Hypoxia Drives AML Proliferation in the Bone Marrow Microenvironment Via Macrophage Inhibitory Factor

Introduction

Hypoxia is an important component of the bone marrow microenvironment and the hematopoietic stem cell niche. Studies have shown that hypoxia contributes to the development and maintenance of acute myeloid leukemia (AML) cells within the bone marrow microenvironment. Hypoxia is principally maintained by members of the hypoxia-inducible factor (HIF), in particular HIF1a and its target genes, including MIF. We have previously shown that AML cells express constitutively high macrophage migration inhibitory factor (MIF) which drives IL-8 expression by the BM-MSC which in turn supports AML cell survival and proliferation. The aim of the present study is to determine if there is a connection between the role of hypoxia in regulating AML survival and MIF survival signals. Furthermore we investigate the role BM-MSC in regulating the hypoxic response.

Methods

Primary AML and BM-MSC were isolated from AML patients following informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). AML cell lines and primary AML blasts were cultured under normoxic (20% oxygen) or hypoxic conditions (1% oxygen) for 4 - 24 hours, mRNA expression of MIF, HIF1a, VEGF and IL-8 were determined by RT-PCR. MIF and IL-8 protein was determined using target specific ELISA. HIF1a protein expression was determined by western blotting. Hypoxia-mimetic agents, cobalt chloride (CoCl2) and desferrioxamine (DFO) were used. Cell proliferation was determines using CellTiter Glo and trypan blue exclusion. CFU-assays were performed using complete methylcellulose media.

Results

To determine If MIF is regulated by HIF1a in AML cells, we mimicked hypoxic conditions using CoCl2 and DFO in AML cells. Both CoCl2 and DFO upregulate MIF transcription and protein expression in OCI-AML3 cell lines and in primary AML blasts. Moreover, hypoxia increases both MIF mRNA expression and MIF chemokine expression compared to normoxic conditions. Lentiviral mediated knockdown of MIF in AML cells show significantly reduced cell proliferation and colony formation in methylcellulose media. Recombinant MIF induced interleukin-8 in AML blasts and the MIF inhibitor blocked MIF induced IL-8 release. Lentiviral mediated KD of HIF1a decreased MIF expression in human AML cells and a significant reduced their proliferative capacity. Finally we found that hypoxia increased MIF in AML blasts which was further increased when in co-culture with BM-MSC.

Conclusions

The results reported here suggest that hypoxia significantly affects the expression of the survival cytokine MIF in AML blasts. Furthermore, we show that BM-MSC regulate HIF1a induced MIF expression in AML under hypoxic conditions. We propose this hypoxia regulated HIF1a/MIF axis is essential to blast survival in the bone marrow niche.