Fig 1.
Expression of HMX genes in AML cell lines.
(A) Gene expression values (DESeq2 normalized count data) are shown according to RNA-seq data from the LL-100 dataset for HMX2 (above) and HMX3 (below). The color-code indicates AML-subgroups: erythroid (brown), megakaryocytic (yellow), monocytic (orange), myelocytic (violet); CML-subtypes: myeloid (green), lymphoid (blue), myeloproliferativ neoplasm (just cell line SET-2). Of note, the scale illustrating the transcript amount is much smaller for HMX1. According to the setting of the cut-off at 500 normalized counts no cell line is positive for HMX1. The expression of HMX2 (B) and HMX3 (C) was analyzed in selected cell lines and primary samples from granulocytes by RQ-PCR and Western blot (inserts). TUBA served as loading control. Significant expression was detected in cell lines EOL-1, MOLM-13 and MV4-11 which all carry KMT2A aberrations. Of note, HL-60 expressed low levels of HMX3 but no HMX2 RNA. WT: wild type.
Fig 2.
Regulation of HMX2 and HMX3 by KMT2A and ERK-signalling.
(A) RQ-PCR analysis of EOL-1 (left) and MV4-11 (middle) treated for siRNA-mediated knockdown of KMT2A demonstrates raised expression levels of HMX2 and HMX3, indicating a repressive impact of KMT2A. This treatment resulted in enhanced expression of HMX3 in HL-60 cells which did not express HMX2 (right). Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. (B) RQ-PCR analysis of KMT2A of EOL-1 cells treated for siRNA-mediated knockdown of HMX2 (left) and HMX3 (right) showed unaltered KMT2A expression levels, discounting any regulatory impact. (C) Quantification of KMT2A transcripts in AML cell lines and primary granulocytes showed low expression levels in EOL-1, MV4-11 and in eosinophils. (D) RQ-PCR analysis of HMX2 showed elevated expression levels in EOL-1 cells treated with PDGFRA-inhibitor dasatinib. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (DMSO) and pharmacological treatments. (E) Treatment of EOL-1 cells with ERK-inhbitor PD98059 resulted in reduced levels of phospho-ERK as analyzed by Western blot (left), and in elevated transcript levels of HMX2 (middle) and HMX3 (right) as analyzed by RQ-PCR. (F) Treatment of EOL-1 cells with ERK-activator FLT3LG resulted in elevated levels of phosphorylated ERK (above) and in reduced transcript levels of HMX2 and HMX3 (below).
Fig 3.
Identification of upstream factors regulating HMX2 and HMX3.
(A) Treatment of EOL-1 cells with IL7 resulted in increased transcript levels of HMX2 and HMX3 as analyzed by RQ-PCR. Asterisks indicate calculated p-values obtained by t-Test analysis of medium controls and interleukin stimulation. (B) Treatment of EOL-1 and MV4-11 cells with WNT3A and WNT5B resulted in increased expression levels of HMX2 (left) and HMX3 (right). (C) Treatment of EOL-1 cells with NFkB-activator TNFa resulted in reduced expression levels of HMX2 and HMX3 (left). Treatment of EOL-1 cells with NFkB-inhibitor resulted in increased expression of HMX2 and HMX3 (middle). STAT5A served as marker for NFkB-activity which showed low expression levels and increased activity after stimulation with TNFa (right). (D) A genomic map of the locus for HMX2/3 was obtained from the UCSC genome browser, showing potential transcription factor binding sites including those for IRF1, IRF2 and NKX2-5. (E) RQ-PCR analysis of EOL-1 cells treated for siRNA-mediated knockdown of IRF8 resulted in elevated expression levels of HMX2 (left) and HMX3 (right), indicating an activating impact of IRF8. (F) Quantification of IRF8 by RQ-PCR in primary granulocytes and monocytes (left) and selected AML cell lines (right) demonstrated significant levels in EOL-1 and MV4-11. (G) RQ-PCR analysis of EOL-1 (left) and MV4-11 (right) treated for siRNA-mediated knockdown of HMX3 and HMX2 demonstrated slightly reduced expression levels of HMX2 and HMX3, respectively, indicating mutual activation.
Fig 4.
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.
Fig 5.
(A) Treatment of EOL-1 cells with DMSO for three days induced morphological alterations as shown by Giemsa-May-Grünwald staining. The nuclei became typically indented like kidney beans which can be interpreted as eosinophilic differentiation. (B) The same treatment resulted in decreased expression of HMX2 (left) and HMX3 (right) as analyzed by RQ-PCR. SiRNA-mediated knockdown of HMX2 (C) or treatment with TNFa (D) generated the same nuclear indentation as visible after DMSO-treatment, indicating that HMX2/3 inhibited eosinophilic differentiation. (E) Marker expression. (F) Marker expression. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown.
Fig 6.
Regulation of EPX by HMX2 and HMX3.
(A) Quantification of EPX expression in primary granulocytes (neutrophils, eosinophils, basophils) and selected cell lines by RQ-PCR. (B) SiRNA-mediated knockdown of HMX2 (left) and HMX3 (right) resulted in elevated expression levels of EPX. Reduced expression of HMX2 and HMX3 and elevated expression of EPX was also shown by Western blot analysis. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. (C) Forced expression of HMX2 in HL-60 cells resulted in reduced expression level of EPX (left). SiRNA-mediated knockdown of EPX resulted in activation of CD11B expression (right). (D) A genomic map of the locus for EPX was obtained from the UCSC genome browser, showing potential transcription factor binding sites including one for HMX1. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing the identified HMX-site. Forced expression of HMX2 resulted in decreased reporter-gene activity, demonstrating a direct repressive impact. (F) Forced expression of EPX in EOL-1 slightly reduced the expression level of CD11B. Microscopical inspection of Giemsa-May-Grünwald-stained EOL-1 cells indicated strongly induced apoptosis by forced EPX expression as possible consequence of continued differentiation. Living cells resemble differentiated cells after DMSO-induction.
Fig 7.
Regulation of HTR7 by HMX2 and HMX3.
(A) Quantification of HTR7 expression in primary granulocytes (neutrophils, eosinophils, basophils), monocytes, and selected cell lines by RQ-PCR. (B) Genomic profiling data demonstrated a duplication in EOL-1 which encompassed the flanking regulatory region of HTR7. (C) A genomic map of the locus for HTR7 was taken from the UCSC genome browser, showing potential transcription factor binding sites. The indicated duplication detected in EOL-1 containes a potential HMX1-site. (D) SiRNA-mediated knockdown of HMX2 (left) and HMX3 (middle) resulted in reduced expression levels of HTR7, indicating an activating impact. Asterisks indicate calculated p-values obtained by t-Test analysis of controls (siCTR) and siRNA-targeted knockdown. Forced expression of HMX2 in HL-60 resulted in elevated transcript levels of HTR7 (right), supporting an activating impact. (E) Reporter-gene assay in NIH-3T3 cells using a fragment containing the identified HMX1-site. Forced expression of HMX2 resulted in decreased reporter-gene activity, demonstrating a direct regulatory impact. (F) Treatment of EOL-1 cells with HTR7-inhibitor MMS and HTR7-activator LP211 resulted in decreased and increased levels of ERK-phosphorylation, respectively, as shown by Western blot. (G) Treatment of EOL-1 cells with HTR7-inhibitor MMS and HTR7-activator LP211 resulted in decreased and increased levels of DLX2-transcription, respectively, as shown by RQ-PCR.
Fig 8.
Gene regulatory network for HMX2 and HMX3 in AML.
This diagram summarizes the results of this study, showing a gene regulatory network. HMX2 and HMX3 are located centrally, activating and repressing factors and pathways are indicated above, and the target genes EPX and HTR7 below. Deregulation of EPX and HTR7 indicates aberrant effects for differentiation and proliferation. Genomic rearrangements targeting KMT2A or PDGFRA are indicated.