CML CP progenitors integrate signals by BCR-ABL and exogenous cytokines to sustain GSK3βpY²¹⁶ activation. (A) Normal (CTR, n = 3) and CML CP (n = 5) CD34⁺ cells were exposed to IM (1μM) or SB216763 (5μM) for 4 hours in serum-free medium (SFM) containing 5GF concentrations. Representative immunoblots for total and pSer⁹ and pY²¹⁶ of GSK3β, active β-catenin (non-pS/T β-Cat), phosphorylated β-catenin (non-pS/T β-Cat), and total and phosphorylated BCR-ABL (pY²⁴⁵ BCR-ABL) in one healthy and one CML CP patient sample are reported. (B) CML CP cells (n = 5) were cultured for 4 hours in the presence of 5GF, either without inhibitors (−) or in the presence of 1μM IM, 25μM UO126 (MAPK inhibitor), 30μM LY294002 (PI3K/AKT signaling inhibitor), or 5μM SB216763 or combinations as indicated. Representative immunoblots for total and pSer⁹ and pY²¹⁶ levels of GSK3β, active β-catenin, dual phosphorylated MAPKs (pT²⁰²Y²⁰⁴MAPKs), and pSer⁴⁷³AKT are shown. (C) CML CP cells (n = 5) were exposed to IM (1μM), SB216763 (5μM), or the combination for 4 hours in the absence of serum and cytokines (−5GF) or in the presence of high 5GF-concentrations (+5GF), and then analyzed with the indicated Abs. Representative immunoblots for one CML CP sample are shown. (D) CML CP progenitors were treated with IM (1μM), dasatinib (0.15μM) or SB216763 (5μM) for 4 hours in SFM with 5GF. Representative immunoblots are indicated. (E). BCR-ABL or GF receptors both signal through MAPK and SRC kinases. SRC signaling activated by exogenous cytokines is not affected by IM. Conversely, both BCR-ABL– and GF-dependent activation of SRC and MAPKs is inhibited by dasatinib.