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T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients’ prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect. CA extern

Simple Summary Approximately 60% of all melanomas are associated with a constitutive activating BRAF mutation. Inhibition of BRAF downstream signaling by targeted therapies significantly improved patient outcomes. However, most patients eventually develop resistance. Here we identified miR-129-5p as a novel tumor suppressor in BRAF mutated melanoma, which expression is increased during response to BRAF inhibition, but repressed in an EZH2 dependent manner during activated BRAF signaling. Overexpression of miR-129-5p decreases melanoma cell proliferation and improves response to BRAF inhibition by targeting SOX4. Taken together our results emphasize SOX4 as a potential therapeutic target in BRAF driven melanoma which could be attacked by pharmaceutically. Abstract Many melanomas are associated with activating BRAF mutation. Targeted therapies by inhibitors of BRAF and MEK (BRAFi, MEKi) show marked antitumor response, but become limited by drug resistance. The mechanisms for this are not fully revealed, but include miRNA. Wishing to improve efficacy of BRAFi and knowing that certain miRNAs are linked to resistance to BRAFi, we wanted to focus on miRNAs exclusively associated with response to BRAFi. We found increased expression of miR-129-5p during BRAFi treatment of BRAF- mutant melanoma cells. Parallel to emergence of resistance we observed mir-129-5p expression to become suppressed by BRAF/EZH2 signaling. In functional analyses we revealed that miR-129-5p acts as a tumor suppressor as its overexpression decreased cell proliferation, improved treatment response and reduced viability of BRAFi resistant melanoma cells. By protein expression analyses and luciferase reporter assays we confirmed SOX4 as a direct target of mir-129-5p. Thus, modulation of the miR-129-5p-SOX4 axis could serve as a promising novel strategy to improve response to BRAFi in melanoma.

Additional file 8: Figure S3. (A-B) GSEA of RANBP3L dependent genes shows enrichment of the REACTOME Paythways “degradation of extracellular matrix” and “collagen formation” in RANBP3L-KO cells. (C) Mean log2 fold change of 1771 overlapping genes with prognostic favorable (blue), unfavorable (yellow) and uncategorized (black) genes in RCC. (D-E) RT-qPCR validation of different RANBP3L-KO clones (each in triplicates) for (D) Sparc, Col4a1, Col4a2 and (E) Sh3gl2, Aldh2 and Hoxa7. Values represent mean ± SEM (error bars), n.s. > 0.05, *, p

Additional file 7: Figure S2. (A) Phase contrast images of Scr, R1 and R3 mpkCCD cells with 300 mosmol/kg and 600 mosmol/kg. Arrowheads indicate colony-detaching cells. Scale bar: 400 μm (B) Fluorescence of F-Actin staining in Scr, R1 and R3 mpkCCD cells using Phalloidin coupled to Alexa-568 and DAPI for nucleus visualization (Scale bar: 100 μm). (C-D) TIDE (Tracking of Indels by Decomposition) analysis of R1 (C) and R3 (D) single clone revealing INDELs in Ranbp3l target site [26]. (E-F) Proliferation analyses of Scr, R1 and R3 mpkCCD cells. Cells were cultivated in 96-well plates and the proliferation was measured by live-cell imaging using the IncuCyte S3 system taking an image every 4 h for 24 h. The relative cell density was calculated by IncuCyte S3 software and normalized to the cell density at 0 h. The data points were fitted by nonlinear exponential growth equation using GraphPad Prism to calculate the mean doubling times. Values represent mean ± SEM (error bars). n = 3 (with 4 technical replicates) n.s.,p > 0.05, **, p < 0.01; ***, p < 0.001, 1-way ANOVA.

Additional file 11: Figure S6. (A) RT-PCR of different RCC cell lines for RANBP3L and GAPDH. The cell lines were incubated in isoosmolar or hyperosmolar medium before RNA isolation. As a control normal kidney tissue and GAPDH was used. (B) Immunofluorescence images of different RCC cell lines showing NFAT5 nuclear translocation under hyperosmolar cultivation. Scale bar: 100 μm.

Additional file 6: Figure S1. (A) Schematic representation of domain organization in RANBP3L and RANBP3 (AS sequence in Table S1). (B) Median gene expression of RANBP3 and RANBP3L in a bodymap from the gene expression profiling interactive analysis tool (GEPIA2) [33]. (C) Schematic representation of IMCD isolation and culturing from murine kidneys for next-generation sequencing (NGS). (D-E) Analysis of NFAT5 intensity in mpKCCD immunofluorescence images (D) and in IMCD cell immunofluorescence images (E) shown in Fig. 2A. Scale bar 100 μm. (F-G) The NFAT5 fluorescence intensities of cells were plotted on a line graph. Lines correspond to the relative fluorescence of cells marked with a white arrow. F corresponds to signal intensity in mpkCCD cell while G corresponds to signal intensity of IMCD cells. (H) TIDE analysis of N1 and N3 single clones reveal INDELs in the Nfat5 target sites [26]. (I) Selected distribution of RNA-Seq reads across the Nfat5 gene for control and 4-OH-TM treated cells shows loss of exon 4. (J) Screen capture of human RANBP3L promotor region from the ECR Browser website with evolutionary conserved regions (ECR) in the genomes of rat, mouse, canis, macaca and pan. The lines in pink (arrows ahead) show ECR between human and the indicated mammals [28]. (K) 800 bp region uptream from RANBP3L start site is conserved in the indicated mammals and contains one conserved NFAT5 consensus sequence (NNTTTCCA is indicated in yellow) and one non-conserved (nucleotides are indicated in red). (L) Relative promotor activity of all Ranbp3l promotor constructs normalized to the isoosmolar control (n = 4, each with duplicates). n.s. > 0.05, *, p

Additional file 9: Figure S4. (A-C) Somatic mutation analysis of RANBP3L in KIRC (A), KICH (B) and KIRP (C). (D) RNA-Seq analysis of WT (wildtype) and Vhl∆/∆Trp53∆/∆Rb1∆/∆ mice samples from Frew et al. showing the normalized reads of Ranbp3l [56]. ***, p