Journal: bioRxiv

Article Title: Rewiring the fusion oncoprotein EWS/FLI1 in Ewing sarcoma with bivalent small molecules

doi: 10.1101/2025.03.14.643353

Figure Lengend Snippet: (A) Schematic of EB-TCIP mechanism of action. EB-TCIP induces a ternary complex between FKBP F36V tagged EWS/FLI1 and BCL6, which leads to activation of BCL6 target gene transcription. Image made with Biorender. (B) Structures of compounds used in this work. (C) EB-TCIP increases the association of BCL6 with EB-TCIP in a dose dependent manner in EWS502 FKBP-E/F cell lysates while NEG-1 (D) does not induce a ternary complex. The association is reversible as excess BI3812 (C) and excess OAP (free acid) (D) abrogate ternary complex formation. GAPDH was probed to determine if unbound proteins were removed by washing. EB-TCIP dose dependently increases SOCS2 (E) and CISH (F) expression by RT-qPCR. (G) SOCS2 protein levels dose dependently increase while BCL6 protein levels dose dependently decrease after EB-TCIP treatment. EB-TCIP induces higher SOCS2 (H) and CISH (I) transcript levels than chemical inhibition with BI3812 or chemically induced degradation with BI3802 (DEG) . (J) SOCS2 protein levels are highest in EB-TCIP treated cells compared to BI3812 , BI3802 (DEG) , or negative control compounds that do not form ternary complexes. Immunoblotting is representative of three biological replicates and GAPDH serves as a loading control. All experiments were performed in FKBP-E/F expressing EWS502 cells. RT-qPCR experiments show one experiment with technical triplicate that is representative of three biological replicates. Means of SOCS2 and CISH expression were compared using one-way ANOVA with multiple comparisons; NS = not significant, *** p < 0.005, **** p < 0.001. All error bars in the figure indicate mean ± SD. Unless indicated with brackets, significance above each condition indicates comparison of that mean to the mean of DMSO.

Article Snippet: The following primary antibodies were used at the following dilutions: rabbit monoclonal anti-SOCS2 (Abcam, ab109245) at 1:1000, rabbit monoclonal anti-BCL6 (CST, 14895) at 1:1000, rabbit monoclonal anti-FLI1 (Abcam, ab133485) at 1:1000, rabbit monoclonal anti-HA (CST, 3724) at 1:1000, and rabbit monoclonal anti-GAPDH (at 1:2000).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Inhibition, Negative Control, Western Blot, Control, Comparison

Journal: Cell Reports Methods

Article Title: Refined culture conditions with increased physiological relevance uncover oncogene-dependent metabolic signatures in Ewing sarcoma spheroids

doi: 10.1016/j.crmeth.2025.100966

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: monoclonal anti-FLI1 raised in rabbit (1:1,000, EPR4646, ab133485, Abcam), monoclonal anti-ERG raised in rabbit (1:1,000, EPR3864, ab92513, Abcam), monoclonal anti-GAPDH raised in rabbit (1:2,000, GAPDH 14C10, #2118, Cell Signaling), and polyclonal anti-rabbit IgG-HRP raised in goat (1:2,000, cat # 31460, Thermo Fisher Scientific).

Techniques: Recombinant, Electrophoresis, Reverse Transcription, Cell Culture, Sequencing, Software

Journal: bioRxiv

Article Title: Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts

doi: 10.1101/2024.10.27.620498

Figure Lengend Snippet: A) Schematic depicting SMASh and AID based degron approaches for depletion of endogenous EWSR1-FLI1. B) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or IAA (100 μM) for 24 hours prior to collection. C) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or IAA (100 μM) for the indicated time prior to collection. D) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or danoprevir (1 μM) for 24 hours prior to collection. E) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or danoprevir (1 μM) for the indicated time prior to collection.

Article Snippet: Antibodies used were anti-FLI1 rabbit [ERP4646] mAb (Abcam #133485), and anti-β-actin (8H10D10) mouse mAb (#3700, Cell Signaling Technologies).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts

doi: 10.1101/2024.10.27.620498

Figure Lengend Snippet: A) Population doublings after 6 days of treatment. Indicated cell lines were exposed to vehicle or 1 μM danoprevir (DSV). B) Population doublings after 6 days of treatment. Indicated cell lines were exposed to DMSO or IAA (100 μM). C) Immunoblot for EWSR1-FLI1 (FLI1) in TC32 EF SMASh cells with indicated pLVX constructs. Cell lines were exposed to either Vehicle or danoprevir (1 μM) for 24 hours prior to collection. D) Population doublings after 6 days of treatment. TC32 EF SMASh cells with indicated pLVX constructs (C) were exposed to vehicle or 1μM danoprevir. E-F) Cell cycle analysis using propidium iodide. Flow cytometry plots (left two panels) for indicated cell lines treated with either vehicle or 1 μM danoprevir for 72 hours before cells were collected. Plot (left panel) of cells (percentage) in each phase of the cell cycle based on flow cytometry data.

Article Snippet: Antibodies used were anti-FLI1 rabbit [ERP4646] mAb (Abcam #133485), and anti-β-actin (8H10D10) mouse mAb (#3700, Cell Signaling Technologies).

Techniques: Western Blot, Construct, Cell Cycle Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: CD44 Modulates Cell Migration and Invasion in Ewing Sarcoma Cells

doi: 10.3390/ijms241411774

Figure Lengend Snippet: Genes regulated by EWSR1::FLI1 in Ewing sarcoma cell line A673. ( A ) A673/TR/shEF cells were cultured in the absence or presence of doxycycline (DOX, 1 µg/mL, 72 h) and analyzed by RNAseq to identify EWSR1::FLI1-regulated genes. ( B ) Genes regulated by EWSR1::FLI1 were cross-referenced with the PubMed database using the terms invasion, migration, and metastasis. The graph represents the fold change and adjusted p -value for each upregulated and downregulated gene and the number of citations for each gene with these terms (which is proportional to the circle areas).

Article Snippet: Primary antibodies were as follows: anti-FLI1 rabbit monoclonal antibody (#ab133485), anti-CD44 rabbit monoclonal antibody (#ab157107, against the intracellular domain of CD44), and HRP-anti-α-tubulin antibody (#ab185067), all purchased from Abcam (Cambridge, UK).

Techniques: Cell Culture, Migration

Journal: International Journal of Molecular Sciences

Article Title: CD44 Modulates Cell Migration and Invasion in Ewing Sarcoma Cells

doi: 10.3390/ijms241411774

Figure Lengend Snippet: CD44s expression correlates negatively with EWSR1::FLI1 levels in Ewing sarcoma cell line A673 and is not expressed in other Ewing sarcoma cell lines (EWSR1::FLI1 high phenotype). ( A ) CD44s mRNA expression (RT-qPCR) in A673/TR/shEF cell cultures in the absence (EWSR1::FLI1 high ) or presence (EWSR1::FLI1 low ) of doxycycline (DOX 1 µg/mL, 72 h). CD44s mRNA levels impressively increased upon EWSR1::FLI1 knockdown (mean ± SD, three experiments performed in triplicate) (*** p < 0.001, **** p < 0.0001; Student’s t -test). ( B ) Western blot analysis confirmed the upregulation of CD44s protein upon EWSR1::FLI1 knockdown. ( C ) A673/TR/shEF cells were cultured in the absence or presence of doxycycline, and CD44s protein was detected by immunofluorescence. CD44s is located in the extracellular membrane, and its expression is notably increased when cells are stimulated with doxycycline (Scale bar: 100 µm). ( D ) CD44s mRNA expression (RT-qPCR) in several Ewing sarcoma, osteosarcoma, chondrosarcoma, and fibrosarcoma cell lines (mean ± SD). ( E ) Western blot analysis of CD44s and EWSR1::FLI1 in several Ewing sarcoma, osteosarcoma, chondrosarcoma, and fibrosarcoma cell lines. CD44s was not detected in any of the Ewing sarcoma cell lines tested, which expressed high levels of the EWSR1::FLI1 protein. By contrast, CD44 was expressed in cell lines derived from other sarcoma types, both at the mRNA ( D ) and protein levels ( E ). CADO-ES1 is an Ewing sarcoma cell line that expresses the chimeric EWSR1::ERG protein instead of EWSR1::FLI1, which is not recognized by the anti-FLI1 antibody used.

Article Snippet: Primary antibodies were as follows: anti-FLI1 rabbit monoclonal antibody (#ab133485), anti-CD44 rabbit monoclonal antibody (#ab157107, against the intracellular domain of CD44), and HRP-anti-α-tubulin antibody (#ab185067), all purchased from Abcam (Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Immunofluorescence, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: CD44 Modulates Cell Migration and Invasion in Ewing Sarcoma Cells

doi: 10.3390/ijms241411774

Figure Lengend Snippet: Characterization of Ewing sarcoma cell models generated to study the role of CD44s in Ewing sarcoma. A673, MHH-ES1, and CADO-ES1 Ewing sarcoma cell lines were engineered to express the CD44s isoform through the control of a doxycycline-inducible system. ( A ) mRNA expression levels (RT-qPCR) of CD44s and EWSR1::FLI1 (A673, MHH-ES1) or EWSR1::ERG (CADO-ES1) upon stimulation with doxycycline (DOX, 1 µg/mL, 72 h) (mean ± SD, three experiments performed in triplicate); **** p < 0.0001, ns: not significant, Student’s t -test. ( B ) Western blot analysis of CD44s and EWSR1::FLI1 in the same cell lines confirms the upregulation of CD44s upon doxycycline stimulation. EWSR1::FLI1 protein levels were not affected by CD44s overexpression. EWSR1::ERG fusion protein is not recognized by the anti-FLI1 antibody used to detect EWSR1::FLI1. ( C ) The three Ewing sarcoma cell lines were cultured in the absence or presence of doxycycline, and CD44 protein was detected by immunofluorescence. CD44s is located in the cell membrane, and its expression is remarkably increased when cells are stimulated with doxycycline (Scale bar: 100 µm).

Article Snippet: Primary antibodies were as follows: anti-FLI1 rabbit monoclonal antibody (#ab133485), anti-CD44 rabbit monoclonal antibody (#ab157107, against the intracellular domain of CD44), and HRP-anti-α-tubulin antibody (#ab185067), all purchased from Abcam (Cambridge, UK).

Techniques: Generated, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Cell Culture, Immunofluorescence