Mouse Model Of Medulloblastoma Research Articles

A novel indole derivative, 2-{3-[1-(benzylsulfonyl)piperidin-4-yl]-2-methyl-1H-indol-1-yl}-1-(pyrrolidin-1-yl)ethenone, suppresses hedgehog signaling and drug-resistant tumor growth.

The Hedgehog (Hh) signaling pathway plays important roles in various physiological functions. Several malignancies, such as basal cell carcinoma (BCC) and medulloblastoma (MB), have been linked to the aberrant activation of Hh signaling. Although therapeutic drugs have been developed to inhibit Hh pathway-dependent cancer growth, drug resistance remains a major obstacle in cancer treatment. Here, we show that the newly identified, 2-{3-[1-(benzylsulfonyl)-1,2,3,6-tetrahydropyridin-4-yl]-2-methyl-1H-indol-1-yl}-1-(pyrrolidin-1-yl)ethenone analog (LKD1214) exhibits comparable potency to vismodegib in suppressing the Hh pathway activation. LKD1214 represses Smoothened (SMO) activity by blocking its ciliary translocation. Interestingly, we also identified that it has a distinctive binding interface with SMO compared with other SMO-regulating chemicals. Notably, it maintains an inhibitory activity against the SmoD477H mutant, as observed in a patient with vismodegib-resistant BCC. Furthermore, LKD1214 inhibits tumor growth in the mouse model of MB. Collectively, these findings suggest that LKD1214 has the therapeutic potential to overcome drug-resistance in Hh-dependent cancers.

MDB-79. THE DEUBIQUITINATING ENZYME USP7 COOPERATES WITH SONIC HEDGEHOG SIGNALING TO PROMOTE MEDULLOBLASTOMA

Abstract BACKGROUND Constitutive activation of the Sonic Hedgehog (SHH) signaling pathway during cerebellar development may lead to medulloblastoma (MB), one of the most common malignant pediatric brain tumor. Clinically, SHH-MB with mutations downstream of SMO or TP53-mutated are a real challenge and alternative targeted therapies are sorely needed for this group of patients. In SHH-MB, the transcription factor Atoh1 is constitutively overexpressed and essential for tumor initiation and progression. Previous work demonstrated that proteasomal-mediated degradation of Atoh1 induces tumor regression in vitro and in vivo, therefore deciphering mechanisms controlling Atoh1 stabilization should provide new avenues for drug development in SHH-MB. METHODS Using a proteomic approach, we identified the deubiquitinating enzyme Usp7 as a positive regulator of Atoh1 in MB cells. We then applied a SILAC-based quantitative ubiquitinomics to investigate the Usp7 target landscape in SHH-MB. We also employed several biochemical approaches to demonstrate the functional interaction between Atoh1 and Usp7 in SHH-MB. In parallel, we validated the impact of Usp7 depletion in vitro and in vivo using relevant SHH-MB models. Last, we investigated large transcriptomic and proteomic datasets to unveil the prognostic value of Usp7 in SHH-MB human tumors. RESULTS We demonstrate that Usp7 binds and accumulates Atoh1 protein by counteracting its ubiquitylation. Post-natal electroporation in vivo directly into the developing cerebella also indicates that overexpression of Usp7 promotes proliferation, whereas in the absence of Atoh1 Usp7 was no longer able to spread cell growth, demonstrating a functional link between the Usp7-Atoh1 axis. In vivo, disruption of Usp7 expression in SHH-MB blocks tumor proliferation. Pharmacological inhibition of Usp7 triggers tumor growth in a syngeneic SHH MB mouse model. Lastly, SHH-MB cells with either TP53 or SMO downstream mutations are sensitive to Usp7 inhibition. CONCLUSIONS Overall, our data pinpoint Usp7 as a promising target against Atoh1, a master regulator of SHH-MB.

GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Abstract 4599: A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most prevalent malignant childhood brain tumour. Although prognosis has improved over time, 95% of children with high-risk or relapsed MB eventually succumb to the disease. A key driver gene in high-risk MB is the c-MYC oncogene, for which there are currently no approved inhibitors. Histone deacetylases (HDACs) are transcriptional repressors that are dysregulated in many cancers, making them an attractive therapeutic target. Although several HDAC inhibitors have been FDA-approved for cancer treatment, they have been associated with high toxicity and limited efficacy as single agents. Therefore, simultaneously targeting c-MYC and HDACs may result in synergistic effects that could overcome drug resistance in high-risk MB. Methods and Results: We previously reported a novel pyrido-benzimidazole analogue, SE486-11, which enhanced the therapeutic effect of HDAC inhibitors in MYCN-driven cancers. We recently developed analogues (UNSW-SC compounds) with more potent activity (IC50: 0.017 to 3.70 µM). Here, we showed that these compounds significantly reduced cell viability and induced apoptosis in MB cells. MYC status was a key determinant of sensitivity of UNSW-SC compounds, with more than 3-fold higher cytotoxicity in high MYC compared to low MYC MB cells. The lead compound, UNSW-SC-22, was shown to reduce MYC protein expression and was found to be highly synergistic in enhancing the efficacy of HDAC inhibitors in MB cells. Most importantly, we demonstrated that UNSW-SC-22 was able to freely cross the blood brain barrier, reaching a concentration of 10.8 μM, with a half-life of 1.22 hours. UNSW-SC-22 directly bound to c-MYC and MYCN proteins at low micromolar equilibrium dissociation constant (KD) values which was demonstrated by surface plasmon resonance assay. In a MYCN-driven MB mouse model, we found that UNSW-SC-22 as a single agent at 90 mg/kg decreased tumor growth and prolonged survival. Through RNA-sequencing, we identified 512 differentially expressed genes after in vitro treatment with UNSW-SC-22. Importantly, GSEA analysis revealed MYC targets and cell cycle pathways as highly downregulated pathways, and upregulation in the p53 pathway. Several genes, such as MELK and USP1 were validated as down-stream targets of UNSW-SC-22. As UNSW-SC-22 is a first-in-class anticancer drug, which we are currently testing extensively in other MYC-driven MB mice models. Conclusion: Collectively, our data strongly suggest that c-Myc and MYCN are the molecular targets of UNSW-SC compounds, and these compounds have strong potential to serve as specific targeted therapy to treat subgroup 3 and 4 of MB patients with c-Myc and MYCN overexpression and amplification. Citation Format: Sin Wi Ng, Satyanarayana Gadde, Qian Wang, Belamy B. Cheung, Glenn M. Marshall. A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4599.

Abstract 6505: Targeting PINK1 reduces medulloblastoma progression in animal models

Abstract Medulloblastoma is a brain cancer that mainly arises during infancy and childhood. Medulloblastoma is the most common pediatric malignant brain tumor worldwide. It comprises of approximately 20% of all childhood brain tumors and 63% of intracranial embryonic tumors. One of the major effects of proliferating cancer cells is altered mitochondrial metabolism, as well as aberrant metabolism. Medulloblastoma patients suffer severe complications due to intensive and long-term treatments. Thus, we aim to improve applications against medulloblastoma by targeting the PINK1 gene. We hypothesize that PINK1 inactivation slows down medulloblastoma progression. PINK1 is a mitochondrial serine/threonine protein kinase that protects cells by controlling mitochondria quality in response to cellular stress. PINK1 recruits parkin to target damaged mitochondria for degradation through autophagy. In this study, PINK1 has been knocked-out using gene editing technology (CRISPR/Cas9) in two commercial medulloblastoma cell lines (DAOY, and D283 cell lines). We validated PINK1 editing using RT-PCR and Western blot. Then, cell viability was studied in comparison to the control sample. The control sample contained scramble fragments with empty CRISPR/Cas9. We studied apoptosis and performed cell cycle analysis using flow cytometry. Next, orthotopic medulloblastoma mouse model was generated which showed differential survivals comparing the PINK1 knockout group and control. The results showed a low expression of PINK1 within the PINK1 knockout group. In addition, the Western blot showed reductions in PINK1 expression in the knockout group. The knockout group showed more apoptosis (cell death) 40% more than the control. The mouse model showed increased survival of the PINK1 knockout group compared to the control with a p-value of 0.0288 (log-rank). Our analysis shows that survival differences approximately extend human life by one year. We conclude that inhibiting the PINK1 gene in medulloblastoma decreases cell metabolic activities. PINK1 Knockout in medulloblastoma enhances the apoptosis pathways. Engrafted medulloblastoma mouse model treated with PINK1 inhibitor showed superior survival than the control. These results qualify PINK1 to be used as potential therapeutic target for medulloblastoma. The Future work needs additional preclinical studies to investigate the proper dose of PINK1 inhibitor and measurement of toxicity index. Citation Format: Reema N. Alsubaiee, Amani Alghamdi, Bahauddeen M. Alrfaei. Targeting PINK1 reduces medulloblastoma progression in animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6505.

Active targeting of nanotherapeutics using power cavitation imaging with a linear array transducer

Power cavitation imaging (PCI) is an emerging strategy for monitoring blood–brain barrier (BBB) opening, enabling spatial discrimination of cavitation intensity through power Doppler-analogous processing of passive cavitation images. Single-element cavitation detectors, while beneficial in signal monitoring for cavitation controllers, provide limited spatial information, conferring the need for an image-guided approach to ensure accurate cavitation localization and regulation. This study aims to evaluate the capability of PCI to spatially correlate real-time acoustic cavitation emissions with mechanical bioeffects as a predictor of P-selectin-targeted nanocarrier delivery. Preliminary sonoporation experiments were performed in vitro with brain tissue derived mouse endothelial cells and 3 kDa tetramethylrhodamine (TMR)-dextran. A focused ultrasound transducer (0.5 MHz, 5000 cycles/pulse, 5 Hz PRF, 30 second treatment duration) was used to sonicate cells in small volume (25 μl) cell suspension. Homogenous distribution of TMR-dextran in the cytosol and nucleus was observed via fluorescence microscopy at lower pressures (7% ± 2% fluorescent cells), while higher pressures had no significant differences above control (3% ± 2% vs 1% nontreated) due to microbubble destruction. Future experiments will correlate sonoporation with P-selectin expression and validate a linear array PCI system against contrast-enhanced MRI as a guide for P-selectin-targeted nanocarrier delivery in mouse models of medulloblastoma.

10205-TB-8 ABSENCE OF RNF213 IN MEDULLOBLASTOMA INDUCES STARVATION RESISTANCE VIA GAS6 AND RESULTS IN TUMOR PROGRESSION

Abstract RNF213 gene, initially identified as a disease-causing gene for moyamoya disease, has recently been recognized as a tumor regulator. We investigated the functions of RNF213 in tumorigenicity of medulloblastoma. Knockout of Rnf213 in Ptch1+/- spontaneous medulloblastoma mouse model increased the incidence of SHH type medulloblastoma from 19.8% to 76.5% (p<0.001) at 9 months. Subcutaneous implantation of the tumors showed that depletion of Rnf213 increased tumor formation frequency and tumor size. Unexpectedly, knockout of RNF213 in human Daoy cells decreased tumor growth and invasion in vitro. However, they were resistant to serum starvation and showed higher growth rate when implanted subcutaneously in nude mice. Transcriptome analysis of mouseRnf213-/- cells indicated that downregulation of Igf2 decreased tumor growth and upregulation of Gas6 increased resistance to starvation. In fact, GAS6 was upregulated in serum-free conditions and further upregulated in RNF213-/- cells. Survival advantage in serum starved conditions was lost by GAS6 knock-down in RNF213-/- Daoy cells. In conclusion, the effect of RNF213 depletion was context dependent. Resistance to starvation seems to outweigh growth retardation in normal conditions and is important for tumorigenicity and aggressiveness in vivo.

Single-fraction Radiation Treatment Dose Response in a Genetically Engineered Mouse Model of Medulloblastoma.

Medulloblastoma is the most common malignant brain tumor of children. Although standard of care radiotherapy for pediatric medulloblastoma (PM) can lead to long-term remission or cure in many patients, it can also cause life-long cognitive impairment and other adverse effects. The pathophysiological mechanisms involved in radiation-induced cerebral damage are incompletely understood, and their elucidation may lead to interventions that mitigate radiation toxicity. To explore the mechanisms of radiation-induced cerebral damage, transgenic mouse models of PM and non-tumor-bearing controls were exposed to radiation doses that ranged from 0 to 30 Gy. Between 0-20 Gy, a significant dose-dependent reduction in tumor-associated hydrocephalus and increase in overall survival were observed. However, at 30 Gy, hydrocephalus incidence increased and median overall survival fell to near-untreated levels. Immunohistochemistry revealed that both tumor-bearing and non-tumor-bearing mice treated with 30 Gy of radiation had significantly more reactive astrocytes and microvascular damage compared to untreated controls. This effect was persistent across mice that were given 1 and 2 weeks of recovery time after irradiation. Our data suggest that radiation therapy promotes neural death by inducing long-term neuroinflammation in PM, suggesting radiation delivery methods that limit inflammation may be effective at widening the therapeutic window of radiation therapy in PM patients.

EXTH-25. IN VIVO TWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing B7-H3 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. B7-H3-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against B7-H3 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

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.

Intraventricular SHH inhibition proves efficient in SHH medulloblastoma mouse model and prevents systemic side effects.

Medulloblastoma (MB) is the most common malignant brain tumor in children and requires intensive multimodal therapy. Long-term survival is still dissatisfying and, most importantly, survivors frequently suffer from severe treatment-associated morbidities. The sonic hedgehog pathway (SHH) in SHH MB provides a promising target for specific therapeutic agents. The small molecule Vismodegib allosterically inhibits SMO, the main upstream activator of SHH. Vismodegib has proven effective in the treatment of MB in mice and in clinical studies. However, due to irreversible premature epiphyseal growth plate fusions after systemic application to infant mice and children, its implementation to pediatric patients has been limited. Intraventricular Vismodegib application might provide a promising novel treatment strategy for pediatric medulloblastoma patients. Infant medulloblastoma-bearing Math1-cre::Ptch1Fl/Fl mice were treated with intraventricular Vismodegib in order to evaluate efficacy on tumor growth and systemic side effects. We show that intraventricular Vismodegib treatment of Math1-cre::Ptch1Fl/Fl mice leads to complete or partial tumor remission only 2 days after completed treatment. Intraventricular treatment also significantly improved symptom-free survival in a dose-dependent manner. At the same time, intraventricular application prevented systemic side effects in the form of anatomical or histological bone deformities. We conclude that intraventricular application of a SHH pathway inhibitor combines the advantages of a specific treatment agent with precise drug delivery and might evolve as a promising new way of targeted treatment for SHH MB patients.

OS09.1.AIN VIVOTWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing EphA2 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. EphA-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against EphA2 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

Oncolytic virus-driven immune remodeling revealed in mouse medulloblastomas at single cell resolution

Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert Tcell-dependent efficacy in mouse models of glioblastoma, exerts Tcell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma.

Low-dose radiation followed by on-target inhibition of Galectin-3 in combination with anti-4-1BB monoclonal antibody regulates immune responses in Group 3 and Group 4 medulloblastoma mouse model (P11-13.001)

<h3>Objective:</h3> Investigation novel treatment options for Group 3 and Group 4 medulloblastoma patients <h3>Background:</h3> Group 3 and 4 medulloblastomas (MB) have a significantly worse prognosis when compared to the sonic hedgehog (SHH) or wingless (WNT) -activated subtypes and are also the most resistant to therapy. Current aggressive radiation/chemotherapy practices though improving survival, have potential long-term comorbidities with potential neurocognitive deficits impacting patients’ overall quality of life. A key factor limiting the therapeutic success of single-agent immune therapies is the poor penetration of tumor-specific immune cells into tumor microenvironments. Recent studies have demonstrated that galectin-3 (Gal-3) accelerates M2 macrophage-infiltration and restricts T cell receptor (TCR)-mediated signaling. Immunotherapy targeting CD8⁺ T cells with agonistic anti-4-1BB (CD137) monoclonal antibody (mAb) activates CD8⁺ T cells, promoting their survival and acquiring potent cytolytic properties. Building on these findings, we hypothesize that a novel treatment paradigm with immune-priming using sub-lethal low-dose radiation in combination with a Gal-3 inhibitor (anti-Gal-3 mAb) plus anti-4-1BB mAb will synergistically deplete tumor associated macrophages, restore CD8+ T cell migration, and promote autoimmune responses into tumor islets with resultant survival benefits in preclinical immunocompetent Group 3 and Group 4 MB mouse models. <h3>Design/Methods:</h3> Treatment effects were assessed using western blot, flow cytometry, H&amp;E, immunofluorescence imaging and ELISA SPOT. <h3>Results:</h3> Our data demonstrated higher levels of Gal-3 expression in Group 3 and 4 patient derived tumor tissue, as compared to non-tumor tissue. We observed that Gal-3 shifted cytokine production via switching microglia polarization to the M2 subtype. Furthermore, H&amp;E-stained tumor sections following treatment with combination low-dose radiation, Gal-3 inhibitor and anti-4-1BB mAb showed a reduction of tumor size by ~70% when compared to untreated controls. <h3>Conclusions:</h3> The results of these investigations provide critical preliminary data for further trials examining the effectiveness of immune-priming using sub-lethal low-dose radiation followed by combination treatment using a Gal-3 inhibitor plus anti-4-1BB mAb. <b>Disclosure:</b> Dr. Cachia has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for RedHill Biopharma Ltd. Dr. Cachia has received personal compensation in the range of $500-$4,999 for serving as a Consultant for GuidePoint Global LLC. Dr. Cachia has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Dedham Group. Dr. Cachia has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Massachusetts Neurological Society. Dr. Eskandari has nothing to disclose. Mr. McDonald has nothing to disclose. Dr. Infinger has nothing to disclose. William A. Vandergrift has nothing to disclose. Abhay K. Varma has nothing to disclose. Sunil J. Patel has nothing to disclose. Dr. Zukas has nothing to disclose. Dr. Lindhorst has nothing to disclose. Dr. DAS has nothing to disclose.

EXTH-84. A NOVEL DUAL-SPECIFIC CHIMERIC ANTIGEN RECEPTOR T CELL WITH HIGH SPECIFICITY FOR EGFR AND EGFRVIII IMPROVES SURVIVAL IN EGFR EXPRESSING MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma is the most common malignant brain tumor in children 0-19 years of age and current treatment requires surgical resection, followed by high dose chemotherapy and radiotherapy. Therapy carries high morbidity, with late effects including neurocognitive decline, endocrine dysfunction, and subsequent malignancies. Chimeric antigen receptor (CAR) T cell therapy presents the potential for less toxic and more effective treatment for medulloblastoma. Epidermal growth factor (EGFR) is expressed in medulloblastoma derived cell lines and appears to serve an important role in the metastatic potential of this tumor type. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) with dual specificity, binding to wild type EGFR (EGFRwt) and its mutant EGFR variant III (EGFRvIII). We previously developed a novel, third-generation chimeric antigen receptor T-cell construct utilizing the D2C7 scFv and performed analysis on both glioblastoma and medulloblastoma tumor models. METHODS U87 and U87vIII glioblastoma and DAOY medulloblastoma cell lines were characterized by flow cytometry to evaluate for EGFRwt and EGFRvIII expression. Cytotoxicity assays were performed utilizing flow cytometry on serially diluted effector: target ratios of U87, U87vII, and DAOY mixed with D2C7 CAR. DAOY was orthotopically implanted into the frontal lobe of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. After tumor implantation, D2C7 CAR was introduced within the tumor bed. RESULTS EGFR was detected in U87, U87vIII, and DAOY on flow analysis. EGFRvIII was expressed only on U87vIII. D2C7 CAR demonstrated effective in vitro cytotoxicity against all three cell lines with &amp;gt;90% cytotoxicity achieved at an effector: target ratio of 2.5:1. In vivo, D2C7 CAR treatment significantly prolonged survival compared to treatment with mock CAR. CONCLUSION D2C7 CAR demonstrates efficacy in vitro and in mouse models of medulloblastoma and glioblastoma. Future steps in our work will set out to assess the D2C7 CAR in NSG mice with DAOY in the posterior fossa.

EXTH-79. HIJACKING A NEURODEVELOPMENTAL EPIGENOMIC PROGRAM IN METASTATIC DISSEMINATION OF MEDULLOBLASTOMA

Abstract Normal brain development relies on precise genetic and epigenetic spatiotemporal regulation of gene expression. How dysregulation of neurodevelopment relates to medulloblastoma, the most common pediatric brain tumor, remains elusive. Here, we uncovered a novel neurodevelopmental epigenomic program that regulates Purkinje cell migration in developing cerebellum is hijacked to induce tumor metastatic dissemination in medulloblastoma. Integrating publicly available datasets with our in-house data, unsupervised analyses revealed that BAF60C/SMARCD3, a subunit of SWI/SNF chromatin remodeling complex, promotes tumor cell migration in vitro and metastasis in vivo. Based on analyzing the single-cell RNAseq data of cerebellum developmental trajectory in mice and humans, aligning with the medulloblastoma patients’ datasets, we found that BAF60C/SMARCD3 regulated DAB1-mediated Reelin signaling is involved in Purkinje cell positioning during cerebellum development and medulloblastoma metastasis by orchestrating the cis-regulatory elements (CREs) at the DAB1 gene locus. Moreover, analysis of spatiotemporal gene expression and chromatin architecture in the human and mouse cerebellum demonstrated that transcription activity of the BAF60C/SMARCD3-DAB1 circuit is downregulated in a mature state of cerebellar development, however, is upregulated in metastatic medulloblastoma. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), bi-directionally control BAF60C/SMARCD3 transcriptional regulation by coordinating with the CREs at the BAF60C/SMARCD3 gene locus to form a chromatin hub during developing cerebellar development and medulloblastoma metastatic dissemination. Highly expressed BAF60C/SMARCD3 activates the Reelin/DAB1 signaling pathway downstream Src kinase, which was validated in the pair-wised primary and metastatic tumors from medulloblastoma patients. Preclinical medulloblastoma mouse models revealed that inhibiting Src activity reduces tumor cell migration and metastatic dissemination at a lower and safe dose. Together, these data deepen our understanding of how the developmental program influences disease progression and provide an opportunity for the development of therapeutics for this devastating brain cancer in children.

O-GlcNAcylation promotes cerebellum development and medulloblastoma oncogenesis via SHH signaling

Sonic hedgehog (Shh) signaling plays a critical role in regulating cerebellum development by maintaining the physiological proliferation of granule neuron precursors (GNPs), and its dysregulation leads to the oncogenesis of medulloblastoma. O-GlcNAcylation (O-GlcNAc) of proteins is an emerging regulator of brain function that maintains normal development and neuronal circuitry. Here, we demonstrate that O-GlcNAc transferase (OGT) in GNPs mediate the cerebellum development, and the progression of the Shh subgroup of medulloblastoma. Specifically, OGT regulates the neurogenesis of GNPs by activating the Shh signaling pathway via O-GlcNAcylation at S355 of GLI family zinc finger 2 (Gli2), which in turn promotes its deacetylation and transcriptional activity via dissociation from p300, a histone acetyltransferases. Inhibition of OGT via genetic ablation or chemical inhibition improves survival in a medulloblastoma mouse model. These data uncover a critical role for O-GlcNAc signaling in cerebellar development, and pinpoint a potential therapeutic target for Shh-associated medulloblastoma.

Abstract 663: Systemic targeting of therapeutic RNA to cancer via a novel, cell-penetrating and nucleic acid binding, monoclonal antibody

Abstract There is intense interest in the development of nucleic acid ligands for immune stimulation of the tumor microenvironment via pattern recognition receptors (PRRs), particularly for the treatment of “cold” tumors. However, delivery of these ligands in a tumor-specific manner to avoid systemic toxicity has been a challenge. Many current efforts rely on direct intra-tumoral injection of RNAs or other stimulatory immune ligands, which is therapeutically sub-optimal, especially for metastatic disease. Here we report studies evaluating the utility of a lupus-derived, cell-penetrating antibody, to deliver nucleic acids to tumors in vivo. This antibody, a modified version of 3E10-D31N, designated GMAB, forms non-covalent complexes with RNAs and can mediate highly specific delivery into tumors via intravenous injection by targeting the nucleoside transporter ENT2, which is highly expressed in tumor cells. Studies with labeled RNAs showed tumor specific delivery and functional expression by imaging, with minimal delivery to healthy tissues. Following these proof-of-concept studies, we investigated whether our antibody could be used to deliver RIG-I agonists to tumors. Using a known agonist of RIG-I, 3p-hpRNA, we demonstrated single agent activity of our GMAB/RNA complexes in multiple tumor models, including a mouse model of melanoma (B16) and an orthotopic model of pancreatic cancer (KPC). Measuring 3p-hpRNA by RT-PCR, there was a 1000X fold difference in RNA uptake in KPC tumor cells relative to CD45+ cells isolated from tumors. In addition, synergy between our GMAB/RNA complexes and anti-PD-1 was additionally observed in mouse models of breast (EMT6) and colon (MC38) cancer. Given the expression of ENT2 along the blood brain barrier (BBB), we demonstrate single agent activity of our GMAB/RNA complexes in an orthotopic mouse model of medulloblastoma, resulting in suppression of tumor growth and spinal metastases. Together, these studies demonstrate that the GMAB antibody can 1) localize to orthotopic and flank tumor models, 2) cross the BBB, and 3) deliver RNA payloads to tumors, providing a novel platform for delivery of immunogenic RNAs, as well as mRNAs and siRNAs, directly to tumors with high specificity following systemic administration. Citation Format: Elias Quijano, Diana Martinez Saucedo, Minsoo Khang, Yanfeng Liu, Dale Ludwig, Bruce C. Turner, Stephen Squinto, Ranjit Bindra, W. Mark Saltzman, Luisa Escobar-Hoyos, Peter M. Glazer. Systemic targeting of therapeutic RNA to cancer via a novel, cell-penetrating and nucleic acid binding, monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 663.

Three dimensional reconstruction of the mouse cerebellum in Hedgehog-driven medulloblastoma models to identify Norrin-dependent effects on preneoplasia

Spontaneous mouse models of medulloblastoma (MB) offer a tractable system to study malignant progression in the brain. Mouse Sonic Hedgehog (Shh)-MB tumours first appear at postnatal stages as preneoplastic changes on the surface of the cerebellum, the external granule layer (EGL). Here we compared traditional histology and 3DISCO tissue clearing in combination with light sheet fluorescence microscopy (LSFM) to identify and quantify preneoplastic changes induced by disrupting stromal Norrin/Frizzled 4 (Fzd4) signalling, a potent tumour inhibitory signal in two mouse models of spontaneous Shh-MB. We show that 3DISCO-LSFM is as accurate as traditional histology for detecting Norrin/Fzd4-associated changes in PNL formation in Ptch+/− mice and EGL hyperplasia in Neurod2-SmoA1+/− mice. Moreover, we show that the anti-tumour effect of Norrin/Fzd4 signalling is restricted to the posterior region of the cerebellum and is characterized by defective neural progenitor migration away from the EGL. In conclusion, 3DISCO-LSFM is a valid way to monitor tumour initiation events in mouse MB models and reveals an unanticipated regional restriction of stromal signalling in constraining tumour initiation.

OTHR-42. Missing data tolerant integration of proteomic datasets enables the identification and characterization of brain cancer subtypes

Investigating the proteome can add a significant layer of information to manifold existing methylation, mutation, and transcriptome data on brain tumors as proteins represent the pharmacologically addressable phenotype of a disease. Small cohorts limit the usability and validity of statistical methods, and variable technical setups and high numbers of missing values make data integration from public sources challenging. Using a newly developed framework being able to reduce batch effects without the need for data reduction or missing value imputation, we show –based on in-house and publicly available datasets- successful integration of proteomic data across different tissue types, quantification platforms, and technical setups. Exemplarily, data of a Sonic hedgehog (Shh) medulloblastoma mouse model were analyzed, showing efficient data integration independent of tissue preservation strategy or batch. We further integrated batches of publicly available data of human brain tumors, confirming proposed proteomic cancer subtypes correlating with clinical features. We show that, missing value tolerant reduction of technical variances may be helpful to identify biomarkers, proteomic signatures, and altered pathways characteristic for molecular brain cancer subtypes.