Hh/IGF-1R/PI3K/Akt/MRP1 Pathway Induce Refractory Acute Myeloid Leukemia and Its Targeting Therpy

Hh/IGF-1R/PI3K/Akt/MRP1 pathway induce refractory acute myeloid leukemia Background: Although a great progress has made in the treatment of acute myeloid leukemia(AML), chemoresistance and relapse due to multidrug resistance (MDR) remains a therapeutic challenge. We have proven that LBH589 combined with Bortezomib had synergistic effects on proliferation, apoptosis and sensitivity to cytotoxic drugs via AKT and NF-κB pathways in chemoresistant HL60/ADR cells and refractory AML primary cells. Using gene chip analysis, we found that among those disregulated pathways, Hedgehog(Hh) signaling pathway was probably the upstream paths which could regulate others.

Aims: The purpose of the study was to investigate the expression of Hh pathway in AML including primary cells and cell lines, analyze the relationship between Hh expression and clinical prognosis. Also investigate the biological effect of Hh pathway inhibitor NVP-LDE225 in reversing drug-resistance in HL60/adriamycin-resistant(ADR) cells and refractory AML primary cells and the underlying mechanisms. Besides, animal experiments were done to verify the results in vitro.

Methods: Western blot assay were used to determine the protein expression of Ptch–Smo–Gli-1–Shh in HL60 and HL60/ADR cell lines and refractory or non-refractory primary AML cells. Kaplan–Meier curves were used to estimate Relapse-free survival(RFS) and Over-all survival(OS). HL60/ADR cells and refractory primary cells were treated with adriamycin or combined with NVP-LDE225 concentration among 0-10µM). Proliferation were evaluated by 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay and cell apoptosis were analyzed by Annexin V-FITC/PI staining through flow-cytometry(FCM). Intercellular adriamycin accumulation(MFI) were analyzed by FCM. The changes in protein levels of Gli-1–IGF-1R–p-IGF-1R–IRS-1–Akt–p-Akt–MRP-1–Bcl-2 were detected by Western blot. Besides, we used a nude mouse xenograft model to verify the anti-proliferative effects in vivo.

Results: We found that the refractory primary AML cells and HL60/ADR cells correlated with higher activation of the Hh pathway, however in non-refractory primary cells or chemosensitive cell lines HL60, such activation was less pronounced. Higher protein expression of this pathway was related to higher recurrence rate and associated with poor relapse free survival (RFS) and poor overall survival (OS)(P=0.002)(a). NVP-LDE225, a potent and selective Hh inhibitor, significantly reverted resistance of adriamycin(reversal fold was 3.75), induced cell apoptosis(P=0.007), increased the intracellular adriamycin accumulation(P=0.000), decreased protein expression of p-IGF-1R, IRS-1, p-Akt, Bcl-2, MRP1 also Gli-1(b). These effects were likely to be mediated via inhibition of IGF-1R/PI3K/Akt/MRP1 pathway. Besides, in a AML xenograft mouse model, NVP-LDE225 combined with ADM resulted in significant tumor regression(P=0.002) (c).

Conclusions: These findings provided evidence that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance in myeloid leukemia.

Keywords: Hedgehog signaling, acute myeloid leukemia, IGF-1R/PI3K/Akt signaling