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@PHDTHESIS{Jenseit:294895,
      author       = {A. L. Jenseit$^*$},
      title        = {{D}eveloping targeted therapies for pediatric {PFA}
                      ependymoma},
      school       = {Universität Heidelberg},
      type         = {Dissertation},
      publisher    = {Heidelberg University Library},
      reportid     = {DKFZ-2024-02605},
      year         = {2024},
      note         = {Dissertation, Universität Heidelberg, 2024},
      abstract     = {PFA ependymomas are highly aggressive brain tumors
                      typically arising in young children (median age 3 years) and
                      characterized by a poor outcome (10-year OS < $60\%).$
                      Standard of care therapy for PFA ependymoma consists of
                      surgery and radiotherapy, while chemotherapy has been
                      largely ineffective and proposed targeted therapies have not
                      yet translated to the clinics. This is also routed in the
                      fact that for a long time, no genetic driver mutations could
                      be identified for PFA ependymoma. Recently, aberrant EZHIP
                      overexpression has been identified as likely driver of PFA
                      ependymoma. By inhibiting EZH2, the catalytic subunit of the
                      polycomb repressive complex 2 (PRC2), EZHIP prevents the
                      distribution of the epigenetic repressor mark H3K27me3,
                      resulting in de-repression of PRC2 target genes and
                      corresponding expression changes. However, without any known
                      enzymatic function, EZHIP cannot serve as the drug target,
                      which is so urgently needed. Therefore, in my thesis I
                      focused on the identification, mechanistic characterization
                      and validation of potential alternative treatments for PFA
                      ependymoma. Firstly, published IP-MS data from non-PFA
                      ependymoma cells was analyzed to identify druggable
                      interaction partners of EZHIP. After the identification of
                      the deubiquitinase USP7, the interaction of USP7 and EZHIP
                      in the nucleus was confirmed in PFA ependymoma cells by
                      co-IP and immunofluorescence, and was found to be
                      independent of EZH2, a separate interaction partner of both
                      proteins. Functionally, USP7 interacts with EZHIP via the
                      USP7 TRAF-like domain, resulting in deubiquitination of
                      EZHIP, which prevents it from being degraded. This
                      stabilization of EZHIP is mediated by the six lysine
                      residues of EZHIP. Previously described somatic EZHIP
                      mutations in patients were not found to interfere with the
                      EZHIP-USP7 interaction. Genetic and pharmacological
                      interventions were used to illustrate the vulnerability of
                      PFA ependymoma cells to a loss of USP7 in vitro. PFA
                      ependymoma cells were sensitive to USP7 inhibitors, while
                      normal healthy human astrocytes were not affected, thereby
                      presenting a potential therapeutic window. However, in vivo
                      validation treatments in the PFA ependymoma PDOX model BT232
                      using a maximally tolerated dosing of the USP7 inhibitors
                      P22077 and P005091 did not result in tumor growth inhibition
                      or improved survival of the animals. The reason for this is
                      unclear but could be due to limited brain penetrance or too
                      low exposure of the USP7 inhibitors. Therefore,
                      mid-throughput drug library screening of PFA ependymoma
                      cells was applied to identify drugs that improve their
                      performance in combination with USP7 inhibitors. Based on
                      the appearance of multiple BET inhibitors as top hits of the
                      screen, the drug class of BET inhibitors was identified and
                      their synergy with USP7 inhibitors in PFA ependymoma was
                      validated in vitro. Treatments of subcutaneous PFA
                      ependymoma PDX models with the BET inhibitor OTX015, alone
                      and in combination with P005091, are ongoing. Secondly,
                      epigenetic drug library screening was performed in the PFA
                      ependymoma cell line EPD210FH and two ST-ZFTA ependymoma
                      cell lines (EP1NS, BT165) to identify drugs that would
                      target ependymoma. Overall, BET and HDAC inhibitors showed
                      high potencies. The 10 drugs showing sensitivity in the PFA
                      ependymoma cells included four BET inhibitors, and three
                      drugs were selective for the PFA subtype of ependymoma:
                      Curcumin, the histone methyltransferase inhibitor Chaetocin
                      and the EZH2 inhibitor UNC199. Abstract II Thirdly, three
                      publication-informed targets were tested in in vitro drug
                      treatments of different PFA ependymoma cell lines. EZH2
                      inhibitors were evaluated for their ability to further
                      unbalance the PRC2 complex. While UNC199 affected EPD210FH
                      viability at micromolar potency, DZNep significantly reduced
                      PFA ependymoma viability at nanomolar levels in vitro. In
                      contrast, although PPARγ was found to be specifically
                      overexpressed in PFA ependymoma in a cohort of pediatric
                      brain tumors and in PFA ependymoma cell lines, PPARγ
                      agonists failed to reduce PFA ependymoma survival in vitro.
                      Moreover, As EZHIP might play a role in DNA damage repair,
                      EZHIP-expressing cells were tested for their acclaimed
                      sensitivity to PARP inhibition. Of the four tested PARP
                      inhibitors, Talazoparib affected PFA ependymoma cells at
                      nanomolar potencies, but also affected human astrocytes or
                      ST-ZFTA cells. Furthermore, USP7 and PARP inhibitors were
                      tested in combination for potential synergism, but this did
                      not appear to be the case for PFA ependymoma. Taken
                      together, my work presents USP7 as a potential target for
                      PFA ependymoma. I identified the stabilization of the
                      oncogenic driver EZHIP through deubiquitination by USP7.
                      Targeting USP7 genetically or pharmacologically reduced
                      EZHIP levels and affected PFA ependymoma survival, rendering
                      USP7 inhibitors a mechanistically sound option to further
                      validate for PFA ependymoma therapy. Additionally, further
                      experiments should be performed to explore EZH2 and PARP
                      inhibitors as therapeutic options for PFA ependymoma.},
      keywords     = {500 Natural sciences and mathematics (Other)},
      cin          = {B062},
      cid          = {I:(DE-He78)B062-20160331},
      pnm          = {312 - Funktionelle und strukturelle Genomforschung
                      (POF4-312)},
      pid          = {G:(DE-HGF)POF4-312},
      typ          = {PUB:(DE-HGF)11},
      doi          = {10.11588/HEIDOK.00035281},
      url          = {https://inrepo02.dkfz.de/record/294895},
}