Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis

Alejandra M Petrilli,Marga Bott,Cristina Fernández-Valle

doi:10.18632/oncotarget.1422

Abstract

Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

Highlights

Neurofibromatosis type 2 (NF2) is a benign tumor disorder of the nervous system caused by mutations in the NF2 gene that encodes a tumor suppressor called schwannomin or merlin
Merlin-mutant MSC were created by in vitro adeno-Cre transduction of mouse Schwann cells isolated from sciatic nerves of homozygous Nf2flox2/flox2 mice as previously described [2224]
We found that AGK2 did not significantly alter the distribution of diploid cells in the cell cycle, there was a tendency to slightly increase the percentage of cells in the G2/M phase (Fig. 3e)

Summary

INTRODUCTION

Neurofibromatosis type 2 (NF2) is a benign tumor disorder of the nervous system caused by mutations in the NF2 gene that encodes a tumor suppressor called schwannomin or merlin. Many schwannomas are inoperable and surgery often causes complete loss of nerve function, while radiosurgery carries an increased risk of a future secondary malignancy [2]. The pleotropic effect of merlin has made it difficult to identify the most relevant drug targets. As an alternative approach to drug discovery, we conducted an unbiased high-throughput screen of the library of Pharmacologically Active Compounds (LOPAC) using viability of merlin-mutant mouse Schwann cells (MSC) as a phenotypic assay to identify potential compounds and pathways relevant to NF2 schwannoma treatment. We validate AGK2 as a compound that selectively reduces viability of merlin-mutant MSC compared to normal MSCs. we demonstrate increased expression levels of SIRT2 in merlin-mutant versus normal MSCs that are associated with a general reduction in lysine acetylation. Phenotypic mechanism of action studies suggests that inhibition of SIRT2 in merlinmutant SCs triggers a necrotic pathway

RESULTS

DISCUSSION

MATERIALS AND METHODS