PU.1 Is a Downstream Target of C/EBPα in Normal Hematopoiesis and Acute Myeloid Leukemia

Abstract 1353

The transcription factors PU.1 and C/EBPα are key regulators of hematopoietic cell differentiation. Tight and coordinated regulation of these factors is essential for normal hematopoiesis and even moderate alterations can lead to acute myeloid leukemia (AML). Previous studies established that in PU.1 knockout mice myeloid differentiation is blocked at an earlier stage compared to C/EBPα knockouts, consistent with PU.1 acting upstream of C/EBPα during hematopoietic differentiation. Recently, however, we and others identified a PU.1 upstream regulatory element (URE) which contains potential C/EBP binding sites. C/EBPα binds to the PU.1 URE in vitro and in vivo. Furthermore, C/EBPα transactivated the PU.1 proximal promoter in a URE dependent manner. We, therefore, hypothesized that PU.1 is a target gene of C/EBPα in hematopoietic cells.

To assess the role of PU.1 as a downstream target of C/EBPα in normal hematopoiesis we performed gene expression analysis in immature hematopoietic cells of conditional C/EBPα knockout mice (Mx1-Cre). Of note, we observed a strong reduction of PU.1 expression in hematopoietic stem cells (HSCs: CD150+CD48-LSK) after excision of C/EBPα, corroborating that PU.1 is a target of C/EBPα in murine HSCs in vivo. Moreover, lentiviral PU.1 expression alleviated the myeloid differentiation block of C/EBPα^−/− KSL cells as evidenced by the differentiation to Gr-1 and Mac1 positive myeloid cells.

Targeted deletion of the PU.1 URE reduces PU.1 expression and induces myeloid leukemia. Additionally, inactivation of C/EBPα by various mechanisms is a common observation in many AML subtypes. Therefore, we tested if dysregulation of C/EBPα is associated with decreased PU.1 expression. Gene expression studies in several human AML cell lines revealed a positive correlation between C/EBPα and PU.1 expression. Furthermore, we analyzed expression of C/EBPα and PU.1 in a well characterized cohort of 285 AML patients. Importantly, PU.1 expression was strongly reduced in cases with either C/EBPα mutations or C/EBPα promoter silencing compared to other AML subtypes.

Taken together, our data support that PU.1 is a downstream target gene of C/EBPα in normal hematopoiesis as well as human leukemia. We currently develop a mouse model containing targeted mutations of three C/EBP binding sites in the PU.1 URE. This model will help to further pinpoint the functional impact of C/EBPα mediated regulation of PU.1 in different hematopoietic populations and to determine how this regulation may contribute to leukemia development in vivo.

The first two authors contributed equally to this work.