Figure 5.

SYK inhibition down-regulates Mcl-1 via STAT3. (A) Chronic lymphocytic leukemia (CLL) cells were co-cultured with B-cell activating factor (BAFF)-expressing stroma for 24 hours (h), followed by incubation with the indicated drugs for 24 h. Cells were also treated off stroma for 24 h. Apoptosis within CD19⁺ subset of cells was determined by Annexin V and 7-AAD staining (n=6). Data are presented as mean±Standard Error (SE). *P<0.05, **P<0.01, compared to ‘off stroma’ control, or as shown. (B and C) CLL cells were co-cultured with BAFF-expressing stroma for 24 h, followed by incubation with the indicated drugs for 24 h in the presence of caspase inhibitor QVD-OPh (1 μM). Cells were lysed and subjected to immunoblotting. Densitometry chart (C) represents data from 6 individual CLL samples. Data are presented as mean±Standard Error (SE). *P<0.05 compared to control. (D) CLL cells (4 individual samples) were co-cultured with BAFF-expressing stroma for 24 h, followed by addition of 100 μg/mL cycloheximide and 1 μM entospletinib or vehicle control. Cells were lysed at the indicated time points and subjected to immunoblotting. (E and F) CLL cells (n=4) were co-cultured with BAFF-expressing stroma for 24 h, treated with SYK inhibitors (entospletinib, R406), JAK1/2 inhibitor (ruxolitinib), BTK inhibitor (ibrutinib) or PI-3Kδ inhibitor (idelalisib) for 24 h in the presence of caspase inhibitor QVD-OPh (1 μM), followed by collection of mRNA and protein.