Mithramycin analogues with unique mechanism of action in Ewing sarcoma
Authors: Hayden Reiya , Mandal Abhishek , Liu Yang , Hou Caixia , Thorson Jon , Rohr Jurgen , Tsodikov Oleg , Leggas Markos 
Published: April 2020

IntroductionChromosomal translocations involving ETS family proteins are common in Ewing Sarcoma Family of Tumors (ESFT) and form aberrant ETS‐fusion transcription factors. EWS‐FLI1 and EWS‐ERG are the most common transcription factors in ESFT and function by binding DNA at microsatellite regions, characterized by GGAA repeats, which are found at promoters of genes that drive ESFT growth. These fusion products also interact with the DNA damage response protein and transcriptional co‐regulator PARP‐1, which allows for the development and combination treatments with novel targeted therapeutics in ESFT. Mithramycin (MTM) has been identified as a potent inhibitor of EWS‐ETS, but this drug has a very narrow therapeutic window and its clinical use is marked by severe liver and hematological toxicities; likely the result of interference with the ubiquitously expressed Sp1 transcription factor as well as the pharmacokinetic and biodistribution characteristics of MTM. Here, we sought to identify analogues of MTM that selectively perturb the ETS transcription factor complex and afford efficacy in Ewing sarcoma as a monotherapy and potentially in combination with PARP inhibitors.MethodsQualitative interactions between drug‐DNA‐protein were assessed by electrophoretic mobility shift assays (EMSA). Time‐resolved fluorescence energy transfer (TR‐FRET) assays were used to quantitatively determine DNA bound ERG and Sp1 perturbation by MTM and analogues. Cellular thermal shift assays (CETSA) were used to semi‐quantitatively assess the physical interaction of compounds with EWS‐FLI1 and Sp1. 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. mRNA expression of genes involved in DNA damage repair (DDR) was determined by RT‐PCR. Cell viability, by aerobic respiration, in single and combination treatments with a PARP inhibitor were assessed with resazurin.ResultsAnalogues with hydrophobic 3‐side chain modifications showed increased interaction with DNA bound ERG by TR‐FRET, CETSA, and luciferase reporter assays. Specifically, CETSA demonstrated that MTM analogues physically interact and stabilize EWS‐FLI1, but not with Sp1, which is a unique mechanism of action. MTM analogues enhanced DNA damage in Ewing Sarcoma cells and inhibited RNAP2 phosphorylation. As compared to MTM, treatment with analogues resulted in higher expression of DNA damage markers and down regulation of DDR genes specifically in cell lines containing EWS‐ETS translocations in a concentration dependent manner. Cell viability assays showed strong synergy when MTM analogues were combined with Olaparib, a PARP inhibitor. This effect was only seen in Ewing sarcoma cells and was abolished with EWS‐FLI1 silencing.ConclusionThese studies demonstrate that novel MTM analogues selectively perturb the EWS‐ETS transcription factor complex function. This interaction affects transcription of downstream genes and the DDR apparatus specifically in EWS‐ETS expressing cell lines. The recruitment of PARP to DNA damage sites induced by MTM analogues provides the opportunity for a rational and synergistic treatment combination with a PARP inhibitor.