Introduction: We previously showed that up-regulation and release of HML-2 subfamily of human endogenous retrovirus K envelope protein (HERVK ENV) due to loss of a chromatin remodeling protein, SWI/SNF matrix-associated actin-dependent regulator of chromatin sub-family B member 1 (SMARCB1), maintains pluripotency and syncytial properties characteristic of atypical teratoid rhabdoid tumor (ATRT). Here, we investigated the regulation of intracellular HML-2 ENV and demonstrated two potential therapeutic strategies—1) inhibition of calcium influx by ouabain, a cardiac glycoside that is toxic to neural stem cells, and 2) targeted inhibition of cyclin-dependent kinase 5 (CDK5), which is restricted to neurons by p35, its activator protein, by TP5— to decrease intracellular HML-2 ENV. 

 
Methods: ATRT cell lines (CHLA02 and CHLA04) and tumor tissue obtained from patients were confirmed for SMARCB1 loss and increased HML-2 ENV with immunohistochemistry and immunofluorescence. Cell viability and HML-2 ENV concentration in the intracellular compartment were measured after treatment with ouabain and TP5 by Alamar blue assay and western blot, respectively. We evaluated the calcium-mediated effect of ouabain on HML-2 intracellular concentration by treating the cells with ouabain, the calcium chelators calcimycin and EGTA, and calpeptin, an inhibitor of calpain, which activates CDK5, and measuring HML-2 ENV and p35. We evaluated HML-2 ENV for a CDK5 consensus phosphorylation site and performed co-immunoprecipitation to evaluate the potential interaction. We evaluated activity of CDK5 in ATRT cell lines by autoradiogram. 

 
Results: Both Ouabain and TP5 cause a decrease in cell viability in a dose-dependent manner. Further, ouabain treatment decreases HML-2 ENV intracellular concentration. We found that HML-2 ENV contains a consensus phosphorylation site for CDK5. We demonstrated that HML-ENV binds to CDK5. We established that ATRT cell lines have hyperactive CDK5. Finally, we established that the effect of ouabain on HML-2 ENV is due to indirect inhibition of calcium-mediated activation of calpain and thus CDK5. 

 
Conclusions : Here we demonstrated that ouabain and TP5 decrease ATRT cell line viability and are potential therapeutic strategies for decreasing HERV-K ENV, which we have shown is necessary for tumor survival. We showed the effect of ouabain is indirect through calcium mediated activation of CDK5. Therefore, ouabain and TP5 are potential indirect and direct therapeutic strategies, respectively, to target HML-2 ENV production.