXCL12 also has a distinctive role in metastasis of MB to CXCL12-enriched tissues such as bone and liver. The Shh pathway enhances CXCR4 surface localization, and synergizes to promote tumor growth. Therefore, combined Shh and CXCR4 antagonism may provide therapeutic benefits [44]. Interleukins are cytokines secreted by immune cells and activate target cells via cell-surface receptors. Interleukins possess a variety of immunomodulatory functions by directly stimulating immune effector and stromal cells at the tumor site [45–48], and can have both inflammatory and anti-inflammatory actions. These cytokines also control cellular differentiation, proliferation, and antibody secretion. The involvement of Interleukins in MB has been shown in many studies. Ashour et al. demonstrated that thymoquinone suppresses the growth of MB via inhibition of NF-kB and of IL-8 and its receptors [49]. Bhoopathi et al. showed involvement of IL-6 in MB cells. Overexpression of SPARC (secreted protein acidic and rich in cysteine) increased the expression of neuronal markers NeuN, nestin, neurofilament, and MAP-2 in MB cells. Their

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study shows that SPARC induces the expression of neuronal markers in MB cells via IL-6. Expression of SPARC decreased IL-6 production and suppressed Notch signaling, increasing the expression of neuronal markers [42]. In response to receptor activation, CD4+ type 2 T helper (Th2) cells, basophils, and mast cells produce the immune regulatory cytokine IL-4 which controls T cell differentiation in response to antigen. IL-4 functions through binding to its receptors (IL-4Rs) expressed mainly on hematopoietic, endothelial, muscular, and neuronal cells. Upon ligand binding, IL-4R initiates cellular proliferation and target gene expression. IL-4R expression by MB cells may serve as a target for future treatment. A study by Joshi et al. concluded that cpIL4-PE protein, an IL-4Ra antagonist, is cytotoxic to MB cell lines without affecting the growth of normal cells [50,51]. In other studies it was shown that IL-17 produced by Th17 cells can inhibit MB growth in mice [52], and that patients with MB display elevated blood levels of Th17 cells and IL-17 [53]. Together, these studies suggest that interleukins are likely to play a role in MB tumorigenesis.

Cellular Origin of MB Various reports highlight the importance of the cellular origin and the stage of their differentiation in determining tumor phenotype in MB [54]. Dysregulated Shh functions as a potent mitogen for granule neuron precursors (GNPs) and results in proliferation. GNP-specific deletion of Ptch1 or activation of Smo results in MB formation [55]. Furthermore, activation of the Shh pathway in ventricular zone-derived stem cells also results in MB tumors. However, stem cells must differentiate to the granule lineage by activating Atoh1 before MB generation. Grammel et al. showed that Shh-type MB could also arise from granule neuron precursors of the cochlear nucleus of the brainstem, which have the same origin as cerebellar GNPs  the rhombic lip  and also express Atoh1 [56]. These studies suggest that Shh-type MB originates from Atoh1+ neuronal progenitor cells. Wnt-type MB is often located within the IV ventricle and infiltrates into the dorsal surface of the brainstem. Thus, cells surrounding the IV ventricle including dorsal brainstem progenitors of cochlear, mossy-fiber, and climbing-fiber neurons may give rise to Wnt MB [57]. Their transcriptional profile also resembles that of dorsal brainstem progenitors. In mouse, activating mutations in catenin b1 (Ctnnb1) and Tp53 deletion in Blbp+ radial glial cells resulted in the abnormal accumulation of cells in the embryonic dorsal brainstem. Moreover, a proportion of mice with Tp53 deletion generated Wnt-type MB. Concomitant activation of the PI3K pathway in Blbp+ cells significantly increases tumor incidence [58]. Based on these results, it can be concluded that Wnt-type MB originates from dorsal brainstem progenitor cells. Two independent studies reported at least two cell types as the origin of group 3 MB. Overexpression of MYC is characteristic of this subtype of MB. Pei et al. infected prominin 1+lineage cerebellar stem cells with retroviruses expressing MYC and a mutant form of p53 (DNp53) [59]. Orthotopic transplantation of these cells into the cerebellum of immunocompromised mice generated tumors with a histological and molecular resemblance to human group 3 MB and gene expression profiles of MYC-driven MB [59]. In another study, Kawauchi et al. transplanted MYC-overexpressing and p53-deficient cerebellar granule neuron precursors into the cerebellum. These cells were able to form tumors, but lost granule lineage markers, suggesting that GNPs may dedifferentiate during transformation [60]. Furthermore, group 3 MB express markers of GABAergic neurons, which suggests that GABAergic progenitors can also be the cells of origin for this subtype of MB [61]. Group 4 MB is a heterogeneous disease, and multiple cell types can give rise to tumors of this subtype. Swartling et al. showed that mice expressing MYCN under the control of a glutamate transporter-1 (Glt1) promoter develop tumors that resemble group 4 MB at the molecular level.

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Glt1 is expressed in progenitors and stem cells in the developing cerebellum, and it is therefore reasonable to assume that the cells of origin for these tumors may be a primitive progenitor or stem cell. Interestingly, similar tumors were produced by overexpression of MYCN in postnatal cerebellar stem cells, but not in the embryonic cerebellum. Further, many group 4 MBs express markers of glutamatergic neurons (GNPs), and therefore these are possible cells of origin.

Enhancers and Superenhancers in MB Tumorigenesis Many mutations often dysregulate enhancers and superenhancers in MB that control the signal-dependent expression of growth-related genes. Changes in these regulatory sequences can affect the expression of oncogenes, which is well evidenced by the discovery of the MYC oncogene which was translocated close to enhancer region of the immunoglobulin heavy chain locus, leading to MYC overexpression [62,63]. MYC expression is involved in many different cancers [64,65] and in all subgroups of MB, with maximum overexpression in group 3 and minimal expression in group 4 (Figure 1). To gain insight into the mechanism of gene regulation, Boulay et al. performed genome-wide chromatin and expression profiling of an aggressive subtype of MB, group 3. Their study demonstrates that transcription factor OTX2 in cooperation with NEUROD1 controls the regulatory landscape of group 3 MB through cooperative activity at enhancer elements that contributes to the expression of target genes [66]. The FOXF1 gene is involved in epithelium– mesenchyme transition (EMT) as a downstream target of the Shh pathway. FOXF1 overexpression is regulated by tissue-specific enhancers, and has been reported in many cancers including MB [67]. A study by Northcott et al. demonstrated that activation of the GFI1 (growth factor independent 1) family protooncogenes, GFI1 and GFI1B, is driven by active enhancer and superenhancer element in group 3 and group 4 MB [68]. Superenhancers are involved in controlling cell identity [69]. Superenhancers function as active enhancer element that has cell type-specific OCT4-dependent functions. They are frequently occupied by terminal transcription factors of the Wnt, TGF-b, and LIF signaling pathways. Manipulation of these three developmental pathways preferentially affects the expression of the regulatory element-associated genes. Wnt signaling involves regulatory elements at key genes associated with tumorigenesis [70]. Because Wnt signaling is often dysregulated in MB, it is possible that this superenhancer is centrally involved in the Wnt group of MB. Detailed information about the involvement of enhancers/superenhancers in MB transcriptional landscapes and their role in the developmental origin of MB subtypes is lacking. To gain insight into this Lin et al. performed chromatin immunoprecipitation and deep sequencing (ChIP-seq) to identify active enhancers containing acetylated histone 3 lysine 27 (H3K27ac) and bromodomain containing 4 (BRD4) protein in MB. They compared ChIP-seq data with DNA methylation and transcriptome data to identify enhancers of MB [71]. TGFB was identified as an oncogenic enhancer in group 3. Several subgroup-specific superenhancers were also found, including SMO and NTRK3 in the Shh MB subtype, ALK in the Wnt MB subtype, LMO1, LMO2, and MYC in group 3, and ETV4 and PAX5 in group 4 MB. Superenhancers regulate several transcription factors, and the regulatory networks are MB subtype-specific. For example, LMX1A as a master regulator of group 4 regulates cerebellar upper rhombic lip transcriptional programming [54].

Barriers to MB Therapy MB tumors possess characteristics distinct from peripheral tumors, and many factors such as the BBB, the tumor microenvironment, and responses of stem cells (versus bulk tumor cells) must be taken into consideration for effective MB targeted drug delivery. For the treatment of MB, drug delivery and nanomedicines have attracted significant attention in recent years.

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Physiological Barriers The protective barrier that lines the blood vessels of the brain to avoid penetration of toxic xenobiotics and other harmful compounds from the bloodstream into the brain tissue is known the BBB. The BBB limits the transport of many hydrophilic, protein-bound drugs, especially when their molecular weight exceeds 400 Da. The BBB is a specialized system of brain endothelial cells which are connected by tight junctions and are partially covered by pericytes and basement membrane. Astrocytes surrounding the BBB make it almost 98% impermeable to small molecules and almost 100% impermeable to large molecules, thus precluding delivery of recombinant proteins and genes. However, some endogenous compounds and drugs may cross the BBB by passive diffusion, GLUT1-mediated carrier transport, endocytosis, and active transport [72–76]. Chemoresponsiveness in MB varies greatly among subtypes owing to the different composition of the BBB in different tumor subtypes. A study by Phoenix et al. showed that the composition of the BBB in MB subtypes is determined by their patterns of gene expression. It was found that the Wnt MB subtype has a better response to chemotherapy because it has a fenestrated vasculature that permits the accumulation of high levels of intratumor chemotherapeutics, leading to a robust therapeutic response by b-catenin. By contrast, non-Wnt MB subtypes have an intact BBB, rendering them impermeable and resistant to chemotherapy. Their study concludes that MB genotype dictates the tumor microenvironment and subsequent blood vessel phenotype [77]. Several strategies are being explored to overcome the BBB including barrier disruption, blocking of efflux pumps, utilization of transporters, and local drug delivery. For effective drug delivery to the brain, many types of active targeting strategies have been developed. These include absorption-mediated transcytosis (AMT), transporter-mediated transcytosis, and receptor-mediated endocytosis (RME). The tumor BBB (BBTB) is another important physiological barrier to drug delivery to MB. It is located between highly specialized endothelial cells of blood vessels and tumor tissues. This microenvironment around the tumor tissue possesses characteristics that are very distinct from normal tissues. The BBTB limits the delivery of most hydrophilic molecules to tumor tissues. During the development of brain tumors, tumor cells disrupt the connectivity between astrocytes and brain endothelial cells, allowing tumor cells to invade the surrounding normal brain tissues. Thus the BBB is damaged, leading to formation of the BBTB [78]. The microenvironment formed by BBTB has three main hallmarks: low oxygen tension or hypoxia, high interstitial fluid pressure (IFP), and low extracellular pH. The pore size for the BBTB in solid malignant orthotopic RG-2 rat glioma microvasculature is approximately 12 nm [79]. With the progression of brain tumors, angiogenesis, and gradual impairment of BBB, the BBTB becomes the main obstacle to drug delivery. Stem Cells Stem cells are undifferentiated cells with remarkable potency to develop into many different cell types. There is evidence that cancer stem cells (CSCs) have self-renewal ability and can differentiate to give rise to a variety of heterogeneous progeny that can form the bulk of the tumor [80]. CSC dissemination poses great challenges to the long-term survival of patients [81]. Like other types of CSCs, medulloblastoma stem cells (MBSCs) are also responsible for therapeutic resistance and invasion. MBSCs are responsible for quiescence, activation of pro-survival/anti-apoptosis pathways, and interaction with microenvironmental factors, thus contributing to treatment failure. This has been well documented in Shh group MB [55]. Further, MB group 3, the most aggressive subgroup of MB and which is characterized by high levels of MYC amplification, appears to derive from cerebellar stem cells [82].

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Studies aiming to target MBSCs have focused on targeting their various signaling pathways such as the Hh, PI3K/AKT, Stat3, and Notch pathways. In MB the majority of cells have an undifferentiated stem-or progenitor-like appearance. These cells have the ability to differentiate into neurons, glia, or other cell types. The cerebellar surface is covered by two primary germinal epithelia: (i) the external germinal layer (EGL), and (ii) the deep-seated ventricular zone (VZ) [33,83]. The VZ germinal matrix gives rise to neuronal and glial cells in cerebellum, while the EGL produces granule cells. The group 3 MB arises from cerebellar stem cells [59,60]. Further studies support that MB primarily originates from the cerebellar stem and progenitor cell population [84]. The VZ marker protein calbidin D is not found in EGL and is overexpressed in half of MB cases, mainly in non-nodular type MB [85]. However, the EGL marker p75 is commonly expressed in nodular MB [86]. These reports further confirm that MB tumors arise from either VZ or EGL cerebellar stem cells. Many of the pathways associated with neuronal stem cells are also highly activated in MB. This dysregulation of signaling pathways in MB subtypes further correlates with tumor-initiating stem or progenitor cell populations as the main origins of MB. Thus, a molecular understanding of cerebellar development and its relation to tumor formation has potential in the development of new therapeutics for MB. MBSCs have a higher level of Hh signaling than non-CSC counterparts, and targeting the Shh pathway is an effective way to eradicate MBSCs. Flora et al. demonstrated that deletion of the gene encoding transcription factor Math1 prevents the development of Shhdriven MB [87]. Math1 is also responsible for maintaining granule neuron precursors in a Shhresponsive state by regulating genes that promote neuronal differentiation [88]. The use of a smallmolecule inhibitor targeting transcription factors Math1, Gli1, or Gli2 could inhibit the constitutive activation of the Shh pathway and thereby prevent MB tumor formation. Histone deacetylase 1 (HDAC1) is an enzyme which modulates Shh signaling through deacetylation of Gli1 and Gli2 proteins [89]. The use of HDAC inhibitors may represent novel therapeutic agents targeting Shhmediated MB. One of the HDAC inhibitor proteins, termed potassium channel tetramerization domain (KCTD), has demonstrated a reduction in Shh-driven MB tumorigenesis [90]. The Wnt signaling pathway regulates the proliferation of MBSCs. Various Wnt ligands activate Wnt signaling which regulates stemness and stem cell niches in normal cells [91,92]. The Wnt inhibitory factor 1 (WIF1) induces cellular senescence, hence impeding stemness and tumor growth [93]. In addition, myriad studies have shown the involvement of Wnt signaling in various brain tumors [94,95]. Membrane coreceptor low-density lipoprotein receptor-related proteins (LRPs) have been shown to be involved in the Wnt/b-catenin pathway. Furthermore, it was reported that MB Daoy CD133+ cells mainly express LRP5/8. Under nutrient-deficient conditions these cells showed an increase of CD133+ cells expressing LRP1/1b/5, indicating that the expression of LRP is associated with MBSC adaptation to nutrient derivation [96]. Most MB tumors develop resistance to radiotherapy and chemotherapy, and activation of Wnt signaling is one factor that determines resistance against radiotherapy and chemotherapy [12]. MBSC resistance may be overcome by the use of ATP-binding cassette (ABC) transporter inhibitors, aldehyde dehydrogenase (ALDH) inhibitors, and/or via targeting CSC-specific Wnt, Hh, and Notch pathways. MBSCs are frequently enriched in tumors following chemotherapy. In addition, some of the markers of MBSCs are activation molecules that may be increased by chemotherapy. Consequently, it is likely that properly planned and administered combination therapies of chemotherapy and MBSC inhibitors will be needed.

Role of MicroRNA in MB MicroRNAs (miRNAs) are small (19–25 nt in length) endogenous non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. miRNAs

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resemble small interfering RNA (siRNA) except that miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, whereas siRNAs derive from longer regions of double-stranded RNA. miRNAs bind within the 30 -untranslated regions (UTRs) of mRNAs in a sequence-specific manner and regulate gene expression post-transcriptionally [97,98]. Dysregulation of miRNA expression has been implicated in many diseases including MB (Figure 3). miRNAs in MB regulate the expression of both oncogenes and tumor-suppressor genes, and thus represent a potential target in cancer therapy. To reveal the role of miRNAmediated signaling cascade involved in MB, Ferretti et al. carried out miRNA profiling and identified a downregulated miRNA signature pattern [99]. These profiling experiments identified a set of 248 miRNAs, of which 34 miRNAs were dysregulated significantly between Shh and non-Shh driven MB. miR-324-5p, miR-125b, and miR-326 could inhibit the activation of SMO by binding to the 3'-UTR of SMO mRNA. In addition, miRNA 324-5p targets the downstream target GLI1. Transfection with these miRNAs in Daoy cells inhibited cell proliferation. These miRNAs were also upregulated in granule progenitor cell (GPC) differentiation and inhibited growth, thus allowing cell maturation. Taken together, these findings suggest that deregulation of specific miRNAs is actively involved in abnormal activation of Shh signaling [99]. A comparative miRNA analysis approach of Uziel et al. between mature rodent cerebellum and tumor cells isolated from spontaneous MB of Ink4c/Ptch1+/ and Ink4c/Tp53/ mice revealed 26 upregulated and 24 downregulated miRNAs [100]. It was also reported that miR183, miR-96, and miR-182 were highly upregulated in non-Shh MB, and miR-182 was upregulated in metastatic tumors [101]. miR-128a was significantly downregulated in MB relative to normal neuronal differentiated cells [102]. The role of miR-124 was found to be crucial for neurogenesis and neuronal differentiation, and it was the most abundant miRNA found in neuronal cells [103–105]. In addition, the expression level of miR-124 was significantly downregulated in MB, and a similar pattern has been reported for other adult brain tumors [106]. Indeed, ectopic expression of miR-124a leads to reduced expression of CDK6 protein [107]. Ferretti et al. used the expression level of Gli1 to differentiate between Shh-driven MB and nonShh driven MB. miRNA profiling results for 250 miRNAs allowed MBs to be divided into Gli1high and Gli1-low groups. In Gli1-high tumors, miR-125b, miR-324-5p, and miR-326 were

Pathways in medulloblastoma

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Figure 3. Representative miRNAs Dysregulated in Medulloblastoma (MB). Aberrant expression of miRNAs is implicated in MB. They are involved in various MB pathways (Wnt, Shh, Notch, p53, PI3K) which regulate multiple functions including angiogenesis, apoptosis, cell cycle, and migration. miRNAs can act both as oncogenes (miR-21, miR-30b, miR-204) and tumor suppressors (miR-124, miR-9, miR-326). In-depth mechanistic understanding of the role of miRNAs in MB can further facilitate the discovery of novel therapies.

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poorly expressed and were chosen for further studies to confirm whether they could inhibit SMO and Gli1 expression. Their in vitro results showed that these three miRNAs could downregulate SMO levels when overexpressed in Daoy MB cells. It was also shown, in MB cells, that Stat3 activation induced miR-21 expression which in turn suppressed PIAS3 activity by epigenetic regulation, and this activated downstream proliferation and anti-apoptotic genes that promoted tumor proliferation and survival.

Small-Molecule Drugs for Treatment of MB In pediatric patients, chemotherapy and radiation are used to kill residual MB cells which remain after surgery. The anticancer drugs used for the treatment of MB can be broadly categorized into the following groups. Topoisomerase Inhibitors Topoisomerase I (Topo I) and topoisomerase II (Topo II) are enzymes which help in unwinding and rewinding of DNA by relieving the torsional strain on the DNA molecule during replication. Inhibition of these enzymes results in partial or complete inhibition of DNA replication and subsequent death of the cells [108]. Two FDA-approved topoisomerase inhibitors, irinotecan and topotecan, are also effective for treating MB [109]. Topotecan and irinotecan both are analogs of the plant alkaloid, camptothecan (CPT), and are effective in a variety of human tumors. The antitumor activity of topoisomerase inhibitors against brain metastases has been investigated in several studies [110,111]. CPT and its analogs bind at the free 30 -phosphate of DNA molecule and stabilize the cleavable complex with Topo I enzyme. Enzyme-linked DNA breaks cannot be regulated/initiated in the presence of these drugs [112]. This leads to S-phase cytotoxicity and DNA damage due to the interaction between replication fork and cleavable complex within DNA molecules [113]. CPT is a pentacyclic ring-like structure. For optimal interaction with Topo I enzyme, CPT requires an intact a-hydroxyl lactone group in the E-ring (Figure 4) [114]. The E-ring of CPT is labile in aqueous solutions and can be converted into inactive open-ring carboxylate form by pHdependent hydrolysis. The solubility of CPT can be altered by substitutions at the C-7 and C-10 positions without affecting its biological activity. Structure–activity relationship (SAR) studies of

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Figure 4. Structures of Some of Topoisomerase Inhibitors.

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CPT resulted in various analogs with improved drug-like properties. The substitution patterns on R1, R2, and R3 are as follows: in topotecan [R1 = OH, R2 = CH2N(CH3)2, and R3 = H], irinotecan (R1 = C11H20N2O2, R2 = H, R3 = CH2CH3), 9-amino camptothecin [R1 = H, R2 = CH2N(CH3)2, R3 = H], and in 7-ethyl-10-hydroxycamptothecin (R1 = OH, R2 = H, R3 = CH2CH3) (Figure 4). Irinotecan acts as a prodrug for 7-ethyl-10-hydroxycamptothecin (SN-38) and is converted to its active form by esterase-mediated enzymatic hydrolysis. The SN38 molecule binds reversibly to the ternary complex of DNA and Topo I enzyme and inhibits DNA replication which finally leads to cell death [115]. Although topotecan and irinotecan both share the same specific mechanism of cell killing, they have distinct pharmacokinetic properties. While most of the drugs suffer from poor penetration through the BBB, topotecan can freely cross the BBB owing to its low protein-binding affinity in serum. A clinical trial for topotecan in MB has been completed and showed prolonged survival of MB patients. Other topoisomerase inhibitors currently in clinical trials are NB-506, X-8951f, and tothecin. These analogs show remarkable potency in vitro against a variety of adult and pediatric cell lines [116– 118]. The first indenoisoquinoline compound reported to have anticancer activity was NSC 314622 (Figure 4) and was synthesized in 1978 [119]. The cytotoxicity profile was similar to those of irinotecan and topotecan. Owing to the poor biological activity of indenoisoquinolines, the structure was optimized and various derivatives were synthesized. Optimizing the side-chain group on 3-substituted indenoisoquinoline resulted in indimitecan (LMP 776) and indotecan (LMP 400) with improved efficacy [120]. Both drugs are currently in Phase I clinical trials at the National Cancer Institute. Zhao et al. designed and synthesized isoquinoline derivatives by substituting the nitro group. The compounds with acetylamino at R1 and 4-methylpiperazinyl at R2 were found to have maximum anticancer properties in various cancer cell lines [121]. In another study, Majumdar et al. synthesized a series of hydantoin (glycolylurea) and thiohydantoin derivatives by substituting thiophenyl as the central moiety [122]. The other Topo inhibitors used in MB therapy include doxorubicin, etoposide, cisplatin, vincristine and teniposide. Hh Inhibitors One of the first chemotherapeutic approaches to MB treatment involved Hh inhibitors. Many Hh pathway inhibitors have been investigated for their potential anticancer activities. The first Hh pathway inhibitor discovered to have an anticancer effect was the naturally occurring plant alkaloid, cyclopamine. Structurally, cyclopamine consists of a hexacyclic framework of four annulated carbocycles. One of them belongs to five-membered ring (C ring) and others are six-membered rings (A, B, and D rings; Figure 6). Cyclopamine binds to the heptahelical transmembrane domain of SMO and prevents the conformation shift that is necessary to activate the protein, and thus suppresses the proliferation of cancer cells. Berman et al. also reported its ability to induce neural differentiation of murine MB cells in vitro and in tumor allografts [123]. Despite high efficiency in preclinical studies, cyclopamine failed in clinical development owing to its suboptimal pharmacokinetic characteristics, low metabolic stability, low potency, and related toxicities. However, the potential of cyclopamine led to the development of many small molecules with improved drug-like properties, potency, and bioavailability. These include vismodegib (GDC-0449), saridegib (IPI-926), erismodegib (LDE-225), TAK-441, XL-139 (BMS-833923), PF-04449913, and PF-5274857. Vismodagib (GDC-0449) is an SMO antagonist and is the first FDA-approved drug as a Hh inhibitor for advanced and metastatic basal cell carcinoma [124]. In MB, patients treated with vismodegib showed a remarkable response and regression in tumor size in Shh-driven MB

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[125]. A variety of heterocyclic group replacements of the 2-pyridyl biphenyl amide scaffold led to 2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide (GDC-0449). Furthermore, Castanedo et al. synthesized various analogs of GDC-0449 by optimizing the polar group in the para position of 2-pyridyl biphenyl amide [126]. When the trifluoro methyl nicotinic amide of GDC-0449 was modified into 2-chloro-4-methane sulfonyl benzamide, there was an improvement in the solubility of the molecule while retaining potency in a Ptch+/+ MB allograft model. Therefore, they focused their SAR study around the aryl amide group and generated various GDC-0449 analogs. Recently, Kumar et al. synthesized a new and potent analog of GDC-0449 that exhibited better activity in a pancreatic cancer model and thus could be an attractive agent for MB [127]. Hyman et al. developed a series of benzimidazolephenylbisamide analogs showing enhanced activity compared to GDC-0449 against MGC803, HT29, and MKN45 gastrointestinal cancer cell lines [128]. SAR analysis resulted in the compound MRT-10, which was an attractive molecule with a balanced structure displaying two hydrophobic zones at the terminal ends, demarcated by a polar connector, allowing H-bond interactions at the SMO receptor level (Figure 5). Based on the structural features of MRT-10, Sun et al. designed a target compound which consisted of three parts: A, B, and connector (Figure 5). Part A contains the benzimidazole moiety, part B contains the 4-benzyloxyphenyl moiety, and the connector includes acylthiourea or acylurea groups. By replacing the moieties at the 4-positions of the A-ring (part A) and B-ring (part B) with methyl or chlorine they synthesized various analogs of N-3benzimidazolephenylbisamide (Figure 5). Saridegib (IPI-926) is a semi-synthetic derivative of cyclopamine that has been clinically investigated in metastatic solid tumors [129,130]. It is an orally bioavailable drug which

Vismodegib (GDC-0449) Cyclopamine

Taladegib (LY-2940680)

TAK-441 Sonidegib (LDE-225)

MRT-10 Lapanib Part B

Connector Part A

Glasdegib

Figure 5. Representative Structures of Hh Signaling Pathway Inhibitors.

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Taladegib

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Figure 6. Structures of Some Small-Molecule Protein Kinase Inhibitors Used in Cancer Therapy.

produced tumor regression in a Shh-driven MB mouse model. Erismodegib (NVP-LDE225) is another potent SMO inhibitor that has been shown to inhibit tumor progression and Shh signaling in the Ptch+/Tp53+/ MB mouse model [131]. Its antitumor activity has also shown in advanced basal cell carcinoma (BCC) and is currently in a Phase I study. Other potent inhibitors of the Shh pathway include LDE-225 and BMS-833923, but their effects on MB have not been tested. Cells with mutant SMO proteins developed resistance to these inhibitors. Therefore, molecules acting downstream of the Hh pathway were recently developed. These include GANT61 and HPI-1 [132,133]. Tyrosine Kinase Inhibitors (TKIs) Tyrosine kinases catalyze tyrosine phosphorylation through the transfer of the g-phosphate of ATP to tyrosine residues on protein substrates. They activate diverse proteins and participate in many key cell functions including cell signaling, growth, and division. TKIs can inhibit specific signal transduction by blocking phosphorylation, and thus are widely used in cancer therapy [134]. Most TKIs target oncogene growth factors, including Bcr–Abl, Her2, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-I), which modulate many cancer progression pathways in MB including cell proliferation, death, differentiation, and metastasis [135]. TKIs in clinical use include imatinib, gefitinib, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib, sunitinib, leflunomide (SU101), and erlotinib [136,137]. Imatinib has shown to block the migration and invasion properties of MB cells by blocking the PDGF receptor [138]. Imatinib prevents ATP binding to the Abl domain via a six hydrogen bond

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interaction, and it was the first inhibitor of Bcr–Abl tyrosine kinase family [139]. The principle hydrogen bonds involved are between aminopyrimidine and the side-chain hydroxyl of Thr315, pyridine-N and the backbone-NH of Met318, amide-NH and the side-chain carboxylate of Glu285, the protonated methylpiperazine with the backbone-carbonyl atoms of Ile360 and His361, and the carbonyl with the backbone-NH of Asp381. Efficient binding was also supported by several van der Waals interactions [140]. Imatinib is a derivative of 2-phenylamino-pyrimidine (PAP) that is approved for treating patients with chronic myeloid leukemia (CML). The derivative compounds obtained from PAP were STI-571 {4-(4-methylpiperazin-1ylmethyl)-N-[4-methyl-3-(4-pyridine-3-yl-pyrimidin-2-ylamino)phenyl] benzamide mesylate: imatinib mesylate, Gleevec} and showed potent efficacy in chronic phase CML patients [136]. The addition of a nitro group and subsequent methylation, benzoylation, and oxidation yielded a series of PAP derivatives. All the compounds were assayed for the inhibition of tyrosine kinases. Among them phenylamino-pyrimidine l was found to be an attractive lead. With the introduction of a methyl group at the 6-position of the phenyl ring, the potency for inhibition of PDGFR autophosphorylation was significantly enhanced. SAR studies of PAP derivatives resulted in N-acyl and N-aroyl amide derivatives in which replacement of the imidazole with a benzamido group maintained the activity of the compound as a kinase inhibitor. Introducing a methyl substituent ortho to the pyrimidinyl-amino group inhibited tyrosine kinase activity [136]. This is due to the orthogonal orientation of pyrimidine and phenyl rings, and steric clash with Met470 and Leu134, respectively, on PAP-derivative compounds. These amides are highly insoluble and thus lack oral bioavailability. Introducing a hydrophilic moiety such as an N-methylpiperazine group resulted in the discovery of imatinib [141]. The increase in PDGF inhibition via pharmacophore was defined by Druker et al. as follows: phenylaminopyrimidine with a pyridine attached to the 4-position of the pyrimidine ring [136]. The substitution pattern of the phenyl group needs to be as follows: a methyl- or chlorosubstituent at the 6-position (R2 = CH3 or CI) and a substituted benzamide [R4 = C(O)-phenyl] with a free NH-group (R3 = H) at the 3-position. Benzamide is preferentially substituted again with a small lipophilic substituent (chlorine or methyl) at the 4-position of the benzamide moiety. The compound STI-571 was found to be the best and most specific PDGF-dependent tumor growth inhibitor in vivo. SAR analysis of the Abl–imatinib interaction had a tremendous influence in the area of kinase inhibitor design. Several other TKIs have been developed including sorafenib [142], BIRB796 [143], lapatinib (Tykerb, GW572016) [144], nilotinib (AMN 1070), dasatinib (BMS-345825), ponatinib (AP24534), and bafetinib (INNO-406) (Figure 6). Later, in 2008 Liu et al. developed a mild, convenient, and inexpensive approach for the preparation of imatinib and its nine analogs with improved efficacy [19]. Imatinib can inhibit the in vitro and in vivo growth and invasiveness of human MB cell lines Daoy and D556 [138]. This drug can also cross the BBB efficiently. Another tyrosine kinase receptor protein, c-MET, is overexpressed in human MB. MB tumor growth was inhibited by the SPINT2 serine protease inhibitor [145]. Furthermore, it was shown that TKI SGX523 could effectively decrease the growth and migration of Daoy cells [146]. These findings collectively suggest that TKIs which target c-MET signaling could be effective alternatives in the treatment of MB. PI3K/AKT Signaling Inhibitors PI3K signaling plays a crucial role in controlling cell proliferation, survival, and motility/metastasis, and is considered as a promising therapeutic target for cancer treatment. Inhibition of PI3K and IGF-1R signaling pathways in combination has been shown to be effective in MB. Anna et al. reported the antiproliferative potential of combination therapy involving humanized

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anti-IGF-1R antibody R1507 in conjunction with PIK75, a class IA PI3K inhibitor, in MB and neuroblastoma cell lines [148]. Angiogenesis and Anti-Angiogenesis Agents in MB One of the striking histopathological characteristics of MB in the Ptch1/Tp53/ mouse model is the significant increase in the density of small blood vessels in the tumor compared to normal medulla. One of the downstream targets of Wnt and Shh is the VEGF pathway, and VEGFR is expressed abundantly in some MB and may be associated with gadolinium enhancement in MRI [149]. Gao et al. showed that the expression levels of VEGF and miR210 were upregulated in MB and metastatic MB [150]. These data suggest that anti-angiogenic therapies may have value in MB. Bai et al. showed that mebendazole had low toxicity in the Ptch1/Tp53/ mouse model and significantly extended the survival of mice with tumors [151]. Other Small Molecules Altered ion channel activity has been reported in a variety of cancers. The voltage-gated potassium channel EAG2 (ether-a-go-go 2) and potassium channel KCNT2 cooperatively promote tumor growth and metastasis in MB cells. Inhibition of EAG2 with thioridazine decreased cell viability and motility in a metastasis MB xenograft model [152]. Radiotherapy may cause severe long-term side effects that significantly impact on the quality of life in young patients. Thus, new chemotherapeutics that can sensitize the tumor to irradiation may reduce both the total dose and the side effects. A screen of 960 chemical compounds for sensitization of MB cells to irradiation found that quercetin is a novel radiosensitizer for the MB cell lines Daoy, D283-med, and, to a lesser extent, D458-med. Administration of quercetin at the time of irradiation significantly prolonged survival in orthotopically xenografted mice [153].

Drug Delivery Systems and Ongoing Clinical Trials A distinct feature of brain tumors is that it is difficult to achieve sufficient drug delivery. Furthermore, the use of chemotherapy in children has always been a challenge. For example, although Hh pathway inhibition is a promising strategy for MB treatment, its direct inhibition can result in a severe adverse effect on developmental processes in children [154]. Therefore, drug delivery systems can play a crucial role in safe and effective therapy (Figure 7). Several methods and delivery systems including nanotechnology have been applied for safe and efficacious therapy. Delivery of anticancer drugs across the BBB remains a challenge. Several methods have been applied in the past to facilitate drug partitioning into the brain. Low-density lipoprotein receptorrelated protein (LRP) mediates the transport of aprotinin with a Kunitz protease inhibitor (KPI) sequence across the BBB. Peptides based on the amino acid sequence of aprotinin with a KPI domain of human proteins (Angiopeps) were developed. In one study, Angiopep-2 polypeptide was conjugated covalently to etoposide and doxorubicin via hydrolyzable glutaric acid and succinic acid linkers, respectively. Angiopep-2 is a 19 amino acid peptide with very high transport properties into the brain by crossing the BBB [155]. These conjugates showed decreased toxicity. Biodistribution studies in a mouse model of brain tumors showed that distribution of peptide-conjugated drugs was higher in the brain than were unconjugated counterparts. The biodegradable and biocompatible properties of albumin make it an attractive material to prepare nanoparticles (NPs). Albumin NPs are readily taken up by cancer cells to fulfill their increased need for amino acids and energy. Furthermore, albumin NPs interact electrostatically with cell membranes and readily cross the BBB via adsorptive transcytosis. Catanzaro et al. developed glutathione (GSH)-responsive disulfide bond crosslinked bovine serum albumin

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Nanoparcles

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Figure 7. Drug Formulation and Targeted Delivery to Brain. The nanocarrier system has potential to enhance the circulation time of encapsulated drugs, allowing effective drug accumulation at the target site. Further receptor-mediated transcytosis can be an effective means of active transport for nanoparticle entry into the brain. Imaging and sensing agents can also be encapsulated to generate multifunctionality in the treatment of medulloblastoma (MB).

(BSA) NPs for CPT delivery to MB cells. These NPs were evaluated for effective uptake and in vitro cytotoxicity in healthy human keratinocyte (HaCaT) and human MB (Daoy) cell lines [156]. Radiation therapy generates double-stranded DNA breaks and results in cytotoxicity. In human cells a protein Ape1 can initiate the repair of DNA breaks, leading to radiation resistance. Suppression of Ape1 expression sensitizes MB cells to radiation as well as to abasic siteinducing alkylating agents. Methoxyamine, a small molecule and indirect inhibitor of Ape1, has progressed to Phase I and II clinical trials, but has low potency and lacks specificity. Kievit et al. recently reported RNAi-based suppression of Ape1 in vivo. The authors developed NPs comprising an iron oxide (Fe3O4) core coated with a polymeric shell composed of chitosan, low molecular weight (1200 kDa) polyethyleneimine (PEI), and polyethylene glycol (PEG). NPs containing siApe1 suppressed Ape1 expression and abasic lesion endonuclease activity in MB cells. The authors also showed that siRNA-mediated Ape1 suppression increases abasic lesion and double-strand break abundance, and reduces tumor cellular resistance to 137Cs g-rays [156]. NPs encapsulating magnetic materials such as iron oxide and a drug can be directed remotely to the disease site. However, poor penetration depth and diffusion of the released drug at the disease site have limited their use. The addition of receptor-specific ligands to magnetic NPs for active targeting can significantly increase their efficacy. Chlorotoxin (CTX), a peptide from scorpion venom, exhibits a high affinity for brain tumor cells. Sun et al. conjugated CTX to iron oxide NPs and the drug methotrexate (MTX) via a PEG linker to serve as both a diagnostic and therapeutic agent. The targeting potential of this NP–MTX–CTX conjugate was evaluated for preferential accumulation in tumor cells in vitro and in vivo. MRI performed on xenograft tumorbearing mice revealed decreased T2 signal in tumor regions of mice receiving NP–MTX–CTX,

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suggesting persistent accumulation and binding of these NPs in MB tumors [157]. Omid et al. synthesized a gene carrier P-PEG–AF–CTX with branched polyethyleneimine (PEI) shielded by PEG and functionalized with targeting peptide CTX and Alexa fluor 647 (AF) dye. The specificity and transfection of the system was studied with MMP-2-positive C6 and Daoy cells. The incorporation of CTX significantly increased transfection efficiency and the addition of PEG reduced the toxicity of PEI [158]. Upon entering the systemic circulation, NPs become coated with non-specific proteins, leading to loss of ligand-mediated targeting potential. Li et al. synthesized a copolymer consisting of blocks of PEG and allyl glycidyl ether (PEG-b-AGE) for coating magnetic iron oxide nanoparticles (IONPs) to reduce non-specific protein adhesion. PEG-b-AGE-coated IONPs showed significantly reduced antibiofouling properties, and improved targeting of transferrin (Tf) PEG-bAGE-coated IONPs to Tf receptors in D556 and Daoy MB cancer cells [159]. Absorption of specific proteins on particle surfaces can also be utilized for targeting. PEG-decorated NPs tend to adsorb LDLs such as ApoB-100 and ApoE proteins on their surface. LDL receptorexpressing neurons and glial cells recognize these adsorbed proteins and can help NPs to penetrate the brain epithelium [160]. Statins (simvastatin, mevastatin) reduce the levels of drug exporters such as P-glycoprotein and breast cancer resistance protein (BCRP), resulting in increased penetration of drugs across the BBB. Based on these observations, Pinzón-Daza et al. designed an LDL receptor-targeted liposome-encapsulated doxorubicin system. Higher transport of apo-Lipodox was observed across the BBB than of the free doxorubicin in cells exposed to statins [161]. The meninges of the brain and spinal cord are continuous, and this delivery route may avoid the BBB and represent a rationale for using intrathecal (IT) therapy. However, to maintain cytotoxic concentrations of drugs in CSF with conventional drugs requires frequent repeated lumbar punctures and may pose technical difficulties and discomfort. Sustained-release formulations prolong cytotoxic concentrations and are an attractive alternative. In a case study, a liposomal formulation containing cytarabine (half-life 100–263 h) was administered intrathecally for the treatment of recurrent MB. The formulation was administered every 15 days at 2 mg/kg in conjunction with systemic cisplatin, lomustine, and vincristine chemotherapy(every 6 weeks). The treatment was well tolerated with no severe/life-threatening toxicities, and all patients achieved progression-free survival ranging from 4 to 11 months [162]. Combination therapy of temozolomide (TMZ) and oral etoposide (VP-16) showed a synergistic effect in children with progressive or recurrent MB. TMZ is a bioavailable oral brain-penetrating alkylating agent, and etoposide as discussed before is a Topo II enzyme inhibitor. The combination therapy was well tolerated and demonstrated high antitumor activity [25]. Failure to eliminate MB CSCs is believed to be a major cause of tumor recurrence. Treatment strategies to suppress stem cell-related signaling pathways may affect normal stem cells which play a vital role in hemostasis. Therefore, new therapeutics which can differentiate CSCs versus non-tumor stem cells are desirable. Litian et al. used Seneca Valley virus-001 (SVV-001) to kill CSCs in 10 primary tumor-based orthotopic xenograft mouse models. SVV-001 is a naturally occurring oncolytic picornavirus which can infect and eliminate CD133-expressing tumor cells (CSCs). Furthermore, SVV-001 crossed the BBB and resulted in a significant increase in the survival (2.2- to 5.9-fold) of animals [163]. MBs frequently disseminate to the leptomeninges and are a sanctuary from systemic therapy, resulting in poor prognoses. Cobo et al. developed a wireless-operated, refillable, and implantable micropump. This device can inject multiple drug infusions directly into the brain ventricles. The main innovations of this pump are the combination of wireless control, programmability, and miniaturized packaging. This refillable pump can reliably deliver hourly infusions into the CSF over prolonged periods. This pump was used to

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deliver topotecan into the CSF, and this resulted in complete remission in mice with leptomeningeal MB. Intraperitoneal administration of the same dose was ineffective; furthermore, in addition to chemotherapeutics, the pump is capable of delivering anti-GD2-mediated immunotherapeutic agents [164]. The most lethal MB subtype exhibits high expression of the g-aminobutyric acid (GABAA) receptor a5 subunit gene and MYC amplification. Benzodiazepines are a5-GABAA receptor ligands. Jonas et al. synthesized new a5-GABAA agonists and analyzed their efficacy using a microscale implantable device that allowed drug delivery into small distinct regions of tumors. One benzodiazepine derivative, KRM-II-08, was found to be a potent inhibitor of several a5GABAA receptor-expressing tumor models. This was the first instance of in vivo testing of several benzodiazepine derivatives and standard chemotherapeutic drugs within the same tumor. Podoplanin is a 162 amino acid type I transmembrane sialomucin-like glycoprotein consisting of serine- and threonine-rich extracellular domains. Podoplanin overexpression is observed in 27% cases of MB, making it an attractive target for brain tumor therapy. Yukinari et al. developed anti-podoplanin antibody NZ-1 to target radionuclides to malignant gliomas [165]. Furthermore, Chandramohan and colleagues constructed a recombinant pseudomonas exotoxin-based IT therapeutic, NZ-1-(scdsFv)-PE38KDEL, from the NZ-1 mAb. IT delivery of NZ-1-(scdsFv)-PE38KDEL was shown to inhibit growth of brain tumors [166]. Overexpression of cell-surface integrins on tumors and their vasculature represents an attractive target for anti-angiogenesis, drug delivery, and cancer imaging. Several potent small molecules and peptides interacting with integrins have been studied in clinical trials. Kishima et al. developed monoclonal antibody ONS-M21 Mab which reacts with a surface antigen of most gliomas and MB [167]. A head-to-tail cyclized RGD-based antagonist of integrins avb3, Cilengitide, was developed for blocking the growth of brain tumors and angiogenesis. Targeting ligands with broad specificity for multiple tumor-associated integrins is expected to have higher sensitivity and selectivity compared to ligands targeting a single type of receptor. Recently, Moore et al. engineered the cystine-knot (knottin) peptide EETI 2.5F (3.5 kDa) with high affinity for avb3, avb5, and a5b1 integrins which are highly expressed in MB, and tested its targeting ability in the Ptch+/ mouse model of MB. The dye-conjugated EETI 2.5F accumulated in tumors and the intensity of the imaging signal correlated with the tumor volume. EETI 2.5F binds to a5b1 integrin in addition to avb3 and avb5, and therefore is superior to several known integrin-binding peptides such as AgRP 7C, EETI 2.5D, and c(RGDfK). Further, to check the effect of molecular weight on EETI 2.5F binding, EETI 2.5F was fused to the Fc region of a mouse IgG2a fragment (60 kDa). Dye-labeled AF680–EETI 2.5F–Fc exhibited robust targeting of intracranial MB in the orthotopic Med1-MB model, compared to control construct AF680– EETI RDG–Fc, and highlighted the targeting potential of specific integrin-binding interactions [168].

Concluding Remarks and Future Directions MB is highly aggressive tumor of the cerebellum. Treatment of MB using a combination of surgery, chemotherapy, and irradiation remains a challenge. Improved knowledge of aberrant signaling pathways in MB has provided novel therapeutic targets for drug development that include Hh, Wnt, tyrosine kinases, and their downstream effectors such as PI3K/AKT, Gli1, MYC, and STAT3, which can be pharmacologically targeted to regress MB growth. Owing to toxicity and off-target effect of various drugs, efforts are being made to refine the MB treatment strategy. Molecular classification of MB has opened a new door for developing drugs which target specific MB subtypes. Various small-molecule inhibitors are now available for Wnt and

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Outstanding Questions Can miRNAs targeting MBSCs improve the treatment of MB? Will the molecular classification of medulloblastoma based on extensive genomic analysis help in designing new therapeutic agents? Would a combination therapy of miRNA and a small-molecule inhibitor be more effective against the chemoresistance displayed by stem cells in medulloblastoma therapy? Will a nanomedicine carrying the therapeutic agents cross the blood–brain barrier (BBB) treat the disease more effectively? Will nanoparticle systems minimize the toxicity and off-target effects of existing drugs?

Shh subgroups of MB. However, the Wnt and Shh pathways account for only
40% of all MB cases, and the remaining 60% are still poorly understood and have a poor prognosis. Therefore, the use of specific signaling pathway inhibitors in preclinical models, miRNA, and combination therapies have been shown to have improved antineoplastic effects (see Outstanding Questions). However, BBB and high heterogeneity limit the successful application of most existing therapies. Therefore, the use of polymeric nanomedicines carrying miRNA and small molecules has potential to effectively cross the BBB, which will result in effective treatment of MB. The use of polymeric nanomedicines carrying small molecules and miRNA exemplifies the exciting possibilities for targeted therapy of MB. Despite significant advances in surgery, radiotherapy, and drug therapy, effective treatment of MB remains a challenge. To improve the outcome it is therefore crucial to develop a drug based on comprehensive understanding of MB subtypes. Acknowledgments We greatly acknowledge the financial support of the Pediatric Cancer Research Group of the University of Nebraska Medical Center and Children’s Hospital (LB805) Omaha, Nebraska and The Team Jack Foundation for this work. We also acknowledge the help of William G. Glass and Paul Dye, graphic arts visualization specialists at the Interprofessional Experiential Center for Enduring Learning (iEXCEL)-Academic Affairs, University of Nebraska Medical Center, for helping in making the illustrations.

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