Abstract 2090: GLI3 repressor levels determine Hedgehog pathway activity and predict response to Smoothened antagonist in acute myeloid leukemia

The Hedgehog (Hh) signaling pathway is activated in most hematological and solid cancers and is a promising target for therapeutic development. Deletions in the receptor Patched (PTCH) or activating mutations in the upstream positive regulator Smoothened (SMO) have been reported in a few cancers such as basal cell carcinoma and medulloblastoma, but are largely absent in most tumor types; therefore, the mechanism of pathway activation in most cancers remains unknown. In normal tissues, Hh pathway activation via PTCH/SMO causes an increase in the downstream activating transcription factor GLI1 and a decrease in the transcriptional repressor GLI3R. We confirm that the Hh pathway is active in acute myeloid leukemia (AML) and is required for cell survival, however, this activity is largely independent of the upstream activator SMO. Using siRNA knock down of GLI1, GLI3 and SMO in AML cell lines, Gli3 conditional knock out mice, and primary AML samples, we are able to show that loss of the GLI3R repressor function is sufficient to activate Hh target gene expression independent of ligand binding, and GLI3R levels correlate inversely with GLI1 levels. Importantly, we show that GLI3R is required for the therapeutic effect of SMO antagonists in AML samples and restoration of GLI3R restores sensitivity to SMO antagonists in AML cell lines. Epigenetic and gene expression analysis of the TCGA AML data set reveals that the GLI3 expression is silenced in most AML patient samples. We demonstrate in vitro and in patients that treatment with hypomethylating agents restores GLI3R levels and sensitivity to SMO antagonists in AML. In a transgenic mouse model of myeloproliferative disease (vav-Jak2V617F, gift of Joe Zhao, OUHSC) effective treatment with SMO antagonists are associated with increases in GLI3R but no changes in GLI1. Finally we show that GLI3R can directly repress expression of AKT and in vivo responses to SMO antagonists are correlated with changes in AKT expression, independent of GLI1 levels. In summary, this study provides the first evidence that GLI3R plays an essential role in SMO independent Hh signaling in AML, and suggests that GLI3R could serve as a potential biomarker for patient selection in SMO antagonist clinical trials. Furthermore, these data provide important mechanistic data to support rational combinations of hypomethylating agents with SMO antagonists in clinical trials. Citation Format: Parvesh Chaudhry, Mohan Singh, Tim Triche, Aparna Jorapur, Parkash S. Gill, Akil Merchant. GLI3 repressor levels determine Hedgehog pathway activity and predict response to Smoothened antagonist in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2090. doi:10.1158/1538-7445.AM2015-2090