Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors.

Background: Medulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. Methods: To determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Children's Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. Results: Our RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. Conclusions: These results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key Points: Several Type I MAGE CTAs are expressed in >60% of group 3 MBs. Type I MAGEs affect MB cell proliferation and apoptosis. MAGEs are potential biomarkers and therapeutic targets for group 3 MBs. Importance of the Study: This study is the first comprehensive analysis of all Type I MAGE CTAs ( MAGEA , -B , and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options.

Group 3 medulloblastoma transcriptional networks collapse under domain specific EP300/CBP inhibition.

Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.

CAR T-cell Design-dependent Remodeling of the Brain Tumor Immune Microenvironment Modulates Tumor-associated Macrophages and Anti-glioma Activity.

Understanding the intricate dynamics between adoptively transferred immune cells and the brain tumor immune microenvironment (TIME) is crucial for the development of effective T cell-based immunotherapies. In this study, we investigated the influence of the TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3-specific CAR T-cells. Using an immunocompetent glioma model, we evaluated a panel of seven fully murine B7-H3 CARs with variations in transmembrane, costimulatory, and activation domains. We then investigated changes in the TIME following CAR T-cell therapy using high-dimensional flow cytometry and single-cell RNA sequencing. Our results show that five out of six B7-H3 CARs with single costimulatory domains demonstrated robust functionality in vitro. However, these CARs had significantly varied levels of antitumor activity in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 41BB and CD28 costimulation through transgenic expression of 41BBL on CD28-based CAR T-cells. This CAR design was associated with significantly improved anti-glioma efficacy in vitro but did not result in similar improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the TIME, with the recruitment and activation of distinct macrophage and endogenous T-cell subsets crucial for successful antitumor responses. Indeed, complete brain macrophage depletion using a CSF1R inhibitor abrogated CAR T-cell antitumor activity. In sum, our study highlights the critical role of CAR design and its modulation of the TIME in mediating the efficacy of adoptive immunotherapy for high-grade glioma. SIGNIFICANCE: CAR T-cell immunotherapies hold great potential for treating brain cancers; however, they are hindered by a challenging immune environment that dampens their effectiveness. In this study, we show that the CAR design influences the makeup of the immune environment in brain tumors, underscoring the need to target specific immune components to improve CAR T-cell performance, and highlighting the significance of using models with functional immune systems to optimize this therapy.

Intrathecal delivery of nanoparticle PARP inhibitor to the cerebrospinal fluid for the treatment of metastatic medulloblastoma.

The morbidity associated with pediatric medulloblastoma, in particular in patients who develop leptomeningeal metastases, remains high in the absence of effective therapies. Administration of substances directly into the cerebrospinal fluid (CSF) is one approach to circumvent the blood-brain barrier and focus delivery of drugs to the site of tumor. However, high rates of CSF turnover prevent adequate drug accumulation and lead to rapid systemic clearance and toxicity. Here, we show that PLA-HPG nanoparticles, made with a single-emulsion, solvent evaporation process, can encapsulate talazoparib, a PARP inhibitor (BMN-673). These degradable polymer nanoparticles improve the therapeutic index when delivered intrathecally and lead to sustained drug retention in the tumor as measured with PET imaging and fluorescence microscopy. We demonstrate that administration of these particles into the CSF, alone or in combination with systemically administered temozolomide, is a highly effective therapy for tumor regression and prevention of leptomeningeal spread in xenograft mouse models of medulloblastoma. These results provide a rationale for harnessing nanoparticles for the delivery of drugs limited by brain penetration and therapeutic index and demonstrate important advantages in tolerability and efficacy for encapsulated drugs delivered locoregionally.

CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity.

Understanding interactions between adoptively transferred immune cells and the tumor immune microenvironment (TIME) is critical for developing successful T-cell based immunotherapies. Here we investigated the impact of the TIME and chimeric antigen receptor (CAR) design on anti-glioma activity of B7-H3-specific CAR T-cells. We show that five out of six B7-H3 CARs with varying transmembrane, co-stimulatory, and activation domains, exhibit robust functionality in vitro. However, in an immunocompetent glioma model, these CAR T-cells demonstrated significantly varied levels of anti-tumor activity. We used single-cell RNA sequencing to examine the brain TIME after CAR T-cell therapy. We show that the TIME composition was influenced by CAR T-cell treatment. We also found that successful anti-tumor responses were supported by the presence and activity of macrophages and endogenous T-cells. Together, our study demonstrates that efficacy of CAR T-cell therapy in high-grade glioma is dependent on CAR structural design and its capacity to modulate the TIME.

Class I HDAC inhibitor entinostat synergizes with PLK1 inhibitors in MYC-amplified medulloblastoma cells.

PURPOSE: We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy. METHODS: We used a gene expression dataset to identify PLK1 as a second target in MB cells and validated the relevance of PLK1 in MB. We measured cell metabolic activity, viability, and cycle progression in MB cells after treatment with PLK1-specific inhibitors (PLK1i). Chou-Talalay synergy calculations were used to determine the nature of class I HDACi entinostat and PLK1i interaction which was validated. Finally, the clinical potential of the combination was assessed in the in vivo experiment. RESULTS: MYC-amplified tumor cells are highly sensitive towards treatment with ATP-competitive PLK1i as a monotherapy. Entinostat and PLK1i in combination act synergistically in MYC-driven MB cells, exerting cytotoxic effects at clinically relevant concentrations. The downstream effect is exerted via MYC-related pathways, pointing out the potential of MYC amplification as a clinically feasible predictive biomarker for patient selection. While entinostat significantly extended survival of mice implanted with orthotopic MYC-amplified MB PDX, there was no evidence of the improvement of survival when treating the animals with the combination. CONCLUSION: The combination of entinostat and PLK1i showed synergistic interaction in vitro, but not in vivo. Therefore, further screening of blood-brain barrier penetrating PLK1i is warranted to determine the true potential of the combination as no on-target activity was observed after PLK1i volasertib treatment in vivo.

Atypical teratoid/rhabdoid tumoroids reveal subgroup-specific drug vulnerabilities.

Atypical teratoid/rhabdoid tumors (ATRTs) represent a rare, but aggressive pediatric brain tumor entity. They are genetically defined by alterations in the SWI/SNF chromatin remodeling complex members SMARCB1 or SMARCA4. ATRTs can be further classified in different molecular subgroups based on their epigenetic profiles. Although recent studies suggest that the different subgroups have distinct clinical features, subgroup-specific treatment regimens have not been developed thus far. This is hampered by the lack of pre-clinical in vitro models representative of the different molecular subgroups. Here, we describe the establishment of ATRT tumoroid models from the ATRT-MYC and ATRT-SHH subgroups. We demonstrate that ATRT tumoroids retain subgroup-specific epigenetic and gene expression profiles. High throughput drug screens on our ATRT tumoroids revealed distinct drug sensitivities between and within ATRT-MYC and ATRT-SHH subgroups. Whereas ATRT-MYC universally displayed high sensitivity to multi-targeted tyrosine kinase inhibitors, ATRT-SHH showed a more heterogeneous response with a subset showing high sensitivity to NOTCH inhibitors, which corresponded to high expression of NOTCH receptors. Our ATRT tumoroids represent the first pediatric brain tumor organoid model, providing a representative pre-clinical model which enables the development of subgroup-specific therapies.

From PROTAC to inhibitor: Structure-guided discovery of potent and orally bioavailable BET inhibitors.

An X-ray structure of a CLICK chemistry-based BET PROTAC bound to BRD2(BD2) inspired synthesis of JQ1 derived heterocyclic amides. This effort led to the discovery of potent BET inhibitors displaying overall improved profiles when compared to JQ1 and birabresib. A thiadiazole derived 1q (SJ1461) displayed excellent BRD4 and BRD2 affinity and high potency in the panel of acute leukaemia and medulloblastoma cell lines. A structure of 1q co-crystalised with BRD4-BD1 revealed polar interactions with the AZ/BC loops, in particular with Asn140 and Tyr139, rationalising the observed affinity improvements. In addition, exploration of pharmacokinetic properties of this class of compounds suggest that the heterocyclic amide moiety improves drug-like features. Our study led to the discovery of potent and orally bioavailable BET inhibitor 1q (SJ1461) as a promising candidate for further development.

Targeting protein folding in N-Myc-driven medulloblastoma.

Selective targeting of N-Myc-driven Sonic hedgehog (SHH) medulloblastoma has been a challenge for many years and, despite decades of research, few targeted therapy opportunities exist. Recently, Kuzuoglu-Ozturk et al. characterized the translatome of N-Myc-driven medulloblastoma as a promising therapeutic target. The study showed that N-Myc controls a subset of members of the protein folding machinery that could be inhibited pharmacologically and validated a subset of Hsp70 functions as required for medulloblastoma progression in vitro and in vivo.

Loss of phosphatase CTDNEP1 potentiates aggressive medulloblastoma by triggering MYC amplification and genomic instability.

MYC-driven medulloblastomas are highly aggressive childhood brain tumors, however, the molecular and genetic events triggering MYC amplification and malignant transformation remain elusive. Here we report that mutations in CTDNEP1, a CTD nuclear-envelope-phosphatase, are the most significantly enriched recurrent alterations in MYC-driven medulloblastomas, and define high-risk subsets with poorer prognosis. Ctdnep1 ablation promotes the transformation of murine cerebellar progenitors into Myc-amplified medulloblastomas, resembling their human counterparts. CTDNEP1 deficiency stabilizes and activates MYC activity by elevating MYC serine-62 phosphorylation, and triggers chromosomal instability to induce p53 loss and Myc amplifications. Further, phosphoproteomics reveals that CTDNEP1 post-translationally modulates the activities of key regulators for chromosome segregation and mitotic checkpoint regulators including topoisomerase TOP2A and checkpoint kinase CHEK1. Co-targeting MYC and CHEK1 activities synergistically inhibits CTDNEP1-deficient MYC-amplified tumor growth and prolongs animal survival. Together, our studies demonstrate that CTDNEP1 is a tumor suppressor in highly aggressive MYC-driven medulloblastomas by controlling MYC activity and mitotic fidelity, pointing to a CTDNEP1-dependent targetable therapeutic vulnerability.

Combination of Ribociclib with BET-Bromodomain and PI3K/mTOR Inhibitors for Medulloblastoma Treatment In Vitro and In Vivo.

Despite improvement in the treatment of medulloblastoma over the last years, numerous patients with MYC- and MYCN-driven tumors still fail current therapies. Medulloblastomas have an intact retinoblastoma protein RB, suggesting that CDK4/6 inhibition might represent a therapeutic strategy for which drug combination remains understudied. We conducted high-throughput drug combination screens in a Group3 (G3) medulloblastoma line using the CDK4/6 inhibitor (CDK4/6i) ribociclib at IC20, referred to as an anchor, and 87 oncology drugs approved by FDA or in clinical trials. Bromodomain and extra terminal (BET) and PI3K/mTOR inhibitors potentiated ribociclib inhibition of proliferation in an established cell line and freshly dissociated tumor cells from intracranial xenografts of G3 and Sonic hedgehog (SHH) medulloblastomas in vitro. A reverse combination screen using the BET inhibitor JQ1 as anchor, revealed CDK4/6i as the most potentiating drugs. In vivo, ribociclib showed single-agent activity in medulloblastoma models whereas JQ1 failed to show efficacy due to high clearance and insufficient free brain concentration. Despite in vitro synergy, combination of ribociclib with the PI3K/mTOR inhibitor paxalisib did not significantly improve the survival of G3 and SHH medulloblastoma-bearing mice compared with ribociclib alone. Molecular analysis of ribociclib and paxalisib-treated tumors revealed that E2F targets and PI3K/AKT/MTORC1 signaling genes were depleted, as expected. Importantly, in one untreated G3MB model HD-MB03, the PI3K/AKT/MTORC1 gene set was enriched in vitro compared with in vivo suggesting that the pathway displayed increased activity in vitro. Our data illustrate the difficulty in translating in vitro findings in vivo. See related article in Mol Cancer Ther (2022) 21(8):1306-1317.

Primary cilia control translation and the cell cycle in medulloblastoma.

The primary cilium, a signaling organelle projecting from the surface of a cell, controls cellular physiology and behavior. The presence or absence of primary cilia is a distinctive feature of a given tumor type; however, whether and how the primary cilium contributes to tumorigenesis are unknown for most tumors. Medulloblastoma (MB) is a common pediatric brain cancer comprising four groups: SHH, WNT, group 3 (G3), and group 4 (G4). From 111 cases of MB, we show that primary cilia are abundant in SHH and WNT MBs but rare in G3 and G4 MBs. Using WNT and G3 MB mouse models, we show that primary cilia promote WNT MB by facilitating translation of mRNA encoding ß-catenin, a major oncoprotein driving WNT MB, whereas cilium loss promotes G3 MB by disrupting cell cycle control and destabilizing the genome. Our findings reveal tumor type-specific ciliary functions and underlying molecular mechanisms. Moreover, we expand the function of primary cilia to translation control and reveal a molecular mechanism by which cilia regulate cell cycle progression, thereby providing new frameworks for studying cilium function in normal and pathologic conditions.

Combination of Ribociclib and Gemcitabine for the Treatment of Medulloblastoma.

Group3 (G3) medulloblastoma (MB) is one of the deadliest forms of the disease for which novel treatment is desperately needed. Here we evaluate ribociclib, a highly selective CDK4/6 inhibitor, with gemcitabine in mouse and human G3MBs. Ribociclib central nervous system (CNS) penetration was assessed by in vivo microdialysis and by IHC and gene expression studies and found to be CNS-penetrant. Tumors from mice treated with short term oral ribociclib displayed inhibited RB phosphorylation, downregulated E2F target genes, and decreased proliferation. Survival studies to determine the efficacy of ribociclib and gemcitabine combination were performed on mice intracranially implanted with luciferase-labeled mouse and human G3MBs. Treatment of mice with the combination of ribociclib and gemcitabine was well tolerated, slowed tumor progression and metastatic spread, and increased survival. Expression-based gene activity and cell state analysis investigated the effects of the combination after short- and long-term treatments. Molecular analysis of treated versus untreated tumors showed a significant decrease in the activity and expression of genes involved in cell-cycle progression and DNA damage response, and an increase in the activity and expression of genes implicated in neuronal identity and neuronal differentiation. Our findings in both mouse and human patient-derived orthotopic xenograft models suggest that ribociclib and gemcitabine combination therapy warrants further investigation as a treatment strategy for children with G3MB.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Patient-derived models recapitulate heterogeneity of molecular signatures and drug response in pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. Here we report establishment of 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulate histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and include rare subgroups not well-represented by existing models. We deploy 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predict variable in vivo response to PI3K/mTOR and MEK pathway inhibitors. These unique new models and an online interactive data portal for exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.

Medulloblastoma recurrence and metastatic spread are independent of colony-stimulating factor 1 receptor signaling and macrophage survival.

PURPOSE: Tumor infiltration by immunosuppressive myeloid cells or tumor-associated macrophages (TAMs) contributes to tumor progression and metastasis. In contrast to their adult counterparts, higher TAM signatures do not correlate with aggressive tumor behavior in pediatric brain tumors. While prominent TAM infiltrates exist before and after radiation, the degree to which irradiated macrophages and microglia support progression or leptomeningeal metastasis remains unclear. Patients with medulloblastoma often present with distant metastases and tumor recurrence is largely incurable, making them prime candidates for the study of novel approaches to prevent neuroaxis dissemination and recurrence. METHODS: Macrophage depletion was achieved using CSF-1 receptor inhibitors (CSF-1Ri), BLZ945 and AFS98, with or without whole brain radiation in a variety of medulloblastoma models, including patient-derived xenografts bearing Group 3 medulloblastoma and a transgenic Sonic Hedgehog (Ptch1+/-, Trp53-/-) medulloblastoma model. RESULTS: Effective reduction of microglia, TAM, and spinal cord macrophage with CSF-1Ri resulted in negligible effects on the rate of local and spinal recurrences or survival following radiation. Results were comparable between medulloblastoma subgroups. While notably few tumor-infiltrating lymphocytes (TILs) were detected, average numbers of CD3+ TILs and FoxP3+ Tregs did not differ between groups following treatment and tumor aggressiveness by Ki67 proliferation index was unaltered. CONCLUSION: In the absence of other microenvironmental influences, medulloblastoma-educated macrophages do not operate as tumor-supportive cells or promote leptomeningeal recurrence in these models. Our data add to a growing body of literature describing a distinct immunophenotype amid the medulloblastoma microenvironment and highlight the importance of appropriate pediatric modeling prior to clinical translation.

LC-MS/MS method for quantitation of gemcitabine and its metabolite 2',2'-difluoro-2'-deoxyuridine in mouse plasma and brain tissue: Application to a preclinical pharmacokinetic study.

A simple, sensitive, and relatively fast assay was developed and validated for the quantitation of gemcitabine (dFdC) and its major metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in mouse plasma and brain tissue. The assay used a small sample (25 µL plasma and 5 mg brain) for extraction by protein precipitation. After dilution of the supernatant extract, 1 µL was injected into HPLC system for reverse phase chromatographic separation with a total run time of 8 min. Chromatographic resolution of dFdC and dFdU was achieved on a Gemini C18 column (50 × 4.6 mm, 3 µm) utilizing gradient elution. Multiple reaction monitoring (MRM) with positive/negative ion switching was performed for detection of dFdC and its internal standard (dFdC-IS) in positive ion mode and dFdU and its IS (dFdU-IS) in negative ion mode. Two calibration curves ranging from 5-2000 ng/mL and 250-50,000 ng/mL were generated for dFdC and dFdU in mouse plasma, respectively. For measurement of dFdC and dFdU in mouse brain tissue, another two curves were used ranging from 0.02 to 40 ng/mg and 1-40 ng/mg, respectively. This assay demonstrated excellent precision and accuracy within day and between days for simultaneous measurement of dFdC and dFdU at all the concentration levels in both matrices. The other parameters such as selectivity, sensitivity, matrix effects, recovery, and storage stability were also assessed for both analytes in each matrix. Compared to the previously reported methods, the sample extraction in the current assay was simplified significantly, and the analysis time was greatly shortened. We successfully applied the validated method to the analysis of dFdC and dFdU in mouse plasma, brain, and brain tumor tissue in a preclinical pharmacokinetic study.

Thrombospondin-1 mimetics are promising novel therapeutics for MYC-associated medulloblastoma.

BACKGROUND: Medulloblastoma (MB) comprises four subtypes of which group 3 MB are the most aggressive. Although overall survival for MB has improved, the outcome of group 3 MB remains dismal. C-MYC (MYC) amplification or MYC overexpression which characterizes group 3 MB is a strong negative prognostic factor and is frequently associated with metastases and relapses. We previously reported that MYC expression alone promotes highly aggressive MB phenotypes, in part via repression of thrombospondin-1 (TSP-1), a potent tumor suppressor. METHODS: In this study, we examined the potential role of TSP-1 and TSP-1 peptidomimetic ABT-898 in MYC-amplified human MB cell lines and two distinct murine models of MYC-driven group 3 MBs. RESULTS: We found that TSP-1 reconstitution diminished metastases and prolonged survival in orthotopic xenografts and promoted chemo- and radio-sensitivity via AKT signaling. Furthermore, we demonstrate that ABT-898 can recapitulate the effects of TSP-1 expression in MB cells in vitro and specifically induced apoptosis in murine group 3 MB tumor cells. CONCLUSION: Our data underscore the importance of TSP-1 as a critical tumor suppressor in MB and highlight TSP-1 peptidomimetics as promising novel therapeutics for the most lethal subtype of MB.

Small-molecule screen reveals synergy of cell cycle checkpoint kinase inhibitors with DNA-damaging chemotherapies in medulloblastoma.

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 cell cycle checkpoint kinase (CHK1/2) inhibition 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 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.