Effect of the combined action (synergism) of dasatinib and MP07-66 on CLL cell survival. (A) Freshly isolated CLL cells from all the 42 patients recruited in this study (see supplemental Table 1 for details) were cultured in the absence or presence of increasing concentrations of MP07-66 at different time points, and cell apoptosis was analyzed by annexin V–PI flow cytometry. Quadrant analysis after flow cytometry was histogrammed and expressed as mean percentage ± SD of early and late apoptosis from 3 separate experiments performed in triplicate on samples from 20 CLL patients. Compared with the control, changes were statistically significant (* P < .01). (B) Freshly isolated CLL cells were cultured in the absence or presence of increased concentration of MP07-66 at different time points. After such treatment, cells were lysed and analyzed by western blot (Wb) analysis with anti-PARP antibody, as well as anti-β-actin antibody as a loading control. (C) Freshly isolated CLL cells were cultured in the absence or presence of 0.1 µM dasatinib, 5 µM MP07-66, or a combination of 0.1 µM dasatinib and 5 µM MP07-66 at different time points, and cell apoptosis was analyzed by annexin V–PI flow cytometry. Quadrant analysis after flow cytometry was histogrammed and expressed as mean percentage ± SD of early and late apoptosis from 3 separate experiments performed in triplicate on samples from 20 CLL patients. Compared with the effect of the compounds used alone, changes were statistically significant (*P < .01). (D) Freshly isolated CLL cells were cultured as described in (C) in the absence or presence of 5 nM of OA for 6 hours, and cell apoptosis was analyzed by annexin V–PI flow cytometry. Quadrant analysis after flow cytometry was histogrammed and expressed as mean percentage ± SD of early and late apoptosis from 3 separate experiments performed in triplicate on samples from 20 CLL patients. Compared with the effect of the compounds used alone, changes were statistically significant (* P < .01). (E) Freshly isolated CLL cells were cultured in the absence or presence of 5 nM OA and treated with 1 µM dasatinib, 5 µM MP07-66, or both, for 6 hours. After such treatment, cells were lysed and analyzed by Wb analysis with antibodies against the phosphorylated form and, after stripping, the nonphosphorylated form of Akt, GSK-3, and SHP-1. (F) Freshly isolated CLL cells were cultured in the presence of 5 nM OA and treated with 1 µM dasatinib, 5 µM MP07-66, or both, for 6 hours. After such treatment, cells were lysed and underwent a phosphatase activity assay by using pAkt immunoprecipitated from CLL cells, in the absence or presence of 5 nM OA for 10 minutes at 37°C. After such treatment, the samples underwent Wb analysis with anti-pAkt and anti-Akt antibodies. Pooled densitometric analyses (arbitrary units) of the Wb bands of the immunoblots are shown. Compared with the control, changes were statistically significant (*P < .01). (G) A schematic representation of the altered signaling network sustained by the Lyn-PP2A axis in CLL. Lyn, by phosphorylating PP2A, stabilizes the SET/PP2A complex and prevents dephosphorylation of downstream players (Akt, GSK-3, and SHP-1) and ultimately the onset of apoptosis. This latter event is initiated by small molecules affecting either Lyn activity (dasatinib), the stability of the aberrant cytosolic Lyn/Hsp90 complex (geldanamycin), or of the SET/PP2A complex (MP07-66), eventually resulting in the degradation of antiapoptotic proteins, which in turn triggers caspase-dependent apoptosis. Solid lines with arrows denote action; dotted lines with arrows denote process; solid lines with bars denote inhibition.