ABSTRACT Glioblastoma, the most common primary malignant brain cancer, remains uniformly fatal despite current standard of care consisting of maximal safe surgical resection followed by chemotherapy and radiation. Glioblastoma is characterized by a high degree of heterogeneity, including the presence of therapeutically resistant cancer stem cells (CSCs) that drive tumor recurrence after therapy. CSCs are greatly influenced by cues received from the surrounding tumor microenvironment, but the results from studies on the role of direct cell-cell communication through gap junctions in regulating GBM cell fate remain inconsistent. Gap junctions have canonically been thought of as tumor suppressive due to frequent loss of connexin expression in tumor cells. However, work from our laboratory and others now shows that connexins can also be tumor promoting in CSCs in a context- and connexin subunit-specific manner. We have previously reported tumor-promoting roles for connexin 46 (Cx46) in glioblastoma, Cx26 in triple-negative breast cancer, and Cx25 in leukemia. Here, in preliminary data, we now show that glioblastoma CSCs from patient-derived xenografts in fact express higher levels of Cx43 than Cx46, and Cx43 is required for their survival. Mechanistically, Cx43 is required to maintain expression of MYC, a critical factor for CSCs. This relationship that has not previously been described. Phosphorylation of the kinase WNK lysine-deficient protein kinase 1 (WNK1) occurs downstream of Cx43 and is also required for MYC expression. Based on these preliminary data, we hypothesize that Cx43 activates WNK1 to maintain GBM CSC proliferation, survival, and self-renewal by driving gene expression of MYC. We will test this hypothesis through the following specific aims: 1) test the hypothesis that Cx43 is required for phosphorylation of WNK1 in an AKT-dependent manner and 2) test the hypothesis that the catalytic activity of WNK1 is required for Cx43-mediated MYC expression. With these aims, we will continue to uncover a novel Cx43-WNK1-MYC signaling axis. Due to the difficulty in specifically targeting connexin proteins, the long-term goal of this project is to identify downstream signaling nodes that are amenable to targeting to develop new therapeutic options for patients with glioblastoma.