Abstract 3414: High-throughput screening identifies mithramycin as a potent inhibitor of the EWS-FLI1 transcription factor and a strong cytotoxic agent in Ewing's sarcoma cells
Authors: Grohar Patrick J. , Woldemichael Girma M. , Griffin Laurie B. , Yeung Choh , Chen Qing-Rong , Khanna Chand , Khan Javed , McMahon James B. , Helman Lee J. 
Published: February 2011

Background:

Chromosomal translocations involving the EWS gene and one of the members of the ETS family of transcriptions factors are the hallmark of the Ewings Sarcoma Family of Tumors (ESFT). The most common translocation, EWS-FLI1, is found in 85% of the tumors and plays an important role in malignant transformation and maintenance of the oncogenic phenotype. Multiple groups have demonstrated that continued expression of this transcription factor is required for cellular survival.

Methods:

High throughput screening of a library of more than 50,000 pure compounds was utilized to identify inhibitors of a known EWS-FLI1 downstream target, NR0B1, luciferase promoter construct. The top compounds were subsequently validated and sorted by using a novel 17 gene multiplex PCR assay that quantitates a gene signature of EWS-FLI1. The results were confirmed by quantitative PCR and western blot analysis. The resulting compounds were analyzed for cytotoxicity and selectivity in a panel of ESFT cell lines and compared to cell lines that do not contain the EWS-FLI1 transcription factor.

Results

Several compounds have been identified that appear to modulate the activity of the EWS-FLI1 transcription factor. These compounds include microtubule inhibitors, topoisomerase inhibitors, compounds of unknown mechanism and an sp1 inhibitor. One of the most potent inhibitors identified was Mithramycin, a natural product known to inhibit sp1. Mithramycin inhibits expression of luciferase in a dose dependent fashion in the NR0B1 promoter reporter construct at concentrations between 10 and 100 nM without decreasing expression in a constitutively active CMV promoter construct. This effect translates into an LD50 of 10 to 25 nM in a panel of ESFT cell lines and increased selectivity relative to osteosarcoma, breast carcinoma and ovarian carcinoma cell lines. Mithramycin also inhibits the expression of the gene signature of EWS-FLI1 as determined by multiplex PCR and confirmed by quantitative PCR. Western blot analysis confirms the inhibition of the downstream target ID2 with a corresponding induction of apoptosis as demonstrated by an increase in cleaved PARP. A review of the literature identified a small panel of patients treated with Mithramycin in the 1960s with at least one documented durable complete response.

Conclusion:

We have developed a method to screen for transcription factor inhibitors and have utilized it to identify inhibitors of the EWS-FLI1 transcription factor. The most potent inhibitor was Mithramycin which inhibits the expression of a gene signature of EWS-FLI1, has a nanomolar LD50 in ESFT cells and shows some selectivity compared to cells that do not contain EWS-FLI1. Work continues to define the relationship between EWS-FLI1 and sp1 and evaluate the safety and efficacy profile in xenograft models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3414.