BM-MSC Hhip Induced Microenvironment Protection from Chemotherapy in AML Via Ptch/Smo/Gli Pathwa

Introduction. The mechanism of drug resistance is complicated in acute myeloid leukemia (AML), bone microenvironment provides protection niche for leukemia stem cells (LSC), and considered as an internal environment for AML cells to escape from chemotherapeutics cytotoxicity, but mechanism of drug resistance induced by microenvironment mesenchymal stem cell (MSC) isn't completely clear. In prophase study, it was found that Ptch/Smo/Glis pathway was the key in the network of AML drug resistance. Smo inhibition improved the survival of AML-bearing mice. Low expression of HHIP was found in refractory AML MSC and bone morrow liquid, overexpression of Smo and Gli-1 was verified in AML cells, and negatively correlated with prognosis in AML patients. Chemotherapeutic drug sensitivity was decreased in AML cells after co-cultured with AML MSC in vitro.

Methods. Bone morrow samples from AML patients and normal donors were collected to culture MSC cells. HL60/ADM and Kasumi-1 cells as well as HL60、KG-1cells were treated with daunomycin (DNR) or cytosine arabinoside (Ara-C) when cocultured with MSC. Cell cycle and apoptosis were determined by flow cytometry. The expression of HHIP was determined by confocal image and western blotting. Smo and Gli-1 activity was determined by western blotting.

Result. We took advantage of AML cells cocultured with MSC to imitate leukemic microenvironment in vitro. Co-culture with AML BM-MSC decreased the sensitivity to DNR or Ara-C compared with normal BM-MSC in HL60/ADM or Kasumi-1 cells. Flow cytometry analysis showed that cells in S phase and percentage of apoptosis cells were increased after co-culture with AML MSC. Western blotting also determined that low expression of HHIP was detected in refractory AML BM-MSC, Smo and Gli-1 expression were increased in HL60/ADM or Kasumi-1 cells after co-cultured with MSC. Confocal analysis also confirmed that the combination of HHIP and Ptch was decreased in AML MSC co-culture, Smo/Gli-1 pathway was activated through decreased inhibition of Shh in refractory AML cells co-cultured with MSC. Samples from AML patients also demonstrated that HHIP expression in AML BM-MSC was lower than that in normal BM-MSC, especially in refractory AML samples. HHIP expression in BM-MSC was negatively related to Smo and Gli-1 activity. Clinic data also showed that AML patients with overexpression of HHIP had a worse prognosis.

Conclusion. Our study demonstrated expression of BM-MSC HHIP was negatively related to activity of Ptch/Smo/Glis in AML. Low expression of BM-MSC HHIP resulted in activating Ptch/Smo/Gli pathway, and indicated drug resisitance and bad prognosis in AML. This project is connected with basic research and clinic, and provides support for diagnosis and targeted therapy in AML.