Drug resistance is dramatically restored by hedgehog inhibitors in CD34+ leukemic cells

Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers and in cancer stem cells. However, the contribution of Hh signaling to leukemic cell regulation has remained unclear. In this study, we assessed the possibility that Hh pathway activation contributes to the survival and drug resistance of cluster of differentiation (CD)34(+) leukemia cells. Hh signaling in leukemic cell lines and primary leukemic cells was screened by reverse transcription - polymerase chain reaction (RT-PCR) and a Hh signaling reporter assay. We found that Hh signaling is active in several human acute myeloid leukemia (AML) cells, especially primary CD34(+) leukemic cells and cytokine-responsive CD34+ cell lines such as Kasumi-1, Kasumi-3 and TF-1. These CD34+ cells express the downstream effectors glioma-associated oncogene homolog (GLI)1 or GLI2, indicative of active Hh signaling. Moreover, inhibition of Hh signaling with the naturally derived Smoothened antagonist cyclopamine, endogenous Hh inhibitor hedgehog-interacting protein or anti-hedgehog neutralizing antibody induced apoptosis after 48 h of exposure, although these CD34(+) cell lines exhibited resistance to cytarabine (Ara-C). In contrast, cyclopamine failed to affect growth or survival in U937 and HL-60 cell lines that lack expression of Hh receptor components, confirming that the effect of Hh inhibition is specific. Furthermore, combination with 10 mu M cyclopamine significantly reduced drug resistance of CD34(+) cell lines and primary CD34(+) leukemic cells to Ara-C. These results suggest that aberrant Hh pathway activation is a feature of some CD34(+) myeloid leukemic cells and Hh inhibitors may have a therapeutic role in the treatment of AML. (Cancer Sci 2009; 100: 948-955).