NF2/Merlin Michael Anderson

Hepatocellular carcinomas Diseases Schwannomas are hallmark trait Rare NF2 encoded tumor suppressor Merlin Neurofibromatosis 2 NF2 mutations found in sporadic mesotheliomas associated with asbestos exposure Mesotheliomas NF2 mutations discovered more recently Help reveal additional cell types affected by NF2 loss Thyroid carcinomas Hepatocellular carcinomas Perineurial tumors

Neurofibromatosis 2 Hallmark of disease are schwann cell tumors (schwannomas) Rare Occurrence 1/25,000 individuals Autosomal dominant genetic disease Tumors are benign and slow growing, responding poorly to chemotherapy Schwannomas most often found on vestibular branch of both 8th cranial nerves Highly Penetrant >95%

Loss of Merlin leads to SC hyperproliferation and tumorigenesis in animal models Stained section of schwannoma emanating from the trigeminal nerve of a NF2 mutant mouse Schwann cell hyperplasia in the sciatic nerve of a NF2 mutant mouse Loss of Merlin leads to SC hyperproliferation and tumorigenesis in animal models. (A) Hematoxylin and eosin (H&E)-stained section of frank schwannoma emanating from the trigeminal nerve of a P0- Cre;Nf2flox2/flox2 mouse (*). (B) H&E-stained section depicting SC hyperplasia in the sciatic nerve of a P0-Cre;Nf2flox2/flox2 mouse. (C) Malignant peripheral nerve sheath tumor in Nf2a-mutant zebrafish. Zebrafish bearing a germline mutation in one allele of the NF2a gene develop spindle cell tumors that have features in common with nerve sheath tumors from other species (image kindly provided by K. Edepli) (Amsterdam et al. 2004). Malignant peripheral nerve sheath tumor (MPNST) in NF2 mutant zebrafish. McClatchey A. I., Giovannini M. Genes Dev. 2005;19:2265-2277

NF2 NF2 is located on Chromosome 22 Tumor suppressor First tumor suppressor gene to encode more of a structure protein than an inhibitor Encodes Protein Merlin Genetics Home Reference, 2007.

Merlin Amino Acid similar to Ezrin, Radixin, Moesin (ERM) proteins Blocks cell proliferation Located near membrane Regulates linkage between membrane associated proteins and cytoskeleton Responsible for receiving and interpreting signals from extracellular milieu

Merlin Serine Residue 518 (S518) phosphorylation weakens self-association inactivating the growth suppressing Merlin Activation of the small GTPase Rac1 is most common method of phosphorylation of S518 In folded conformation it is the N-terminal and C-terminal that associate Fagan T., Conjuring with Merlin, 2001

Merlin-organized complexes prevent mitogenic signaling and contact-dependent inhibition of proliferation Merlin-organized complexes prevent mitogenic signaling and contact- dependent inhibition of proliferation. (Left) At low cell density in cell culture, Merlin is predominantly hyperphosphorylated and inactive and mitogenic signaling proceeds. Similarly, upon cell reattachment to certain ECM substrates, a pool of Merlin is rapidly phosphorylated. (Right) At high cell density in cell culture, Merlin is hypophosphorylated, self-associated, and active in mediating contact- dependent inhibition of proliferation. Merlin is recruited to nascent cell:cell boundaries where it appears to stabilize AJs between cells, perhaps by inhibiting Rac/Pak signaling and/or stabilizing the actin cytoskeleton. Under these conditions, Merlin may inhibit signaling from AJ-associated growth-factor receptors. Hypophosphorylated Merlin can also interact with cell:ECM receptors such as CD44, which binds to hyaluronic acid (HA); increased CD44:HA interaction with increased cell density leads to high levels of active Merlin, which may inhibit associated growth-factor receptors. Notably, complete cell detachment also leads to hypophosphorylation and activation of Merlin. Perhaps inhibited mitogenic signaling from multiple complexes accumulates, reaching a threshold that halts proliferation. The study and manipulation of cell:cell contact in two dimensions in cell culture is nonphysiological; in vivo cells in a tissue are contacting other cells and can override contact-dependent inhibition of proliferation under certain normal conditions or in the context of a tumor. Merlin may coordinate the receipt of physical information from the extracellular milieu with the receipt and processing of mitogenic stimuli. McClatchey A. I., Giovannini M. Genes Dev. 2005;19:2265-2277

Curto M., et al., Contact Dependent Inhibition, 2007.

Mutations of NF2 Rare Often Deletions Nonsense Missense

Type of Mutation Number of people Number of Families Nonsense 293 232 Frameshift deletion 159 128 Frameshift insertion 47 41 Indel 10 Splice donor site 174 88 Splice acceptor site 150 86 Missense 35 In-frame deletion 18 In-frame insertion 13 4 Large deletion 141 80 Large insertion 3 Chromosomal translocation 15 Total 1,112 738 Baser M. E., NF2 Mutation Databases, 2004.

Oncogenic Properties Blocks cell proliferation Contact Inhibition Inactivated merlin leads to hyperproliferation Contact Inhibition Merlin regulates contact-dependent inhibition of proliferation Phosphorylation regulated by cellular density Interaction with Epidermal Growth Factor Receptor Merlin can inhibit EGFR upon cell to cell contact

Summary NF2 encodes protein Merlin Merlin acts as a tumor suppressor by regulating proliferation First tumor suppressor to act as more of a structural protein Merlin located near cell membrane and regulates contact-dependent inhibition of proliferation Absence of Merlin causes over proliferation and most often Neurofibromatosis Neurofibromatosis is autosomal dominant most often resulting in schwannomas