Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2′-oxime (MTM ox ) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM ox analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusionindependent/dependent selectivity indices for select 2′-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTM ox 32E (a Phe−Trp dipeptide-based 2′-conjugate) for in vivo testing. Relative to MTM, MTM ox 32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.
IntroductionChromosomal translocations involving the ETS family of transcription factors are common in Ewing sarcoma, prostate cancer, and leukemia. These translocations lead in overexpression of aberrant ETS transcription factors, which drive tumorigenesis. Mithramycin (MTM) was shown to inhibit EWS/FLI1, the most common ETS related transcription factor in Ewing sarcoma, presumably through interference at its DNA binding sites on promoter regions. However, MTM has a short half‐life and a narrow therapeutic window marked by severe liver and hematological toxicities. Considering that MTM has a pKa of 5, we hypothesized that its rapid pharmacokinetic (PK) clearance is attributed to liver uptake by organic anion transporter polypeptides (OATP), which may also contribute to the observed hepatotoxicity. Here, we sought to develop analogues with specificity toward ETS transcription factors and reduced toxicity by attempting to modify the physicochemical properties of MTM.MethodsA series of analogues were obtained by modifying the 3‐side chain of MTM to increase its pKa. Specificity toward cells expressing ETS transcription factors was determined by cytotoxicity studies in ETS expressing cancer cells (n=9) vs. cancer cell lines (n=9) lacking ETS. Luciferase reporter constructs were used to indirectly evaluate if MTM analogues affected interaction of EWS/FLI1 and Sp1 binding to promoters. Gene expression was tested by qRT‐PCR and interaction at specific promoter sites was determined by ChIP‐qRT‐PCR. The PK of MTM was determined in the presence and absence of rifampin pretreatment and in OATP1B1/1B3 transgenic mice. Select analogues were tested in vivo to identify the maximum tolerated dose and determine pharmacokinetics (PK).ResultsSemisynthetic substitutions using aromatic amino acids at the 3‐side chain of MTM lead to a significant increase (0.5–1.5 units) of the pKa. Growth inhibition assays identified several MTM analogues with > 10 fold selectivity toward ETS cell lines and this was corroborated by reporter and expression assays. The clearance of mithramycin was significantly decreased by rifampicin pretreatment and MTM exposure was significantly higher in Oatp1a/1b KO mice expressing OATP1B3 (transgenic) vs KO mice expressing OATP1B1. Select analogues were further tested to determine relative toxicity in mice and were found to be tolerated at 10–30 fold higher doses than MTM. Significantly, the presumed shift in pKa afforded a dramatic difference in pharmacokinetic properties resulting in relative drug exposures (area under the concentration‐time curves) that were 50–400 higher than MTM at their respective maximum tolerated doses.ConclusionThese studies show that substitutions at the 3‐side chain of MTM yield analogues that are selectively more cytotoxic against tumor cell lines expressing aberrant ETS transcription factors. Further, significantly altered pharmacokinetics of these analogues may expand the therapeutic window and lead to the development of a new generation of MTM analogues amenable for targeted treatment of ETS expressing cancers.Support or Funding InformationThis work was partially funded by DoD grant W81XWH‐16‐1‐0477; projects PC150300P1, PC150300P2 and by funds from the DanceBlue Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introduction: Chromosomal translocations of ETS family transcription factors (TF) are found in Ewing sarcoma, prostate cancer, and leukemia and lead to expression of aberrant ETS transcription factors driving tumorigenesis. Mithramycin (MTM) inhibits EWS-FLI1, the most common ETS-related TFs in Ewing sarcoma through interference at its DNA binding sites on promoters. However, MTM has a narrow therapeutic window marked by severe liver and hematologic toxicities and poor pharmacokinetic (PK) properties, as demonstrated recently in a pediatric clinical trial with Ewing sarcoma patients. Here, we sought to develop analogues with specificity toward ETS family TFs and improved pharmacologic properties. Methods: Guided by crystal structures of MTM/DNA/ETS, we used semisynthetic approaches to prepare a library of analogs by substitution on the 3-side chain of MTM-SA and MTM (analogs). To select analogs with specific activity toward cells with ETS-related chromosomal translocations, analog cytotoxicity was determined in TC32 cells before and after EWS-FLI1 silencing. To confirm selectivity, analogs were tested in a panel of Ewing sarcoma cell lines (n=8) expressing EWS-ETS translocations and compared to a panel of non-Ewing cell lines (n=9) that do not express these translocations. CETSA, FRET, and luciferase reporter assays were used to evaluate interactions with EWS-FLI1 and SP1 proteins and gene promoter regions they bind. Inhibition of select transcriptionally regulated genes was determined by qRT-PCR and immunoblotting. Select analogs were tested in vivo to assess PK in mice and primates, identify the maximum tolerated dose (MTD), and determine dose-dependent efficacy in mouse xenograft models. Results: MTM analogs had higher GI50 in silenced EWS-FLI1 cells and > 10-fold lower GI50 in ETS-dependent cell lines vs. non-ETS dependent cancer cells, as compared to ~3-fold for MTM. Target engagement was demonstrated in dose-dependent manner using a luciferase reporter TC32 cell line under control of the NR0B1 promoter, which is regulated by EWS-FLI1, and CETSA experiments showed increased physical interaction of the analogs with EWS-FLI1 as compared to MTM. Treatment with MTM and analogs produced comparable dose-dependent displacement of an ETS DNA binding domain from GGAA (microsatellite-like) DNA fragments as demonstrated by FRET. Exposure to analogs led to concentration-dependent downregulation of EWS-FLI1 and Sp1 regulated genes. In vivo studies showed that MTM analogs attained higher exposure at their respective MTD and had lower clearance in mice and primates. Unlike MTM, analogs reversed tumor growth and increased survival at the MTD and 2/3 of MTD. Conclusion: We demonstrated that MTM analogs are selectively more cytotoxic in tumor cell lines dependent on ETS family TFs and have vastly improved PK properties as confirmed in a primate model. Given their effectiveness at 2/3 of the MTD, they are expected to have an improved therapeutic window. Citation Format: Markos Leggas, Joseph Eckenrode, Yang Liu, Abhisek Mandal, Reiya Hayden, Oleg Tsodikov, Jon Thorson, Jurgen Rorh. Novel mithramycin analogues with improved pharmacologic profile and efficacy in ETS transcription factor-driven tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A41.
Introduction Chromosomal 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. Methods Qualitative 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. Results Analogues 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. Conclusion These 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 specific...
IntroductionEwing 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.MethodsQualitative 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.ResultsUsing 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.ConclusionThese 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.
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