SHH-driven Medulloblastoma Research Articles

DIPG-89. ENHANCEMENT OF BLOOD-BRAIN BARRIER PENETRATION AND TUMOR TARGETED DRUG DELIVERY FOR DMG USING A P-SELECTIN TARGETED NANOMEDICINE APPROACH

Abstract The blood-brain barrier (BBB) is one of the greatest barriers for the effective treatment of brain tumors, including H3K27-altered diffuse midline glioma (DMG), a near universally fatal childhood brain cancer. The BBB typically requires drugs to be given at maximally tolerated doses that are limited by systemic toxicities, particularly in settings of combination therapy. We have developed a clinically compatible fucoidan nanoparticle (Fi-NP) that homes to P-selectin on tumor vasculature after low-dose radiation (RT) to breach the BBB through an active caveolin-1-dependent mechanism and deliver several classes of targeted therapies. In non-CNS cancer xenograft models and a transgenic mouse model of SHH-driven medulloblastoma with an intact BBB, this approach improved survival while eliminating on-target systemic toxicities. We have now applied this approach to both brainstem and non-brainstem RCAS-TVA mouse models of DMG with an intact BBB. Specifically, we have found that DMG tumor vasculature expresses P-selectin, and that a single low-dose 2 Gy fraction of ionizing radiation further enhances it in a time-dependent manner up to 24 hours post-treatment. Importantly, we have also found that this P-selectin targeted drug delivery approach facilitates DMG tumor localization of Fi-NP encapsulated EZH2 inhibitor tazemetostat (EPZ-6438) as well as larger macromolecules including the PROTAC BET degrader dBET6, two promising targeted therapies for DMG limited by extremely poor BBB-penetration, while sparing drug delivery to non-tumor healthy brain regions. Work to measure PK, biodistribution, survival benefit, and drug target inhibition is ongoing. Our findings will provide the foundation for this P-selectin targeted Fi-NP approach to be evaluated in clinical trials for children with this lethal cancer.

High-throughput neural stem cell-based drug screening identifies S6K1 inhibition as a selective vulnerability in sonic hedgehog-medulloblastoma.

Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current treatments have increased overall survival but can lead to devastating side effects and late complications in survivors, emphasizing the need for new, improved targeted therapies that specifically eliminate tumor cells while sparing the normally developing brain. Here, we used a sonic hedgehog (SHH)-MB model based on a patient-derived neuroepithelial stem cell system for an unbiased high-throughput screen with a library of 172 compounds with known targets. Compounds were evaluated in both healthy neural stem cells (NSCs) and tumor cells derived from the same patient. Based on the difference of cell viability and drug sensitivity score between normal cells and tumor cells, hit compounds were selected and further validated in vitro and in vivo. We identified PF4708671 (S6K1 inhibitor) as a potential agent that selectively targets SHH-driven MB tumor cells while sparing NSCs and differentiated neurons. Subsequent validation studies confirmed that PF4708671 inhibited the growth of SHH-MB tumor cells both in vitro and in vivo, and that knockdown of S6K1 resulted in reduced tumor formation. Overall, our results suggest that inhibition of S6K1 specifically affects tumor growth, whereas it has less effect on non-tumor cells. Our data also show that the NES cell platform can be used to identify potentially effective new therapies and targets for SHH-MB.

PRC2 disruption in cerebellar progenitors produces cerebellar hypoplasia and aberrant myoid differentiation without blocking medulloblastoma growth

We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects.

PATH-44. VARIANT ALLELIC FREQUENCY OF DRIVER MUTATIONS PREDICTS SUCCESS OF GENOMIC METHYLATION CLASSIFICATION IN CNS TUMORS

Abstract Whole genome CpG DNA methylation profiling is an extremely valuable tool in the workup of central nervous system (CNS) tumors. Reliability of such profiling depends on sufficient tumor cellularity. Many neoplastic entities have well-known driver mutations that occur in virtually 100% of tumor cells, and next-generation sequencing (NGS) assays can detect those mutations and report their relative amounts in the form of Variant Allelic Frequency (VAF). Since NGS and methylation profiling are often done on the same tumor block, we sought to determine whether driver mutation VAF affects the accuracy of methylation profiling, and whether VAF can help establish more rigorous cutoffs for quality assurance. Using NGS and Infinium Epic850K methylation arrays, we evaluated 153 CNS neoplasms representing a range of entities, including TERT promoter-mutant glioblastoma, IDH-mutant astrocytoma, IDH-mutant oligodendroglioma, SHH-driven medulloblastoma, and CTNNB1-driven adamantinomatous craniopharyngioma. VAFs of each driver mutation ranged between 1-60%. One hundred eleven of 153 cases (73%) had a methylation classification score ≥ 0.9, the most widely accepted cutoff for a successful result. A fit-of-mixture analysis via CutoffFinder (PMID: 23251644) suggested that the optimal VAF cutoff=31%, generating an AUC of 0.87. Ninety-six of 107 (89%) cases with a VAF of 31% or higher had a methylation classification score ≥ 0.9, whereas only 15/46 (33%) below 31% were classifiable with methylation profiling (P< 0.0001 by Fisher’s exact test). An independent validation cohort from NYU (N=50) showed nearly identical results, with 37/50 (74%) of cases having a methylation classification score ≥ 0.9, an optimal cutoff=0.32 with an AUC=0.84, and % classification above and below the 0.32 cutoff being 30/34 (88%) and 7/16 (44%), respectively (P=0.002) These data indicate that VAF of driver mutations can serve as a useful predictor of classification success via methylation profiling, and should be taken into account when interpreting methylation profiling results.

Variant allelic fraction of driver mutations predicts success of genomic methylation classification in CNS tumors.

e14035 Background: Whole genome CpG DNA methylation profiling is proving to be an extremely valuable tool in the diagnostic and prognostic classification of central nervous system (CNS) tumors, and is especially adept at discriminating between very different tumors, or tumor subtypes, that are histologically similar. However, like any bulk assay, reliability of methylation-based classification depends on sufficient tumor cellularity, as admixed nonneoplastic elements (e.g., neurons, glial cells, inflammatory cells) have their own unique methylation profiles, and thus can skew the results. Currently, it is recommended that at least 70% of the analyzed specimen be tumor; however, this is based on subjective estimates from light microscopy. Many neoplastic entities have well-known driver mutations that occur in virtually 100% of tumor cells, and next-generation sequencing (NGS) assays can detect those mutations and report their relative amounts in the form of Variant Allelic Fraction (VAF). Since NGS and methylation profiling are now frequently being done on the same tumor block, we sought to determine whether driver mutation VAF affects the accuracy of methylation profiling, and whether VAF can help establish more rigorous cutoffs for quality assurance. Methods: Using NGS and Infinium Epic850K methylation arrays, we evaluated 62 CNS neoplasms representing a range of entities, including TERT promoter-mutant glioblastoma, IDH-mutant astrocytoma, IDH-mutant oligodendroglioma, SHH-driven medulloblastoma, and CTNNB1-driven adamantinomatous craniopharyngioma. Results: VAFs of each driver mutation ranged between 1-60%. Forty-four of 62 cases (71%) had a methylation classification score ≥0.9, the most widely accepted cutoff for a successful result. A fit-of-mixture analysis via CutoffFinder (PMID: 23251644) suggested that the optimal VAF cutoff is 21%. Forty-one of 46 (89%) cases with a VAF of 21% or higher had a methylation classification score ≥0.9, whereas only 3/16 (19%) at or below 21% were classifiable with methylation profiling ( P< 0.0001 by Fisher’s exact test). Nonlinear regression of VAF versus methylation score by least squares fit produced R2= 0.54, with sum of squares = 2.0. Conclusions: These data indicate that VAF can serve as a useful predictor of classification success via methylation profiling, and should be taken into account when interpreting methylation profiling results.

Enhancing CDK4/6 inhibitor therapy for medulloblastoma using nanoparticle delivery and scRNA-seq-guided combination with sapanisertib.

The therapeutic potential of CDK4/6 inhibitors for brain tumors has been limited by recurrence. To address recurrence, we tested a nanoparticle formulation of CDK4/6 inhibitor palbociclib (POx-Palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma, alone or in combination with specific agents suggested by our analysis. Nanoparticle encapsulation reduced palbociclib toxicity, enabled parenteral administration, improved CNS pharmacokinetics, and extended mouse survival, but recurrence persisted. scRNA-seq identified up-regulation of glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes in proliferating medulloblastoma cells in POx-Palbo–treated mice, suggesting mTORC1 signaling suppression, subsequently confirmed by decreased 4EBP1 phosphorylation. Combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects and prolonged mouse survival compared to either agent alone, contrasting markedly with other tested drug combinations. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment and identify POx-Palbo + Sapanisertib as effective combinatorial therapy for SHH medulloblastoma.

IMMU-48. IMMUNOMODULATION WITH RESIQUIMOD REPOLARIZES THE IMMUNE MICROENVIRONMENT TO INHIBIT MEDULLOBLASTOMA PROGRESSION

Abstract BACKGROUND New, non-cytotoxic treatments may improve outcomes for medulloblastoma, the most common malignant pediatric brain tumor. Sonic hedgehog (SHH) subgroup medulloblastoma, which includes subtypes with poor prognosis, can be modeled in immunocompetent, transgenic mice. These models are ideal for preclinical testing of immunotherapies. Here, we show that immune stimulation using resiquimod, a small molecule agonist of Toll-like receptor 7 and 8 (TLR 7/8), alters myeloid populations in medulloblastoma and significantly reduces tumor growth. METHOD We generated mice with medulloblastoma by interbreeding the hGFAP-Cre and SmoM2 mouse lines. The resultant hGFAP-Cre/SmoM2 (G-Smo) mice develop SHH medulloblastoma with 100% frequency. We analyzed myeloid populations and demonstrated TLR7/8 expression patterns in G-Smo tumors. We then compared survival of untreated G-Smo mice versus G-Smo mice treated with three doses of resiquimod at postnatal days 10, 12, and 14. We also assessed pharmacodynamic effects at progressive intervals after a single dose. RESULTS Approximately 10% of cells in G-Smo medulloblastomas were myeloid cells, and these cells were the only cells that expressed TLR7/8. Resiquimod slowed tumor growth and increased the survival of mice with medulloblastoma. Untreated median survival was 14.5 days (n=12), compared to resiquimod-treated median survival of 37 days (n=10; p=0.0003). All treated mice eventually demonstrated tumor progression. Immunohistochemistry for IBA1, a pan-macrophage marker, demonstrated significant increase in myeloid cells within the tumor by 24 hours after treatment (p=0.0178), however the IGF1+ fraction of myeloid cells decreased (p=0.0275). CONCLUSION Resiquimod prolongs survival in mice with SHH-driven medulloblastoma, demonstrating the potential for therapies that target myeloid cells to produce significant anti-tumor effects. Myeloid-derived IGF-1 has been shown to support tumor progression and resiquimod may act by disrupting this paracrine signaling.

MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis.

Simple SummaryIn eukaryotes, MAPK15 controls the assembly of primary cilia, which are microtubule-based cell surface organelles necessary for sensing and processing developmental signals as well as for transducing tumorigenic Hedgehog signaling in medulloblastomas and basal cell carcinomas. The aim of this study was to evaluate the role of MAPK15 in regulating Hedgehog signaling in medulloblastoma cells. Indeed, we demonstrated strict dependency on this kinase of medulloblastoma ciliogenesis and Hedgehog signaling, which resulted in a reduced cancer stem cell compartment. Based on the scarce therapeutic options available for medulloblastoma patients, our data support the possibility of exploiting novel pharmacological approaches targeting this often-underestimated MAP kinase.In medulloblastomas, genetic alterations resulting in over-activation and/or deregulation of proteins involved in Hedgehog (HH) signaling lead to cellular transformation, which can be prevented by inhibition of primary ciliogenesis. Here, we investigated the role of MAPK15 in HH signaling and, in turn, in HH-mediated cellular transformation. We first demonstrated, in NIH3T3 mouse fibroblasts, the ability of this kinase of controlling primary ciliogenesis and canonical HH signaling. Next, we took advantage of transformed human medulloblastoma cells belonging to the SHH-driven subtype, i.e., DAOY and ONS-76 cells, to ascertain the role for MAPK15 in HH-mediated cellular transformation. Specifically, medullo-spheres derived from these cells, an established in vitro model for evaluating progression and malignancy of putative tumor-initiating medulloblastoma cells, were used to demonstrate that MAPK15 regulates self-renewal of these cancer stem cell-like cells. Interestingly, by using the HH-related oncogenes SMO-M2 and GLI2-DN, we provided evidences that disruption of MAPK15 signaling inhibits oncogenic HH overactivation in a specific cilia-dependent fashion. Ultimately, we show that pharmacological inhibition of MAPK15 prevents cell proliferation of SHH-driven medulloblastoma cells, overall suggesting that oncogenic HH signaling can be counteracted by targeting the ciliary gene MAPK15, which could therefore be considered a promising target for innovative “smart” therapies in medulloblastomas.

Tumor cells generate astrocyte-like cells that contribute to SHH-driven medulloblastoma relapse.

Astrocytes, a major glial cell type in the brain, play a critical role in supporting the progression of medulloblastoma (MB), the most common malignant pediatric brain tumor. Through lineage tracing analyses and single-cell RNA sequencing, we demonstrate that astrocytes are predominantly derived from the transdifferentiation of tumor cells in relapsed MB (but not in primary MB), although MB cells are generally believed to be neuronal-lineage committed. Such transdifferentiation of MB cells relies on Sox9, a transcription factor critical for gliogenesis. Our studies further reveal that bone morphogenetic proteins (BMPs) stimulate the transdifferentiation of MB cells by inducing the phosphorylation of Sox9. Pharmacological inhibition of BMP signaling represses MB cell transdifferentiation into astrocytes and suppresses tumor relapse. Our studies establish the distinct cellular sources of astrocytes in primary and relapsed MB and provide an avenue to prevent and treat MB relapse by targeting tumor cell transdifferentiation.

EMBR-19. SHH-DRIVEN MEDULLOBLASTOMA WITH CONCURRENT UNILATERAL RENAL AGENESIS

Case PresentationA 3-year-old female with insignificant past medical history presented with 6 weeks of headaches, emesis, and lethargy. MR imaging identified a heterogeneously enhancing right cerebellar hemispheric mass and obstructive hydrocephalus. Gross total resection was performed without complications; pathology revealed classical WHO grade 4 medulloblastoma (MB). MR imaging of the spine and cerebrospinal fluid testing were negative for disseminated disease. Treatment for standard risk medulloblastoma was initiated, comprising proton craniospinal irradiation with posterior fossa boost and concurrent vincristine, followed by adjuvant chemotherapy with vincristine, lomustine, cisplatin, and cyclophosphamide as standard of care. Next generation sequencing of the tumor tissue performed using a high-multiplex PCR-based NGS panel was negative for alteration of the SMO, PTCH1 and CTBNN1 genes. Further molecular characterization via methylation profiling demonstrated the sonic hedgehog (SHH) molecular subtype. Prior to initiation of chemotherapy, renal ultrasound was performed and identified congenitally absent right kidney; audiology evaluation was unremarkable. DiscussionIn patients with Gorlin syndrome, cases of unilateral renal agenesis in association with germline SHH-pathway mutations have been reported [1]. SHH signaling is implicated in multiple steps in the development of the urinary system [2]. Outside Gorlin syndrome, however, to the best of our knowledge unilateral renal agenesis coinciding with SHH-driven MB has not been reported. Our patient notably lacks any clinical stigmata of Gorlin syndrome (skeletal abnormalities, skin pits, macrocephaly) and does not exhibit the characteristic germline genetic abnormalities that define GS (PTCH1 mutation or 9q22.3 microdeletion). There are important treatment implications for patients with the constellation of abnormalities we describe here, particularly regarding the requisite frequent monitoring of renal function during multi-agent chemotherapy courses. Our patient tolerated chemoradiation well, and is currently on maintenance chemotherapy with favorable course to date.

DDRE-34. TARGETING RESISTANCE IN MEDULLOBLASTOMA

Medulloblastoma is the most commonly diagnosed pediatric brain tumor. Although therapeutic advances have improved survival from this cancer, they result in devastating sequelae and, additionally, have proven inadequate in metastatic disease and recurrence where survival remains <5%. Effective therapies are urgently needed to improve outcomes in medulloblastoma. Medulloblastoma development is driven by dysregulation of normal cerebellar proliferation. Mutations in the sonic hedgehog (Shh) pathway are found in ~30% of these tumors and responsible for their aggressive growth. The poor outcomes in Shh-driven medulloblastoma have prompted the evaluation of Shh-targeting agents in their treatment – with limited success likely attributable in part to the upregulation of alternate survival pathways (e.g. Ras/MAPK and HIF-1α). These alternate mechanisms stimulate glycolysis, in part by increasing the activity of the 6-phosphofructo-2-kinase/fructose-2,6 bisphosphatases (PFKFB1-4) to produce fructose-2,6-bisphosphate (F26BP), a potent activator of the rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase. In recent studies, we have determined that the PFKFB4 enzyme is highly expressed in patient-derived Shh medulloblastomas. We have found that hypoxia, through HIF-1α, strongly induced PFKFB4 expression in Shh-driven medulloblastoma cells and that silencing PFKFB4 suppressed F26BP, glycolysis and proliferation in normoxia and, more markedly, in hypoxia, indicating that PFKFB4 may be required for growth under hypoxia. We found that simultaneously silencing PFKFB4 and Shh pathway effectors significantly reduced cell survival and that co-targeting PFKFB4 (with a novel inhibitor) and Shh effectors synergistically decreased cell viability. In order to simulate Shh antagonist resistance, we have now subjected Shh medulloblastoma cells to prolonged Shh inhibitor exposure and found that these cells exhibit increased proliferation, glycolysis and PFKFB4. Studies are underway to delineate their metabolic alterations. Taken together, our data indicate that targeting PFKFB4 may be a valid therapeutic option in aggressive, treatment-resistant medulloblastoma and strongly support the further examination of PFKFB4 inhibitors in these tumors.

MBRS-08. SONIC HEDGEHOG SIGNALING PRIMES CEREBELLAR GRANULE NEURON PROGENITORS, THE CELL OF ORIGIN FOR MEDULLOBLASTOMA, FOR APOPTOSIS BY INDUCING PRO APOPTOTIC BIM

Medulloblastomas, unlike other malignant brain tumors, are typically sensitive to radiation therapy, but the mechanisms that mediate this sensitivity are unclear. Cerebellar granule neuron progenitors (CGNPs), the cell of origin for SHH-subgroup medulloblastoma, are also highly sensitive to radiation. In early life, CGNPs proliferate in response to Sonic Hedgehog (SHH) signaling, and hyperactivation of SHH signaling in CGNPs can lead to the development of SHH-subgroup medulloblastoma. We propose that SHH activation induces radiation sensitivity along with tumorigenesis. We have previously shown that the proapoptotic protein BAX is required for radiation sensitivity of both SHH-driven medulloblastomas and CGNPs in mice, and that BCL-xL supplies critical regulation of BAX, preventing spontaneous cell death. Here, we show that SHH signaling increases the radiation sensitivity of CGNPs by inducing the proapoptotic protein BIM. We found that BIM expression depends on SHH activity, and that genetic deletion of Bim decreases the radiation-sensitivity of CGNPs. Mechanistically, we show that BIM binds to anti-apoptotic proteins BCL-xL and MCL-1, where it may alter the balance of BAX and BCL-xL interactions. Consistent with our mechanistic model, human medulloblastoma patients with high BIM expression show a better prognosis. Based on these observations, we propose that SHH-induced BIM mediates the typical radiation sensitivity of SHH-driven medulloblastoma. Finding ways to enhance BIM activity may open new opportunities for targeted medulloblastoma therapy.

MBRS-64. STUDY OF ARGININE METHYL TRANSFERASES IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant pediatric cancer and a leading cause of childhood cancer-related mortality. There is dire need for new therapies. Molecular sub-classification of medulloblastomas has identified chromatin modifiers as potential drivers of tumorigenesis. Expression of the RE1 Silencing Transcription Factor (REST), a hub for assembly of repressive chromatin remodelers and a known regulator of neurogenesis, is elevated in a subset of human sonic hedgehog (SHH) subgroup medulloblastomas, and is associated with poor prognosis. Using a novel transgenic mouse model, we showed REST to be a driver of medulloblastoma development. Surprisingly, our studies also revealed a role for REST in promoting proliferation of granule cell progenitors (GCPs), the cells of origin of SHH-driven medulloblastoma, and a concomitant loss of telomeres and increased genomic instability. We performed a gain of function screen using a library of chromatin modifiers to understand the mechanism by which proliferative potential was maintained, despite the loss of telomeres. This screen identified the Protein Arginine Methyltransferase 6 (PRMT6) as a high confidence hit. PRMT6 upregulation caused a reduction in CDKN2A, an important regulator of replicative senescence. Evasion of senescence is frequently implicated in tumor progression. Using a chemical screen, we also identified a novel, selective, reversible and competitive inhibitor of PRMT6. The consequence of genetic and pharmacological inhibition of PRMT6 on cell proliferation and senescence will be reported. Thus, our studies are the first to demonstrate a role for arginine methyl transferase family of chromatin modifiers in medulloblastoma genesis.

Modeling SHH-driven medulloblastoma with patient iPS cell-derived neural stem cells

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.

ScRNA-seq in medulloblastoma shows cellular heterogeneity and lineage expansion support resistance to SHH inhibitor therapy

Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.

PDTM-05. SONIC HEDGEHOG SIGNALING PRIMES CEREBELLAR GRANULE NEURON PROGENITORS AND MEDULLOBLASTOMA CELLS FOR APOPTOSIS BY INDUCING PRO-APOPTOTIC BIM

Abstract Medulloblastomas, unlike other malignant brain tumors, are typically sensitive to radiation therapy (xRT). However, although xRT allows medulloblastoma patients to survive, it can also confer significant long term cognitive sequelae. Understanding the mechanisms of radiation sensitivity in medulloblastoma may identify ways to increase this sensitivity through targeted therapy. Cerebellar granule neuron progenitors (CGNPs), the cells of origin for SHH-subgroup medulloblastoma, are also sensitive to xRT. We have shown that SHH signaling, which induces CGN proliferation and in excess can cause medulloblastoma, induces the pro-apoptotic protein BIM, resulting in increased radiation sensitivity. SHH-stimulated CGNPs show robust BIM expression, which is blocked by the SMO inhibitor vismodegib. CGNPs in BIM null mice show normal SHH-driven proliferation, but are markedly less sensitive to radiation. Protein studies show that BIM binds to the anti-apoptotic proteins BCL-xL and MCL-1, suggesting a mechanism for increasing the sensitivity to radiation by lowering the apoptotic threshold. On-going studies will determine whether BIM is required for radiation sensitivity in SHH-driven medulloblastoma. If validated, the BIM interactions with BCL-xL and MCL-1 may be novel mechanisms to be targeted to improve medulloblastoma therapy.

SCIDOT-03. HYPERLOADED POLY(2-OXAZOLINE) MICELLES AS PERSONALIZED DRUG CARRIERS FOR BRAIN TUMORS

Abstract Systemic therapies for brain tumors are complicated by dose-limiting extraneural toxicity, serum protein binding and restricted blood-brain barrier (BBB) permeability. For patients with SHH-subgroup medulloblastoma, SHH-pathway inhibition may be more effective and less toxic than current non-targeted therapy. However, vismodegib, which effectively targets SHH-driven basal cell carcinoma, has limited benefit for patients with SHH-driven medulloblastoma. Formulating vismodegib in poly(2-oxazoline) micelles (POx-vismo) improved delivery and efficacy, using medulloblastoma-prone hGFAP-Cre/SmoM2 (G-Smo) mice for a preclinical model. Compared to conventional vismodegib (c-vismo), POx-vismo increased maximum tolerated dose (MTD), tumor:serum ratio and penetration to brain and tumor. Further studies have shown the ability of POx micelles to load CDK4/6 inhibitor. Loading into POx micelles improved MTD and efficacy of palbociclib. Taken together, our results show the potential for poly(2-oxazoline) micelles delivery to make failed brain tumor treatments newly effective. This work was supported by the NCI Alliance for Nanotechnology in Cancer (U54CA198999, Carolina Center of Cancer Nanotechnology Excellence), by NINDS (R01NS088219, R01NS102627) and by the St. Baldrick’s Foundation.

No REST for medulloblastoma

Cancer Medulloblastoma is a pediatric brain tumor with few therapeutic options. Attempts to block the Sonic hedgehog (SHH) pathway that drives various tumor subtypes have been unsuccessful clinically. Dobson et al. found a mechanism by which SHH signaling may be increased in some patients. In a mouse model, increased expression of the transcriptional repressor REST in the disease-causing cells triggered epigenetic silencing of an inhibitor of SHH signaling. These findings suggest potential therapeutic targets for patients with high-REST, SHH-driven medulloblastoma. Sci. Signal. 12 , eaan8680 (2019).

GSK-3 modulates SHH-driven proliferation in postnatal cerebellar neurogenesis and medulloblastoma.

Cerebellar development requires regulated proliferation of cerebellar granule neuron progenitors (CGNPs). Inadequate CGNP proliferation causes cerebellar hypoplasia whereas excessive CGNP proliferation can cause medulloblastoma, the most common malignant pediatric brain tumor. Although sonic hedgehog (SHH) signaling is known to activate CGNP proliferation, the mechanisms downregulating proliferation are less defined. We investigated CGNP regulation by GSK-3, which downregulates proliferation in the forebrain, gut and breast by suppressing mitogenic WNT signaling in mouse. In striking contrast to these systems, we found that co-deleting Gsk3a and Gsk3b blocked CGNP proliferation, causing severe cerebellar hypoplasia. The GSK-3 inhibitor CHIR-98014 similarly downregulated SHH-driven proliferation. Transcriptomic analysis showed activated WNT signaling and upregulated Cdkn1a in Gsk3a/b-deleted CGNPs. Ctnnb co-deletion increased CGNP proliferation and rescued cerebellar hypoproliferation in Gsk3a/b mutants, demonstrating physiological control of CGNPs by GSK-3, mediated through WNT. SHH-driven medulloblastomas similarly required GSK-3, as co-deleting Gsk3a/b blocked tumor growth in medulloblastoma-prone SmoM2 mice. These data show that a GSK-3/WNT axis modulates the developmental proliferation of CGNPs and the pathological growth of SHH-driven medulloblastoma. The requirement for GSK-3 in SHH-driven proliferation suggests that GSK-3 may be targeted for SHH-driven medulloblastoma therapy.

Abstract 4138: CK1δ-BRD4 pathway as novel therapeutic target for SHH subtype of medulloblastoma

Abstract Background: Medulloblastoma is the most common malignant pediatric brain tumor with variable prognosis due to its clinical and genomic heterogeneity. Despite decades of treatment advances, nearly 40% of children experience tumour recurrence, and 30% will die from this disease. Thus, new drugs and drug combinations need to be developed that effectively treat medulloblastoma. Casein kinase 1δ (CK1δ) is a serine/threonine kinase that controls cell cycle progression, signal transduction and neurogenesis, and we previously reported high levels of CK1δ in mouse models of medulloblastoma. BRD4 is an epigenetic reader protein that controls expression of several oncogenes. We wanted to determine the mechanism through which CK1δ regulates BRD4 activity in medulloblastoma and assess whether a combination of CK1δ and BRD4 inhibitors could be more effective in decreasing medulloblastoma progression in vivo. Methods: Granule cell progenitors (GCPs), medulloblastoma Ptch-/-P53-/- mouse cells and SUFU-/- mouse embryonic fibroblasts were incubated with the CK1δ inhibitor SR-1277 and the phosphorylation levels of BRD4 were determined by WB and its binding to chromatin by ChIP. In vitro phosphorylation studies with human BRD4 purified from E. coli were performed and whether genetic disruption of CK1δ reduces BRD4 phosphorylation was tested. We combined BRD4 and CK1δ inhibitors (JQ1 and SR-1277, respectively) and analysed Gli1 mRNA expression and EdU incorporation. We deleted CK1δ in GCPs in Ptch-/-;P53-/- mouse models of medulloblastoma by breeding these mice with a Tg(Atoh1-Cre);CK1δfl/fl strain. Furthermore, we intracranially transplanted human SHH medulloblastoma PDX cells expressing luciferase in mice, to validate the combination therapy. Results: CK1δ inhibitor treatment and CK1δ knockout reduced BRD4 phosphorylation, suggesting that CK1δ phosphorylates BRD4 and is required for BRD4 recruitment to chromatin. In vitro phosphorylation studies with purified human BRD4 and CK1δ confirmed CK1δ-mediated BRD4 phosphorylation and CK1δ is required for BRD4 phosphorylation at serine 492 and 494 in vivo. Using loss of function studies in GCPs, both CK1δ and BET inhibition reduced BRD4 association with Gli1 promoter, thereby reducing Gli1 mRNA levels. In addition, SR-1277 suppressed SHH signaling downstream of SUFU of medulloblastoma cells, in vitro and in vivo. We also found synergy by combining these compounds in vitro and in reducing tumor burden in the in vivo model with intracranial allografts. Conclusion: Together, our studies validate the CK1δ-BRD4 pathway as a novel target in medulloblastoma. The significance of our work is underscored by the possibility that simultaneous CK1δ-BRD4 inhibition could overcome the resistance observed with BRD4 inhibitors and enhance therapeutic benefit to patients. We are also the first group to demonstrate that CK1δ acts downstream of SHH signalling in SHH-driven medulloblastoma. Citation Format: Sze Kiat Tan, Clara Penas, Cheng-Ming Chiang, David Robbins, Nagi Ayad. CK1δ-BRD4 pathway as novel therapeutic target for SHH subtype of medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4138.