Molecular Mechanism and Optimal Treatment Strategy in Acute Myeloid Leukemia with Resistance to Drugs and Radiation By NVP-LED225

PURPOSE: Total body irradiation (TBI) combined with chemotherapy is currently the most effective procedure of traditional preparative myeloablative regimen. However, resistance to chemotherapy in refractory acute myeloid leukemia (AML) is associated with short-time recurrence after Allo-HSCT. To investigate the mechanism of Hedgehog signaling pathway resulting in resistance, we used primary AML cells originated in refractory patients and 3 cell lines including HL60, HL60/ADR (a adriamycin-resistant cells), and HL60/RX (a radiation-resistant cell line established from HL60) as cellular modes to examine the expression of p-IGF-1R, IRS-1, p-Akt, Gli-1, MRP1, Bcl-2, and explored the safety and efficacy of NVP-LDE225 (an inhibitor of Hedgehog pathway) for enhancing the sensitivity to treatment in refractory leukemia in vitro and in vivo. Methods: The expression of Hedgehog signaling pathway was measured in 3 leukemia cell lines, and primary leukemia cells originated in refractory AML patients and non-refractory AML patients using western blot technique. In in vitro experiments, HL60/ADR cells and HL60/RX cells were treated with DMSO (control) or NVP-LDE225 for 48 h, and then assigned in six groups respectively: (a) Control, (b) ADM alone, (c) Radiation alone (d) NVP-LDE225 alone, (e) NVP-LDE225+ADM, (f) NVP-LDE225+Radiation. Flow cytometry, MTT assay and Western-Blot were performed separately to detect apoptosis, adriamycin uptake rate, proliferation inhibit rate and the expression of MRP1, p-IGF-1R, IRS-1, p-Akt, Gli-1, and Bcl-2. Furthermore, based on the results in vitro, the serial tumor volumes, general condition of the mice, complete blood counts, multiple organs injury, and the expression levels of hedgehog signaling pathway were used to detect the antitumor efficacy and toxicity of NVP-LDE225 in HL60/ADR and HL/RX xenograft model. Results: Our result showed that, compared with leukemia cells in non-refractory AML patients and HL60 cells, the expression of hedgehog signaling pathway protein was significantly elevated in refractory AML, HL60/ADR and HL60/RX cells(P Conclusion: The findings from this study demonstrated that Gli-1/p-Akt/NF-kb pathway play a key role for resistance to both drug and irradiation, and suggested that the combination of hedgehog pathway inhibitor (NVP-LDE225) with chemotherapy or radiotherapy could significantly enhance the antitumor activity via overcoming chemoresistance or radioresistance. The myelosuppression and toxicity could be well tolerated Disclosures No relevant conflicts of interest to declare.