Figure 3. mTOR inhibition with AZD8055 or rapamycin treatment overcomes the pro-survival effect of BCR crosslinking in primary CLL cells and in a poor prognostic CLL mouse model in vitro.

CLL cells were pre-treated with 100 nM AZD8055 (AZD) or 10 nM rapamycin (RAP), or left untreated (NDC) and then BCR was ligated with addition of F(ab’)₂ fragments for 1 or 48 h or left unstimulated (US), as indicated. A. Cell viability was analyzed by flow cytometry after 48 h incubation (n=14). p value generated by a paired two tailed t test; B. Western blotting of the anti-apoptotic protein Mcl-1 was performed after 48 h incubation; C. Western blotting was performed to determine the phosphorylated levels of mTORC1 downstream targets (S6^S235/236 and 4EBP1^T37/46) and mTORC2 substrate (AKT^S473) and pERK (pERK1/2^T202/Y204) together with the respective total proteins and a GADPH loading control, 1 h post drug treatment; D. MIEV- or PKCα-KR-HSPC-derived mouse cells from OP9 co-cultures (post d14) were treated for 24 h in the presence of 100 nM AZD8055 or 10 nM rapamycin or left untreated as indicated. Western blotting was performed to determine the phosphorylated levels of mTORC1 downstream targets (S6^S235/236 and 4EBP1^T37/46) and mTORC2 substrate (AKT^S473) together with the respective total proteins or GAPDH. E. 5 x 10⁴ d15 MIEV- or PKCα-KR-HSPC-derived cells were plated on OP9 co-cultures and treated for 48 h in the presence of 200 nM AZD8055 or 10 nM rapamycin or left untreated as indicated, and cells were counted. Data are represented as mean (± SEM) of at least 3 biological replicates. F. Apoptosis was analyzed by flow cytometry after 48 h treatment of MIEV- or PKCα-KR-HSPC-derived cells. Annexin V⁺7AAD^- cells represent early apoptotic cells. Data are represented as mean (± SEM) of at least 4 biological replicates. Significance was determined by a 2-way ANOVA.