Transcriptional Landscape of CUT-Class Homeobox Genes in Blastic Plasmacytoid Dendritic Cell Neoplasm.

Homeobox genes encode developmental transcription factors regulating tissue-specific differentiation processes and drive cancerogenesis when deregulated. Dendritic cells (DCs) are myeloid immune cells occurring as two types, either conventional or plasmacytoid DCs. Recently, we showed that the expression of NKL-subclass homeobox gene VENTX is restricted to conventional DCs, regulating developmental genes. Here, we identified and investigated homeobox genes specifically expressed in plasmacytoid DCs (pDCs) and derived blastic plasmacytoid dendritic cell neoplasm (BPDCN). We analyzed gene expression data, performed RQ-PCR, protein analyses by Western blot and immuno-cytology, siRNA-mediated knockdown assays and subsequent RNA-sequencing and live-cell imaging. Screening of public gene expression data revealed restricted activity of the CUT-class homeobox gene CUX2 in pDCs. An extended analysis of this homeobox gene class in myelopoiesis showed that additional CUX2 activity was restricted to myeloid progenitors, while BPDCN patients aberrantly expressed ONECUT2, which remained silent in the complete myeloid compartment. ONECUT2 expressing BPDCN cell line CAL-1 served as a model to investigate its regulation and oncogenic activity. The ONECUT2 locus at 18q21 was duplicated and activated by IRF4, AUTS2 and TNF-signaling and repressed by BMP4-, TGFb- and IL13-signalling. Functional analyses of ONECUT2 revealed the inhibition of pDC differentiation and of CDKN1C and CASP1 expression, while SMAD3 and EPAS1 were activated. EPAS1 in turn enhanced survival under hypoxic conditions which thus may support dendritic tumor cells residing in hypoxic skin lesions. Collectively, we revealed physiological and aberrant activities of CUT-class homeobox genes in myelopoiesis including pDCs and in BPDCN, respectively. Our data may aid in the diagnosis of BPDCN patients and reveal novel therapeutic targets for this fatal malignancy.

Establishment of the lymphoid ETS-code reveals deregulated ETS genes in Hodgkin lymphoma.

The human family of ETS transcription factors numbers 28 genes which control multiple aspects of development, notably the differentiation of blood and immune cells. Otherwise, aberrant expression of ETS genes is reportedly involved in forming leukemia and lymphoma. Here, we comprehensively mapped ETS gene activities in early hematopoiesis, lymphopoiesis and all mature types of lymphocytes using public datasets. We have termed the generated gene expression pattern lymphoid ETS-code. This code enabled identification of deregulated ETS genes in patients with lymphoid malignancies, revealing 12 aberrantly expressed members in Hodgkin lymphoma (HL). For one of these, ETS gene ETV3, expression in stem and progenitor cells in addition to that in developing and mature T-cells was mapped together with downregulation in B-cell differentiation. In contrast, subsets of HL patients aberrantly overexpressed ETV3, indicating oncogenic activity in this B-cell malignancy. Analysis of ETV3-overexpressing HL cell line SUP-HD1 demonstrated genomic duplication of the ETV3 locus at 1q23, GATA3 as mutual activator, and suppressed BMP-signalling as mutual downstream effect. Additional examination of the neighboring ETS genes ETS1 and FLI1 revealed physiological activities in B-cell development and aberrant downregulation in HL patient subsets. SUP-HD1 showed genomic loss on chromosome 11, del(11)(q22q25), targeting both ETS1 and FLI1, underlying their downregulation. Furthermore, in the same cell line we identified PBX1-mediated overexpression of RIOK2 which inhibited ETS1 and activated JAK2 expression. Collectively, we codified normal ETS gene activities in lymphopoiesis and identified oncogenic ETS members in HL.

Chromosomal Aberration t(14;17)(q32;q21) Simultaneously Activates HOXB5 and miR10a in Triple-Hit B-Cell Lymphoma.

BCL2, BCL6 and MYC are major oncogenes in B-cell lymphoma. Their aberrant activation frequently occurs via chromosomal translocations which juxtapose light or heavy chain immunoglobulin (IG) genes to BCL2 and MYC or fuse diverse partner genes with BCL6. So-called double-hit lymphomas usually carry BCL2 and MYC rearrangements, while triple-hit lymphomas additionally bear BCL6-fusions. All these translocations are of diagnostic relevance and usually denote poor prognosis. Here, we genomically characterized classic follicular lymphoma (FL) cell line SC-1, thereby identifying t(14;18)(q32;q21) juxtaposing IGH and BCL2, t(8;14)(q24;q32) juxtaposing IGH and MYC, and t(3;3)(q25;q27) fusing MBNL1 to BCL6. In addition, we found that SC-1 carries a novel chromosomal rearrangement, t(14;17)(q32;q21), which, though present at establishment, has remained unreported until now. We further show that t(14;17)(q32;q21) juxtaposes IGH with the HOXB gene cluster at 17q21 and affect the oncogenic activation of both homeobox gene HOXB5 and neighboring micro-RNA gene miR10a. Moreover, we detected aberrant overexpression of HOXB5 in subsets of Burkitt lymphoma, FL, and multiple myeloma patients, confirming the clinical relevance of its deregulation. In SC-1, HOXB5 activation was additionally supported by co-expression of hematopoietic stem cell factor ZNF521, indicating an aberrant impact in cell differentiation. Functional investigations showed that HOXB5 represses the apoptotic driver BCL2L11 and promotes survival in the presence of etoposide, and that miR10a inhibits BCL6 and may thus play an oncogenic role in later stages of lymphomagenesis. Collectively, we characterize triple-hit B-cell line SC-1 and identify the aberrant expression of HOXB5 and miR10a, both novel oncogenes in B-cell lymphoma.

Establishment of the Myeloid TBX-Code Reveals Aberrant Expression of T-Box Gene TBX1 in Chronic Myeloid Leukemia.

T-box genes encode transcription factors, which control developmental processes and promote cancer if deregulated. Recently, we described the lymphoid TBX-code, which collates T-box gene activities in normal lymphopoiesis, enabling identification of members deregulated in lymphoid malignancies. Here, we have extended this analysis to cover myelopoiesis, compiling the myeloid TBX-code and, thus, highlighting which of these genes might be deregulated in myeloid tumor types. We analyzed public T-box gene expression datasets bioinformatically for normal and malignant cells. Candidate T-box-gene-expressing model cell lines were identified and examined by RQ-PCR, Western Blotting, genomic profiling, and siRNA-mediated knockdown combined with RNA-seq analysis and live-cell imaging. The established myeloid TBX-code comprised 10 T-box genes, including progenitor-cell-restricted TBX1. Accordingly, we detected aberrant expression of TBX1 in 10% of stem/progenitor-cell-derived chronic myeloid leukemia (CML) patients. The classic CML cell line K-562 expressed TBX1 at high levels and served as a model to identify TBX1 activators, including transcription factor GATA1 and genomic amplification of the TBX1 locus at 22q11; inhibitors, including BCR::ABL1 fusion and downregulated GNAI2, as well as BMP, FGF2, and WNT signaling; and the target genes CDKN1A, MIR17HG, NAV1, and TMEM38A. The establishment of the myeloid TBX-code permitted identification of aberrant TBX1 expression in subsets of CML patients and cell lines. TBX1 forms an integral part of an oncogenic regulatory network impacting proliferation, survival, and differentiation. Thus, the data spotlight novel diagnostic markers and potential therapeutic targets for this malignancy.

Normal and Aberrant TALE-Class Homeobox Gene Activities in Pro-B-Cells and B-Cell Precursor Acute Lymphoblastic Leukemia.

Homeobox genes encode transcription factors regulating basic developmental processes. They are arranged according to sequence similarities of their conserved homeobox in 11 classes, including TALE. Recently, we have reported the so-called TALE-code. This gene signature describes physiological expression patterns of all active TALE-class homeobox genes in the course of hematopoiesis. The TALE-code allows the evaluation of deregulated TALE homeobox genes in leukemia/lymphoma. Here, we extended the TALE-code to include the stages of pro-B-cells and pre-B-cells in early B-cell development. Detailed analysis of the complete lineage of B-cell differentiation revealed expression of TALE homeobox genes IRX1 and MEIS1 exclusively in pro-B-cells. Furthermore, we identified aberrant expression of IRX2, IRX3 and MEIS1 in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) which originates from early B-cell progenitors. The data showed correlated activities of deregulated TALE-class members with particular BCP-ALL subtype markers, namely IRX2 with TCF3/E2A-fusions, IRX3 with ETV6/TEL-fusions, and MEIS1 with KMT2A/MLL-fusions. These correlations were also detected in BCP-ALL cell lines which served as experimental models. We performed siRNA-mediated knockdown experiments and reporter gene assays to analyze regulatory connections. The results showed mutual activation of IRX1 and TCF3. In contrast, IRX2 directly repressed wild-type TCF3 while the fusion gene TCF3::PBX1 lost the binding site for IRX2 and remained unaltered. IRX3 mutually activated fusion gene ETV6::RUNX1 while activating itself by aberrantly expressed transcription factor KLF15. Finally, KMT2A activated MEIS1 which in turn supported the expression of IRX3. In summary, we revealed normal TALE homeobox gene expression in early B-cell development and identified aberrant activities of IRX2, IRX3 and MEIS1 in particular subtypes of BCP-ALL. Thus, these TALE homeobox genes may serve as novel diagnostic markers and therapeutic targets.

Genomic Aberrations Generate Fusion Gene FOXK2::TP63 and Activate NFKB1 in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a severe lymphoid malignancy with a worse prognosis lacking curative treatment regimens. Several gene mutations and deregulated pathways, including NFkB signaling, have been implicated in its pathogenesis. Accordingly, CTCL cell line HUT-78 reportedly contains mutated NFKB2, which is constitutively activated via partial gene deletion, also demonstrating that genomic rearrangements cause driving mutations in this malignancy. Here, along with HUT-78, we analyzed CTCL cell line HH to identify additional aberrations underlying gene deregulation. Karyotyping and genomic profiling of HH showed several rearrangements worthy of detailed investigation. Corresponding to the established karyotype, RNA-seq data and PCR analysis confirmed the presence of t(3;17)(q28;q25), generating a novel fusion gene, FOXK2::TP63. Furthermore, chromosomal rearrangement t(1;4)(p32;q25) was connected to amplification at 4q24-26, affecting aberrant NFKB1 overexpression thereat. Transcription factor binding-site analysis and knockdown experiments demonstrated that IRF4 contributed to NFKB1 expression. Within the same amplicon, we identified amplification and overexpression of NFkB signaling activator CAMK2D (4q26) and p53-inhibitor UBE2D3 (4q24). Genomic profiling data for HUT-78 detailed a deletion at 10q25 underlying reported NFKB2 activation. Moreover, amplifications of ID1 (20q11) and IKZF2 (2q34) in this cell line drove overexpression of these NK cell differentiation factors and possibly thus formed corresponding lineage characteristics. Target gene analysis for NFKB1 via siRNA-mediated knockdown in HH revealed activation of TP63, MIR155, and NOTCH pathway component RBPJ. Finally, treatment of HH with NFkB inhibitor demonstrated a role for NFkB in supporting proliferation, while usage of inhibitor DAPT showed significant survival effects via the NOTCH pathway. Collectively, our data suggest that NFkB and/or NOTCH inhibitors may represent reasonable treatment options for subsets of CTCL patients.

Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL.

The Hematopoietic TALE-Code Shows Normal Activity of IRX1 in Myeloid Progenitors and Reveals Ectopic Expression of IRX3 and IRX5 in Acute Myeloid Leukemia.

Homeobox genes encode transcription factors that control basic developmental decisions. Knowledge of their hematopoietic activities casts light on normal and malignant immune cell development. Recently, we constructed the so-called lymphoid TALE-code that codifies expression patterns of all active TALE class homeobox genes in early hematopoiesis and lymphopoiesis. Here, we present the corresponding myeloid TALE-code to extend this gene signature, covering the entire hematopoietic system. The collective data showed expression patterns for eleven TALE homeobox genes and highlighted the exclusive expression of IRX1 in megakaryocyte-erythroid progenitors (MEPs), implicating this TALE class member in a specific myeloid differentiation process. Analysis of public profiling data from acute myeloid leukemia (AML) patients revealed aberrant activity of IRX1 in addition to IRX3 and IRX5, indicating an oncogenic role for these TALE homeobox genes when deregulated. Screening of RNA-seq data from 100 leukemia/lymphoma cell lines showed overexpression of IRX1, IRX3, and IRX5 in megakaryoblastic and myelomonocytic AML cell lines, chosen as suitable models for studying the regulation and function of these homeo-oncogenes. Genomic copy number analysis of IRX-positive cell lines demonstrated chromosomal amplification of the neighboring IRX3 and IRX5 genes at position 16q12 in MEGAL, underlying their overexpression in this cell line model. Comparative gene expression analysis of these cell lines revealed candidate upstream factors and target genes, namely the co-expression of GATA1 and GATA2 together with IRX1, and of BMP2 and HOXA10 with IRX3/IRX5. Subsequent knockdown and stimulation experiments in AML cell lines confirmed their activating impact in the corresponding IRX gene expression. Furthermore, we demonstrated that IRX1 activated KLF1 and TAL1, while IRX3 inhibited GATA1, GATA2, and FST. Accordingly, we propose that these regulatory relationships may represent major physiological and oncogenic activities of IRX factors in normal and malignant myeloid differentiation, respectively. Finally, the established myeloid TALE-code is a useful tool for evaluating TALE homeobox gene activities in AML.

NKL Homeobox Genes NKX2-3 and NKX2-4 Deregulate Megakaryocytic-Erythroid Cell Differentiation in AML.

NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte-erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.

Establishment of the TBX-code reveals aberrantly activated T-box gene TBX3 in Hodgkin lymphoma.

T-box genes encode transcription factors which control basic processes in development of several tissues including cell differentiation in the hematopoietic system. Here, we analyzed the physiological activities of all 17 human T-box genes in early hematopoiesis and in lymphopoiesis including developing and mature B-cells, T-cells, natural killer (NK)-cells and innate lymphoid cells. The resultant expression pattern comprised six genes, namely EOMES, MGA, TBX1, TBX10, TBX19 and TBX21. We termed this gene signature TBX-code which enables discrimination of normal and aberrant activities of T-box genes in lymphoid malignancies. Accordingly, expression analysis of T-box genes in Hodgkin lymphoma (HL) patients using a public profiling dataset revealed overexpression of EOMES, TBX1, TBX2, TBX3, TBX10, TBX19, TBX21 and TBXT while MGA showed aberrant downregulation. Analysis of T-cell acute lymphoid leukemia patients indicated aberrant overexpression of six T-box genes while no deregulated T-box genes were detected in anaplastic large cell lymphoma patients. As a paradigm we focused on TBX3 which was ectopically activated in about 6% of HL patients analyzed. Normally, TBX3 is expressed in tissues like lung, adrenal gland and retina but not in hematopoiesis. HL cell line KM-H2 expressed enhanced TBX3 levels and was used as an in vitro model to identify upstream regulators and downstream targets in this malignancy. Genomic studies of this cell line showed focal amplification of the TBX3 locus at 12q24 which may underlie its aberrant expression. In addition, promoter analysis and comparative expression profiling of HL cell lines followed by knockdown experiments revealed overexpressed transcription factors E2F4 and FOXC1 and chromatin modulator KDM2B as functional activators. Furthermore, we identified repressed target genes of TBX3 in HL including CDKN2A, NFKBIB and CD19, indicating its respective oncogenic function in proliferation, NFkB-signaling and B-cell differentiation. Taken together, we have revealed a lymphoid TBX-code and used it to identify an aberrant network around deregulated T-box gene TBX3 in HL which promotes hallmark aberrations of this disease. These findings provide a framework for future studies to evaluate deregulated T-box genes in lymphoid malignancies.

Identification of cell lines CL-14, CL-40 and CAL-51 as suitable models for SARS-CoV-2 infection studies.

The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.

NKL Homeobox Gene VENTX Is Part of a Regulatory Network in Human Conventional Dendritic Cells.

Recently, we documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in human myelopoiesis including monocytes and their derived dendritic cells (DCs). Here, we enlarge this map to include normal NKL homeobox gene expressions in progenitor-derived DCs. Analysis of public gene expression profiling and RNA-seq datasets containing plasmacytoid and conventional dendritic cells (pDC and cDC) demonstrated HHEX activity in both entities while cDCs additionally expressed VENTX. The consequent aim of our study was to examine regulation and function of VENTX in DCs. We compared profiling data of VENTX-positive cDC and monocytes with VENTX-negative pDC and common myeloid progenitor entities and revealed several differentially expressed genes encoding transcription factors and pathway components, representing potential VENTX regulators. Screening of RNA-seq data for 100 leukemia/lymphoma cell lines identified prominent VENTX expression in an acute myelomonocytic leukemia cell line, MUTZ-3 containing inv(3)(q21q26) and t(12;22)(p13;q11) and representing a model for DC differentiation studies. Furthermore, extended gene analyses indicated that MUTZ-3 is associated with the subtype cDC2. In addition to analysis of public chromatin immune-precipitation data, subsequent knockdown experiments and modulations of signaling pathways in MUTZ-3 and control cell lines confirmed identified candidate transcription factors CEBPB, ETV6, EVI1, GATA2, IRF2, MN1, SPIB, and SPI1 and the CSF-, NOTCH-, and TNFa-pathways as VENTX regulators. Live-cell imaging analyses of MUTZ-3 cells treated for VENTX knockdown excluded impacts on apoptosis or induced alteration of differentiation-associated cell morphology. In contrast, target gene analysis performed by expression profiling of knockdown-treated MUTZ-3 cells revealed VENTX-mediated activation of several cDC-specific genes including CSFR1, EGR2, and MIR10A and inhibition of pDC-specific genes like RUNX2. Taken together, we added NKL homeobox gene activities for progenitor-derived DCs to the NKL-code, showing that VENTX is expressed in cDCs but not in pDCs and forms part of a cDC-specific gene regulatory network operating in DC differentiation and function.

[Implementation and development of a center for hereditary hemorrhagic telangiectasia]. / Aufbau und Entwicklung eines interdisziplinären Zentrums für hereditäre hämorrhagische Teleangiektasie.

OBJECTIVE: Hereditary Hemorrhagic Telangiectasia (HHT) is a rare and systemic disorder which is characterized by recurrent epistaxis, mucocutaneous telangiectases, and visceral arteriovenous malformations (AVM). An interdisciplinary concept is recommended. MATERIAL AND METHODS: We performed a retrospective review of consecutive patients who were referred to our newly established HHT Center of Excellence (HHT COE) for evaluation and treatment between April 2014 and August 2019. RESULTS: A network of over 20 departments was established at the University Hospital Essen. In 261 of the 282 patients (93 %), who were referred to the hospital's COE, the HHT diagnosis was at least possible. Most patients suffered from several symptoms (epistaxis and / or telangiectasia: > 80 %, visceral involvement: 65 %) and received a variety of treatments, often in a multidisciplinary setting. Alongside this direct treatment, the COE leader manages the coordination of the center and its public relations, which involves more than 900 e-mails per year. International collaboration and exchanges of expertise within the European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN) can improve the treatment of patients with HHT particularly where these cases are complex. CONCLUSIONS: An HHT COE provides an interdisciplinary network where highly specialized diagnostic and therapeutic processes can be updated and optimized continuously.

Establishment of the TALE-code reveals aberrantly activated homeobox gene PBX1 in Hodgkin lymphoma.

Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation and are grouped into classes and subclasses according to sequence similarities. Here, we analyzed the activities of the 20 members strong TALE homeobox gene class in early hematopoiesis and in lymphopoiesis including developing and mature B-cells, T-cells, natural killer (NK)-cells and innate lymphoid cells (ILC). The resultant expression pattern comprised eleven genes and which we termed TALE-code enables discrimination of normal and aberrant activities of TALE homeobox genes in lymphoid malignancies. Subsequent expression analysis of TALE homeobox genes in public datasets of Hodgkin lymphoma (HL) patients revealed overexpression of IRX3, IRX4, MEIS1, MEIS3, PBX1, PBX4 and TGIF1. As paradigm we focused on PBX1 which was deregulated in about 17% HL patients. Normal PBX1 expression was restricted to hematopoietic stem cells and progenitors of T-cells and ILCs but absent in B-cells, reflecting its roles in stemness and early differentiation. HL cell line SUP-HD1 expressed enhanced PBX1 levels and served as an in vitro model to identify upstream regulators and downstream targets in this malignancy. Genomic studies of this cell line therein showed a gain of the PBX1 locus at 1q23 which may underlie its aberrant expression. Comparative expression profiling analyses of HL patients and cell lines followed by knockdown experiments revealed NFIB and TLX2 as target genes activated by PBX1. HOX proteins operate as cofactors of PBX1. Accordingly, our data showed that HOXB9 overexpressed in HL coactivated TLX2 but not NFIB while activating TNFRSF9 without PBX1. Further downstream analyses showed that TLX2 activated TBX15 which operated anti-apoptotically. Taken together, we discovered a lymphoid TALE-code and identified an aberrant network around deregulated TALE homeobox gene PBX1 which may disturb B-cell differentiation in HL by reactivation of progenitor-specific genes. These findings may provide the framework for future studies to exploit possible vulnerabilities of malignant cells in therapeutic scenarios.

Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia.

The NKL-code describes normal expression patterns of NKL homeobox genes in hematopoiesis. Aberrant expression of NKL homeobox gene subclass members have been reported in several hematopoietic malignancies including acute myeloid leukemia (AML). Here, we analyzed the oncogenic role of the HMX-group of NKL homeobox genes in AML. Public expression profiling data-available for HMX1 and HMX2-indicate aberrant activity of HMX2 in circa 2% AML patients overall, rising to 31% in those with KMT2A/MLL rearrangements whereas HMX1 expression remains inconspicuous. AML cell lines EOL-1, MV4-11 and MOLM-13 expressed both, HMX2 and neighboring HMX3 genes, and harbored KMT2A aberrations, suggesting their potential functional association. Surprisingly, knockdown experiments in these cell lines demonstrated that KMT2A inhibited HMX2/3 which, in turn, did not regulate KMT2A expression. Furthermore, karyotyping and genomic profiling analysis excluded rearrangements of the HMX2/3 locus in these cell lines. However, comparative expression profiling and subsequent functional analyses revealed that IRF8, IL7- and WNT-signalling activated HMX2/3 expression while TNFa/NFkB- signalling proved inhibitory. Whole genome sequencing of EOL-1 identified two mutations in the regulatory upstream regions of HMX2/3 resulting in generation of a consensus ETS-site and transformation of a former NFkB-site into an SP1-site. Reporter-gene assays demonstrated that both mutations contributed to HMX2/3 activation, modifying ETS1/ELK1- and TNFalpha-mediated gene regulation. Moreover, DMSO-induced eosinophilic differentiation of EOL-1 cells coincided with HMX2/3 downregulation while knockdown of HMX2 induced cell differentiation, collectively supporting a fundamental role for these genes in myeloid differentiation arrest. Finally, target genes of HMX2/3 were identified in EOL-1 and included suppression of differentiation gene EPX, and activation of fusion gene FIP1L1-PDGFRA and receptor-encoding gene HTR7, both of which enhanced oncogenic ERK-signalling. Taken together, our study documents a leukemic role for deregulated NKL homeobox genes HMX2 and HMX3 in AML, revealing molecular mechanisms of myeloid differentiation arrest.

The NKL-code for innate lymphoid cells reveals deregulated expression of NKL homeobox genes HHEX and HLX in anaplastic large cell lymphoma (ALCL).

NKL homeobox genes encode developmental transcription factors and display an NKL-code according to their physiological expression pattern in hematopoiesis. Here, we analyzed public transcriptome data from primary innate lymphoid cells (ILCs) for NKL homeobox gene activities and found that ILC3 expressed exclusively HHEX while in ILC1 and ILC2 these genes were silenced. Deregulation of the NKL-code promotes hematopoietic malignancies, including anaplastic large cell lymphoma (ALCL) which reportedly may derive from ILC3. Accordingly, we analyzed NKL homeobox gene activities in ALCL cell lines and investigated their role in this malignancy. Transcriptome analyses demonstrated low expression levels of HHEX but powerfully activated HLX. Forced expression of HHEX in ALCL cell lines induced genes involved in apoptosis and ILC3 differentiation, indicating tumor suppressor activity. ALCL associated NPM1-ALK and JAK-STAT3-signalling drove enhanced expression of HLX while discounting HHEX. Genomic profiling revealed copy number gains at the loci of HLX and STAT3 in addition to genes encoding both STAT3 regulators (AURKA, BCL3, JAK3, KPNB1, NAMPT, NFAT5, PIM3, ROCK1, SIX1, TPX2, WWOX) and targets (BATF3, IRF4, miR135b, miR21, RORC). Transcriptome data of ALCL cell lines showed absence of STAT3 mutations while MGA was mutated and downregulated, encoding a novel potential STAT3 repressor. Furthermore, enhanced IL17F-signalling activated HLX while TGFbeta-signalling inhibited HHEX expression. Taken together, our data extend the scope of the NKL-code for ILCs and spotlight aberrant expression of NKL homeobox gene HLX in ALCL. HLX represents a direct target of ALCL hallmark factor STAT3 and deregulates cell survival and differentiation in this malignancy.

NKL homeobox gene activities in normal and malignant myeloid cells.

Recently, we have documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis, which spotlights genes deregulated in lymphoid malignancies. Here, we enlarge this map to include normal NKL homeobox gene expressions in myelopoiesis by analyzing public expression profiling data and primary samples from developing and mature myeloid cells. We thus uncovered differential activities of six NKL homeobox genes, namely DLX2, HHEX, HLX, HMX1, NKX3-1 and VENTX. We further examined public expression profiling data of 251 acute myeloid leukemia (AML) and 183 myelodysplastic syndrome (MDS) patients, thereby identifying 24 deregulated genes. These results revealed frequent deregulation of NKL homeobox genes in myeloid malignancies. For detailed analysis we focused on NKL homeobox gene NANOG, which acts as a stem cell factor and is correspondingly expressed alone in hematopoietic progenitor cells. We detected aberrant expression of NANOG in a small subset of AML patients and in AML cell line NOMO-1, which served as a model. Karyotyping and genomic profiling discounted rearrangements of the NANOG locus at 12p13. But gene expression analyses of AML patients and AML cell lines after knockdown and overexpression of NANOG revealed regulators and target genes. Accordingly, NKL homeobox genes HHEX, DLX5 and DLX6, stem cell factors STAT3 and TET2, and the NOTCH-pathway were located upstream of NANOG while NKL homeobox genes HLX and VENTX, transcription factors KLF4 and MYB, and anti-apoptosis-factor MIR17HG represented target genes. In conclusion, we have extended the NKL-code to the myeloid lineage and thus identified several NKL homeobox genes deregulated in AML and MDS. These data indicate a common oncogenic role of NKL homeobox genes in both lymphoid and myeloid malignancies. For misexpressed NANOG we identified an aberrant regulatory network, which contributes to the understanding of the oncogenic activity of NKL homeobox genes.

Deregulated expression of NKL homeobox genes in T-cell lymphomas.

Recently, we have presented a scheme, termed "NKL-code", which describes physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis including main stages of T-, B- and NK-cell development. Aberrant activity of these genes underlies the generation of hematological malignancies notably T-cell leukemia. Here, we searched for deregulated NKL homeobox genes in main entities of T-cell lymphomas comprising angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), hepatosplenic T-cell lymphoma (HSTL), NK/T-cell lymphoma (NKTL) and peripheral T-cell lymphoma (PTCL). Our data revealed altogether 19 aberrantly overexpressed genes in these types, demonstrating deregulated NKL homeobox genes involvement in T-cell lymphomas as well. For detailed analysis we focused on NKL homeobox gene MSX1 which is normally expressed in NK-cells. MSX1 was overexpressed in subsets of HSTL patients and HSTL-derived sister cell lines DERL-2 and DERL-7 which served as models to characterize mechanisms of deregulation. We performed karyotyping, genomic and expression profiling, and whole genome sequencing to reveal mutated and deregulated gene candidates, including the fusion gene CD53-PDGFRB. Subsequent knockdown experiments allowed the reconstruction of an aberrant network involved in MSX1 deregulation, including chromatin factors AUTS2 and mutated histone HIST1H3B(K27M). The gene encoding AUTS2 is located at chromosome 7q11 and may represent a basic target of the HSTL hallmark aberration i(7q). Taken together, our findings highlight an oncogenic role for deregulated NKL homeobox genes in T-cell lymphoma and identify MSX1 as a novel player in HSTL, implicated in aberrant NK- and T-cell differentiation.

Epstein-Barr virus (EBV) activates NKL homeobox gene HLX in DLBCL.

NKL homeobox genes encode developmental transcription factors regulating basic processes in cell differentiation. According to their physiological expression pattern in early hematopoiesis and lymphopoiesis, particular members of this homeobox gene subclass constitute an NKL-code. B-cell specific NKL-code genes generate a regulatory network and their deregulation is implicated in B-cell lymphomagenesis. Epstein-Barr virus (EBV) infects B-cells and influences the activity of signalling pathways including JAK/STAT and several genes encoding developmental regulators. Therefore, EBV-infection impacts the pathogenesis and the outcome of B-cell malignancies including Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL). Here, we isolated EBV-positive and EBV-negative subclones from the DLBCL derived cell line DOHH-2. These subclones served as models to investigate the role of EBV in deregulation of the B-cell specific NKL-code members HHEX, HLX, MSX1 and NKX6-3. We showed that the EBV-encoded factors LMP1 and LMP2A activated the expression of HLX via STAT3. HLX in turn repressed NKX6-3, SPIB and IL4R which normally mediate plasma cell differentiation. In addition, HLX repressed the pro-apoptotic factor BCL2L11/BIM and hence supported cell survival. Thus, EBV aberrantly activated HLX in DLBCL, thereby disturbing both B-cell differentiation and apoptosis. The results of our study appreciate the pathogenic role of EBV in NKL homeobox gene deregulation and B-cell malignancies.

NKL homeobox gene activities in B-cell development and lymphomas.

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.