Inactivation of RUNX3/p46 Promotes Cutaneous T-Cell Lymphoma Ahmed Haider, Anne Steininger, Reinhard Ullmann, Michael Hummel, Lora Dimitrova, Marc Beyer, Staffan Vandersee, Dido Lenze, Wolfram Sterry, Chalid Assaf, Markus Möbs  Journal of Investigative Dermatology  Volume 136, Issue 11, Pages 2287-2296 (November 2016) DOI: 10.1016/j.jid.2016.05.126 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Structure of the human RUNX3 locus and the transcripts coding for RUNX3/p47 and RUNX3/p44. The boxes represent the different exons. Translated regions are marked in light grey. Dark grey areas indicate the localization of the DNA-binding Runt domain. Dotted lines indicate that the untranslated part of the last exon is not entirely displayed. ∗Initiation codons. P1, distal promoter; P2, proximal promoter. Journal of Investigative Dermatology 2016 136, 2287-2296DOI: (10.1016/j.jid.2016.05.126) Copyright © 2016 The Authors Terms and Conditions

Figure 2 RUNX3 is inactivated by genomic deletion and promoter methylation but not by cytoplasmic retention. (a) Physical position and frequency of genomic aberrations at chromosome 1 in tumor cells of 23 SS patients. Gains and losses at chromosome 1 are plotted according to their frequency (%) to the right and left of the idiogram, respectively. Aberrations within the region at 22–31 Mb (hg18) are enlarged at the left panel with losses (red) and gains (green) depicted as bars. Color saturation represents the average log2 ratio of aberrations. The arrow indicates the genomic position of RUNX3. (b) FISH analysis of PBMCs from SS patients and CTCL cell lines using a gene-specific probe for RUNX3 (red) and a centromeric probe for chromosome 1 (green) (scale bar = 25 μm). Cell nuclei are stained with DAPI (blue). (c) Bisulfite sequencing results for the distal RUNX3 promoter. Chromatograms of bp –200 to –181 relative to ENST00000399916 are shown for methylated control DNA, for two representative SS patients showing either complete methylation (SS_#21) or mixed methylation of CpG sites (SS_#4), and one control, as indicated. CpG sites are marked by red arrows. (d) Western blots of nuclear (N) and cytoplasmic (C) extracts from four CTCL cell lines. Whole-cell lysate of SeAx cells has been used as a positive control (+). Because of strong signals and overlapping sizes of target proteins, three different membranes were loaded with the same amount of the identical lysates and were incubated with the respective primary antibodies as depicted. GAPDH and TBP served as cytoplasmic and nuclear markers, respectively. (e) Immunofluorescence staining of RUNX3 protein in CTCL cell lines (scale bar = 25 μm). Formaldehyde-fixed CTCL cell lines were incubated with RUNX3 antibody and counterstained with an Alexa Fluor 594 coupled secondary antibody (red). Nuclei were visualized with DAPI. Original magnification ×400. bp, base pairs; chr., chromosome; CTCL, cutaneous T-cell lymphoma; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBMC, peripheral blood mononuclear cell; SS, Sézary syndrome; TBP, TATA-box binding protein. Journal of Investigative Dermatology 2016 136, 2287-2296DOI: (10.1016/j.jid.2016.05.126) Copyright © 2016 The Authors Terms and Conditions

Figure 3 RUNX3 gene expression analysis in SS tumor cells. (a) Quantification of RUNX3/p46 mRNA levels in CD4-enriched blood circulating SS cells from 11 patients and CD4+ memory T cells from healthy volunteers, as indicated. RUNX3 mRNA expression from the distal promoter was analyzed relative to TBP by quantitative real-time PCR using primers recognizing only RUNX3/p46. Error bars indicate standard error of the mean normalized expression values of three technical replicates. The presence of RUNX3 deletions, methylation of the distal promoter (P1 meth.), and presence of rs6672420 (SNP) is depicted below each SS sample. (b) RUNX3 mRNA expression was analyzed as in a, with primers recognizing RUNX3/p46 and RUNX3/p44. (c) Whisker-box plots of the relative RUNX3 mRNA expression ratios between SS samples and healthy control samples as measured in a (left) and b (right). The expression ratios between both groups as well as P-values were calculated with REST 2009, version 2.0.13. The dotted line represents the median relative expression value in SS tumor cells, the box area encompasses 50% of all observations, and the whiskers represent the outer 50% of observations. (d) Quantification of RUNX3 mRNA levels in four CTCL cell lines and the T-ALL cell line Jurkat. Relative expression to TBP was analyzed by real-time PCR with primers recognizing both RUNX3 transcripts (RUNX3, black bars) or only RUNX3/p46 mRNA (white bars). Error bars indicate standard error of the mean normalized expression values of three technical replicates. Note the different scaling of y-axes in the subfigures. CTCL, cutaneous T-cell lymphoma; P1 meth., methylation of the distal promoter; SNP, single-nucleotide polymorphism; SS, Sézary syndrome; T-ALL, T-cell acute lymphoblastic leukemia; TBP, TATA-box binding protein. Journal of Investigative Dermatology 2016 136, 2287-2296DOI: (10.1016/j.jid.2016.05.126) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Reconstitution of RUNX3/p46 expression in MyLa cells. (a) MyLa cells were transfected with a C-terminally HA-tagged RUNX3/p46 expression construct or empty vector (MOCK). At 48 hours after transfection, the proportion of dead cells was visualized by trypan blue staining. The y-axis shows the percentage of trypan blue-positive cells. Error bars indicate the standard deviation of five independent experiments performed in triplicate. (b) MyLa cells were treated as in a, and the percentage of apoptotic cells was determined by staining of ethanol-fixed cells with propidium iodide and subsequent flow cytometry. Values are given as percentage of apoptotic cells (SubG1 population). Error bars indicate standard deviation of four independent experiments performed in triplicate. (c) Histogram plots of one representative experiment, as shown in b, also showing the fraction of cells in the different cell cycle phases depicted in percentage of overall cells. (d) Western blot analysis of RUNX3/p46 or MOCK transfected MyLa cells. Cells were treated as in a. Upper lane, analysis of RUNX3/p46 protein expression by anti-HA antibody showing a robust expression of the transgene. Middle lane, analysis of caspase-3 cleaving products (p19, p17) showing a further procession of p19 to the highly active p17 cleavage product in RUNX3/p46 transfected MyLa cells. Lower lane, GAPDH protein expression showed equal loading of both samples. All proteins were visualized on one identical membrane after stripping and re-incubation with the respective primary antibodies. (e) Equal numbers of untransfected, MOCK transfected, and RUNX3/p46 transfected trypan blue-negative cells were cultivated for another 24 hours and the numbers of viable cells were determined by WST-1. Values are given as percentages relative to untransfected cells. Error bars indicate standard deviation of four independent experiments performed in triplicates. ∗P ≤ 0.05. ∗∗P ≤ 0.01. All P-values were calculated by two-sided t test. Csp-3, caspase-3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOCK, empty vector control; WST-1, water soluble tetrazolium-1. Journal of Investigative Dermatology 2016 136, 2287-2296DOI: (10.1016/j.jid.2016.05.126) Copyright © 2016 The Authors Terms and Conditions