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Mithramycin analogues disrupt ETS transcription factor DNA binding
Author(s) -
Hayden Reiya Cherron,
Hou Caixia,
Mandal Abhisek,
Mitra Prithiba,
Thorson Jon,
Rohr Jurgen,
Tsodikov Oleg,
Leggas Markos
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.674.18
Subject(s) - transcription factor , ets transcription factor family , electrophoretic mobility shift assay , dna damage , chemistry , dna , microbiology and biotechnology , transcription (linguistics) , biology , cancer research , gene , biochemistry , linguistics , philosophy
Ewing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcription factors drive tumorigenesis. The transcription factors EWS‐FLI1 and EWS‐ERG are common translocations in Ewing sarcoma and bind DNA at GGAA repeats leading to expression of genes that drive tumor growth. Pharmacologic inhibition of EWS‐FLI1 with mithramycin (MTM) was shown to inhibit expression of downstream genes and tumor growth in mice. But despite this specific inhibitory activity, MTM has a narrow therapeutic window with hematologic and hepatic toxicity attributed to displacement of the ubiquitously acting Sp1 transcription factor. Thus, a synthetic effort was initiated to develop MTM analogues with reduced toxicity and increased specificity for ETS binding sites. Structural studies informed the design of MTM analogues that may stabilize transcriptional complexes leading to the disruption of transcriptional activity and DNA damage. In vitro cytotoxicity assays demonstrated that MTM analogues have significantly higher cytotoxicity in EWS‐ETS expressing cell lines. Here we present mechanistic evidence for the differences in biochemical activity among MTM and its novel analogues. Methods Qualitative interactions between drug‐DNA‐protein were assessed and optimized by electrophoretic mobility shift assays (EMSA). Time‐resolved fluorescence energy transfer (TR‐FRET) assays were used to quantitatively determine ERG displacement from DNA in the presence of MTM and analogues. Expression of proteins indicating DNA damage (c‐PARP, γ‐H2AX) and phosphorylation at the C‐terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines. Results Using TR‐FRET, we observed that MTM displaced DNA bound ERG more potently and in a concentration dependent manner as compared to MTM analogues. As compared to MTM, treatment with MTM analogues resulted in higher expression of DNA damage markers, γ‐H2AX and c‐PARP, specifically in cell lines containing EWS‐ETS translocations in a concentration dependent manner. Conclusion These studies provide insights regarding differences among MTM and analogues in DNA binding and interactions with DNA associated proteins in the presence and absence of EWS‐ETS expression. Our results suggest that MTM analogues may bind and stabilize transcriptional complexes. These differences will provide the basis for structure activity relationships and for the development of analogues with decreased in vivo toxicity. Future work will incorporate co‐immunoprecipitation studies to determine if physical protein interactions are being disrupted by MTM and analogues and cellular thermal shift assays to directly probe drug interactions with EWS‐ETS proteins and with RNAPII. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .