EPEN-07. USP7 IS AN INTERACTION PARTNER OF EZHIP AND POTENTIAL DRUGGABLE TARGET IN PFA EPENDYMOMAS

Abstract

Abstract BACKGROUND Ependymomas (EPN) arise in the supratentorial brain (ST-EPN), posterior fossa (PF-EPN), or the spinal cord (SP-EPN), in children and adults. Among the three molecular PF-EPN groups, PFAs are characterized by young median age at diagnosis, a balanced genome and poor outcome. Recently, enhancer of zeste inhibiting protein (EZHIP) has been identified as potential driver of PFAs. By inhibiting EZH2, EZHIP prevents the distribution of the epigenetic repressor mark H3K27me3. Without known enzymatic functions though, EZHIP isn´t the urgently needed drug target. We therefore focused on essential and potentially druggable interaction partners. METHODS We investigated protein-protein interactions using co-immunofluorescence, co- immunoprecipitation and proximity-based assays. We utilized CRIPSR/Cas9 and shRNAs-based methods, mutagenesis and PROTAC treatments followed by Western Blot or mass spectrometry for functional analyses. Viability and apoptosis after drug treatments was assessed in vitro by metabolic readouts, synergy was determined using matrix layouts. RESULTS We identified ubiquitin-specific protease 7 (USP7), a known cancer regulator with multiple inhibitors available as direct interaction partner of EZHIP in PFA. We confirmed their interaction in PFA cells and showed that the EZHIP-USP7 interaction is independent of EZH2, a separate interactor of both. Functionally, we show that USP7 de-ubiquitinates EZHIP, preventing its degradation and thus stabilizing it, mediated via the six lysine residues present in EZHIP. We reveal a vulnerability of PFA cells to genetically engineered losses of EZHIP and USP7. Moreover, USP7 inhibitors highly affected the survival and induced apoptosis in different PFA cell lines at a low micromolar IC50 in vitro. Mid-throughput drug library screens additionally identified BET inhibitors as synergistic partners. In vivo treatments with the combination of USP7 and clinically achievable doses of BET inhibitors in PFA PDX models are ongoing. CONCLUSIONS USP7 de-ubiquitinates and thus stabilizes EZHIP, which qualifies it as a candidate therapeutic target in pediatric PFA ependymoma.