SAT-369 - Structure-Based Mechanistic Principles for the Paradoxical Effects of Pathological RET Mutations

Background:
The RET receptor tyrosine kinase transmembrane protein regulates cell growth, differentiation, and survival in diverse tissues, including the thyroid and enteric nervous system. Mutations that reduce RET levels or function can cause Hirschsprung's (HSCR) disease, characterized by abnormal distal colon innervation in the developing embryo. In contrast, mutations that constitutively activate RET can lead to tumorigenesis, most notably in multiple endocrine neoplasia type 2 (MEN2) syndromes usually identified in the thyroid. Paradoxically, some RET mutations can both reduce RET activity in enteric ganglia, and increase signaling in other tissues, resulting in co-occurrence of both HSCR and MEN2A. Although extensive research has been conducted on RET mutations, the structural and mechanistic bases underlying these paradoxical effects remain unclear.

Objective:
To define the structural and mechanistic principles by which extracellular RET missense mutations lead to loss-of-function, gain-of-function, or paradoxical dual phenotypes associated with HSCR and MEN2A.

Methods:
We curated a comprehensive dataset of 70 positions in RET extracellular domains where point-mutations were associated with HSCR, MEN2A, or both. Using 3D structure-based approaches, we predict the potential structural effects of mutations in these positions.

Results:
Our analysis suggests that approximately 90% of positions associated with HSCR disease can, upon mutation, disrupt intramolecular interactions that stabilize RET tertiary structure: residues buried in the protein core, calcium-binding sites, or residues participating in stabilizing intramolecular electrostatic/covalent bonds. A smaller subset of mutations involves substitutions to/from glycines or prolines in key positions. Only a small minority of HSCR-associated positions affect protein-protein interactions needed for signal activation. On the other hand, our analysis showed that ~75%
of mutations in positions that cause MEN2A lead to an unpaired cysteine that can form an intermolecular disulfide bond between two RET monomers. Other mutations that cause MEN2A are also predicted toenhance RET homodimerization via extracellular domains that are proximal to the membrane. Importantly, a substitution leading to an unpaired cysteine that concurrently destabilizes RET tertiary structure is predicted to cause the paradoxical co-occurrence of MEN2A and HSCR.

Conclusions:
Our findings suggest a mechanistic basis for almost all identified pathological mutations in RET and suggest that therapeutic strategies for targeting RET activity in HSCR and MEN2A may need to be orthogonal.

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