Stromal cells activate HH signaling in CLL cells and induce SMO inhibitor resistance. (A) GLI1 and PTCH1 transcript levels compared with GAPDH with (right) and without (left) stromal (M2-10B4) support in 8 CLL samples (trisomy 12, n = 4; 13q14, n = 4). Figure shows up-regulation of the HH target genes GLI (n = 8; P = .0391, Student t test) in 7 of 8 samples and PTCH1 (n = 8; P = .0072, Student t test) in 8 of 8 samples (analyzed patients 1, 7, 18, 19, 61, 62, 63, and 64). (B) Percentage of viable cells measured by annexin V/7-AAD staining after treatment with different concentrations of cyclopamine compared with the DMSO control. CLL cells were either cultivated on murine (M2-10B4) or human (HS-5) stroma or without stroma. Presence of stromal cells reduces sensitivity of CLL cells toward SMO inhibitors (trisomy 12, n = 3; responsive del 13q14, n = 3). (C) Inhibition of SMO in M2-10B4 cells and primary mouse stromal cells reduces transcript levels for Gli1 measured by qPCR and relative to Gapdh and results in a simultaneous up-regulation of Dhh.