Novel activating STAT3 mutations in the coiled-coil domain and clinical phenotype of patients harboring specific STAT3 somatic mutations. (A) Lollipop plot of STAT3 somatic mutations detected in the 105-patient LGL leukemia cohort. Annotations for individual domains are described at the bottom. (B) STAT3 expression vectors, Q160P, L287F, D170Y, and Y640F as well as empty vector and WT STAT3 were transfected into HEK293 cells along with STAT3 luciferase reporter. Shown is a quantification of the relative ratio between firefly STAT3 responsive element against cytomegalovirus-controlled renilla luciferase. Two independent experiments were performed. Shown is 1 representative experiment. (C) Western blot of phospho-Y705 STAT3, total STAT3, mCherry, and vinculin from parallel whole-cell lysates obtained from transfection of empty vector, WT, and mutant STAT3 constructs as in panel B. Two independent experiments were performed. Shown is 1 representative experiment. (D-E) Plots of hematologic parameters ANC and HGB segregated by STAT3 WT, Y640F, D661Y, and N647I mutations. This includes 5 additional patients with N647I mutations identified using Sanger sequencing outside of the original 105-patient cohort. For panel D, Dunnett’s test with WT as a comparison control, P = .045, P = .059, and P = .853 for Y640F, D661Y, and N647I mutations, respectively. For panel E, Dunnett’s test with WT as a comparison control, P = .999, P = .074, and P = .047 for Y640F, D661Y, and N647I mutations, respectively. P values are indicated as follows: *P < .05; **P < .01; ***P < .001.