Stroma-induced SMO inhibitor resistance mediated by noncanonical HH signaling can be overcome by the HH-blocking Ab 5E1 or ERK inhibitors. (A) Percentage of viable CLL cells compared with the DMSO control after 48 hours of treatment with the SMO inhibitor cyclopamine or the HH ligand–blocking Ab 5E1 or a combination of cyclopamine and the 5E1 Ab. The HH-blocking Ab 5E1 overcomes SMO inhibitor resistance induced by stromal cells (patient 1). (B) Percentage of viable cells after treatment of primary CLL cells with the SMO inhibitor cyclopamine and the HH-blocking Ab for 48 hours compared with control. Although in both karyotypes SMO inhibitor treatment with 10μM cyclopamine cannot significantly reduce viable cells (trisomy 12: n = 4; P = .1539, Student t test; del 13q14: n = 3; P = .0799, Student t test), the HH-blocking Ab 5E1 can significantly reduce the percentage of viable cells (trisomy 12: n = 4; 20 μg/mL 5E1 P = .0131, Student t test; Del 13q14: 40 μg/mL; n = 3; P = .0075, Student t test; patients 1, 7, 18, 50, 60, 66, 68, 69). (C) Numbers of viable CLL cells compared with the DMSO control in cells treated with cyclopamine or the ERK inhibitor U0126 or a combination of both for 48 hours and measured by flow cytometry after annexin V/7-AAD staining. Combination of SMO and ERK inhibitors shows a significant increase in apoptosis compared with single treatment alone (n = 7; P = .0007, Student t test). (D) Diagram shows the interaction between stromal cells and CLL cells and changes in canonical and noncanonical HH signaling on treatment with the HH ligand–blocking Ab 5E1 (left) or a combination of SMO and ERK inhibitors (right).