SMO inhibition in primary human CLL samples (n = 60) resulted in 3 different response groups. (A) Apoptosis assay showing annexin V–positive cells after treatment of primary CLL cells with the SMO inhibitor NVP-LDE225 (0μM, 1μM, and 3μM) for 24 hours. Top panel shows one representative example for a SMO inhibitor–sensitive CLL (patient 2), bottom panel an example for a SMO inhibitor–resistant CLL (patient 9). (B) Viable cell counts from the same 2 patients with the use of Guava ViaCount after 24 hours of treatment with the SMO inhibitors cyclopamine and NVP-LDE225 in increasing concentrations. Left side shows CLL cells treated with cyclopamine; right side CLL cells treated with NVP-LDE225. Top panel shows a SMO inhibitor–sensitive example (patient 2), bottom panel shows a SMO inhibitor–resistant CLL (patient 9). (C) Diagram displays the distribution of 60 CLL patient samples (supplemental Table 2; patients 1-60) among the 3 different response groups to SMO inhibitor treatment. Response was determined as good if the CLL sample responded to all 3 SMO inhibitors and with IC₅₀s for cyclopamine < 5μM and for NVP-LDE225 and IPI-926 < 2.5μM. The intermediate response group was represented by patients who responded to only 1 or 2 of the 3 used SMO inhibitors, and nonresponders showed no response to any SMO inhibitor or only responded at very high concentrations. (D) Treatment of CLL cells with 5μM cyclopamine and extraction of RNA after defined treatment periods. qPCR for GLI1 and PTCH1 in comparison to GAPDH and relative to the time point before treatment were performed in triplicates and show a significant reduction of GLI1 transcript levels after 6 hours (n = 3; P = .0045, Student t test) and of PTCH1 transcript levels after 2 hours (n = 3; P = .0041, Student t test). Target gene reduction stays significant until 72 hours.