Supplemental Figures 1-6. Supplemental figure S1: Semi-quantitative analysis of GLI2 expression in AML cohort A; Supplemental figure S2: Relative gene expression of Hedgehog family members in AML patient cohort A; Supplemental figure S3: Michael Friendly's fourfold displays for Hedgehog family members; Supplemental figure S4: Impact of GLI1 expression on clinical outcome in cohort B (n=290); Supplemental figure S5: Impact of GLI2 expression on clinical outcome in FLT3 subgroups of cohort B; Supplemental figure S6: BCL2 mRNA expression in GLI1/2 knockdown cells.
ARTICLE ABSTRACT
Purpose: The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute myeloid leukemia (AML).Experimental Design: Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo.Results: Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice.Conclusions: GLI expression is a negative prognostic factor and might represent a novel druggable target in AML. Clin Cancer Res; 21(10); 2388–98. ©2015 AACR.
