NPM-ALK phosphorylates STAT1 at Y701 and promotes its downregulation. (A) SupM2 cells were treated with different doses of the ALK inhibitor crizotinib for 5 hours. The decrease in pALK supports the effectiveness of the inhibitor. In the same experiment, pSTAT1 decreased, whereas the total STAT1 level gradually increased in a dose-dependent manner. (B) Time course experiment showed that pSTAT1 decreased along with pALK almost simultaneously. Furthermore, total STAT1 increased after the downregulation of pALK. (C) Transfection of NPM-ALK in GP293 cells led to a marked increase in STAT1 phosphorylation, which correlated with a dramatic decrease in the total STAT1 level. Importantly, treatment of 5 μM MG132 for 6 hours largely abrogated the downregulation of STAT1 by NPM-ALK, even though the pSTAT1 level increased dramatically. (D) Compared with the co-transfection of NPM-ALK and wild-type STAT1, co-transfection of NPM-ALK and mutant STAT1^Y701F resulted in a higher total STAT1 level, indicating that the phosphorylation of Y701 is crucial for the downregulation of STAT1 by NPM-ALK. (E) With the siRNA knockdown of STAT3, the NPM-ALK–mediated downregulation of STAT1 was largely abrogated. Correlating with this finding, IRF-1 (a STAT1 downstream target) was higher when STAT3 was silenced in the presence of NPM-ALK. (F) Time course experiment with siRNA knockdown of STAT3 in the two ALK+ ALCL cell lines correlated with the reciprocal increase in the total STAT1 level. EV, empty vector.