STAT3 SH2 Domain Aspartic Acid 661 Mutations Activate Immune Gene Programs

ABSTRACT The conserved aspartic acid residue D661 within the STAT3 SH2 domain is a recurrent mutational hotspot in hematologic malignancies, including T‐cell large granular lymphocytic leukaemia, myelodysplastic syndromes and acute lymphoblastic leukaemia. To define the functional consequences of distinct STAT3 D661 variants, we integrated computational, structural and in vitro and in vivo genetic approaches. AlphaMissense and PolyPhen‐2 classified all four STAT3 D661 variants (D661Y, D661V, D661N and D661H) as pathogenic. ClinVar classified D661Y and D661V as variants of uncertain significance. AlphaFold 3‐based modelling predicted that D661Y and D661V strongly promoted SH2‐TAD‐mediated dimerization, while D661N and D661H exerted weaker structural effects. Functional in vitro assays in Stat3 ‐deficient T cells demonstrated a gain‐of‐function (GOF) hierarchy of the STAT3 variants (D661Y ≈ V > H > N ) resulting in activation of canonical STAT3 target genes and immune transcriptional programs. In vivo, only STAT3 D661H mice were viable, displaying reduced CD4 + T cells, expansion of memory CD8 + T cells and enhanced immune gene expression. Collectively, our findings define a gradient of STAT3 D661 GOF variants, consistent with in vitro and in vivo experiments. D661Y and D661V mutants exhibited stronger transcriptional activity in T cells with impaired viability of mice carrying these variants.