Noncanonical HH signaling in CLL cells is partially mediated by ERK phosphorylation. (A) Intracellular p-ERK staining and measurement by flow cytometry in 4 patients with trisomy 12 and 4 patients with del 13q14 in the absence of stromal cells. Comparison of pERK baseline levels show a statistically significant increase in trisomy 12 patient samples compared with del 13q14 (n = 8; P = .0331, Student t test; analyzed patients 1, 7, 44, 50, 60, 66, 68, 69). (B) Intracellular pERK staining shows induction of ERK phosphorylation in CLL cells on coculture with stromal cells measured by the mean fluorescence intensity (n = 7; P = .0029, Student t test). Although 4 hours of treatment with 5μM cyclopamine could not significantly reduce ERK phosphorylation (n = 7; P = .2173, Student t test), the ERK inhibitor U0126 could significantly reduce stroma-induced ERK phosphorylation (n = 7; P = .0043, Student t test; patients 3, 7, 44, 50, 60, 64, 65). (C) Treatment of stromally cocultured CLL cells with the HH-blocking Ab 5E1 could block stroma-induced ERK phosphorylation in CLL cells (n = 8; P = .0285, Student t test; patients 1, 7, 18, 50, 60, 66, 68, 69). (D) Diagram shows the interaction in between stromal cells and CLL cells mediated by HH ligands with and without SMO inhibitor treatment.