ZNF521 Is a Zinc Finger Protein That Prevents Differentiation Of Human MLL-AF9-Positive Myeloid Leukemic Cells

Abstract

IntroductionMLL- rearranged acute myeloid leukemia (AML) remains a type of leukemia difficult to treat, for which alternative and more adequate treatment options are still urgently needed. The zinc finger protein 521 (ZNF521/EHZF) is a protein with multiple zinc finger domains that plays an essential role in the homeostasis of the hematopoietic stem/progenitor cell compartment. ZNF521 mRNA is highly expressed in hematopoietic stem and progenitor cells and its levels rapidly decrease during cell differentiation. Gene expression studies demonstrated that ZNF521 transcripts are abundantly expressed in MLL-rearranged AML. However, the functional significance of this up-regulation in AML with MLLrearrangements remains largely unknown. Methods and resultsQuantitative RT-PCR analysis (qRT-PCR) confirmed that ZNF521 expression was significantly up regulated in a cohort of MLL-rearranged pediatric AML patients (n=80) compared with other AML subtypes (n=30) together with healthy bone marrow samples (n=7, p<0.0001), and AML cell lines with MLL fusion (THP-1, NOMO-1, ML2). To investigate the role of ZNF521, we performed shRNA-mediated knockdown of ZNF521 in MLL-rearranged AML cell lines THP-1 and NOMO-1, in which a reduced transcript levels of 60-70% resulted in a severe reduction of proliferation and largely abolished colony formation in methylcellulose, where residual colonies were also smaller than control. Next, flow-cytometry analysis showed that observed proliferation reduction was associated with a block of cell cycle in G1 phase (shRNA/control vs shRNA/ZNF521 G1=45.4% vs G1=60.4%, n=3, p<0.05) and a late cell death induction (Annexin/PI positive cells: shRNA/control 21.4±0.5% vs shRNA/ZNF521 30.1±0.3% 3 weeks, n=3, p<0.0001). Subsequently, morphology analysis of May-Grunwald-Giemsa stained cytospins identified a large number of monocytic cells differentiating into macrophages. This observation was further validated by flow-cytometry analysis that confirmed the increased levels of CD11b and CD14 antigens as sign of monocytic differentiation, indicating a tendency of ZNF521 when over-expressed to prevent cell differentiation. Then, we treated THP-1 cells with ATRA to induce differentiation and demonstrated by qRT-PCR a significant reduction of ZNF521 mRNA expression, supporting its role in blocking differentiation in MLL-rearranged leukemic cells. Hence, to validate the specificity of these effects in vivo, we transplanted leukemic bone marrow cells from different MLL-AF9 AML pediatric patients (n=4) into immunodeficient mice. All mice developed AML by 7 to 9 weeks after transplantation. Then, the ex vivo cells were transduced with ZNF521 shRNA and showed morphology changes similar to those observed in vitro, with typical features of maturation. ConclusionsCollectively, these results demonstrate that ZNF521 plays an important role in MLL-AF9-induced AML leukemia wherein it maintains a block of differentiation. Thus, our findings make ZNF521 a novel attractive target for therapeutic intervention in MLL-rearranged AML. However, further studies on the function of ZNF521 in MLL-rearranged AML and on the possibility to block it are required. Disclosures:No relevant conflicts of interest to declare.