CANCER-ASSOCIATED MISSENSE SINGLE NUCLEOTIDE VARIANTS REGULATE THE STABILITY AND SUBCELLULAR LOCALIZATION OF NF2/MERLIN

Abstract BACKGROUND Alterations in the NF2 tumor suppressor gene lead to meningiomas and schwannomas, but the tumor suppressor functions of the NF2 gene product, Merlin, are incompletely understood. To address this problem, we performed a structure-function analysis of Merlin by expressing cancer-associated missense single-nucleotide variants (mSNVs) in primary cancer cells for biochemical and cell biology experiments. METHODS All NF2 mSNVs were assembled from cBioPortal and COSMIC, and modelled on the FERM, a-helical, and C-terminal domains of Merlin (PDB 4ZRJ) using comparative structure prediction on the Robetta server and visually inspected using Pymol. mSNV hotspots were defined from sliding windows with at least 10 mutations within 5 residues in either direction. mSNVs from hotspots in meningiomas, schwannomas, or both, were selected for in vitro mechanistic analyses using immunofluorescence and immunoblotting of whole cell, plasma membrane, cytoskeletal, cytoplasmic, nuclear, and chromatin subcellular fractions from M10G meningioma cells and HEI-193 schwannoma cells. RESULTS We identified the following cancer-associated hotspot mSNVs in NF2, which were over-expressed for mechanistic studies: L46R, S156N, W191R, A211D, V219M, R418C and R462K. Endogenous Merlin was detected in all subcellular compartments, but was enriched in the nucleus. L46R and A211D mapped to hydrophobic pockets in the FERM domain, destabilized Merlin, and excluded Merlin from all subcellular compartments except the cytoskeleton. S156N, W191R and V219M also mapped to the FERM domain, but did not affect Merlin stability, and V219M attenuated chromatin localization, suggesting this motif may be involved in binding events that regulate subcellular localization. R418C and R463K mapped to the a-helical domain, but only R418C destabilized Merlin. CONCLUSION Our results suggest that cancer-associated mSNVs inactive the tumor suppressor functions of NF2 by altering the stability, subcellular localization, or binding partners of Merlin. Further work is required to identify and understand the impact of binding partners and subcellular localization on Merlin function.