Trisomy 12 and elevated GLI1 and PTCH1 transcript levels are biomarkers for Hedgehog-inhibitor responsiveness in CLL

Abstract

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...

Hedgehog Pathway Inhibition in Chondrosarcoma Using the Smoothened Inhibitor IPI-926 Directly Inhibits Sarcoma Cell Growth

“…Recent evidence for constitutive Hh activation was observed in models of small and non-small cell lung cancer, melanoma, and chronic myelogenous and lymphocytic leukemia, in which the pathway is activated in the tumor cells (36)(37)(38)(39)(40). However, the presence of transcripts for Hh target genes does not necessarily represent active signaling, without evidence that the expression can be modulated by pathway inhibition (35,41).…”

Targeting hedgehog in hematologic malignancy

“…In addition, trisomy 12 CLL cells produced DHH, suggesting that autocrine signaling may be important in this subtype. 98 Hh has been implicated in the etiology of T-cell lymphomas. Singh et al demonstrated that constituents of the Hh pathway were overexpressed in ALK ϩ anaplastic large cell lymphoma.…”

“…They found that 60% of their CLL cohort responded to SMO inhibition. 98 Factors that predicted likelihood of response included elevated expression of GLI1 and PTCH1 and carriage of trisomy 12. In addition, trisomy 12 CLL cells produced DHH, suggesting that autocrine signaling may be important in this subtype.…”

PIM Kinases Are Essential for Chronic Lymphocytic Leukemia Cell Survival (PIM2/3) and CXCR4-Mediated Microenvironmental Interactions (PIM1)

“…The interaction between BMSCs and CLL cells induces a protective microenvironment blocking the proapoptotic effect of various chemotherapeutical agents and spontaneous apoptosis in vitro. Pathways involved in this process are the CXCL12-CXCR4 axis, Hedgehog (HH) ligands activating the HH signaling pathway in CLL cells, and various other chemokines and integrins (12,16,43). To evaluate the effect of protective stromal cells on the effect of PIM kinase inhibitors in CLL, we cocultured CLL cells with the BMSC line M2-10B4 (base line viability with and without stroma is shown in Supplementary Fig.…”