**2.2. Sonic hedgehog (SHH) pathway medulloblastomas**

Activation of the sonic hedgehog (SHH) pathway drives tumorigenesis in the SHH group of medulloblastomas. SHH medulloblastomas are frequently found in infant (ages 0–3) and adult (>16 years) but occur less commonly in pediatric cases [25]. The prognosis is similar to Group 4 medulloblastomas.

#### *2.2.1. Molecular basis of SHH medulloblastomas*

Interestingly, known nonsteroidal anti-inflammatory drugs (NSAIDs) have been found to have anti-Wnt pathway activity, possibly explaining in part their antineoplastic properties [27, 34, 35]. *In vitro* studies of colon cancer cells have shown that the NSAID sulindac inhibits canonical Wnt pathway activity via inhibition of cGMP hydrolysis [27]. Sulindac may also affect the Wnt pathway by affecting Dishevelled [34]. Celecoxib and diclofenac have been shown to decrease Wnt pathway signaling in *in vitro* glioblastoma cells [36]. Aspirin too affects the Wnt pathway [37]; in one study, aspirin diminished tumorigenesis in intestinal cells. The possible mechanism for aspirin in this study was downregulation of the expression of *PPARδ*, a growth and antiapoptotic promoting transcription factorthat is a direct product of the Wnt

There are a number of ongoing trials using novel agents targeting the Wnt pathway. These agents include PRI-724, designed by Prism BioLab and which blocks the interaction of βcatenin with cotranscriptional coactivator CBP [29, 31]. A Phase I clinical on the molecule LGK-794, a porcupine inhibitor that inhibits Wnt protein secretion, is currently recruiting patients and will assess the safety profile in patients who carry malignancies that are depend‐ ent on Wnt ligands [29, 31]. It is important to note that these Wnt pathway-targeting com‐ pounds have not been tested in medulloblastomas, which would be the next direction for assessing their efficacy in Wnt medulloblastoma. However, although the Wnt pathway is a potential target for future medulloblastoma therapies, some authors have described poten‐ tial theoretical barriers to the utilization of Wnt-targeted therapy in malignancy [28]. First, the Wnt pathway is crucial to organogenesis and homeostasis, begging the question as to whether altering the Wnt pathway may be detrimental to these processes. Second, some have contest‐ ed the assumption that Wnt pathway antagonism would be desirable as anticancer therapy, given that the Wnt pathway is involved in neural regeneration after brain injury (such as surgery). The ongoing clinical trials using therapies targeting the Wnt pathway will help to

better elucidate the safety and viability of targeting this pathway.

Group Patient epidemiology Prognosis Associated genetic aberration

Best prognosis of all subgroups: 95% 10-year survival in children; 100% 5-year survival in adults

Worst prognosis of all subgroups:

40–60% 5-year survival

75% 5-year survival *Ptch1, Smo, Gli1, Gli2*, and/or

75% 5-year survival 17q chromosome Loss of X

*SUFU*—hyperactivation of sonic hedgehog signaling

β-Catenin—increased MYC

MYC Photoreceptor-associated

chromosome in female

expression

pathways

patients

**Clinical and molecular overview of medulloblastoma subgroups**

SHH Frequent in infants and in

adults but not in pediatric and teenage patients

WNT Rare in infants More common in males than in females

Group 3 Infants and pediatric patients but rare in adults

Group 4 Most prevalent subgroup, 34% of cases Found in all age groups

pathway [38].

388 Neurooncology - Newer Developments

In sonic hedgehog signaling, the receptor Patch (specifically Ptch1) inhibits a G-proteincoupled receptor called Smoothened (Smo) in the absence of Hedgehog ligand. Hedgehog ligand binding to Patch results in disinhibition of Patch from Smo, allowing downstream signaling transduction and the activation of the Gli transcription factors, Gli1, Gli2, and Gli3 (**Figure 4**) [39, 40]. Mutations in Patch, Smo, Gli1, and Gli2 have been shown to initiate medulloblastoma in a variety of models [41–44]. Mutation in SUFU, a negative regulator of SHH signaling, is another initiating mutation [45].

**Figure 4.** Schematic showing sonic hedgehog (SHH) signaling: in the absence of Hedgehog ligand, the Patch receptor (Ptch1) inhibits Smoothened (Smo). Hedgehog ligand binding to Patch results in the disinhibition of Smoothened, leading to downstream activation of the Gli transcription factors. Schematic illustrated by author JYY of this book chapter.

#### *2.2.2. Drug targets in SHH medulloblastomas*

Alkylating agents have long since served in chemotherapy for medulloblastoma, but for the SHH subgroup, inhibitors of Smo are also popular. The compound cyclopamine launched initial interest in targeting SHH signaling which was responsible forthe developmental defects

found in sheep that ingested corn lilies in which cyclopamine was originally discovered [46]. 2004 marked the year that Genentech identified the drug vismodegib in a screen for com‐ pounds that inhibit the SHH pathway [46]. Studies assessed vismodegib initially in ad‐ vanced basal cell carcinoma and were also launched to assess the drug for other cancers [46]. Vismodegib was approved in 2012 by the Food and Drug Administration (FDA) for the treatment of metastatic or recurring BCC [46]. A Phase I study has been undertaken to assess the safety, safe dosing range, and side effects of vismodegib in a population of children with recurrent or refractory medulloblastoma [47]. Out of the 20 patients enrolled for flat-dosage testing (150 mg for smaller body area and 300 mg for larger), only two dose-limiting toxici‐ ties were observed. The study concluded that vismodegib is well tolerated in pediatric patients with recurrent or refractory medulloblastoma and recommended 150 or 300 mg dosage for Phase II trials.

Consequently, a Phase II trial was conducted at this recommended dosage with adult and pediatric patient groups. The study found that vismodegib increased progression-free survival in SHH medulloblastoma group but not in the non-SHH medulloblastoma group. Vismode‐ gib exhibited activity against adult SHH medulloblastoma. However, inadequate sampling size for the pediatric group precluded conclusions about vismodegib efficacy in this group [48]. Therefore, vismodegib appears promising for adult medulloblastoma patients but remains to be further examined for pediatric patients.

In 2015, the FDA approved another Smo inhibitor, sonidegib (also known as LDE225), for use in treating basal cell carcinoma [49]. Sonidegib has been tested in a variety of cancers, including medulloblastoma [50]. Other Smo inhibitors are being tested in other cancers. GANT61 has been tested in a prostate cancer model [51], while BMS-833923 was tested in a gastric and esophageal cancer model [52]. Both remain to be tested in medulloblastoma.

For SHH medulloblastoma, targeting SHH signaling is a more direct therapeutic approach than the use of alkylating agents; however, drug resistance may pose a realistic concern. For example, it has been found that drug resistance can arise from amino acids changes in Smo which leads to a deficiency in drug binding to vismodegib [53]. With the approval of sonide‐ gib, researchers then investigated whetherits usage might improve tumorresponse in patients with basal cell carcinoma who were resistant to vismodegib. They concluded that, unfortu‐ nately, patients with advanced basal cell carcinoma, who were previously resistant to vismodegib, also experienced resistance with sonidegib treatment [54]. So, drug resistance with novel Smo inhibitors remains an ongoing concern.

Toward the goal of developing combination therapies and limiting drug resistance, recent research has progressed to investigating the molecular regulation of proteins within the SHH pathway as potential drug targets. For example, several kinases have been shown to control the activity of Gli1: ribosomal protein S6 activates Gli1 through phosphorylation on its serine 84 [55], while protein kinase A phosphorylation inhibits Gli1's activity [56].

AMP kinase (AMPK), a regulator of cell energy allocation during stress conditions, has been shown to modulate Gli1 activity. Specifically, overexpression of AMPK leads to a decrease in Gli1 expression, while downregulation of AMPK activity increases Gli1 expression [57].

Therefore, suppression of SHH signaling through downregulation of Gli1 may serve as a venue of targeting SHH medulloblastomas. Our group has demonstrated how direct regulation of SHH signaling through AMPK function impacts tumorigenesis. We found that AMPK regulates Gli1 activity by phosphorylating the transcription factor at serines 102 and 408 and threonine 1074. Mutation of these phosphorylation sites to nonphosphorylatable alanine increasesGli1 protein stability, transcriptional activity, andoncogenic potency, suggesting that AMPK phosphorylation reduces Gli1 activity (**Figure 5**). Another group has supported our finding that AMPK phosphorylates and may regulate Gli1 through serine 408. This group found that AMPK promotes Gli1 degradation upon its phosphorylation of serine 408 on Gli1 [58]. Further studies illustrating the effect of modulating the activity of Gli1 regulators on medulloblastoma tumorigenesis in *in vivo* systems will inform whetherthey are potential drug targets.

found in sheep that ingested corn lilies in which cyclopamine was originally discovered [46]. 2004 marked the year that Genentech identified the drug vismodegib in a screen for com‐ pounds that inhibit the SHH pathway [46]. Studies assessed vismodegib initially in ad‐ vanced basal cell carcinoma and were also launched to assess the drug for other cancers [46]. Vismodegib was approved in 2012 by the Food and Drug Administration (FDA) for the treatment of metastatic or recurring BCC [46]. A Phase I study has been undertaken to assess the safety, safe dosing range, and side effects of vismodegib in a population of children with recurrent or refractory medulloblastoma [47]. Out of the 20 patients enrolled for flat-dosage testing (150 mg for smaller body area and 300 mg for larger), only two dose-limiting toxici‐ ties were observed. The study concluded that vismodegib is well tolerated in pediatric patients with recurrent or refractory medulloblastoma and recommended 150 or 300 mg dosage for

Consequently, a Phase II trial was conducted at this recommended dosage with adult and pediatric patient groups. The study found that vismodegib increased progression-free survival in SHH medulloblastoma group but not in the non-SHH medulloblastoma group. Vismode‐ gib exhibited activity against adult SHH medulloblastoma. However, inadequate sampling size for the pediatric group precluded conclusions about vismodegib efficacy in this group [48]. Therefore, vismodegib appears promising for adult medulloblastoma patients but

In 2015, the FDA approved another Smo inhibitor, sonidegib (also known as LDE225), for use in treating basal cell carcinoma [49]. Sonidegib has been tested in a variety of cancers, including medulloblastoma [50]. Other Smo inhibitors are being tested in other cancers. GANT61 has been tested in a prostate cancer model [51], while BMS-833923 was tested in a gastric and

For SHH medulloblastoma, targeting SHH signaling is a more direct therapeutic approach than the use of alkylating agents; however, drug resistance may pose a realistic concern. For example, it has been found that drug resistance can arise from amino acids changes in Smo which leads to a deficiency in drug binding to vismodegib [53]. With the approval of sonide‐ gib, researchers then investigated whetherits usage might improve tumorresponse in patients with basal cell carcinoma who were resistant to vismodegib. They concluded that, unfortu‐ nately, patients with advanced basal cell carcinoma, who were previously resistant to vismodegib, also experienced resistance with sonidegib treatment [54]. So, drug resistance

Toward the goal of developing combination therapies and limiting drug resistance, recent research has progressed to investigating the molecular regulation of proteins within the SHH pathway as potential drug targets. For example, several kinases have been shown to control the activity of Gli1: ribosomal protein S6 activates Gli1 through phosphorylation on its

AMP kinase (AMPK), a regulator of cell energy allocation during stress conditions, has been shown to modulate Gli1 activity. Specifically, overexpression of AMPK leads to a decrease in Gli1 expression, while downregulation of AMPK activity increases Gli1 expression [57].

serine 84 [55], while protein kinase A phosphorylation inhibits Gli1's activity [56].

esophageal cancer model [52]. Both remain to be tested in medulloblastoma.

remains to be further examined for pediatric patients.

with novel Smo inhibitors remains an ongoing concern.

Phase II trials.

390 Neurooncology - Newer Developments

**Figure 5.** AMPK phosphorylation on Gli1 reduces Gli1 activity. During stress conditions, AMPK phosphorylation on Gli1 results in decreased cell growth. Uncontrolled Gli1 activity, which can arise from downregulating AMPK, leads to uncontrolled cell growth such as in medulloblastoma. Schematic adapted from author JYY's work, *AMP-activated pro‐ tein kinase directly phosphorylates and destabilizes hedgehog pathway transcription factor GLI1 in medulloblastoma* (Li et al., Cell Rep. 2015).

Another approach to developing combination drug therapies has been to identify additional signaling pathways that impact SHH-driven medulloblastoma. Research has demonstrated that these pathways play a role in medulloblastoma development:


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 candidates to target.
