Pimozide treatment of primary T-PLL cells to target increased pSTAT5B levels leads to reduced tumor cell growth and apoptosis. (A) Nuclear and cytoplasmic pY699 phosphorylated STAT5 in primary T-PLL samples by immunofluorescence microscopy (representative data are shown for 2 primary T-PLL cases [T-PLL25 and T-PLL38]). (B-C) Effects on HUT78 (pSTAT5-positive) viability following treatment with the JAK3 inhibitor tofacitinib (B; n = 3, P = .1144) and the STAT5 inhibitor pimozide (C; n = 3, asterisk indicates P < .0001). (D-F) Pharmacologic inhibition of pSTAT5 with pimozide leads to decreased viability (D) and diminished pSTAT5 levels (E) in primary T-PLL samples (n = 3 independent replicates for experiments in D; asterisks indicate P < .005; representative data are shown for T-PLL 25 in panel E). HH (pSTAT5 negative) is used as negative control, whereas SUDHL-1 (pSTAT5 positive) is used as a positive control. (F) A pathway diagram illustrates the interaction of IL2RG, JAK1, JAK3, and STAT5B during IL-2 cytokine activation. Cytokine binding to the extracellular portion of membrane-associated IL-2 receptors induces conformational change in the intracellular portion. Associated JAK nonreceptor tyrosine kinases autophosphorylate, leading to STAT recruitment and activation through tyrosine phosphorylation. Activated STAT proteins then dimerize and translocate to the nucleus to regulate transcription of numerous genes involved in differentiation, proliferation, and survival. Mutated components of the IL2R-JAK1-JAK3-STAT5B pathway are highlighted in green. Pimozide treatment inhibits STAT5B phosphorylation, limiting downstream transcriptional activation initiated by mutations in cytokine receptor/JAK-STAT proteins.