Inhibition of Elastase or SDF-1/CXCR4 Axis Promotes Differentiation of Human AML Cells.

Abstract

Elastase, along with other azurophil granule proteins like proteinase 3 regulates normal and leukemic granulopoiesis in an un-defined mechanism. We have recently showed that human acute myeloid leukemic (AML) cells constitutively express and secrete stromal derived factor 1 (SDF-1) dependent cell surface elastase, which regulates their migration and proliferation. To elucidate the molecular events and genes regulated by elastase and SDF-1/CXCR4 axis in AML cells, we examined gene expression of U937 AML cell line treated with neutralizing anti-CXCR4 Abs or elastase inhibitor (EI) compared to untreated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis showed very similar gene expression profiles of EI and anti CXCR4 Abs treated cells as compared to control. 230 of 8400 genes interrogated were repressed, and 164 were induced after culturing AML cells in the presence of EI or anti CXCR4 Abs at different time points as compared to untreated cells. Inhibition of elastase or CXCR4 was accompanied by down regulation of the transcripts of primary granule proteins. Functional classification of elastase or SDF-1/CXCR4 axis regulated genes revealed downregulation of HOXA9, HOXA10, ETS2, as well as other transcription factors that are over expressed in AML and are important for the development of leukemia. Whereas, transcriptional factors and regulators known to be induced during myeloid differentiation like C/EBPε, ID1, RUNX3 and HHEX were up-regulated in treated cells. Expression patterns of apoptosis genes indicated decline in death control by the p53 dependent pathway and a more prominent control by mitochondrial mediated apoptotic pathway like bcl2 related genes. In addition, receptors for interleukins, growth factors (G-CSFR and GM-CSF), complement component (C1QR1) were upregulated in the treated cells. In contrast, FLT-3, a growth factor receptor stimulating growth of early progenitor cells and AML blasts, was down regulated in AML cell treated with EI or anti CXCR4 Abs. These data were confirmed by real time PCR for selected marker genes of granulocytic differentiation. Interestingly, many of the differentially expressed genes were common to the transcriptional program of normal terminal granulocytic differentiation (Theilgaard-Monch & Borregarrd 2005. Blood 105:1785) suggesting that inhibition of elastase may induce differentiation in AML cells. Thus we further analyzed the effect of elastase inhibition on AML cell differentiation and growth. Treatment of HL60 AML cell line with EI triggered a proliferative arrest, apoptosis and mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b expression, and the ability to produce oxidative bursts. In summary, our study showed that inhibition of elastase or SDF-1/CXCR4 axis in AML cells affects similar pathways related to differentiation and malignant transformation, implying a critical role for those molecules in regulating leukemic development. Repression of elastase decreases proliferation and induces differentiation of AML cells, suggesting a potential new therapeutic approach for AML.