Hedgehog (HH) signaling is activated in various lymphoid malignancies, but conflicting results exist about its role in chronic lymphocytic leukemia (CLL). Here, we demonstrate that the expression of essential HH pathway components like GLI1, PTCH1, and the HH ligands is highly diverse in CLL. A subset of 36.7% of 60 tested CLL samples responded to all 3 SMOOTHENED (SMO) inhibitors, whereas 40% were completely resistant. Responsiveness correlated with elevated GLI1 and PTCH1 transcript levels and the presence of trisomy 12, whereas no other karyotype correlated with responsiveness. All trisomy 12 CLLs displayed constitutive HH pathway activation driven by autocrine DESERT HH (DHH) ligand secretion, which could be blocked by the HH-blocking Ab 5E1. Cocultures with DHH-expressing BM stromal cells reduced sensitivity of CLLs to SMOinhibitor treatment by activation of noncanonical ERK phosphorylation directly downstream of the PTCH1 receptor without involvement of SMO and could be overcome by the HH-blocking Ab 5E1 or a combination of SMO and ERK inhibitors. Our results demonstrate that the HHsignaling pathway is an interesting therapeutic target for a subset of patients with CLL, characterized by high GLI1 and PTCH1 transcript levels, and all patients with trisomy 12 and indicate HH-blocking Abs to be favorable over SMO inhibitors in overcoming stroma-mediated protective effects. (Blood. 2012;119(4):997-1007) Introduction B-cell chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in Western countries and is characterized by a progressive accumulation of functionally incompetent Blymphocytes in the peripheral blood, BM, and lymphoid organs. 1,2 Several prognostic markers such as Rai and Binet staging systems, 3,4 immunoglobulin V H gene mutational status, 5 -associated protein 70 (ZAP70) expression 6,7 and cytogenetic abnormalities 8 such as trisomy 12 and del 13q14 can be used to predict the survival outcome and the need for treatment of patients with CLL. 9 Despite encouraging new therapeutic strategies 10 CLL cannot be cured by conventional therapy at present, and only hematopoietic stem cell transplantation has led to complete and ongoing remissions in some patients. 11 The exact pathogenesis of this clinically heterogenous leukemia is still unknown, 12 but it is well established that B-CLL cells are habitually interacting with stromal cells, indicating the importance of the microenvironment for survival of CLL cells. [13][14][15] In our previous work, we demonstrated that the hedgehog (HH) ligands sonic (Shh), indian (Ihh), and desert (Dhh) are produced by stromal cells in lymphoid organs and that the BM promote expansion of murine lymphomas in vitro and in vivo. 16 HH ligands activate the transmembrane receptor PATCHED (PTCH), which abrogates the suppression of the 7-transmembranehelix protein SMOOTHENED (SMO), the key player for signal transduction of the canonical HH pathway. 17 SMO activation leads to production of activating forms of the glioma-associated oncoproteins 1-3 (GLI1-3), the H...