Targeting Group 3 Medulloblastoma by the Anti-PRUNE-1 and Anti-LSD1/KDM1A Epigenetic Molecules.

Medulloblastoma (MB) is a highly malignant childhood brain tumor. Group 3 MB (Gr3 MB) is considered to have the most metastatic potential, and tailored therapies for Gr3 MB are currently lacking. Gr3 MB is driven by PRUNE-1 amplification or overexpression. In this paper, we found that PRUNE-1 was transcriptionally regulated by lysine demethylase LSD1/KDM1A. This study aimed to investigate the therapeutic potential of inhibiting both PRUNE-1 and LSD1/KDM1A with the selective inhibitors AA7.1 and SP-2577, respectively. We found that the pharmacological inhibition had a substantial efficacy on targeting the metastatic axis driven by PRUNE-1 (PRUNE-1-OTX2-TGFß-PTEN) in Gr3 MB. Using RNA seq transcriptomic feature data in Gr3 MB primary cells, we provide evidence that the combination of AA7.1 and SP-2577 positively affects neuronal commitment, confirmed by glial fibrillary acidic protein (GFAP)-positive differentiation and the inhibition of the cytotoxic components of the tumor microenvironment and the epithelial-mesenchymal transition (EMT) by the down-regulation of N-Cadherin protein expression. We also identified an impairing action on the mitochondrial metabolism and, consequently, oxidative phosphorylation, thus depriving tumors cells of an important source of energy. Furthermore, by overlapping the genomic mutational signatures through WES sequence analyses with RNA seq transcriptomic feature data, we propose in this paper that the combination of these two small molecules can be used in a second-line treatment in advanced therapeutics against Gr3 MB. Our study demonstrates that the usage of PRUNE-1 and LSD1/KDM1A inhibitors in combination represents a novel therapeutic approach for these highly aggressive metastatic MB tumors.

Epigenetics and immune cells in medulloblastoma.

Medulloblastoma (MB) is a highly malignant childhood tumor of the cerebellum. Transcriptional and epigenetic signatures have classified MB into four molecular subgroups, further stratified into biologically different subtypes with distinct somatic copy-number aberrations, driver genes, epigenetic alterations, activated pathways, and clinical outcomes. The brain tumor microenvironment (BTME) is of importance to regulate a complex network of cells, including immune cells, involved in cancer progression in brain malignancies. MB was considered with a "cold" immunophenotype due to the low influx of immune cells across the blood brain barrier (BBB). Recently, this assumption has been reconsidered because of the identification of infiltrating immune cells showing immunosuppressive phenotypes in the BTME of MB tumors. Here, we are providing a comprehensive overview of the current status of epigenetics alterations occurring during cancer progression with a description of the genomic landscape of MB by focusing on immune cells within the BTME. We further describe how new immunotherapeutic approaches could influence concurring epigenetic mechanisms of the immunosuppressive cells in BTME. In conclusion, the modulation of these molecular genetic complexes in BTME during cancer progression might enhance the therapeutic benefit, thus firing new weapons to fight MB.

Germline rare variants of lectin pathway genes predispose to asymptomatic SARS-CoV-2 infection in elderly individuals.

PURPOSE: Emerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants - referred as qualifying variants (QVs) - of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. METHODS: Exploiting exome sequencing data and 56 CS genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (aged ≥60 years) and 56,885 European individuals from the Genome Aggregation Database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized patients with COVID-19. RESULTS: We found an enrichment of QVs in 3 genes (MASP1, COLEC11, and COLEC10), which belong to the lectin pathway, in the asymptomatic cohort. Analyses of complement activity in serum showed decreased activity of lectin pathway in asymptomatic individuals with QVs. Finally, we found allelic variants associated with asymptomatic COVID-19 phenotype and with a decreased expression of MASP1, COLEC11, and COLEC10 in lung tissue. CONCLUSION: This study suggests that genetic rare variants can protect from severe COVID-19 by mitigating the activity of lectin pathway and prothrombin. The genetic data obtained through ES of 786 asymptomatic and 147 hospitalized individuals are publicly available at http://espocovid.ceinge.unina.it/.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Metabolic Regulation of the Epigenome Drives Lethal Infantile Ependymoma.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.

Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood-brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.

Recurrent noncoding U1 snRNA mutations drive cryptic splicing in SHH medulloblastoma.

In cancer, recurrent somatic single-nucleotide variants-which are rare in most paediatric cancers-are confined largely to protein-coding genes1-3. Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5' splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5' cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes (PTCH1) and activates oncogenes (GLI2 and CCND2), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer.

Alterations in ALK/ROS1/NTRK/MET drive a group of infantile hemispheric gliomas.

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-ß-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

A Hematogenous Route for Medulloblastoma Leptomeningeal Metastases.

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma.

In vivo bioluminescence imaging using orthotopic xenografts towards patient's derived-xenograft Medulloblastoma models.

BACKGROUND: Medulloblastoma is a cerebellar neoplasia of the central nervous system. Four molecular subgrups have been identified (MBWNT, MBSHH, MBgroup3 and MBgroup4) with distinct genetics and clinical outcome. Among these, MBgroup3-4 are highly metastatic with the worst prognosis. The current standard therapy includes surgery, radiation and chemotherapy. Thus, specific treatments adapted to cure those different molecular subgroups are needed. The use of orthotopic xenograft models, together with the non-invasive in vivo biolumiscence imaging (BLI) technology, is emerging during preclinical studies to test novel therapeutics for medulloblastoma treatment. METHODS: Orthotopic MB xenografts were performed by injection of Daoy-luc cells, that had been previously infected with lentiviral particles to stably express luciferase gene, into the fourth right ventricle of the cerebellum of ten nude mice. For the implantation, specific stereotactic coordinates were used. Seven days after the implantation the mice were imaged by acquisitions of bioluminescence imaging (BLI) using IVIS 3D Illumina Imaging System (Xenogen). Tumor growth was evaluated by quantifying the bioluminescence signals using the integrated fluxes of photons within each area of interest using the Living Images Software Package 3.2 (Xenogen-Perkin Elmer). Finally, histological analysis using hematoxylin-eosin staining was performed to confirm the presence of tumorigenic cells into the cerebellum of the mice. RESULTS: We describe a method to use the in vivo bioluminescent imaging (BLI) showing the potential to be used to investigate the potential antitumorigenic effects of a drug for in vivo medulloblastoma treatment. We also discuss other studies in which this technology has been applied to obtain a more comprehensive knowledge of medulloblastoma using orthotopic xenograft mouse models. CONCLUSIONS: There is a need to develop patient's derived-xenograft (PDX) model systems to test novel drugs for medulloblastoma treatment within each molecular sub-groups with a higher predictive value. Here we show how this technology should be applied with hopes on generations of new treatments to be applied then in human.

Integrated (epi)-Genomic Analyses Identify Subgroup-Specific Therapeutic Targets in CNS Rhabdoid Tumors.

We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.

A therapeutic approach to treat prostate cancer by targeting Nm23-H1/h-Prune interaction.

Nm23-H1 is a metastasis suppressor gene whose overexpression is associated with both reduced cell motility in various cancers and increased metastatic potential in neuroblastomas, osteosarcomas, and hematological malignances. We previously reported that Nm23-H1 exerts tumor suppressor action in prostate cancer cells and that h-Prune, which is overexpressed in various tumor types, binds Nm23-H1. Moreover, blockage of the Nm23-H1/h-Prune interaction with a competitive permeable peptide (CPP) attenuates migration of breast and neuroblastoma cells. This series of events suggests that the Nm23-H1/h-Prune protein complex regulates cancer progression and that its specific impairment could be a new therapeutic strategy in oncology. We found that CPP leads to inhibition of the AKT/mTORv and NF-kBv signaling pathways and also activates apoptosis. To obtain a proof-of-concept of our hypothesis, we used a xenograft model of prostate cancer to evaluate whether impairment of this complex using CPP results in an anti-tumoral effect. Using a mouse orthotopic model with bioluminescent imaging, we show evidences that CPP reduces prostate cancer metastases formation. In conclusion, CPP being able to impair formation of the h-Prune/Nm23-H1 complex holds promise for the treatment of prostate cancer.

MiR-34a deficiency accelerates medulloblastoma formation in vivo.

Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR-34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR-34a in vivo. We generated mice with a constitutive deletion of the miR-34a gene. These mice were devoid of mir-34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re-expression of miR-34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR-34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR-34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR-34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR-34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re-express miR-34a in tumors could, therefore, represent an efficient therapeutic option.

H-Prune through GSK-3ß interaction sustains canonical WNT/ß-catenin signaling enhancing cancer progression in NSCLC.

H-Prune hydrolyzes short-chain polyphosphates (PPase activity) together with an hitherto cAMP-phosphodiesterase (PDE), the latest influencing different human cancers by its overexpression. H-Prune promotes cell migration in cooperation with glycogen synthase kinase-3 (Gsk-3ß). Gsk-3ß is a negative regulator of canonical WNT/ß-catenin signaling. Here, we investigate the role of Gsk-3ß/h-Prune complex in the regulation of WNT/ß-catenin signaling, demonstrating the h-Prune capability to activate WNT signaling also in a paracrine manner, through Wnt3a secretion. In vivo study demonstrates that h-Prune silencing inhibits lung metastasis formation, increasing mouse survival. We assessed h-Prune levels in peripheral blood of lung cancer patients using ELISA assay, showing that h-Prune is an early diagnostic marker for lung cancer. Our study dissects out the mechanism of action of h-Prune in tumorigenic cells and also sheds light on the identification of a new therapeutic target in non-small-cell lung cancer.

Early targets of miR-34a in neuroblastoma.

Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3'-UTR. By combining the proteomics data with Kaplan Meier gene-expression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3'-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-ß, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL.