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Mouse models of medulloblastoma have proven to be instrumental in understanding disease mechanisms, particularly the role of epigenetic and molecular drivers, and establishing appropriate preclinical pipelines. To date, our research community has developed murine models for all four groups of medulloblastoma, each of which will be critical for the identification and development of new therapeutic approaches. Approaches to modeling medulloblastoma range from genetic engineering with CRISPR/Cas9 or in utero electroporation, to orthotopic and patient‐derived orthotopic xenograft systems. Each approach or model presents unique advantages that have ultimately contributed to an appreciation of medulloblastoma heterogeneity and the clinical obstacles that exist for this patient population.

Medulloblastoma (MB) consists of four core molecular subgroups with distinct clinical features and prognoses. Treatment consists of surgery, followed by radiotherapy and cytotoxic chemotherapy. Despite this intensive approach, outcome remains dismal for patients with certain subtypes of MB, namely MYC-amplified Group 3 and TP53-mutated SHH. Using high-throughput assays, six human MB cell lines were screened against a library of 3208 unique compounds. We identified 45 effective compounds from the screen and found that inhibition cell cycle checkpoint kinases (CHK1 and 2) synergistically enhanced the cytotoxic activity of clinically-used chemotherapeutics cyclophosphamide, cisplatin, and gemcitabine. To identify the best-in-class inhibitor, multiple CHK1/2 inhibitors were assessed in mice bearing intracranial MB. When combined with DNA-damaging chemotherapeutics, CHK1/2 inhibition reduced tumor burden and significantly increased survival of animals with high-risk MB, across multiple different models. In total, we tested 14 different models, representing distinct MB subgroups, and data were validated in three independent laboratories. Pharmacodynamics studies confirmed central nervous system penetration. In mice, combination treatment significantly increased DNA damage and apoptosis compared to chemotherapy alone, and studies with cultured cells showed that CHK inhibition disrupted chemotherapy-induced cell cycle arrest. Our findings indicated CHK1/2 inhibition, specifically with LY2606368 (prexasertib), has strong chemosensitizing activity in MB that warrants further clinical investigation. Moreover, these data demonstrated that we developed a robust and collaborative preclinical assessment platform that can be used to identify potentially effective new therapies for clinical evaluation for pediatric MB.

Prexasertib (LY2606368) is a potent and selective small molecule inhibitor of cell-cycle checkpoint CHK1 and CHK2 protein kinases and is currently under clinical evaluation for treatment of pediatric malignancies. As a candidate therapy for pediatric Group 3 medulloblastoma (G3MB), prexasertib CNS penetration was evaluated in mice using cerebral microdialysis and pharmacokinetic modeling. A plasma pharmacokinetic study with a population-based design was performed in CD1 nude mice bearing G3MB orthotopically implanted in the brain and receiving a single dose of prexasertib (10 mg/kg, subcutaneously) to characterize prexasertib disposition and to establish a limited plasma sampling model for the microdialysis studies. The microdialysis studies were performed in both non-tumor bearing mice and in mice bearing G3MB receiving 10 mg/kg prexasertib subcutaneously, for up to 24 hours post-dose. Plasma and extracellular fluid (ECF) concentrations were quantified using validated LC MS/MS methods, and analyzed using a population pharmacokinetic model. Model-derived prexasertib tumor/ECF to plasma partition coefficient K(p,uu) (ratio of tumor/brain ECF to unbound plasma AUC(0–24h)) was significantly greater in G3MB tumor-bearing mice (0.17 ± 0.08) compared to non-tumor bearing mice (0.09 ± 0.04, p=0.04). A pharmacodynamic study was then performed in mice bearing G3MB (20 mg/kg, IV) to evaluate prexasertib-induced target engagement after a single dose. Phosphorylated CHK1 serine 345 (pCHK1 S345), phosphorylated Histone 2A variant (γ-H2AX), and cleaved caspase-3 were quantified in mouse G3MB tumor tissues by immunohistochemistry at different time points up to 24 hours post-dose. The induction of pCHK1 S345 and γ-H2AX peaked at 2 hours after the dose and was elevated above baseline for at least 6 hours, reflecting relevant CHK1 inhibition and DNA damage. Cleaved caspase-3 levels increased at 24 hours suggesting initiation of cell apoptosis. Adequate unbound prexasertib exposure reached the brain tumor site relative to target engagement in G3MB tumor bearing mice at a clinically relevant dosage. These results support further preclinical and clinical development of prexasertib to treat children with medulloblastoma.

Medulloblastoma is an embryonal tumor that shows a predilection for distant metastatic spread and leptomeningeal seeding. For most patients, optimal management of medulloblastoma includes maximum safe resection followed by adjuvant craniospinal irradiation (CSI) and chemotherapy. Although CSI is crucial in treating medulloblastoma, the realization that medulloblastoma is a heterogeneous disease comprising four distinct molecular subgroups (wingless [WNT], sonic hedgehog [SHH], Group 3 [G3], and Group 4 [G4]) with distinct clinical characteristics and prognoses has refocused efforts to better define the optimal role of CSI within and across disease subgroups. The ability to deliver clinically relevant CSI to preclinical models of medulloblastoma offers the potential to study radiation dose and volume effects on tumor control and toxicity in these subgroups and to identify subgroup-specific combination adjuvant therapies. Recent efforts have employed commercial image-guided small animal irradiation systems as well as custom approaches to deliver accurate and reproducible fractionated CSI in various preclinical models of medulloblastoma. Here, we provide an overview of the current clinical indications for, and technical aspects of, irradiation of pediatric medulloblastoma. We then review the current literature on preclinical modeling of and treatment interventions for medulloblastoma and conclude with a summary of challenges in the field of preclinical modeling of CSI for the treatment of leptomeningeal seeding tumors.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is classified into four major molecularly and histopathologically distinct subgroups, among which MYC-driven Group 3 MBs confer a poor prognosis. The Cyclin D/CDK4/CDK6/RB pathway is frequently deregulated in MB leading to uncontrolled cell proliferation, but tumors express an intact RB protein (Northcott et al., Nature, 2017). Therefore, CDK4/6 inhibitor drugs offer a possible therapeutic approach to treat MBs. Because single agent therapy ultimately leads to drug resistance, we initiated in vitro combination drug screens to identify drug classes potentiating CDK4/6 inhibitors. We used Group 3 MB patient-derived orthotopic xenografts (PDOXs), a human cell line (HDMB03), and freshly dissociated tumor cells propagated only in the mouse brain. The drug screen included 90 compounds comprising targeted and cytotoxic drugs that are FDA approved or under active clinical investigation. Using a bioluminescence-based assay that measures ATP consumption (CellTiter-Glo) to evaluate the number of viable cells, these 90 compounds were screened in combination with a fixed concentration of ribociclib, one of the three FDA approved CDK4/6 inhibitors. The primary screen, carried out in HDMB03 cells, revealed several drugs with additive or synergistic potential when combined with ribociclib, including BET inhibitors, MEK inhibitors, PI3K/mTOR inhibitors and gemcitabine. We are currently evaluating the combination of brain penetrant compounds in Group 3 MB PDOXs. The identification of potent drug combinations should provide new therapeutic options for the treatment of Group 3 MB, one of the most difficult to treat.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is characterized by four molecularly and histopathologically distinct groups with different prognoses. With the advent of next generation sequencing, the field has gained new insights into somatically altered genes in MB, many of which are involved in epigenetic gene regulation and chromatin modification. A significant portion of Group 3 MB do not carry any mutations, suggesting that additional, non-mutated, epigenetic regulators may be implicated in shaping chromatin structure and driving tumor pathogenesis. Considering this possibility, we conducted two unbiased screens of epigenetic regulators in G3 MB to identify candidate drivers of medulloblastoma genesis. 1) In collaboration with Dr. Christopher Vakoc, we screened a library of shRNAs targeting 293 known chromatin modifiers in mouse G3 MB cells. Designed to assess reduction in viability, the dropout screen identified putative oncogenes, and of the top hits, SMYD3, a lysine methyltransferase, emerged as the most interesting candidate. SMYD3 is overexpressed and exhibits increased predicted activity in G3 MB compared to other subgroups. Perturbation of SMYD3 using shRNA or CRISPR/Cas9 results in significantly reduced tumor progression and significantly prolonged lifespan in tumor-bearing animals. 2) We also screened a pooled epigenetic CRISPR/Cas9 gRNA library in G3 MB cells where we assessed gRNA enrichment in vivo and thus, identified putative tumor suppressors. Based on biological relevance and computational prediction, PRDM2 was selected as the top candidate. Implicated in other cancers, PRDM2, a histone 3 lysine 9 methyltransferase, exhibits low expression and predicted activity in G3 MB compared to other subgroups. Current work is focused on dissecting the epigenetic, transcriptomic, and phenotypic consequences of PRDM2 perturbation in naïve neural progenitors and G3 MB cells. Taken together, these efforts will help us to understand how changes in chromatin modifiers affect disease pathogenesis and how these changes may represent therapeutic vulnerabilities.