Nix (BNIP3L) Is Downregulated in High-Risk Myelodysplastic Syndromes and Acute Myeloid Leukemia and Its Silencing Enhances Decitabine-Mediated Apoptosis

Background: 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). NIX is a target of HIF-1 alpha and a member of the BH3-only subfamily proteins with a unique role in cell death and survival, through the regulation of both apoptosis and autophagy. NIX is also essential during maturation of reticulocytes and has been shown to be upregulated by decitabine (DAC) treatment in mantle cell lymphoma. Despite of being related to the pathogenesis of several diseases, NIX has never been studied in MDS and AML.

Aims: We aimed to characterize the expression of NIX in bone marrow samples from healthy controls and patients with MDS and AML and the effects of NIX silencing upon DAC treatment in U937 cells.

Patients and Methods: Bone marrow aspirates were obtained from 17 healthy controls, 56 patients at diagnosis of MDS, 12 patients with secondary AML/MDS and 47 patients at diagnosis of de novo AML. MDS patients were grouped in low-risk and high-risk according to WHO (RAUD/RCMD/del5q/=46; RAEB1/RAEB2=17) and IPSS (low/INT1=51; INT2/high=5). NIX expression was evaluated by quantitative PCR. Appropriated statistical analysis was performed and the data is showed as median [max-min]. NIX knockdown was performed in U937 cells with specific shRNA-expressing lentiviral vector. Colony formation was carried out in semisolid methylcellulose medium. Cell viability was evaluated by MTT assays. Apoptosis and autophagy were accessed by flow cytometry. Expression of NIX, Bcl-2, Caspase 3, p62, Beclin and LC3 were investigated by western blot. All assays were performed in lentiviral transduced cells treated or not with 1 µM or 5µM DAC for 72 hours.

Results: NIX expression was significantly decreased in high-risk MDS, AML/MDS and patients with de novo AML, compared to healthy donors (high-risk MDS= 1.28 [2.24-0.69]; AML/MDS=0.52 [2.16-0.19]; AML=1.12 [6.15-0.19] versus healthy donors=2.57 [4.39-0.65]; WHO; P<0.05). Interestingly, 4 out of 5 MDS patients showed a marked decrease in NIX transcripts after disease progression from low-risk to high-risk MDS or from high-risk MDS to AML with myelodysplasia-related changes (P<0.05). In U937 cells, NIX silencing decreased cell viability (MTT) and colony formation (P<0.05). DAC treatment resulted in a pronounced upregulation of NIX. Although NIX silencing itself did not modulate apoptosis, it increased apoptosis induced by DAC treatment (p<0.05), which was confirmed by the reduction of cell viability and increase of the active form of Caspase 3. Autophagy and Bcl-2 expression were not modulated by NIX silencing in U937 cells treated with DAC.

Conclusions:NIX is downregulated in high-risk MDS and AML and after disease progression. Moreover, our findings indicate that NIX has a pro-survival function in U937 cells and is upregulated by decitabine. Therefore, we hypothesize that the combination of NIX inhibition and decitabine would be a more effective strategy to induce apoptosis of malignant cells. Supported by Fapesp and CNPq.