Oncogenic Targets of Hedgehog-GL/1 Activation in Ewing Sarcoma
Ewing Tumors are addicted to aberrant oncogenic transcription factors of the EWS/FLI1 class. Means to target these proteins face substantial technical and economic obstacles. However, my lab and others have shown the importance of Hedgehog-GLI (HH-GLI) signaling in Ewing tumor biology. More recently, data from my lab has shown that a majority of targets transcriptionally deregulated by EWS/FLI1 are co-regulated by GLI1, demonstrating the central importance of HH-GLI in Ewings. We now have evidence that the reason for this tremendous target overlap involves a novel mechanism in which GLI1 interacts with EWS/FLI1 to synergistically upregulate jointly-targeted promoters. This synergism represents a novel aspect of EWS/FLI1 and GLI1 biology that can have tremendous translational implications for Ewing tumors since it will add key details to the only proven driver mutation in this disease. We propose to detail the transcriptional mechanisms by which this interaction occurs. We will analyze specific target promoters and mutagenize specific GLI1 and EWS/FLI1 binding sites to measure their effects on the observed transcriptional synergy. Identified sites will be confirmed by CHIP analysis to demonstrate that this process also occurs in vivo. More extensive analysis with CHIP-Seq will be undertaken, time permitting. We will use the knowledge generated from these studies to devise a reporter system which will permit large scale screening for compounds to interfere with the EWS/FL1-GLI1 interaction. Finally, we will explore the nature of this interaction by confirming our preliminary data that this interaction involves direct physical association between GLI1 and EWS/FLI. Structural mutants will be used to further detail the important moieties involved.
The EWS/FLI1 oncogenic transcription factor is both unique to and indispensable to the development of Ewing’s Sarcoma and peripheral Primitive Neuroectodermal Tumor (collectively known as the Ewing Sarcoma Family Tumors or ESFT). It is imperative that we translate our understanding of EWS/FLI1 biology into improved therapeutics for Ewing Sarcoma. We have recently published findings that EWS/FLI causes increased expression of Gli1 in ESFT in a Hedgehog independent fashion. We have further published data that inhibition of GLI1 expression by shRNA results in decreased tumorigenicity and that several known targets of EWS/FLI1 are diminished in their expression. This suggests that signaling through Gli1 may be a central and critical event in EWS/FLI1 downstream gene activation which is central to this disease. Our preliminary data demonstrates that two newly identified compounds termed GANT58 and GANT61, which inhibit this pathway at the level of GLI1, act to inhibit the HH-GLI1 pathway in Ewings cell lines in vitro. Preliminary data suggest that at least one of these compounds will also be effective in vivo. This proposal will utilize these novel compounds to fully test the potential of GLI1 inhibition in Ewing Sarcoma in vivo models. Both compounds will be tested at full doses in xenograft models of ESFT for their inhibition of tumor growth. We will assess experimental tumors for reduced expression of downstream targets of GLI1 in ESFT. We will also use in vivo imaging systems to assess the effect of the drugs on GLI1 transcriptional activity in vivo. Recently, several prominent tumor types have been shown to depend on GLI1 activation in a Hedgehog independent fashion. As these developments stimulate the development of pathway inhibitors effective in these circumstances, the data generated in this proposal will establish whether such compounds could benefit patients with Ewings Sarcoma in the near future.