Einfluss der Histon 3.3 K27M Mutation auf die CBP- und BET-vermittelte, epigentische Genregulation in pädiatrischen Diffus Intrinsischen Ponsgliomen (DIPG). Entwicklung eines kombinierten, epigenetischen Therapieansatzes für DIPG
Kinder- und Jugendmedizin
Zusammenfassung der Projektergebnisse
Pediatric high grade glioma (gpedHGG) are most aggressive tumors in childhood with almost no prospect of cure. Among them, 85% of all diffuse intrinsic pediatric gliomas (DIPG) and all diffuse midline carry a heterozygous mutation in one histone 3 (H3) gene variant (H3F3A or HIST1H3B/C; mutation: H3K27M). H3K27M mutation disrupts the epigenetic landscape resulting in H3K27 hypotrimetylation (H3K27me3) and compensatory hyperacetylation (H3K27ac). In addition, H3K27M- induced hyperacetylation (H3K27ac) by histone acetyltransferases (HAT) (= epigenetic writer), such as CREB Binding Protein (CBP), might be accompanied by a greater impact of acetylation-dependent epigenetic readers, such as the Bromodomain and Extra Terminal domain protein (BET) BRD4. Thus, targeting both, H3K27ac-specific epigenetic readers and writers, might serve as effective therapeutic treatment option for H3K27M-mutated pedHGG. This project therefore aimed to address (#1) the epigenetic and tumor-biological impact of H3.3K27M-mutation in pedHGG cells lines with regards to (#2) the particular tumor biological function of CBP and BRD4 writer and reader proteins including the identification of small molecules to efficiently inhibit tumor-related CBP/BRD4 functions and the identification of (#3) H3.3K27M-dependend gene expression pattern with regards to the gene-regulatory functions of CBP and BRD4. To find out how H3.3K27M mutation contributes to the malignant phenotype observed in pedHGG, four isogenic cell line pairs with and without H3.3K27M-mutation were generated using CRISPR/Cas9 and analyzed with regard to their tumor-biological characteristics, gene expression pattern and epigenetic landscape. Indeed, introduction of H3.3K27M into pedHGG cells increased cells tumor phenotype with marked invasive, proliferative and stemness potential whereas removal of the mutation resulted in opposite effects. As hypothesized, we found that increased CBP-HAT activity and BRD4-chromatin binding function is responsible for tumor-associated features especially in isogenic H3.3K27M-mutated pedHGG cells compared to their H3WT-counterparts. In agreement, preliminary mRNA sequencing analyses hint towards a more pronounced function of CBP and BRD4 in regulating tumor-associated characteristics primarily in isogenic H3.3K27M-mutated pedHGG cells which in turn showed enrichment in genes associated with cancer pathways. To find out which genes are affected by H3.3K27M-dependent disruption of the epigenetic landscape typical for DIPG/DMG in dependence of CBP and BRD4, chromatin immunoprecipitation (ChIP)-sequencing using antibodies directed against H3K27ac and me3 as well as CBP and BRD4 had been performed following CBP- or BET-inhibition and are currently analyzed together with data derived from global mRNA-sequencing. In addition, we showed for the first time, that CBP is responsible for H3K27ac in H3.3K27M-mutated DIPG, whereas its homologue p300 acetylates H3K27 in pedHGG with H3WT. In ovo tumor growth determined by chorioallantoic membrane (CAM) assays revealed a strong tumor growth reducing potential of CBP- and BET-inhibitor treatment. CAM-pedHGG tumors are currently under investigation with regards to their invasive capacity and stemness-, invasion- and proliferation-marker expression. In summary, H3.3K27M mutation induces a strong tumor phenotype which is mediated by increased contribution of CBP-HAT activity and BET protein function in pedHGG making them a successful therapeutic target for pedHGG treatment.
Projektbezogene Publikationen (Auswahl)
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Combined treatment with CBP and BET inhibitors reverses inadvertent activation of detrimental super enhancer programs in DIPG cells. Cell Death & Disease, 11(8).
Wiese, Maria; Hamdan, Feda H.; Kubiak, Klaudia; Diederichs, Christopher; Gielen, Gerrit H.; Nussbaumer, Gunther; Carcaboso, Angel M.; Hulleman, Esther; Johnsen, Steven A. & Kramm, Christof M.
