Expression Of Hedgehog Pathway Mediator Gli2 Represents a Clinically Negative Prognostic Marker In Acute Myeloid Leukemia and Its Inhibitor GANT61 Exerts Anti-Leukemic Effects In Vitro

Leukemic stem cells depend on signals provided by bone marrow (BM) niche cells. Due to its function in stem cell biology, we investigated expression and prognostic impact of the Hedgehog pathway in acute myeloid leukemia (AML).

Pre-treatment samples from 104 patients with newly diagnosed AML (AMLSG 07-04 trial) were analyzed by quantitative PCR. Expression of receptors Smoothened and Patched and downstream transcription factors Gli1, Gli2 and Gli3 was found in 69%, 41%, 73%, 20% and 26% of cases, respectively. However, no expression of Hedgehog ligands was observed. Gli2 expression had a significant influence on event-free, relapse-free and overall survival (p=0.037, p=0.026 and p=0.013, respectively) and was furthermore correlated to FLT3 mutational status (p<0.001).

To verify our findings in a second, independent patient cohort, microarray-based gene expression data of patients published by Valk et.al.(N Engl J Med. 2004 Apr 15;350(16):1617-28) was used. Clinical data of 291 AML patients from this cohort were available and the negative prognostic impact of Gli2 expression on overall and event-free survival could be confirmed (p=0.003 for overall and p=0.007 for event-free survival, respectively; no data for relapse-free survival available). Additionally, the correlation of Gli2 expression to FLT3 mutational status was also observed in this patient cohort (p=0.003).

Although Hedgehog ligands were not expressed in AML bone marrow aspirates, Desert Hedgehog (DHH) plasma levels were significantly increased in AML patients compared to healthy subjects (p=0.002). Provision of Desert Hedgehog could be ascribed to BM niche cells including endothelial cells and osteoblasts, since DHH expression was found in outgrowth endothelial cells (OECs) and osteoblasts in vitro and on immunohistochemistry of leukemic bone marrow samples in patients.

Next, we investigated the effect of Gli inhibition in vitro. AML cell lines KG-1, OCI-AML5 and UKE-1 as well as freshly isolated primary AML cells were treated with the specific Gli inhibitor GANT61. GANT61 dose-dependently induced apoptosis in all three cell lines investigated. Furthermore, GANT61 treatment resulted in significantly increased apoptosis in primary AML cells upon treatment with 10 µM, 30 µM and 90 µM GANT61 (n=10 pts). Additionally, a pronounced growth inhibitory effect on primary AML cells was observed with a significant reduction in cell count to 82 ± 13%, to 54 ± 11% and to 47 ± 13% upon treatment with 10 µM, 30 µM and 90 µM of GANT61, respectively (n=9 pts). Incubation of KG-1, UKE-1 and OCI-AML5 cells for 7 days with 30 µM GANT61 resulted in reductions in cell numbers compared to the control to 14 ± 2%, to 44 ± 6% and to 31 ± 0%, respectively. Moreover, GANT61 mediated a strong and significant inhibitory effect on the colony formation capacity of AML cell lines and primary AML cells. The average number of colonies was reduced to 44 ± 16% and 4 ± 4% upon treatment with 10 µM and 30 µM GANT61, respectively, while no colony formation was observed with a concentration of 90 µM GANT61 in primary AML cells (n=4 pts).

In conclusion, Gli2 expression is a negative prognostic factor in AML that mirrors activated Hedgehog signaling induced by paracrine stimulation through BM niche cells. Inhibition of Hedgehog signaling by blocking Gli activity might represent a promising therapeutic approach for AML treatment with special regard to patients carrying a mutated FLT3.

Fiedler:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.