The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma

Abstract

Ewing sarcoma is an aggressive bone cancer of children and young adults defined by the presence of a chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the amino-terminal domain of EWS to the carboxyl-terminus of FLI. The EWS portion is an intrinsically disordered transcriptional regulatory domain, while the FLI portion contains an ETS DNA-binding domain and two flanking regions of unknown function. Early studies using non-Ewing sarcoma models provided conflicting information on the roles of each domain of FLI in EWS/FLI oncogenic function. We therefore sought to define the specific contributions of each FLI domain to EWS/FLI activity in a well-validated Ewing sarcoma model and, in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. We analyzed a series of engineered EWS/FLI mutants with alterations in the FLI portion using a variety of assays. Fluorescence anisotropy, CUT&RUN, and ATAC-sequencing experiments revealed that the isolated ETS domain is sufficient to maintain the normal DNA-binding and chromatin accessibility function of EWS/FLI. In contrast, RNA-sequencing and soft agar colony formation assays revealed that the ETS domain alone was insufficient for transcriptional regulatory and oncogenic transformation functions of the fusion protein. We found that an additional alpha-helix immediately downstream of the ETS domain is required for full transcriptional regulation and EWS/FLI-mediated oncogenesis. These data demonstrate a previously unknown role for FLI in transcriptional regulation that is distinct from its DNA-binding activity. This activity is critical for the cancer-causing function of EWS/FLI and may lead to novel therapeutic approaches.

Pioneer factors in development and cancer

“…Perhaps there are severe implications for this conceptual framework as well, such that an oncogene with pioneer function may be independent of the motif abundance or the cell of origin. Increasing evidence suggests that low mutational burden tumors are driven from epigenetic alterations ( Boone et al., 2021 ; Lin et al, 2019 ; Pomella et al., 2021 ; Sunkel et al., 2021 ). It will be important to understand how these chromatin-level changes are translated into stable transcriptional states.…”

“…The expression of SOX2 has been linked to EWS-FLI activity in some reports ( Riggi et al., 2010 , 2014 ). Excitingly, there is evidence that EWS-FLI engages its GGAA response elements even in repressed regions of the genome ( Gangwal et al., 2008 ; Patel et al., 2012 ), though recent findings suggest that while the ETS domain is sufficient for DNA binding, a full-length fusion may be necessary for inaccessible chromatin binding ( Boone et al., 2021 ). Understanding therapeutic mechanisms for destabilizing the motif recognition will continue to be highly impactful ( Harlow et al., 2019 ).…”

“…The inhibition of pioneer function or the co-opting of pioneer function by chimeric translocations represents common epigenetic features of leukemogenesis, with striking parallels to other translocation-driven tumors ( Boone et al., 2021 ; Gryder et al., 2019 ; Sunkel et al., 2021 ). Future studies will be critical to examine the four definitions of pioneer function in the context of AML translocations and the precise mechanisms by which these chimeric oncogenes interfere with PFs in myeloid differentiation blockade.…”

A new phase of the Cancer Moonshot to end cancer as we know it

“…The Fusion Oncoproteins in Childhood Cancers (FusOnC2) Consortium, a diverse group of experts in cancer biology, proteomics, genomics, computational biology and pharmacology, discovered a previously unknown function in the EWS/FLI fusion protein that drives Ewing sarcoma, a rare cancer that occurs in bones or soft tissue around the bones, that may lead to novel therapies 7 . Another group identified TRIM8 as a regulator of EWS/FLI stability, which may provide new therapeutic targets 8 .…”

The oncogenic fusion protein EWS-FLI1 promotes premature ageing of biomolecular condensates by catalyzing fibril formation

“…EWS-FLI1 acts as a pioneering factor aiding in chromatin opening, yet these observations do not fully explain its oncogenic role in EwS. There is mounting evidence that EWS-FLI1 exerts a dominant negative effect on the normal roles of EWS in transcriptional regulation and splicing [2][3][4][5] . Indeed, it was recently noted that the presence of EWS-FLI1 at transcriptionally active sites prevents the release of Breast cancer type 1 susceptibility protein (BRCA1) from DNA-directed RNA polymerase II subunit RPB1 (RNA Pol II), resulting in elevated transcriptional stress and subsequent accumulation of unresolved R-loops 2 .…”