BNIP3 Is Downregulated in Myelodysplastic Syndromes and Acute Myeloid Leukemia and Is a Potential Molecular Target for Decitabine
Authors: Lazarini Mariana , Machado-Neto Joao , Duarte Adriana S. S. , Pericole Fernando V , Favaro Patricia , Barcellos Karin Spat Albino , Traina Fabiola , Saad Sara T. Olalla 
Published: October 2019

Abstract
Abstract 1708
In myelodysplastic syndromes (MDS), changes in the balance between pro- and anti-apoptotic proteins are associated with disease progression and evolution towards acute myeloid leukemia (AML). BNIP3 is a pro-apoptotic protein, member of the Bcl-2 superfamily, associated with the pathogenesis of many diseases, including cancer. More recently, BNIP3 was identified as a potent inducer of autophagy. In primary leukemia cells, BNIP3 expression has been shown to be reduced due to epigenetic modifications; however, there is a lack of studies of BNIP3 in MDS. The aim of this study was to characterize BNIP3 mRNA expression levels and DNA methylation status of its 5' CpG island in bone marrow cells from MDS and AML patients, healthy donors and in leukemia hematopoietic cell lines. We also investigated the effects of BNIP3 silencing upon decitabine (DAC) treatment in U937 cells regarding colony formation, apoptosis and autophagy. Bone marrow aspirates were obtained from 21 healthy donors, 55 patients with MDS and 44 patients with AML at diagnosis. MDS patients were grouped into low-risk and high-risk groups according to FAB (RA/RARS=30; RAEB/RAEBt=25), WHO (RCUD/RCMD/RARS/del5/=29; RAEB1/RAEB2=17) and IPSS (low/INT-1=38; INT2/high=16). The study was approved by the ethics committee and informed written consent was provided. Gene expression was evaluated by qPCR and DNA methylation was performed by Combined Bisulfite Restriction Analysis. BNIP3 knockdown was performed in U937 cells with specific shRNA-expressing lentiviral vector. ShRNA encoding no specific sequence was used as control. Colony formation was carried out in semisolid methylcellulose medium. Apoptosis and autophagy were accessed by flow cytometry. All assays were performed in lentiviral transduced cells treated or not with 5μM DAC for 72 hours. Mann-Whitney and Student's t-test were used for statistical analyses. We observed a significant decrease in BNIP3 mRNA expression in total bone marrow cells from AML and MDS compared to healthy donors (AML= 0.52 [5.27-0.00]; MDS=0.52 [5.25-0.02] versus healthy donors=1.09 [6.04-0.18]; P<0.05). BNIP3 expression did not differ between low-risk and high-risk MDS patients according to FAB and WHO classifications, and IPSS. Methylation of BNIP3 promoter was detected in 19% (4/21; 2/14 low-risk and 2/7 high-risk) MDS and 26% (4/15) AML patients, but not in any of the 6 healthy donors evaluated. BNIP3 expression was detected in all myeloid cell lines studied (K562, HL60, U937 and P39 cells). The lowest expression was found in U937, which presented methylation of BNIP3 promoter. Interestingly, in U937 cells, DAC treatment resulted in upregulation of BNIP3 expression (3.7-fold), total inhibition of colony formation, increased apoptosis and autophagy (50% for both). The effect of DAC on cell apoptosis was reduced by BNIP3 silencing; apoptosis was reduced by 48%±4.3 in shBNIP3 cells compared to 59%±5.7 in shControl cells (p<0.05). Autophagy was not modulated by DAC treatment in BNIP3 silenced cells. In bone marrow cells from MDS and AML patients, the low frequency of methylated BNIP3 promoter suggests that DNA methylation is not the single reason for the decreased BNIP3 expression in these patients. Other mechanisms, such as histone deacetylation, may be involved in aberrant BNIP3 expression. DAC treatment in BNIP3 silenced cells had no effect on autophagy, suggesting that other mechanisms besides increasing BNIP3 expression is involved in this cell function. On the other hand, DAC treatment in U937 cells was able to induce apoptosis, especially in cells expressing BNIP3, supporting the hypothesis that BNIP3 methylation is a molecular target for DAC.

Disclosures:
No relevant conflicts of interest to declare.