Background: Diffuse midline glioma (DMG) is the leading cause of childhood brain cancer deaths due to the absence of effective treatments (1,2). There have not been any advances in brain cancer treatment in 30 years. Anoctamins are a class of 10 calcium-activated chloride ion channels that have recently been implicated in cancer pathogenesis (3). However, their role in DMG has not yet been investigated. The purpose of this work is to characterise the expression of the anoctamin family in DMG. Furthermore, this study aims to investigate the therapeutic benefit of anoctamin-inhibiting drugs for reducing DMG cell survival.
Methods: The mRNA expression of 10 anoctamin genes was confirmed in five DMG patient-derived cell lines using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Anoctamin protein expression was examined using western blotting, immunocytochemistry, and immunohistochemistry. Effects of anoctamin-inhibitors, gallotannin and niclosamide, on DMG cell viability were evaluated using the ViaLightTM assay.
Results: ANO3 and ANO5 gene expression were downregulated in all DMG cell lines relative to total human astrocyte cDNA. ANO1, ANO6, and ANO10 proteins were expressed in DMG patient cells and tumour tissues (p<0.0001, Welch’s ANOVAs, n=6). ANO10 demonstrated consistent nuclear expression in DMG neurospheres and tumour tissues. Anoctamin-inhibitors gallotannin (IC50: 9.29-29.38 μM, n=12) and niclosamide (IC50: 0.28-0.47 μM, n=9) significantly reduced DMG cell viability (p<0.0001, Welch’s ANOVAs).
Conclusion: This project determined the mRNA and protein expression of anoctamins in DMG for the first time. Anoctamin inhibition may be a promising therapeutic avenue that could be utilised in combination therapies for DMG. This work provides the necessary in vitro evidence to continue investigating anoctamins as a therapeutic target in DMG in vivo models.
Key messages: There is an unmet clinical need to explore all potential avenues for efficacious treatments to improve survival outcomes for DMG patients. Anoctamins may have a pathological role in DMG progression, and they are a promising novel therapeutic target that should be explored in future DMG treatments.