Fig. 3. Effect of selected pan-RAF inhibitors on CLL proliferation in co-culture model.
A Schematic overview of the RAF/MAPK/ERK pathway. B Representative immunoblot of CLL cells co-cultured (2 h) with various engineered HS5 cells or their combinations (1:1 ratio). C CLL cells (FarRed labeled) were pre-treated with respective inhibitors for 2 h and then co-cultured in the presence of the inhibitors (7 days) on HS5-CD40L-IL4-IL21 cells. IBRU, 2 µM ibrutinib; IDEL, 2 µM idelalisib; LY30, 2 µM LY3009120; NAPO, 10 µM naporafenib; MEKi, 2 µM PD184352; ERKi, 2 µM LY3214996. D The viability of CLL samples presented in panel [C] after 7 days of the co-culture on HS5-CD40L-IL4-IL21 cells in the presence of the indicated inhibitors (data after 2 or 5 days are in Supplementary Fig. 3B). E Heatmap of differentially expressed mRNAs (Padj < 0.05) in CLL cells co-cultured (3 days, n = 4) on HS5-WT, HS5-CD40L-IL4, HS5-CD40L-IL4-IL21, or on HS5-CD40L-IL4-IL21 in the presence of one of the RAF inhibitors (LY30, 1 µM LY3009120; NAPO, 10 µM naporafenib). One sample cultured on HS5-CD40L-IL4 (same as in Fig. 2E) was excluded due to RNA/library preparation failure. For sample characteristics see Supplementary Table S1 (CLL No. 089, 047, 043, 044). F Representative immunoblot of CLL cells treated (17 h) with inhibitors (as in [C]) and then stimulated with anti-IgM (40 µg/ml, 10 min). G Quantification of phopho-P38 signal from CLL cells cultured (48 h) with LY3009120 (2 µM) or vehicle (DMSO). H CLL cells were co-cultured (48 h; n = 4) on HS5-CD40L-IL4-IL21 cells in the presence of indicated inhibitors (LY30, 1 µM LY3009120; P38inh-1, 2 µM SB202; P38inh-2, 2 µM SB203; P38inh-3, 2 µM SB239; P38inh-4, 2 µM BIRB776.). Cell were harvested after 48 h, and the percentage of cells in the G1 phase of the cell cycle was quantified.