ALBERT

Description: Three NK-like (NKL) homeobox genes, TLX1/HOX11, TLX3/HOX11L2 and NKX2- 5/CSX, have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Here we screened further NKL genes in 24 T-ALL cell lines by RT-PCR and identified common expression of MSX2, highlighting this homeobox gene as a potential physiological family member in T-cells. Subsequent quantification of MSX2 confirmed expression in primary hematopoietic cells demonstrating higher levels in CD34+ stem cells when compared to peripheral blood cells or mature CD3+ T-cells. Analysis of core thymic factors in T-ALL cell lines, including IL7, BMP4, TGFbeta, NOTCH and T-cell receptor signaling, suggests their involvement in MSX2 regulation during T-cell differentiation. Chromosomal and genomic analysis of the MSX2 locus (at 5q35) uncovered deletion in t(5;14)(q35;q32) positive T-ALL cell lines associated with low expression levels of MSX2 and ectopic activation of TLX3 or NKX2-5, respectively. For functional analysis we lentivirally transduced T-ALL cells for overexpression of either MSX2 or oncogenic TLX1 and NKX2-5. These cells displayed transcriptional activation of NOTCH3-signaling, as indicated by expression array profiling and real-time PCR analysis of NOTCH3, HES1 and HEY1. The sensitivities to gamma-secretase inhibitor analyzed by MTT-assay of cells overexpressing MSX2, TLX1 or NKX2-5, respectively, were consistently decreased. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with repressor proteins of the NOTCH-pathway, SPEN/MINT and TLE1/GRG1, as shown by co-immunoprecipitation, probably representing one mechanism of (de)regulation. Elevated expression of NOTCH3 and HEY1 mRNA was detected in TLX1/3 positive T-ALL patients, confirming data obtained from cell lines. In conclusion, we have defined expression patterns, regulation and targets of MSX2 in hematopoietic cells, to reveal a novel modulatory activity in T-cell differentiation operating via NOTCH-signaling, and in leukemogenesis when replaced or supplemented by oncogenic NKL homeodomain proteins.

Description: Abstract 1281 Poster Board I-303 Many oncogenes code for transcription factors involved in regulation of developmental pathways. The activity of these pathways is tissue specific and restricted to certain developmental stages. Here, we searched for T-cell acute lymphoblastic leukemia (T-ALL) oncogenes which physiologically regulate differentiation of natural killer (NK) cells. NK- and T-cells are closely related lymphocytes, sharing the same early progenitors which can differentiate into either lineage. We compared expression profiles of malignant NK- and T-cell lines to identify aberrantly expressed genes in T-ALL. This analysis revealed high expression of HOXA9, HOXA10 and ID2 in NK-cell lines and in one T-ALL line, LOUCY, suggesting leukemic deregulation therein. Subsequently, we analyzed mechanisms underlying their regulation. Overexpression and chromatin immuno-precipitation experiments demonstrated that HOXA9 and HOXA10 directly activate ID2 expression. Analysis of other ALL and acute myeloid leukemia cell lines with and without mixed lineage leukemia (MLL) gene translocations demonstrated a correlated expression of HOXA9/10 and ID2, highlighting ID2 as an indirect target of MLL fusion proteins which deregulate HOXA genes. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including the components EZH2 and HOP of polycomb repressor complex 2 (PRC2), showed downregulation of EZH2 in LOUCY and limited expression of HOP to NK-cell lines. Subsequent treatment of T-ALL cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively, confirming repressive activity of EZH2 in T-cells. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression. Taken together, our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 and may contribute to T-cell leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3636 Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS-box family which is physiologically expressed in hematopoietic stem cells and during development of B-cells. Recently, we identified ectopic expression of MEF2C in T-cell acute lymphoblastic leukemia (T-ALL) cell lines activated either via chromosomally mediated ectopic expression of homeodomain protein NKX2-5 or via deletion of non-coding exon and promoter regions at 5q14, suggesting loss of negative regulatory elements. Our aim was to identify additional transcriptional regulators of MEF2C in T-ALL. Therefore, we analyzed the sequence of the MEF2C 5′-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix (bHLH) proteins, STAT5 and HOXA9/HOXA10. Overexpression studies demonstrated MEF2C activation by GFI1B (strong), LYL1 and TAL1 leukemic bHLH proteins (weak), and inhibition by STAT5 (strong) and HOXA9/HOXA10 (weak). Chromatin-Immuno-Precipitation analysis demonstrated direct binding of GFI1B, LYL1 and STAT5 but not of HOXA10 to the MEF2C 5′-region in T-ALL cells. However, HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of MEF2C regulation. Chromosomal deletion of the 5′-MEF2C STAT5 binding site in LOUCY cells by del(5)(q14), reduced expression levels of STAT5 protein in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5-signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that ectopic expression of MEF2C in T-ALL cells is mainly regulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory STAT5-signaling. Disclosures: No relevant conflicts of interest to declare.

Description: In T-cell acute lymphoblastic leukaemia (T-ALL) alternative t(5;14)(q35;q32.2) forms effect leukemic dysregulation of either TLX3 or NKX2-5 homeobox genes at 5q35 by juxtaposition with 3′-BCL11B at 14q32.2. Putative regulatory sequences underlying ectopic homeobox gene activation in t(5;14), and their mode of action have remained poorly understood mainly because breakpoints at 14q32.2 are widely scattered over the ~1 Mbp genomic desert region. We pooled cytogenetic data from t(5;14) cell lines together with published clinical data to refine the BCL11B downstream breakpoint cluster region (bcr). Ectopic homeobox gene dysregulation was investigated by DNA-i(nhibitory-treatments) with 26-mer double-stranded DNA oligo(nucleotide)s directed against putative enhancers using NKX2-5 expression as endpoints. Enhancer targets were provisionally identified from orphan T-cell DNase-I hypersensive sites (DNaseI-HS) located between 3′-BCL11B and VRK1. NKX2-5 downregulation in t(5;14) PEER cells was almost entirely restricted to DNAi targeting enhancers within the distal bcr and was dose- and sequence-dependent. Interestingly, enhancers near 3′-BCL11B regulated that gene only. These data imply that enhancer-promoter distances and/or locations are important for long-range gene regulation. Chromatin immunoprecipation assays showed that the four most effectual NKX2-5 ectopic enhancers were hyperacetylated. These enhancers clustered ~1 Mbp downstream of BCL11B, within a region displaying multiple regulatory stigmata, including a TCRA-enhancer motif, and abyssal sequence-conservation (“5-Way Regulatory Potential”). Paradoxically, although TLX3/NKX2-5 promoter/exon-1 regions were hypo-acetylated, their expression decreased after TSA treatment, implying extrinsic regulation by factor(s) subject to acetylation-control. PU.1 is known to get transcriptionally repressed by TSA and potentially binds TLX3/NKX2-5 upstream promoter regions. Knockdown of PU.1 effected downregulation of both homeobox genes. Moreover, genomic analysis showed preferential enrichment near validated ectopic enhancers of binding sites for the PU.1-cofactor HMGA1, knockdown of which also inhibited NKX2-5 in PEER cells. Analysis of nuclear matrix attachment (NMA) in PEER cells showed enhanced attachment near to the most effectual enhancer cluster which was alleviated by TSA-treatment. Interestingly, the juxtapositional genomic regions of “active” ins(14;5) rearrangements driving NKX2-5 expression exhibited tight NMA, forming structures reminiscent of “active chromatin hubs”. These findings lead us to propose that HMGA1 and PU.1 co-regulate ectopic homeobox gene expression in t(5;14) T-ALL by interactions mediated at the nuclear matrix, possibly mediated by SATB1 binding. Our data document homeobox gene dysregulation by a novel regulatory region at 3′-BCL11B responsive to HDAC-inhibition and highlight a novel class of potential therapeutic target amid “junk” DNA.

Description: 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. Disclosures: No relevant conflicts of interest to declare.

Description: Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder where the malignant B-cells manifest a “hairy” appearance under the microscope. Recent studies have identified BRAF V600E mutations in most HCL patients, highlighting this abnormality as a molecular hallmark for this disease. However, mutated BRAF occurs widely – already described in several solid tumors, including melanoma, thyroid and colorectal carcinomas, indicating that BRAF V600E is not pathognomonic in HCL. Cell lines originating from HCL patients lack BRAF mutations but retain the typical piliferous morphology and the appropriate HCL immunophenotype (CD19, CD11c, CD103, CD25, CD123), thus constituting tools for identifying alternative mechanisms of leukemogenesis in this disease entity. Genomic aberrations in hematopoietic tumors recurrently target loci bearing genes involved in malignant transformation. These genes may include both candidate biomarkers and potential therapeutic targets. To identify such genes in HCL we here combined genomic profiling and gene expression quantification of a well characterized HCL cell line containing several chromosomal aberrations. The expression levels of genomically targeted genes were compared to HCL control cell lines, identifying 91 deregulated genes. Gene set enrichment analysis of which indicated apoptosis, cell cycle regulation and DNA damage response (DDR) as altered processes in HCL. Accordingly, REL (NFkB and apoptosis), CDK6 and BRAF (cell cycle), ATM and CUTL1 (DDR) comprised prominent target genes overexpressed in this cell line. The same genes were found to be conspicuously expressed in HCL patient samples in silico (Fernandez et al., 2010; Gene Expression Omnibus GSE16455), supporting their clinical significance. Treatments of HCL cell lines for particular siRNA-mediated gene knockdowns and with selective pharmacological inhibitors helped to reveal a regulatory network highlighting NFkB at a central position. Consistently, focused analysis of expression profiling data of several cell lines supported elevated NFkB-pathway activity in HCL and ABC-DLBCL when compared to GC-DLBCL. In conclusion, we identified deregulated genes and multiple mechanisms which contribute to aberrantly activated NFkB-pathway in HCL. Therefore, NFkB may represent a B-cell specific hallmark of HCL and a promising novel therapeutic target most notably in patients lacking BRAF mutations in this entity. Disclosures No relevant conflicts of interest to declare.

Description: In Hodgkin lymphoma (HL) we recently identified deregulated expression of homeobox gene MSX1 which physiologically controls development of the embryonal neural plate border region. This region forms the neural crest and placodes which in turn give rise to fundamental structures and functions of the head including jaw and sensory organs. Here, we examined in HL another homeobox gene, SIX1, an additional regulator of this embryonal region mediating differentiation of placodal precursors. In silico data (GSE12453) show aberrantly activated SIX1 in 12 % of HL patient samples, indicating a pathological role in a subset of this malignancy. In addition, SIX1 expression was detected in HL cell lines which then were used as models to reveal regulators and target genes of this basic developmental factor. Cytogenetics and quantitative genomic PCR revealed copy number gains of the SIX1 locus at chromosomal band 14q23 correlating with enhanced expression while chromosomal translocations were absent. Moreover, comparative expression profiling data and pertinent gene modulation experiments indicated that the WNT-signalling pathway activates and transcription factor MEF2C suppresses SIX1 expression. MAPK7/ERK5 and HDAC9 phosphorylate and deacetylate MEF2C, respectively, boosting or constraining its inhibitory capacity. MEF2C, MAPK7 and HDAC9 show aberrant expression levels in both HL cell lines and patient samples, collectively restricting the inhibitory activity of MEF2C. Genes encoding the transcription factors GATA2, GATA3, MSX1 and SPIB - all basic lymphoid regulators - were identified as targets of SIX1 in HL, contributing to deregulated B-cell differentiation in this malignancy. In addition, cofactors EYA1 and TLE4 contrastingly mediated activation and suppression of SIX1 target gene expression, respectively. Thus, the protein domain interfaces may represent therapeutic targets in SIX1-positive HL subsets to prevent cofactor interactions and subsequent target gene regulation. Collectively, our data reveal a gene regulatory network where SIX1 centrally deregulates lymphoid differentiation. Additionally, our data strengthen the emerging concordance of lymphopoiesis/lymphomagenesis and the neural plate border region ontogeny. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4644 Aberrant activation of various oncogenes, including homeobox genes encoding fundamental transcription factors, during T-cell development contributes to T-cell acute lymphoblastic leukemia (T-ALL). Neoplastic chromosome rearrangements are known to deregulate Antennapedia-class homeobox genes of the NKL-family (TLX1, TLX3, NKX2-5) and HOXA-cluster genes of the Extended-Hox-family. Following analysis of T-ALL cell lines and primary cells, we describe leukemogenic involvement of a third homeobox gene group, the Paired (PRD)-class. Ascertainment was performed in an early stage-arrested T-ALL cell line (LOUCY) which revealed chromosomal deletion at 5q31, removing the downstream regulatory region of the PRD-homeobox gene PITX1. Comparative expression analysis confirmed ectopic PITX1 expression, consistent with aberrant activation by del(5)(q31) which removes a STAT1 binding site. STAT1 mediates repressive IL2-STAT1 signaling, implicating IL2-pathway avoidance as a possible activation mechanism. Furthermore, we detected expression of the physiologically similar PITX2 in 11/24 (46%) T-ALL cell lines, 5 with genomic PITX2 gains. Among primary T-ALL samples, 2/22 (9%) – both pediatric pre-T-ALL - ectopically expressed PITX1 but not PITX2. Forced expression of PITX1 by lentiviral transduction of JURKAT cells and subsequent analysis by expression profiling, prompted upregulation of RUNX2 and JUN and inhibition of RUNX1 and NKX3-1, indicating impaired T-cell differentiation. Taken together, our data show leukemic activation of PITX1, a novice PRD-class homeobox gene in the T-ALL repertoire, which may promote leukemogenesis by inhibiting differentiation. Disclosures: No relevant conflicts of interest to declare.

Description: Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation. Several members of the NKL subclass are deregulated in T-cell progenitors and support leukemogenesis. We have recently described particular expression patterns of nine NKL homeobox genes in early hematopoiesis and T-cell development. Here, we screened NKL homeobox gene activities in normal B-cell development and extended the NKL-code to include this lymphoid lineage. Analysis of public expression profiling datasets revealed that HHEX and NKX6-3 were the only members differentially active in naïve B-cells, germinal center B-cells, plasma cells and memory B-cells. Subsequent examination of different types of B-cell malignancies showed both aberrant overexpression of NKL-code members and ectopic activation of subclass members physiologically silent in lymphopoiesis including BARX2, DLX1, EMX2, NKX2-1, NKX2-2 and NKX3-2. Based on these findings we performed detailed studies of the B-cell specific NKL homeobox gene NKX6-3 which showed enhanced activity in patient subsets of follicular lymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL), and in three DLBCL cell lines to serve as in vitro models. While excluding genomic and chromosomal rearrangements at the locus of NKX6-3 (8p11) promoter studies demonstrated that B-cell factors MYB and PAX5 activated NKX6-3 transcription. Furthermore, aberrant BMP7/SMAD1-signalling and deregulated expression of chromatin complex components AUTS2 and PCGF5 promoted NKX6-3 activation. Finally, NKL homeobox genes HHEX, HLX, MSX1 and NKX6-3 were expressed in B-cell progenitors and generated a regulatory gene network in cell lines which we propose may provide physiological support for NKL-code formation in early B-cell development. Together, we identified an NKL-code in B-cell development whose violation may deregulate differentiation and promote malignant transformation. Disclosures No relevant conflicts of interest to declare.