Interrupting HEG1/Cx43 interaction to promote cuproptosis via inhibiting gap junction to overcome glioblastoma radioresistance.

Abstract

2047 Background: Glioblastoma (GBM) is the most common and aggressive primary malignant tumor of the central nervous system. Its intrinsic radioresistance leads to suboptimal therapeutic outcomes. A prior clinical trial by our team revealed that mutations in the HEG1 gene lead to upregulated protein expression, a key factor limiting the benefit from standard radiotherapy in GBM patients. As a form of non-classical programmed cell death induced by radiotherapy, we hypothesized that high HEG1 expression reduces the susceptibility of GBM cells to cuproptosis, thereby promoting radiotherapy resistance. Methods: Cellular functional assays were performed to evaluate the impact of HEG1 expression on cuproptosis sensitivity and radiotherapy response in GBM cells. Molecular techniques were used to assess intracellular and intercellular copper ion flow. Co-immunoprecipitation and inhibition of protein degradation pathways were employed to investigate the direct interaction between HEG1 and the gap junction protein Connexin 43 (Cx43), and its inhibitory effect on Cx43 degradation via the endolysosomal pathway. Structural modeling combined with virtual screening of a drug library was conducted to identify potential compounds disrupting the HEG1-Cx43 interaction. Site-directed mutagenesis and kinase activity modulation were used to elucidate the mechanism by which c-Kit kinase promotes HEG1-Cx43 binding by phosphorylating HEG1 at tyrosine residue Y1350. Functional validation was performed both in vitro and in vivo using GBM models. Results: High HEG1 expression facilitated intercellular copper ion flow, dampened intracellular copper ion fluctuations, and consequently blocked the propagation of cuproptosis. Mechanistically, HEG1 directly interacted with Cx43, inhibiting its degradation via the endolysosomal pathway and sustaining gap junction establishment. Virtual screening identified several multi-target tyrosine kinase inhibitors capable of disrupting the HEG1-Cx43 interaction. Mechanistic studies revealed that c-Kit kinase promoted this interaction by phosphorylating HEG1 at Y1350. Both in vitro and in vivo functional validation demonstrated that targeting the HEG1-Cx43 interaction reactivated cuproptosis and sensitized GBM to radiotherapy. Conclusions: Our findings elucidate the regulatory role of the "HEG1-Cx43-cuproptosis" axis in GBM radioresistance, offering new insights for treatment. The combination of multi-target tyrosine kinase inhibitors and cuproptosis inducers may represent a promising novel therapeutic strategy for GBM.