MEDB-76. Evaluating the B7-H3 checkpoint in Medulloblastoma

BACKGROUND: There is currently no curative therapy for recurrent/refractory MB. Novel approaches to MB include immunotherapy, such as targeting the immune checkpoint molecule B7-H3. B7-H3 is implicated in tumor metastasis and is highly expressed in MB. This study explores the effects of genetically knocking down B7-H3 in a murine model of recurrent/refractory medulloblastoma. METHODS: Murine MB cells were transduced with a CRISPR/Cas9 lentivirus to create a B7-H3 knockout. Knockout population was sorted twice via FACS by the AECOM flow cytometry CORE and confirmed by western blot and flow cytometry. Three healthy clones were used in subsequent studies, and compared to the wild type and the scramble control. IncuCyte live imaging technology was used to evaluate spheroid growth. Matrigel Boyden chambers were used to evaluate migration. Bulk RNA-seq was performed by the Yale University Core. RESULTS: B7-H3 knockout was successful in the murine MB model. Morphological differences were noted in the B7-H3 knockout cells. Spheroid formation assays show one of the clones with statistically slower growth kinetics compared to controls. Migration results are pending. RNA seq revealed similar clustering amongst knockouts, separate from controls with an enrichment in genes of morphologic development, WNT signaling and amoeboid migration. CONCLUSIONS: The morphologic changes in the B7-H3 knockouts suggest a potential growth differential. Although in vitro growth assays have shown mixed results regarding the effect of knocking out B7-H3 in spheroid formation, B7-H3 has been more directly implicated in migration and immune signaling. If migration is impaired, this will suggest that B7-H3 enhances malignant and metastatic potential in MB. Functional in vivo immune studies in syngeneic mice will investigate immune mediated effects of B7-H3 knockout in this tumor. If our studies support a role for B7-H3 in the development of MB, it may have important clinical implications, particularly for relapsed patients.