The Autophagy Gene ULK1 Plays a Role In AML Differentiation and Is Negatively Regulated by the Oncogenic MicroRNA 106a

Abstract 503

Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents allowing for example survival upon a variety of stress stimuli. Although often referred to as type II programmed cell death, autophagy is primarily a pro-survival mechanism rather than an important cell death effector. During autophagy, part of the cytoplasm or entire organelles are sequestered into double-membraned vesicles, called autophagosomes, that ultimately fuse with lysosomes and degrade their contents. Regarding hematopoiesis there is accumulating evidence that autophagic mechanisms are operative in the development of different hematopoietic lineages. To identify ATG genes with a function in neutrophil differentiation, we employed an shRNA library targeting autophagy regulators in acute myeloid leukemia (AML) cells as a model for neutrophil differentiation. Among others, we found that inhibiting the key autophagy kinase ULK1 (also known as ATG1) using RNAi significantly attenuated all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 and HL60 AML cells. In agreement, we observed significantly elevated ULK1 mRNA and protein levels during neutrophil differentiation of these cells. Moreover, ULK1 expression was significantly reduced in primary AML patient samples as compared to mature myeloid cells from healthy donors. Given the aberrant microRNAs (miRNAs) expression in myeloid malignancies, we wondered if altered miRNA expression in AML might contribute to the low ULK1 levels seen. MiRNAs are 19–25 nucleotide, non-protein coding RNAs that regulate gene expression by directly binding to partially complementary sequences in the 3′-untranslated regions (3′-UTR) of their target mRNAs. Several target prediction software's identified a putative miR-106a binding site in the ULK1 3′-UTR. Interestingly, miR-106a is part of a highly oncogenic miRNA family. We then confirmed direct inhibition of ULK1 by miR-106a using wild-type and miR-106a mutated 3′-UTR ULK1 reporters. Moreover, a decrease of miR-106a expression was seen during ATRA-induced differentiation of AML cells paralleled by increased ULK1 mRNA levels. Lastly, stable overexpression of miR-106a in NB4 cells resulted in decreased ULK1 levels and markedly impaired neutrophil differentiation upon ATRA treatment. In summary, we show for the first time a significant involvement of the autophagy gene ULK1 in neutrophil differentiation and an association of low ULK1 levels with primary AML. Our studies indicate that ULK1 is a putative tumor suppressor in AML that is targeted by the oncogenic miR-106a.