Mithramycin analogues with reduced toxicity for the treatment of ETS transcription factor driven tumors

Abstract

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.