FLT3-ITD induces expression of SOCS family members. (A-B) FLT3 kinase–dependent CIS, SOCS1, SOCS2, and SOCS3 mRNA expression in FLT3-ITD⁺ AML cell lines. Real-time quantitative PCR analysis of CIS and SOCS1-3 mRNA expression levels in MV4-11 (A) and MOLM-13 (B) cell lines. MV4-11 and MOLM-13 cells were incubated overnight with or without the FLT3 kinase inhibitor sorafenib (20nM) as indicated. Normalized mRNA expression is plotted with SD as error bars. Representative data from 1 of the 2 independent experiments is shown. (C) SOCS1 protein expression depends on FLT3 kinase activity. MOLM-13 cells were incubated overnight, with or without FLT3 kinase inhibitor sorafenib (20nM), and SOCS1 protein levels were determined by Western blot. Actin serves as a loading control. From these lysates, FLT3 was immunoprecipitated and pFLT3 was immunoblotted. (D-F) FLT3-ITD expression led to increased CIS, SOCS1, SOCS2, and SOCS3 mRNA levels in BaF/3 and 32D cell lines and in primary murine bone marrow. Real-time quantitative PCR analysis of mRNA expression levels of CIS and SOCS1-3 expression in BaF/3 (D), 32D (E), and murine bone marrow (F). BaF/3 and 32D cell lines and murine bone marrow were transduced with either empty vector (control) or with FLT3-ITD retrovirus and sorted for GFP. RNA was isolated from cytokine and serum-starved BaF/3 and 32D cells and murine primary bone marrow. The normalized mRNA expression is plotted, with SD as error bars. Representative data from 1 of the 3 independent experiments is shown. (G-H) SOCS1 and SOCS3 mRNA is highly expressed in AML patient bone marrow. Bone marrow samples from a cohort of 77 AML patients (38 of FLT3-ITD and 39 FLT3-WT) and 7 healthy CD34⁺ donors were analyzed for mRNA expression of SOCS1 (G) and SOCS3 (H). The normalized mRNA expression is plotted, with SD as error bars (*P < .05).