Aberrant Chromatin Modifications Mediate Ectopic Expression of Homeobox Gene NKX2-1 in B-Cell Lymphoma

Abstract 3494

Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, and when deregulated promote cell transformation in multiple cancers including hematopoietic malignancies. In this context several members of the NKL family of homeobox genes are aberrantly expressed in acute T-cell leukemia by chromosomal aberrations. Here, analysis of 20 cell lines of T- and B-cell leukemia/lymphoma by expression arrays (Affymetrix, HGU133plus2) revealed exclusive activity of NKL homeobox gene NKX2-1 in a diffuse large B-cell lymphoma (DLBCL) cell line. NKX2-1 is physiologically expressed in embryonic lung and thyroid tissues where it regulates differentiation. RQ-PCR analysis of gene expression in primary hematopoietic samples, including bone marrow, lymph node, thymus, peripheral mononuclear blood cells, T-cells and B-cells, confirmed silencing therein highlighting ectopic expression of NKX2-1 in the cell line. Copy number analysis by genomic array data (Broad Institute), spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) excluded chromosomal rearrangements at the NKX2-1 locus in expressing cells. Comparative expression analysis of NKX2-1 negative DLBCL cell lines implicated several candidate genes involved in NKX2-1 regulation, variously encoding transcription factors (TFs), chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed TF HEY1 in ectopic NKX2-1 expression and NKX2-1 in HEY1 expression in DLBCL cells, indicating reciprocal activation of these TFs. Moreover, chromatin immunoprecipitation (ChIP) analysis demonstrated direct binding of NKX2-1 to the HEY1 promoter. HEY1 belongs to the basic helix-loop-helix family disturbing lymphoid differentiation if deregulated. Enhanced expression levels of histone H3K4 methyltransferase MLL correlated with downstream rearrangement and amplification of the MLL-locus at 11q23. SiRNA-mediated knockdown of MLL was accompanied by reduced expression of NKX2-1 but not of HEY1, showing that MLL supports expression of NKX2-1. Furthermore, ChIP analyses demonstrated presence of both activatory H3K4me3 and inhibitory H3K27me3 at the promoter region of NKX2-1, while at the HEY1 promoter only H3K27me3 was detected. Such bivalent histone marks have been described for developmental genes in progenitor cells, indicating a permissive role for aberrant chromatin structures at the NKX2-1 locus in this cell line. Chromosomal alteration del(6p22) as detected by SKY and subsequently mapped by FISH was shown to target the histone gene locus HIST1. Expression analysis at the RNA and protein levels showed elevated expression of core-histones including H2B. Additionally, mono-ubiquitinated H2B was strongly enhanced in this DLBCL cell line when analyzed by Western blot. This histone mark supports the MLL-mediated formation of active chromatin structures, suggesting cooperative action of the chromosomal aberrations targeting MLL and HIST1. Array data also indicated aberrant expression of polycomb repressor complex 2 (PRC2) members which counteract the activity of MLL. Accordingly, siRNA-mediated knockdown analyses demonstrated regulatory impacts of HOPX, E2F6 and JMJD3 in NKX2-1 expression. The potential impact of signaling pathways in NKX2-1 expression, comprising NFkB, SMADs and phosphodiesterases was confirmed by treatments with TNFa, TGFb and cAMP/cGMP, respectively. Taken together, we have identified ectopic expression of NKX2-1 in DLBCL cells, involved in an oncogenic regulative network which may compromise B-cell differentiation via activation of HEY1. Combined analyses of chromosomal alterations and comparative gene expressions identified aberrant chromatin structures underlying expression of NKX2-1, representing the central player in that network. Therefore, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancy.