Overexpression of HOX genes is prevalent in Ewing sarcoma and is associated with altered epigenetic regulation of developmental transcription programs

<div><p>The polycomb proteins BMI-1 and EZH2 are highly overexpressed by Ewing sarcoma (ES), a tumor of stem cell origin that is driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. In the current study we analyzed expression of transcription programs that are controlled by polycomb proteins during embryonic development to determine if they are abnormal in ES. Our results show that polycomb target gene expression in ES deviates from normal tissues and stem cells and that, as expected, most targets are relatively repressed. However, we also discovered a paradoxical up regulation of numerous polycomb targets and these were highly enriched for homeobox (HOX) genes. Comparison of HOX profiles between malignant and non-malignant tissues revealed a distinctive HOX profile in ES, which was characterized by overexpression of posterior HOXD genes. In addition, ectopic expression of EWS-FLI1 during stem cell differentiation led to aberrant up regulation of posterior HOXD genes. Mechanistically, this up regulation was associated with altered epigenetic regulation. Specifically, ES and EWS-FLI1+ stem cells displayed a relative loss of polycomb-dependent H3K27me3 and gain of trithorax-dependent H3K4me3 at the promoters of posterior HOXD genes and also at the HOXD11.12 polycomb response element. In addition, a striking correlation was evident between <i>HOXD13</i> and other genes whose regulation is coordinately regulated during embryonic development by distal enhancer elements. Together, these studies demonstrate that epigenetic regulation of polycomb target genes, in particular HOXD genes, is altered in ES and that these changes are mediated downstream of EWS-FLI1.</p></div

Construction and characterization of the chNKG2D.

<p><i>A,</i> The figure schematically illustrates the domain architecture of wildtype NKG2D and chNKG2D (N = N-terminus, C = C-terminus, CM = cytoplasma membrane). The N-terminal truncation of the extracellular NKG2D domain at aa81 is indicated by a bold bar. The native C-terminal end of NKG2D was fused to the N-terminus of the IgG1-Fc/CD28/CD3ζ transmembrane signaling platform creating a transmembrane receptor of type I orientation in which the relative orientation and positioning of the NKG2D ectodomain has not been changed. <i>B,</i> efficient transfection and strong surface expression of chNKG2D by electroporation of mRNA into primary T cells. The histograms show the surface expression of chNKG2D 24 hours after electroporation of quiescent or anti-CD3-activated human T cells (thick line, open histogram). Analysis was performed by flow cytometry using an anti-CD314 antibody. The expression of endogenous wildtype NKG2D in T transfected with a CMV-gH-specific CAR is shown for comparison (thin line, open histograms; filled histograms = isotype controls). <i>C,</i> chNKG2D triggers cytotoxicity of activated T cells. Quiescent or anti-CD3-activated CD8^pos or CD4^pos T cells were transfected with RNA encoding either the chNKG2D or a CMV-gH-specific control receptor. One day after transfection, the T cells were co-cultured with dye-labeled murine B cells (Ba/F3-MICA) stably expressing the human NKG2D-L MICA. The diagram shows the percentage of Annexin V^pos murine target cells after 5 hours of co-culture at the indicated E∶T ratios (data pooled from three independent experiments using different cell donors; mean ± S.D.). Statistical significance was calculated for the effects of activated T cells expressing the chNKG2D versus T cells expressing the control receptor.</p

Stability of receptor surface expression upon target-cell encounter.

<p>The histograms show the flow cytometric analysis of chNKG2D expression (detected in CD4^pos T cells by an anti-CD314 antibody or in CD8^pos T cells by an anti-human-IgG1-Fc antibody; log scale in all histograms; note: amplification voltage in flow cytometric analysis was lowered for lentivirally transduced T cells due to the strong expression of the receptor). <i>A,</i> comparison of lentivirally transduced and mRNA transfected T cells. 75,000 chNKG2D^pos CD4^pos T cells were cultured 24 hours after electroporation for further 24 hours in presence or absence of 200,000 STA-ET-3 cells (filled grey histograms = presence of STA-ET-3, open histograms bold line = absence of STA-ET-3, open histograms dotted line = T cells not transfected). Shown is one representative experiment out of three independent repetitions. <i>B,</i> chNKG2D down-regulation in response to different ESFT cell lines. 100,000 chNKG2D^pos CD8^pos T cells were co-cultured 50 hours after mRNA electroporation for further 24 hours with 100,000 cells of different ESFT cell lines as indicated (unmodified T cells = “without chNKG2D”; without co-culture = “without target cells”). <i>C,</i> kinetics of chNKG2D down-regulation upon target-cell encounter at different times after transfection. 20,000 chNKG2D^pos CD4^pos T cells were co-cultured with 60,000 STA-ET-3 target cells for different times (1, 4, or 48 hours) before chNKG2D expression was determined (6, 30, or 74 hours after transfection) (one experiment). <i>D,</i> comparison of differentially stabilized mRNA species. ChNKG2D^pos CD4^pos T cells were co-cultured with STA-ET-3 target cells at different effector∶target ratios and at different times after transfection. Target cells were added at the indicated ratios either two or 26 hours after transfection and chNKG2D expression was determined by flow cytometry 6, 30, 50, or 74 hours after transfection. Shown is one out of three experiments. <i>E,</i> effect of patient serum containing soluble NKG2D-Ls. 15,000 chNKG2D^pos CD4^pos T cells were cultured 24 hours after electroporation for further 24 hours with or without serum of two different neuroblastoma patients (final serum concentration 33%).</p

Expression of NKG2D-Ls in ESFT.

<p>The histograms show the surface expression of the respective NKG2D-Ls recognized by specific monoclonal antibodies and analyzed by flow cytometry (filled histograms = specific antibodies; open histograms = isotype controls). The two stably transfected murine B cell lines (Ba/F3) expressing either human MICA or ULBP2 are displayed for comparison.</p

Lentiviral transduction for transfer of chNKG2D.

<p><i>A,</i> antigen-specific enrichment of lentivirally transduced chNKG2D^pos MNCs. On day 13 after transduction the cells were co-cultured either with Ba/F3, ULBP2^pos Ba/F3 or without target cells. FACS analysis was performed 6 days later (open histogram, bold line: co-culture with the ULBP2^pos Ba/F3, 86.7% chNKG2D^pos T cells; filled histogram, dotted line: co-culture with Ba/F3, 60.7% chNKG2D^pos T cells; thin line: cells cultured without target cells, 54.1% chNKG2D^pos T cells; open histogram, dotted line = secondary antibody only). <i>B,</i> cytokine production. The diagram shows TNF secretion of purified CD4^pos T cells lentivirally transduced with either chNKG2D (enriched to 83–92% positive cells) or a similar chimeric CMV-specific control receptor (enriched to 69–81%) in response to the indicated cell lines or only medium as control (measured by ELISA; mean of duplicates). Ba/F3 or ULBP2^pos Ba/F3 cells were used for negative and positive control. Statistical significance was calculated for the effects of T cells expressing the chNKG2D versus T cells expressing the control receptor. <i>C,</i> Comparison of the cytotoxic response triggered by chNKG2D either stably or transiently transferred. CD8^pos T cells either unmodified (“without chNKG2D”) or expressing chNKG2D by lentiviral transduction (“LV”; enriched for chNKG2D^pos >90%) or mRNA transfection (“mRNA”; 20 hours after electroporation) were co-cultured with dye-labeled murine Ba/F3 cells expressing human ULBP2 (ULBP2-Ba/F3) or not (CTRL-Ba/F3). The diagram shows the percentage of Annexin V^pos Ba/F3 target cells after 4 hours of co-culture at the indicated E∶T ratios (mean ± S.D. of triplicates).</p