Inhibition of growth factor receptor expression or signaling reduces JAK/STAT signaling in AML CD34⁺ cells. (A) AML CD34⁺ cells (AML 571, 656, and 704) were exposed to 2 different sets of siRNA targeting different sequences in FLT3 (FLT3 [T] and FLT3 [A]) and c-KIT (c-KIT [T] and c-KIT [A]) and control siRNA (n = 3). Expression of target proteins was evaluated by western blotting. (B-C) Expression of pSTAT3/5 in AML CD34⁺ cells treated with (B) FLT3 and (C) c-KIT siRNA. (D-F) Nucleofected cells were cultured for 72 hours and (D) enumerated and assessed for (E) apoptosis and (F) CFC growth. (G-I) Effect of inhibitor exposure on pSTAT3/5 expression in AML CD34⁺ cells (AML 028, 519, 545, 707, 755, and 813). (G) Effect of AZD1480 (0.1 µM) and the FLT3 inhibitor AC220 (20 nM) on pSTAT3/5 expression after FLT-3 stimulation (n = 6). (H) Effect of AZD1480 (0.1 µM) and the c-KIT inhibitor Imatinib (5 µM) on pSTAT3/5 expression after SCF stimulation (n = 6). (I) Inhibition of pSTAT3/5 after exposure to AC220, Imatinib, and AZD1480 alone or in combination (n = 6). (J-M) Apoptosis and CFC growth of (J-K) FLT3-ITD⁺ AML CD34⁺ cells (n = 4; AML 355, 653, 656, and 868) and (L-M) FLT3 wild-type AML CD34⁺ cells (n = 4; AML 373, 404, 413, and 666), exposed to AZD1480, AC220, or the combination.