The Hedgehog (HH) pathway regulates cell proliferation and survival and contributes to tumorigenesis. However, not much is known about the role of the HH-signaling system in the bone marrow microenvironment during the development of Acute Myeloid Leukemia (AML). The aim of this study was to investigate the anti-leukemic effects of the HH-signaling pathway and molecular mechanisms of apoptosis induction in AML cells, by blocking the hedgehog signaling pathway during co-culture of AML cells with Bone-Marrow Stromal Cells (BMSCs). It was found that the HS-5 line of BMSCs protected AML cells from spontaneous apoptosis via up-regulation of transcription factors GLI1, BCL-2 and BCL-XL. These results indicate that the up-regulation of the transcription factors involved activation of the hedgehog pathway. Thus, targeting a microenvironment-related signaling pathway may be a novel approach to AML therapy.

Small-molecule inhibitors of the Ihh signal transducer SMO have been studied in preclinical models, and have been applied in the treatment of various types of human cancers. Cancers sensitive to SMO inhibitors include Chronic Myeloid Leukemia (CML), some CLLs, and diffuse, large B-cell lymphoma. In other cancer types such as AML and some CLLs, these agents have demonstrated limited clinical activity. However, GANT61, a specific inhibitor of the transcription factors Gli1 and Gli2, is known to mediate strong in vitro and in vivo anti-tumor effects in several murine xenograft models, including neuroblastoma, pancreatic, prostate, and hepatocellular cancers. Several studies reported superior anti-cancer efficacy of GANT61 relative to the SMO inhibitor cyclopamine or GDC-0449. Thus, targeting the GLII genes, which are mechanistically downstream from SMO and constitute the core targets in HH-dependent gene regulation, may provide a significant advantage in eliminating HH signaling.

Human HS-5 fibroblastic stromal cell line was obtained from ATCC (American Type Culture Collection, Manassas, USA). HS-5 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum at 37°C in a humid atmosphere containing 5% CO2 . Human AML cell line HL-60 was obtained from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). HL-60 cells were maintained in RPMI 1640 supplemented with 10% FCS at 37°C in a humid atmosphere containing 5% CO2.

The bone marrow microenvironment plays an important role in the initiation and progression of leukemia, and in the persistence of minimal residual disease and disease relapse. Inhibition of spontaneous apoptosis in leukemic cells by BMSCs, mediated by phosphorylation of the AKT/BCL-2 pathway, has a protective effect. Moreover, MS-5 stromal cells block apoptosis in HL-60 cells and in primary AML blasts via modulation of proteins in the BCL-2 family. Consistent with the above findings, the results of the present showed that HS-5 cells inhibited HL-60 cell apoptosis, and improved HL-60 cells survival. Emerging evidence suggest that HH signaling is aberrantly activated in various cancer types, including gastric, colorectal, prostate, and breast cancers. Therefore, targeting the HH signaling pathway may provide an effective therapeutic approach for the treatment of various cancers. Activation of the HH signaling pathway, which is often associated with stromal cells, has been reported in lymphoma and multiple myeloma, B-CLL; diffuse, large B-cell lymphoma, and myelodysplastic syndrome. Studies have shown that IHH and its signal transducer, SMO are expressed in CD34+ Acute Myeloid Leukemia (AML). Knockdown of HHIP in stromal cells increased their supporting activity although control cells marginally supported SMO+ leukemic cell proliferation. The de-methylating agent, 5-aza-20- deoxycytidine restored HHIP expression via de-methylation of HHIP and reduced the leukemic cell-supporting activity of AML-derived stromal cells. Modulation of HH signaling in B-CLL influenced the survival of B-CLL cells, indicating that HH signaling mediated by stromal cells promotes the survival of B-CLL cells.

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