P10.08.A WOUND HEALING ANALOGY IN MEDULLOBLASTOMA RESPONSE TO GAP JUNCTION INHIBITION AND CYTOTOXIC THERAPY

Abstract BACKGROUND Intercellular communication is a significant factor in chemotherapy resistance in malignant brain tumors. Connexin-43 (Cx43)-based gap junctions (GJs) facilitate this communication by allowing intercellular ion transfer and small molecule exchange. The impact of GJs in medulloblastoma as the most common malignant pediatric brain tumor is unknown. In the present study, we analyzed the potential of GJs as a putative novel therapeutic target in medulloblastoma. Here, we utilized meclofenamate (MFA), an FDA-approved drug with gap junction-inhibitory properties, which had never been used in medulloblastoma research before. MATERIAL AND METHODS Immunofluoresence and Western blot analysis for Cx43 were used to evaluate intercellular GJ expression in human group 3 and 4 medulloblastoma cell lines. A CRISPR/Cas9 gene knockdown of Cx43 was used to explore cellular effects related to the inhibition of GJs. Realtime-imaging fluorescence-guided measurements of GJ-mediated cell-to-cell cytoplasm transfer was performed for the CRISPR/Cas9 Cx43 knockdown model and MFA treatment. We used RNA-sequencing to study downstream signalling cascades in response to GJ inhibition. DNA-fragmentation served as readout for cell death and was assessed by flow cytometric analysis of propidium iodide-stained nuclei. RESULTS All medulloblastoma group 3 and 4 cell lines showed an expression of intercellular Cx43-based GJs. We observed a significant reduction of intercellular cytoplasm transfer via GJs in both the CRISPR/Cas9 Cx43 knockdown model and in MFA-treated medulloblastoma cells. MFA-mediated inhibition of GJs profoundly rendered medulloblastoma cells susceptible for lomustine-mediated cell death. Cytotoxic effects were similar to those observed in CRISPR/Cas9 Cx43 cells. In RNA sequencing, we observed not only an upregulation of programmed cell death pathways but also a significant upregulation of wound healing pathways when MFA was combined with lomustine treatment. CONCLUSION This study suggests that inhibiting intercellular connectivity makes medulloblastoma cells more susceptible to lomustine-induced cytotoxic effects. This disruption of tumor networks can be compared to the initial phase of wound healing, where the cellular network is broken apart. Understanding these processes provides insight into therapeutic strategies that target both the destruction and regrowth phases of tumor cell networks. Consequently, gap junction-targeted approaches might offer a novel treatment strategy for this malignant pediatric brain tumor.