Fig 4 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Regulatory mutations at HMX2/3 in EOL-1. The illustration of RNA-seq data from EOL-1 shows two different mutations at the HMX2/3 locus. (A) An A-to-G mutation in the 5´-region of HMX3 generates a consensus ETS-site. (B) A T-to-C mutation in the 5´-region of HMX2 transforms an NFkB-site into a SP1-site. (C) Quantification of ETS1 (above) and ELK1 (below) in selected AML cell lines by RQ-PCR. (D) Forced expression of ETS1 (above) and ELK1 (below) in EOL-1 cells resulted in respectively reduced and increased expression levels of both HMX2 and HMX3. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (vector) and factor expression. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing a normal (obtained from MV4-11) or mutated (obtained from EOL-1) ETS-site. Forced expression of ELK1 (left) and ETS1 (right) resulted in elevated reporter-gene activity which was higher using ELK1 for the mutated fragment. (F) Reporter-gene assay in HELA cells using a fragment containing a normal (MV4-11) or mutated (EOL-1) NFkB-site. Treatment with NFkB-activator TNFa (left) resulted in reduced reporter-gene activity which was more pronounced using the normal fragment. Forced expression of SP1 (right) resulted in elevated reporter-gene activity which was higher using the mutated fragment.
Source publication
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 pro...
Contexts in source publication
Context 1
... genomic sequence data of the HMX2/3 locus revealed two mutations, respectively altering two different potential transcription factor binding sites in this cell line. First, in the upstream region of HMX3 we identified an A-to-G mutation, generating of a novel consensus ETS-site: ACCGGAA (Fig 4A). Second, within the intergenic region upstream of HMX2 we identified a T-to-C mutation which transforms a potential NFkB-site into a non-consensus SP1-site: GGAGTCGCC (Fig 4B). ...
Context 2
... in the upstream region of HMX3 we identified an A-to-G mutation, generating of a novel consensus ETS-site: ACCGGAA (Fig 4A). Second, within the intergenic region upstream of HMX2 we identified a T-to-C mutation which transforms a potential NFkB-site into a non-consensus SP1-site: GGAGTCGCC (Fig 4B). Interestingly, both alterations chimed with our previous findings, showing that ETS1 levels were reduced (S7B Fig) and that NFkB-activator TNFa inhibited HMX2/3 expression (Fig 3C). ...
Context 3
... analysis confirmed absent expression of ETS1 in EOL-1 and MV4-11 while ETSfactor ELK1 was rather uniformly expressed in AML cell lines (Fig 4C). Consistent with this picture, forced expression of ETS1 in EOL-1 inhibited expression of both HMX2 and HMX3 while ELK1 boosted their activity (Fig 4D). ...
Context 4
... analysis confirmed absent expression of ETS1 in EOL-1 and MV4-11 while ETSfactor ELK1 was rather uniformly expressed in AML cell lines (Fig 4C). Consistent with this picture, forced expression of ETS1 in EOL-1 inhibited expression of both HMX2 and HMX3 while ELK1 boosted their activity (Fig 4D). ChIP-seq data for ELK1 generated in HELA-S3 cells showed strong binding at the transcription start site of HMX3 and weak binding in the upstream region matching the position of the detected mutation in EOL-1 (S9 Fig) [43]. ...
Context 5
... we transfected NIH-3T3 cells with reporter-constructs containing either an incomplete or the novel consensus ETS-site. Additionally, the cells were transfected with an expression-construct for ETS-factor ELK1 (Fig 4E). Our data demonstrated that ELK1 activated the mutated construct significantly more strongly than the wild type sequence. ...
Context 6
... data demonstrated that ELK1 activated the mutated construct significantly more strongly than the wild type sequence. Additional transfection of an expression-construct for ETS1 also activated the mutated construct more strongly, however, this effect was much weaker as compared to ELK1 (Fig 4E). These data show that both, ETS1 and ELK1 are activators of HMX2/3. ...
Context 7
... TNFa-sensitive HELA cells were transfected with reporter-constructs containing normal or mutated NFkB-sites and additionally treated with NFkB-activator TNFa (Fig 4F). The results showed that TNFa inhibited the reporter-gene significantly more via the normal site, suggesting that this mutation reduced binding of suppressive NFkB. ...
Context 8
... results showed that TNFa inhibited the reporter-gene significantly more via the normal site, suggesting that this mutation reduced binding of suppressive NFkB. Furthermore, forced expression of SP1 resulted in elevated reporter-gene activity which was significantly higher using the construct with the mutated site (Fig 4F). Thus, this mutation decreased the inhibitory input of NFkB and simultaneously increased activation by the general transcription factor SP1. ...
Citations
... ᵗ 30 In addition, we found high expression of XAGE1A and XAGE1B, which studies have proposed as potential targets for immunotherapy. 31,32 Based on gene expression, we could not confirm established genetic groupings of KMT2A rearrangements, but rather find KMT2A-MLLT3, KMT2A-MLLT10, and KMT2A-MLLT1 together and KMT2A-MLLT4 separately to form two transcriptional groups, suggesting marked differences in the disease. ...
Subtyping of acute myeloid leukaemia (AML) has made significant progress, exemplified by the recent
classification updates by the World Health Organization and International Consensus Classification.
AML subclassification is predominantly genetics-based, despite research showing the benefits of
transcriptomic profiling on top of known genetic markers. However, a comprehensive survey of
subtypes in AML defined by gene expression has yet to be performed. To this end, we integrated
mRNAseq data from 1337 patients and five studies, with corresponding biological and clinical data. We
defined 19 gene expression-based subtypes, further stratifying AML. We found that KMT2A leukaemias
with fusion partners MLLT3, MLLT10 and MLLT1 clustered together, while KMT2A-MLLT4 displayed a
distinct gene expression pattern, suggesting differences in their aetiology. We discovered a
transcriptional CEBPA subtype, of which only 40% had a CEBPA bZIP indel. Regardless of mutation
status, all patients within this CEBPA cluster had the same favourable outcome. We found four NPM1-
enriched transcriptomic subtypes, each with distinct co-mutation patterns and associated ex-vivo drug
responses. Similarly, we identified nine AML with myelodysplasia-related changes (AML-MRC)
subtypes, dividing a subtype making up one-third of the AML patients into novel groups with different
outcomes and drug response profiles. In conclusion, we provide an unprecedented overview of the
transcriptomic subtypes in AML and illustrate their potential for AML diagnostics.
... Second, we performed ANANSE-CAGE to predict TFs unique to THP-1 by comparing GRNs between primary samples (source type) and THP-1 cells (target type). Surprisingly, most of the top ranked factors were associated with neural and craniofacial development (BARX1, BHLHE22, OTX1, SP9, ALX1, and HMX3) ( Fig. 5b and Supplementary Table S1) 47,48 . These results give us more insights into which TFs regulate key gene programs to keep THP-1 cells indefinite in cell culture, in contrast to primary AML cells. ...
Advanced computational methods exploit gene expression and epigenetic datasets to predict gene regulatory networks controlled by transcription factors (TFs). These methods have identified cell fate determining TFs but require large amounts of reference data and experimental expertise. Here, we present an easy to use network-based computational framework that exploits enhancers defined by bidirectional transcription, using as sole input CAGE sequencing data to correctly predict TFs key to various human cell types. Next, we applied this Analysis Algorithm for Networks Specified by Enhancers based on CAGE (ANANSE-CAGE) to predict TFs driving red and white blood cell development, and THP-1 leukemia cell immortalization. Further, we predicted TFs that are differentially important to either cell line- or primary- associated MLL-AF9-driven gene programs, and in primary MLL-AF9 acute leukemia. Our approach identified experimentally validated as well as thus far unexplored TFs in these processes. ANANSE-CAGE will be useful to identify transcription factors that are key to any cell fate change using only CAGE-seq data as input.
... As analyzed so far, aberrantly expressed NKL homeobox genes show no mutations in their coding regions. Mutations in non-coding regulatory regions have been reported for HMX2 and HMX3 in acute myeloid leukemia (AML), highlighting transcriptional deregulation as a main pathological cause [51]. The following account summarizes deregulating mechanisms and oncogenic functions of selected NKL homeobox genes hitherto described in T-ALL. ...
... However, NKX2-5 is rarely expressed in this malignancy [18,58]. Normally, NKX2-5 plays a key physiological role in the development of the spleen and heart [16,51]. Uniting its physiological and leukemic roles, myocyte-specific enhancer factor 2C (MEF2C) has been shown to serve as a target gene of NKX2-5 in both heart and leukemic T-cells [59,60]. ...
Homeobox genes encode transcription factors controlling basic developmental processes. The homeodomain is encoded by the homeobox and mediates sequence-specific DNA binding and interaction with cofactors, thus operating as a basic regulatory platform. Similarities in their homeobox sequences serve to arrange these genes in classes and subclasses, including NKL homeobox genes. In accordance with their normal functions, deregulated homeobox genes contribute to carcinogenesis along with hematopoietic malignancies. We have recently described the physiological expression of eleven NKL homeobox genes in the course of hematopoiesis and termed this gene expression pattern NKL-code. Due to the developmental impact of NKL homeobox genes these data suggest a key role for their activity in the normal regulation of hematopoietic cell differentiation including T-cells. On the other hand, aberrant overexpression of NKL-code members or ectopical activation of non-code members has been frequently reported in lymphoid and myeloid leukemia/lymphoma, demonstrating their oncogenic impact in the hematopoietic compartment. Here, we provide an overview of the NKL-code in normal hematopoiesis and discuss the oncogenic role of deregulated NKL homeobox genes in T-cell malignancies.
... HMX2 and HMX3 show an absence of chromosomal rearrangements and are activated by IL7-signaling and via the mutation of two transcription factor-binding sites in an AML cell line (EOL-1). One mutation transforms an inhibitory NFkB-site into an activatory SP1-site while the other mutation generates an activating ETS-site [98]. Functional analyses revealed the inhibition of differentiation gene EPX and activation of the receptor HTR7 that drives oncogenic ERK-signaling [98]. ...
... One mutation transforms an inhibitory NFkB-site into an activatory SP1-site while the other mutation generates an activating ETS-site [98]. Functional analyses revealed the inhibition of differentiation gene EPX and activation of the receptor HTR7 that drives oncogenic ERK-signaling [98]. Furthermore, HMX2 and HMX3 inhibit eosinophilic cell differentiation as shown by morphological changes in the AML cell line model [98]. ...
... Functional analyses revealed the inhibition of differentiation gene EPX and activation of the receptor HTR7 that drives oncogenic ERK-signaling [98]. Furthermore, HMX2 and HMX3 inhibit eosinophilic cell differentiation as shown by morphological changes in the AML cell line model [98]. ...
Simple Summary
Gene codes represent expression patterns of closely related genes in particular tissues, organs or body parts. The NKL-code describes the activity of NKL homeobox genes in the hematopoietic system. NKL homeobox genes encode transcription factors controlling basic developmental processes. Therefore, aberrations of this code may contribute to deregulated hematopoiesis including leukemia and lymphoma. Normal and abnormal activities of NKL homeobox genes are described and mechanisms of (de)regulation, function, and diseases exemplified.
Abstract
We have recently described physiological expression patterns of NKL homeobox genes in early hematopoiesis and in subsequent lymphopoiesis and myelopoiesis, including terminally differentiated blood cells. We thereby systematized differential expression patterns of eleven such genes which form the so-called NKL-code. Due to the developmental impact of NKL homeobox genes, these data suggest a key role for their activity in normal hematopoietic differentiation processes. On the other hand, the aberrant overexpression of NKL-code-members or the ectopical activation of non-code members have been frequently reported in lymphoid and myeloid leukemia/lymphoma, revealing the oncogenic potential of these genes in the hematopoietic compartment. Here, I provide an overview of the NKL-code in normal hematopoiesis and instance mechanisms of deregulation and oncogenic functions of selected NKL genes in hematologic cancers. As well as published clinical studies, our conclusions are based on experimental work using hematopoietic cell lines which represent useful models to characterize the role of NKL homeobox genes in specific tumor types.
Hematopoietic cancers (HCs) are a heterogeneous group of malignancies that affect blood, bone marrow and lymphatic system. Here, by analyzing 1,960 RNA-Seq samples from three independent datasets, we explored the co-expression landscape in HCs, by inferring gene co-expression networks (GCNs) with four cancer phenotypes (B and T-cell acute leukemia -BALL, TALL-, acute myeloid leukemia -AML-, and multiple myeloma -MM-) as well as non-cancer bone marrow. We characterized their structure (topological features) and function (enrichment analyses). We found that, as in other types of cancer, the highest co-expression interactions are intra-chromosomal, which is not the case for control GCNs. We also detected a highly co-expressed group of overexpressed pseudogenes in HC networks. The four GCNs present only a small fraction of common interactions, related to canonical functions, like immune response or erythrocyte differentiation. With this approach, we were able to reveal cancer-specific features useful for detection of disease manifestations.
Significance
We demonstrate that gene co-expression is deregulated in four HC, observed by an elevated proportion of intrachromosome interactions in their GCNs with respect to their normal counterparts, and increased interactions between pseudogenes (more evident in AML). This deregulation might be associated with the age of the patients.
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.
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.
