1. Identification and characterization of EWS-FLI1 binding partners
in Ewing sarcoma cell lines
Matthew L. Rotondi and Peter J. Houghton
Ewing sarcoma (ES) is characterized by a reciprocal translocation t(11;22) that results in a
fusion of the EWSR1 and FLI1 genes (EWS-FLI). The objective of this study is to identify protein
binding partners of the purportedly undruggable chimeric transcription factor EWS-FLI1 to serve
as alternative pharmacological targets for potential ES-selective drug therapies. The literature
identifies the orphan nuclear receptor DAX1 (encoded by NR0B1) as a binding partner of EWS-
FLI1 the expression of which is restricted outside ES, thereby providing a target for a potential ES
selective drug. Yeast two-hybrid (Y2H) screening using EWS-FLI1 as the bait was employed to
identify other potential targets. The importance of the binding partners identified by the Y2H
screen was initially assessed by a series of proliferation assays involving the ES cell lines EW8,
ES7 and ES6 as well as rhabdomyosarcoma cell line Rh30 (Non-Ewing control). Prior to
measuring proliferation, cells were transfected a siRNA designed to target the putative binding
partner of EWS-FLI1. These cells were then grown alongside cells transfected with a control
siRNA over a period of 96 hr. The change in cell confluence in the wells was measured by a live-
cell real-time measurement (Incucyte). Gene knockdown was confirmed by RT-PCR and western
blot analysis.
The proliferation assays showed that the knockdown of DAX1 produces a notable
reduction in cell proliferation compared to siRNA control proliferation in all three of the Ewing
cell lines (10% to 25%). No significant DAX1 siRNA based inhibition of cell proliferation was
detected in the Rh30 non-Ewing control cell line. Three of the proteins identified by the Y2H
screen showed significant reductions in ES cell proliferation. Methyl-CpG-Binding Domain
Protein 1 (MBD1) knockdown caused the proliferation of the ES cell lines to level off at 60% to
80% within 2 to 4 days. Proliferation of MBD1 siRNA and control siRNA transfected Rh30 cells
was similar for the full 4 days of the assay. Mannosidase-α-class 2B-member 2 (MAN2B2)
knockdown caused the proliferation of the ES and Rh30 cell lines to level off at 25% to 65% within
1 to 2 days. Mixed-Lineage Leukemia Protein 3 (MLL3) knockdown caused the proliferation of

2. the EW8 and ES7 cell lines to level off at 52% and 70% respectively. MLL3 knockdown did not
inhibit the proliferation of Rh30 cells when compared to siRNA control cells. In contrast, ES6 cell
proliferation was also not inhibited by MLL3 knockdown when compared to their siRNA control
cells.
The knockdowns of DAX1, MLL3 and MBD1 suggest that these EWS-FLI1 binding
partners would likely provide potential targets for ES drug development. The effect of knocking
down Y2H-identified EWS-FLI interactors on colony formation, migration and tumorigenicity is
ongoing.