Novel Role of HDAC Inhibitors in Bone Marrow Microenvironment: Activation of Leukemia Cell Phagocytosis through Annexin A1.

Annexin A1 (ANX-A1) is a calcium-dependent membrane-binding protein involved in the modulation of apoptosis and phagocytosis (FASEB J. 2003;17:1544). We have previously reported that HDAC inhibitor depsipeptide (FK228) caused marked growth inhibition and apoptosis in t(8;21) Kasumi-1 AML cells with up-regulation of 123 genes (by cDNA array) including ANX-A1 (3.5 fold; Tabe, Blood 2004). By chromatin immunoprecipitation (ChIP) assay, FK228 induced H4 and H3-K9 acetylation in the ANX-A1 promoter with corresponding induction of ANX-A1 mRNA (7.2±1.7 fold, TaqMan RT-PCR) and protein (western blot analysis). The markedly increased ANX-A1 protein localized on the cell membrane of Kasumi-1 cells exposed to FK228 was confirmed by immunofluorecence analysis using confocal microscopy. ANX-A1 membrane localization was diminished by treatment with anti-ANX-A1 mAb. To investigate the contribution of ANX-A1 to FK228-induced apoptosis, we neutralized ANX-A1 by anti-ANX-A1 mAb. This moderately decreased FK228 induced apoptosis (36.0±4.1 vs 26.5±3.7% AnnexinV(+)/PI(+) cells, p=0.01). Similarly, Kasumi-1 cells transfected with siRNA/ANX-A1 were less sensitive to FK228-induced cell death compared with nonsense (N) siRNA transfected cells (siRNA 31.2±3.1% vs NsiRNA 39.5±2.9% annexin(+) cells, p=0.03). These data indicate that the upregulation of endogeneous ANX-A1 (either membrane-binding or secreted form) promotes cell apoptosis in an autocrine fashion. Next, we investigated the functional role of ANX-A1 on leukemia cell phagocytosis. The engulfment of Kasumi-1 cells by cocultured human THP-1 monocyte-derived macrophages was evaluated by cell adherence assay. Compared with untreated cells, the exposure to FK228 induced a dramatic increase in Kasumi-1 cells attachment to macrophages (untreated vs FK228 treated; 57 ± 9 cells vs 196 ± 33 cells/ microscopic fields (0.08 mm2/field), n = 5; p=0.01). FK228-induced cell attachment was completely abrogated in the siRNA/ANX-A1 transfected Kasumi-1 cells (60.5% ± 10.5% decrease; n = 5; p<0.001). Consistently, co-treatment with FK228 and anti-ANX-A1 mAb followed by washout of both compounds resulted in significantl repression of FK228-stimulated engulfment of leukemic cells by macrophages (54.1% ± 3.0% decrease; n = 5; p=0.02). This effect was not further enhanced by adding anti-ANX-A1 mAb to the co-culture medium, suggesting that membrane-associated but not soluble ANX-A1 contributes to leukemia cell engulfment by macrophages. Results presented here demonstrate a novel mechanism of action of HDAC inhibitors in the context of bone marrow microenvironment via histone acetylation, increased expression and externalization of ANX-A1, which provides an “eat-me” signal and mediates phagocytic clearance of apoptotic leukemic cells by macrophages. Our data further suggest that ANX-A1 is silenced via histone deacetylation in leukemic cells, and its re-expression by HDAC inhibitors may stimulate apoptosis in an autocrine fashion while diminishing the inflammatory response through activating phagocytosis in the bone marrow microenvironment.