Epigenetic Regulation of Bmi1 by Ubiquitination and Proteasomal Degradation Inhibit Bcl-2 in Acute Myeloid Leukemia

Abstract

Bmi1 is associated with advanced prognosis of acute myeloid leukemia (AML), and polyethylenimine (PEI)-stabilized Bmi1 siRNA-entrapped human serum albumin (HSA) nanocarriers (PEI@HSANCs) were used to protect siRNA from degradation and also to control epigenetic regulation-based AML therapy. The nanoform increased the transfection efficiency of Bmi1 siRNA through caveolae-mediated endocytosis and enhanced Bax translocation into the mitochondria. It enhanced the caspase 3-mediated apoptosis through the Bax activation and Bcl-2 inhibition. The molecular analysis reveals the downregulation of polycomb proteins, Bmi1 and EzH2, along with inhibition of H3K27me3 and H2AK119ub1. The signaling cascade revealed downregulation of Bmi1 through ubiquitin-mediated degradation and is reversed by a proteasome inhibitor. Further mechanistic studies established a crucial role of transcription factor, C-Myb and Bmi1, as its direct targets for maintenance and progression of AML. Chromatin immunoprecipitation (ChIP) assay confirmed Bmi1 as a direct target of C-Myb as it binds to promoter sequence of Bmi1 between -235 to +43 and -111 to +43. The in vivo studies performed in the AML xenograft model evidence a decrease in the population of leukemic stem cells marker (CD45⁺) and an increase in the myeloid differentiating marker expression (CD11b⁺) in the bone marrow after the Bmi1 siRNA nanoconjugated therapy. Activation of apoptotic pathways and withdrawal of epigenetic repression through a ubiquitin proteasomal pathway potentiating a novel antileukemic therapy were established.

Keywords: Bmi1 siRNA; RNAi; acute myeloid leukemia; epigenetics; polycomb; ubiquitin ligase.