STAT5B leukemic mutations, altering SH2 tyrosine 665, have opposing impacts on immune gene programs.

STAT5B is a vital transcription factor for lymphocytes. Here, function of two STAT5B mutations from human T cell leukemias: one substituting tyrosine 665 with phenylalanine (STAT5B ^Y665F ), the other with histidine (STAT5B ^Y665H ) was interrogated. In silico modeling predicted divergent energetic effects on homodimerization with a range of pathogenicity. In primary T cells in vitro STAT5B ^Y665F showed gain-of-function while STAT5B ^Y665H demonstrated loss-of-function. Introducing the mutation into the mouse genome illustrated that the gain-of-function Stat5b ^Y665F mutation resulted in accumulation of CD8 ⁺ effector and memory and CD4 ⁺ regulatory T-cells, altering CD8 ⁺ /CD4 ⁺ ratios. In contrast, STAT5B ^Y665H 'knock-in' mice showed diminished CD8 ⁺ effector and memory and CD4 ⁺ regulatory T cells. In contrast to wild-type STAT5, the STAT5B ^Y665F variant displayed greater STAT5 phosphorylation, DNA binding and transcriptional activity following cytokine activation while the STAT5B ^Y665H variant resembled a null. The work exemplifies how joining in silico and in vivo studies of single nucleotides deepens our understanding of disease-associated variants, revealing structural determinants of altered function, defining mechanistic roles, and, specifically here, identifying a gain-of function variant that does not directly induce hematopoietic malignancy.