Oncogenic potential of NPM1-TYK2 fusion gene product. (A) Presence of the NPM1-TYK2 fusion protein in MyLa and not in other T-cell lines. Individual NPM1 and TYK2 western blot assays show shift in the size of the protein in MyLa as a result of the fusion. (B) Hyperactivation of TYK2 and elevated STAT pathway activation in MyLa cells endogenously expressing NPM1-TYK2 fusion protein as compared with other T-cell lines. The Mac1 cell line that has a JAK2 rearrangement is used as a positive control. The HUT78 cell line exhibits constitutive STAT5 activation. (C) Ectopic expression of NPM1-TYK2 fusion protein in HEK293FT cells reveals activation of STAT proteins in western blot assays. Note the significantly reduced levels of STAT activation in cells expressing NPM1-TYK2 kinase-defective mutant K462R. (D) Exogenously expressed NPM1-TYK2 fusion protein in HEK293FT cells leads to transcriptional activation of STAT1/3/5. Cells expressing kinase-defective mutant K462R NPM1-TYK2 fusion protein show reduced levels of STAT activation indicating a specific effect of TYK2 kinase activity on downstream STAT activation. (E) Diminished STAT pathway activation following knockdown of TYK2 protein in MyLa cell line by shRNA knockdown. (F) shRNA-mediated silencing of TYK2 reduces proliferation of MyLa cells, demonstrating oncogenic potential of NPM1-TYK2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.