Abstract 5739: Small molecule modulation of autoinhibition to inhibit DNA binding of ERG and FLI1 - therapeutic for Ewing sarcoma, prostate cancer, and ALL
Authors: John H. Bushweller, Kiran Srimath Tirumala, Ashish Kabra, Adam Boulton, Karla Rubio, Michael Bond, Anglea Su, Miguel Rivera, Kim Stegmaier
Journal: Cancer Research (2025)

Abstract Targeting transcription factor-DNA interactions presents substantial challenges due to the convex and highly positively charged surface on the DNA binding domains that mediate DNA binding. Such a surface is far from ideal for development of drug-like molecules, making this a challenge. Furthermore, as many transcription factors are members of families with highly conserved DNA binding domains, there are also concerns with respect to what level of specificity could be achieved with an inhibitor targeting the DNA binding domain in a family of transcription factors. The 28 member Ets family of transcription factors has numerous members implicated in various cancers. EWS::FLI1 and EWS::ERG fusions are drivers in Ewing sarcoma, ERG overexpression via the TMPRSS2::ERG fusion is a driver in half of prostate cancer, and ERG has been identified as a marker for poor prognosis in AML patients. Based on this, small molecule inhibitors of DNA binding of ERG and FLI1 could have utility for these cancers.To overcome the challenges mentioned above, we chose to target the autoinhibition of ERG and FLI1 to generate inhibitors of their binding to DNA. Numerous transcription factors modulate their activity via autoinhibition and this has been characterized for many of the members of the Ets family including our own work on ERG. By so doing, we hypothesized we could achieve selectivity within the Ets family due to the differing sequences and mechanisms mediating autoinhibition among the different family members.We screened using fragment libraries for compounds inhibiting DNA binding of an autoinhibited ERG construct but were not active against the uninhibited DNA binding domain. Optimization of these gave a fragment with excellent selectivity but limited potency. We subsequently screened for a molecule that could covalently add to ERG and linked a derivative of this to the autoinhibition fragment. Biochemical assays of effects on DNA binding show low µM IC50 values and a shift in the Kd of DNA binding of ∼2.5-fold for both ERG and FLI1. Importantly, characterization of effects on 7 other Ets family members representing 7 other subfamilies shows the inhibitors are not active against other Ets family members, demonstrating we have achieved a high level of selectivity within the family.We have shown that the ERG/FLI1 inhibitors inhibit the proliferation of Ewing sarcoma cell lines at low µM concentrations and preferentially inhibit Ewing sarcoma cell lines versus other sarcoma cell lines. As EWS::FLI1 is known to bind at enhancers, we evaluated effects on H3K27ac levels at enhancer elements. We observe no global reduction in H3K27ac levels at enhancers but H3K27ac levels at EWS::FLI1 bound enhancers are reduced. In addition, we observe a reduction in the occupancy of RNA Pol II at EWS::FLI regulated genes with inhibitor treatment. Both these results are consistent with an on-target mechanism of action. Citation Format: John H. Bushweller, Kiran Srimath Tirumala, Ashish Kabra, Adam Boulton, Karla Rubio, Michael Bond, Anglea Su, Miguel Rivera, Kim Stegmaier. Small molecule modulation of autoinhibition to inhibit DNA binding of ERG and FLI1 - therapeutic for Ewing sarcoma, prostate cancer, and ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5739.