Epigenetic Dysregulation Of ZNF423 Impedes B-Cell Differentiation and Is Linked To Adverse Outcome Of Childhood B-Precursor ALL

Despite increasing treatment success, acute lymphoblastic leukemia continues to be the most common cancer-related cause of death in childhood. Structural and numerical chromosomal aberrations are considered to be the primary event in disease development followed by a variety of secondary mutational incidents. However, in a large proportion of B-precursor ALL (B-ALL) cases, the cause of differentiation arrest -a hallmark of acute leukemias- has not been attributed to defined genetic aberrations.

To identify differentially regulated genes in B-ALL, comparative intra-individual transcriptome profiling of leukemic versus immunologically matched normal lymphoblasts was employed. In this study, we found the multifunctional transcription modulator Zinc Finger Protein 423 (ZNF423) to be upregulated in the leukemic blasts. Subsequent expression analysis in a cohort of 200 patients corroborated a substantially increased abundance of ZNF423 transcripts compared to normal lymphopoietic cells. Aberrant expression of ZNF423 is maintained in relapsed ALL of initially positive samples. Thus, the transcriptional activity of ZNF423 represents a stable leukemia-associated phenomenon.

Our evaluation of the mechanistic basis of aberrant ZNF423 activation in B-ALL revealed a perturbed epigenetic and transcriptional regulation of ZNF423 as a novel mechanism interfering with B-cell differentiation. Cancer cells show global DNA hypomethylation with concomitant abnormal methylation of cytosines preceding guanosine residues in so-called CpG islands (CGI) (Feinberg and Tycko, 2004). We detected three of these CGIs in the upstream region of the ZNF423 gene locus. Bisulfite sequencing of primary ALL samples (n=58) revealed a significantly lower degree of methylation at multiple CpG positions of the analyzed CGI than in mononuclear control cells. Hence, the hypomethylation of ZNF423 regulatory sequences reflects a dysregulated epigenetic state.

ZNF423 is known to direct bone morphogenetic protein (BMP)-dependent signaling in a ternary SMAD1-SMAD4 transcription factor complex in Xenopus (Hata et al., 2000). Our data demonstrate that ZNF423 not only acts as a binding partner of this complex but is also regulated along BMP2-induced pathways, as BMP2 treatment causes a marked increase of ZNF423 expression.

Moreover, ZNF423 has been implicated as a potent inhibitor of the Early B-cell Factor 1 (EBF-1) in the olfactory system (Tsai & Reed, 1997). In hematopoiesis, EBF-1 controls the expression of essential components of the pre-B-cell receptor (preBCR). We demonstrate that enforced expression of ZNF423 in CD34-positive hematopoietic stem or progenitor cells leads to a significant reduction of transcriptional activation of the EBF-1 target genes CD79a and CD79b, validating the repressive function of ZNF423 on EBF-1 in the hematopoietic context.

In addition, the influence of aberrant ZNF423 expression was assessed in vivo in a xenograft hematopoietic stem cell transplantation model. Analyzing the immunological phenotypes of ZNF423-positive versus control cells after engraftment, a lower frequency of cells expressing mature BCR was found within the ZNF423-transduced cell population. Hence aberrant ZNF423 expression leads to a B-cell maturation arrest in vivo.

Given the impact of ZNF423 on B-cell maturation, we investigated whether presence of ZNF423 is associated with event-free survival of ALL patients. Statistical analyses performed in B-ALL patients (n=190) uncovered high ZNF423 expression to be associated with poor outcome of EVT6-RUNX1-negative patients, whereas low levels of ZNF423 expression were linked to favorable outcome.

We reasoned that -beside the repressive effect of ZNF423 on EBF-1 target gene activation- the heterodimerization of aberrantly expressed ZNF423 with EBF-1 could induce a shift in the target gene spectrum of EBF-1 in leukemic blasts, resulting in a substantial distortion of transcriptional programs in B-ALL patients. Our ongoing studies focus on the identification of the target spectrum of EBF-1 in presence or absence of ZNF423, as well as putative transcriptional targets of the ZNF423-EBF-1 heterodimer and ZNF423 alone, using ChIP-Seq analysis.