ID #475 TRRAP is a novel FOXR2 protein interactor in FOXR2-expressing diffuse midline gliomas

Background Diffuse midline gliomas (DMGs) are universally fatal pediatric brain tumors characterized by a lysine (K) to methionine (M) mutation in histone H3 (H3K27M). The transcription factor Forkhead box R2 (FOXR2) is a pan-cancer oncogene aberrantly expressed in a subset of DMGs but absent in normal brain, representing a therapeutic target. FOXR2 exerts its oncogenic effect by activating ETS transcription factors and physically stabilizing MYC, however this interaction likely requires other proteins that are not well defined and may be relevant for designing therapies targeting FOXR2. Methods Utilizing an isogenic model of HA-tagged FOXR2 in human neural stem cells (hNSCs), FOXR2-expressing patient-derived DMG neurospheres, immunoprecipitation with mass spectrometry (IP-MS), functional genomics, FOXR2 deletion constructs, and RNA-sequencing, we sought to determine FOXR2 protein interactors in the context of DMG. Results We identified protein transformation/transcription domain-associated protein (TRRAP) as a predicted interactor of FOXR2. In IP experiments, FOXR2 binds TRRAP in HA-FOXR2-expressing hNSCs and FOXR2-expressing DMG cells.We next sought to establish the FOXR2 amino acids required for FOXR2-TRRAP binding via IP of HA-tagged FOXR2 deletion constructs expressed in hNSCs followed by immunoblotting for TRRAP. TRRAP is critical for tumorigenesis in MYC-activated cancers through cooperation with MYC to activate MYC target genes. Therefore, FOXR2 deletion constructs that failed to bind TRRAP were used to determine if FOXR2-TRRAP binding is required for MYC stability utilizing cycloheximide chase assays. Importantly, FOXR2-TRRAP is essential for DMG cell survival, as TRRAP knockout by CRISPR-Cas9 diminishes DMG cell viability and neurosphere formation. We are currently interrogating transcriptomic datasets from patient-derived DMG cells with TRRAP deletion to establish if transcriptomic changes associated with TRRAP deletion are consistent with FOXR2 deletion. Conclusion We identified a novel protein interaction between FOXR2 and TRRAP, and understanding this interaction may elicit new therapeutic opportunities for patients with DMG.