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.

FOXR2 Stabilizes MYCN Protein and Identifies Non-MYCN-Amplified Neuroblastoma Patients With Unfavorable Outcome.

PURPOSE: Clinical outcomes of patients with neuroblastoma range from spontaneous tumor regression to fatality. Hence, understanding the mechanisms that cause tumor progression is crucial for the treatment of patients. In this study, we show that FOXR2 activation identifies a subset of neuroblastoma tumors with unfavorable outcome and we investigate the mechanism how FOXR2 relates to poor outcome in patients. MATERIALS AND METHODS: We analyzed three independent transcriptional data sets of in total 1030 primary neuroblastomas with full clinical annotation. We performed immunoprecipitation for FOXR2 and MYCN and silenced FOXR2 expression in two neuroblastoma cell lines to examine the effect on cellular processes, transcriptome, and MYCN protein levels. Tumor samples were analyzed for protein levels of FOXR2 and MYCN. RESULTS: In three combined neuroblastoma data sets, 9% of tumors show expression of FOXR2 but have low levels of MYCN mRNA. FOXR2 expression identifies a group of patients with unfavorable outcome, showing 10-year overall survival rates of 53%-59%, and proves to be an independent prognostic factor compared with established risk factors. Transcriptionally, FOXR2-expressing tumors are very similar to MYCN-amplified tumors, suggesting that they might share a common mechanism of tumor initiation. FOXR2 knockdown in FOXR2-expressing neuroblastoma cell lines resulted in cell cycle arrest, reduced cell growth, cell death, and reduced MYCN protein levels, all indicating that FOXR2 is essential for these tumors. Finally, we show that FOXR2 binds and stabilizes MYCN protein and MYCN protein levels are highly increased in FOXR2-expressing tumors, in several cases comparable with MYCN-amplified samples. CONCLUSION: The stabilization of MYCN by FOXR2 represents an alternative mechanism to MYCN amplification to increase MYCN protein levels. As such, FOXR2 expression identifies another subset of neuroblastoma patients with unfavorable clinical outcome.

Targeting fibroblast growth factor receptors to combat aggressive ependymoma.

Ependymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.

Modeling SHH-driven medulloblastoma with patient iPS cell-derived neural stem cells.

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.

The molecular landscape of ETMR at diagnosis and relapse.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

EZHIP/CXorf67 mimics K27M mutated oncohistones and functions as an intrinsic inhibitor of PRC2 function in aggressive posterior fossa ependymoma.

BACKGROUND: Posterior fossa A (PFA) ependymomas are one of 9 molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis, and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported overexpression of chromosome X open reading frame 67 (CXorf67) as a hallmark of PFA ependymoma and showed that CXorf67 can interact with enhancer of zeste homolog 2 (EZH2), thereby inhibiting polycomb repressive complex 2 (PRC2), but the mechanism of action remained unclear. METHODS: We performed mass spectrometry and peptide modeling analyses to identify the functional domain of CXorf67 responsible for binding and inhibition of EZH2. Our findings were validated by immunocytochemistry, western blot, and methyltransferase assays. RESULTS: We find that the inhibitory mechanism of CXorf67 is similar to diffuse midline gliomas harboring H3K27M mutations. A small, highly conserved peptide sequence located in the C-terminal region of CXorf67 mimics the sequence of K27M mutated histones and binds to the SET domain (Su(var)3-9/enhancer-of-zeste/trithorax) of EZH2. This interaction blocks EZH2 methyltransferase activity and inhibits PRC2 function, causing de-repression of PRC2 target genes, including genes involved in neurodevelopment. CONCLUSIONS: Expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA tumors by mimicking K27M mutated histones. Disrupting the interaction between CXorf67 and EZH2 may serve as a novel targeted therapy for PFA tumors but also for other tumors that overexpress CXorf67. Based on its function, we have renamed CXorf67 as "EZH Inhibitory Protein" (EZHIP).

Heterogeneity within the PF-EPN-B ependymoma subgroup.

Posterior fossa ependymoma comprise three distinct molecular variants, termed PF-EPN-A (PFA), PF-EPN-B (PFB), and PF-EPN-SE (subependymoma). Clinically, they are very disparate and PFB tumors are currently being considered for a trial of radiation avoidance. However, to move forward, unraveling the heterogeneity within PFB would be highly desirable. To discern the molecular heterogeneity within PFB, we performed an integrated analysis consisting of DNA methylation profiling, copy-number profiling, gene expression profiling, and clinical correlation across a cohort of 212 primary posterior fossa PFB tumors. Unsupervised spectral clustering and t-SNE analysis of genome-wide methylation data revealed five distinct subtypes of PFB tumors, termed PFB1-5, with distinct demographics, copy-number alterations, and gene expression profiles. All PFB subtypes were distinct from PFA and posterior fossa subependymomas. Of the five subtypes, PFB4 and PFB5 are more discrete, consisting of younger and older patients, respectively, with a strong female-gender enrichment in PFB5 (age: p = 0.011, gender: p = 0.04). Broad copy-number aberrations were common; however, many events such as chromosome 2 loss, 5 gain, and 17 loss were enriched in specific subtypes and 1q gain was enriched in PFB1. Late relapses were common across all five subtypes, but deaths were uncommon and present in only two subtypes (PFB1 and PFB3). Unlike the case in PFA ependymoma, 1q gain was not a robust marker of poor progression-free survival; however, chromosome 13q loss may represent a novel marker for risk stratification across the spectrum of PFB subtypes. Similar to PFA ependymoma, there exists a significant intertumoral heterogeneity within PFB, with distinct molecular subtypes identified. Even when accounting for this heterogeneity, extent of resection remains the strongest predictor of poor outcome. However, this biological heterogeneity must be accounted for in future preclinical modeling and personalized therapies.

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

Epidemiology, molecular classification and WHO grading of ependymoma.

Ependymoma can arise throughout all compartments of the central nervous system with prevalence for intracranial and spinal location in children and adults, respectively. The current histopathology based WHO grading system distinguishes grade I, II 'classic', and III 'anaplastic' ependymoma. However, analysis of multiple cohorts of intracranial ependymoma demonstrate a wide variance in the utility of the grade II versus grade III distinction as a prognostic marker that may additionally be confounded by the anatomic compartment. Recent (epi)genomic profiling efforts have identified molecularly distinct groups of ependymoma arising from all three anatomic compartments of the central nervous system that outperform the current histopathological classification regarding clinical associations. These advances have led to the cognition that molecular classification should be part of all future clinical trials in ependymoma patients. Clinical management of intracranial ependymomas (WHO Grade II/III) is challenging and molecular classification based risk stratification may help to intensify treatment and surveillance in high-risk patients but to de-escalate therapy in certain patient groups at low risk for recurrence. Finally, experience of neurosurgeons, and other disciplines, as well as intensified co-operation between all stakeholders involved hold promise to finally improve outcome of patients affected with ependymoma.

Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy.

Defensins are small antimicrobial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastrointestinal and genito-urinary tracts. Here, we demonstrate, using patient biopsy specimens, that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ This finding prompted us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 (HAdV3). HAdV3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles, during replication. We have previously shown that PtDd are involved in opening epithelial junctions, thus facilitating lateral spread of de novo-produced virions. Here, we describe a second function of PtDd, namely, the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was an HAdV3 mutant virus in which formation of PtDd was disabled (mut-Ad3GFP, where GFP is green fluorescent protein). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type (wt) form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production of mut-Ad3GFP but not of wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not of wt-Ad3GFP. These data suggest a new mechanism of HAdV3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our manuscript are the following: (i) the discovery of a new mechanism used by human adenovirus serotype 3 to overcome innate antiviral host responses that is based on the capacity of HAdV3 to produce subviral penton-dodecahedral particles that act as decoys for HD5, thus preventing the inactivation of virus progeny produced upon replication; (ii) the demonstration that ectopic HD5 expression in cancer cells decreases the oncolytic efficacy of a serotype 5-based adenovirus vector; and (iii) the demonstration that epithelial ovarian and lung cancers express HD5. The study improves our understanding of how adenoviruses establish infection in epithelial tissues and has implications for cancer therapy with oncolytic adenoviruses.