Developing targeted therapies for pediatric PFA ependymoma

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

The RNA-binding protein SERBP1 functions as a novel oncogenic factor in glioblastoma by bridging cancer metabolism and epigenetic regulation

Abstract Background RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. Results We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. Conclusions SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations