STAT3 SH2 Domain Aspartic Acid 661 Mutations Activate Immune Gene Programs

Area of Science:

• Molecular Biology
• Genetics
• Immunology

Background:

• The STAT3 SH2 domain has a conserved aspartic acid residue D661.
• D661 is a mutational hotspot in hematologic malignancies like T-cell large granular lymphocytic leukemia, myelodysplastic syndromes, and acute lymphoblastic leukemia.

Purpose of the Study:

• To define the functional consequences of distinct STAT3 D661 variants.
• To investigate the impact of these variants on STAT3 function and cellular processes.

Main Methods:

• Computational analysis (AlphaMissense, PolyPhen-2, AlphaFold 3) and structural modeling.
• In vitro functional assays using Stat3-deficient T cells.
• In vivo studies using genetically modified mice.

Main Results:

• All four STAT3 D661 variants (D661Y, D661V, D661N, D661H) were predicted as pathogenic. D661Y and D661V showed stronger predicted promotion of SH2-TAD-mediated dimerization.
• In vitro, a gain-of-function hierarchy was observed: D661Y ≈ V > H > N, activating STAT3 target genes and immune programs.
• In vivo, only STAT3 D661H mice were viable, showing altered CD4+ and CD8+ T cell populations and enhanced immune gene expression. D661Y and D661V mutants impaired mouse viability.

Conclusions:

• A gradient of STAT3 D661 gain-of-function variants was defined, correlating in vitro and in vivo findings.
• Stronger STAT3 D661 variants (D661Y, D661V) exhibit higher transcriptional activity but lead to impaired mouse viability.