Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration.

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

SMARCA4 vulnerability in H3K27M midline glioma: A silver bullet for a lethal disease.

To investigate epigenetic dependencies and identify therapeutic vulnerabilities, Mo et al.1 and Panditharatna et al.2 performed CRISPR screens and show that deadly H3K27M gliomas are dependent on mammalian BAF (SWI/SNF) chromatin remodeling complex.

Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry.

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.

Preventive activity of Copaifera langsdorffii Desf. leaves extract and its major compounds, afzelin and quercitrin, on DNA damage in in vitro and in vivo models.

Copaifera langsdorffii Desf. is a plant found in South America, especially in Brazil. Oleoresin and the leaves of this plant is used as a popular medicinal agent. However, few studies on the chemical composition of aerial parts and related biological activities are known. This study aimed to examine the cytotoxic, genotoxic, and antigenotoxic potential of C. langsdorffii aerial parts hydroalcoholic extract (CLE) and two of its major compounds afzelin and quercitrin. The cytotoxic and antigenotoxic potential of CLE was determined as follows: 1) against genotoxicity induced by doxorubicin (DXR) or methyl methanesulfonate (MMS) in V79 cells; 2) by direct and indirect-acting mutagens in Salmonella typhimurium strains; and 3) by MMS in male Swiss mice. The protective effects of afzelin and quercitrin against DXR or MMS were also evaluated in V79 and HepG2 cells. CLE was cytotoxic as evidenced by clonogenic efficiency assay. Further, CLE did not induce a significant change in frequencies of chromosomal aberrations and micronuclei; as well as number of revertants in the Ames test demonstrating absence of genotoxicity. In contrast, CLE was found to be antigenotoxic in mammalian cells. The results also showed that CLE exerted inhibitory effect against indirect-acting mutagens in the Ames test. Afzelin and quercitrin did not reduce genotoxicity induced by DXR or MMS in V79 cells. However, treatments using afzelin and quercitrin decreased MMS-induced genotoxicity in HepG2 cells. The antigenotoxic effect of CLE observed in this study may be partially attributed to the antioxidant activity of the combination of major components afzelin and quercitrin.

Distinct response to GDF15 knockdown in pediatric and adult glioblastoma cell lines.

INTRODUCTION: Glioblastoma (GBM) is the most common malignant primary brain tumor affecting adults. In pediatric patients, GBM exhibits genetic variations distinct from those identified in the adult GBM phenotype. This tumor exhibits complex genetic changes leading to malignant progression and resistance to standard therapies including radiotherapy and temozolomide treatment. The GDF15 gene codes for a growth factor whose expression is altered in the presence of inflammations and malignancies. GDF15 is associated with a poor prognosis and with radio- and chemoresistance in a variety of tumors. The aim of this study was to compare the response to GDF15 knockdown in adult (U343) and pediatric (KNS42) GBM cell line models. METHODS: The expression of the GDF15 gene was investigated by qRT-PCR and overexpression was identified in both GBM cell lines. The KNS42 and U343 cell lines were submitted to lentiviral transduction with shRNA of GDF15 and validated at the protein level. To understand the difference between cell lines, RNAseq was performed after GDF15 knockdown. RESULTS: The data obtained demonstrated that the pathways were differentially expressed in adult GBM and pediatric GBM cell lines. This was confirmed by functional assays perfomed after independent treatments (radiotherapy and TMZ). CONCLUSION: These results demonstrated that GBM cell lines had distinct responses to GDF15 knockdown, a fact that can be explained by the different molecular profile of pediatric and adult GBM.

The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma.

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

The histone deacetylase inhibitor PCI-24781 as a putative radiosensitizer in pediatric glioblastoma cell lines.

BACKGROUND: Glioblastoma (GBM) is considered to be one of the most aggressive tumors of the central nervous system (CNS). Even with the use of modern treatment protocols, the prognosis remains reserved, with children with GBM having a mean survival of 12-15 months. METHODS: In the present study we investigated the potential radiosensitizing effect of PCI-24781, a potent pan-histone deacetylase inhibitor (HDACi), on the SF188 and KNS42 cell lines of pediatric GBM. Cell proliferation rates, clonogenicity and apoptosis were compared in the presence and absence of treatment with PCI-24781. We also compared the clonogenicity rates of the irradiated SF188 and KNS42 cell lines with or without previous treatment with PCI-24781 at the doses of 0.25-16 µM. In addition, we investigated the effects of PCI-24781 on the expression of some of the main proteins responsible for the repair of double-strand DNA breaks caused by irradiation. RESULTS: The inhibitor blocked cell proliferation, induced death by apoptosis and reduced the colony forming capacity of the cell lines, both of them showing a significant decrease of colony formation at all irradiation doses. The expression of the Rad51 protein, important for the homologous recombination (HR) repair pathway, and of the DNA-PKcs, Ku70 and Ku86 proteins, important for the non-homologous end joining (NHEJ) repair pathway, was more reduced when the irradiated cell line was previously treated with PCI-24781 than when it was treated exclusively with radiotherapy. CONCLUSIONS: These findings demonstrate that HDACi PCI-24781 has a radiosensitizing profile that compromises the repair of double-strand DNA breaks in cells of pediatric GBM treated with radiotherapy.

In vitro cytotoxicity, genotoxicity and antigenotoxicity assessment of Solanum lycocarpum hydroalcoholic extract.

CONTEXT: Solanum lycocarpum A. St.-Hil. (Solanaceae), popularly known as 'fruta-do-lobo' (wolf fruit), 'lobeira' and 'jurubebão', is commonly used by native people of Central Brazil in powder form or as a hydroalcoholic extract for the management of diabetes and obesity and to decrease cholesterol levels. OBJECTIVE: The present study determines the possible cytotoxic, genotoxic and antigenotoxic activities of hydroalcoholic extract of the S. lycocarpum fruits (SL). MATERIALS AND METHODS: The clonogenic efficiency assay was used to determine the cytotoxicity. Three concentrations of SL (16, 32 and 64 µg/mL) were used for the evaluation of its genotoxic and antigenotoxic potential on V79 cells using the micronucleus and comet assays. In the antigenotoxicity assays, the cells were treated simultaneously with SL and the alkylating agent methyl methanesulphonate (MMS, 44 µg/mL for the micronucleus assay and 22 µg/mL for the comet assay) as an inducer of micronuclei and DNA damage. RESULTS: The results showed that SL was cytotoxic at concentrations up to 64 µg/mL. No significant differences in the rate of chromosome or DNA damage were observed between cultures treated with SL and the control group. In addition, the frequencies of micronuclei and DNA damage induced by MMS were significantly reduced after treatment with SL. The damage reduction percentage ranged from 68.1% to 79.2% and 12.1% to 16.5% for micronucleus and comet assays, respectively. DISCUSSION AND CONCLUSION: SL exerted no genotoxic effect and exhibited chemopreventive activity against both genomic and chromosome damage induced by MMS.

Oncohistones in brain tumors: the soil and seed.

Recurrent somatic mutations in histone 3 (H3) variants (termed 'oncohistones') have been identified in high-grade gliomas (HGGs) in children and young adults and induce tumorigenesis through disruption of chromatin states. Oncohistones occur with exquisite neuroanatomical specificity and are associated with specific age distribution and epigenome landscapes. Here, we review the known intrinsic ('seed') and the extrinsic ('soil') factors needed for their optimal oncogenic effect and highlight the many unresolved questions regarding their effects on development and crosstalk with the tumor microenvironment. The 'seed and soil' analogy, used to explain tumor metastatic niches, also applies to oncohistones, which mainly thrive and flourish in specific chromatin states during very narrow windows of development, creating exquisite vulnerabilities, which could provide effective therapies for these deadly cancers.

A Compendium of Syngeneic, Transplantable Pediatric High-Grade Glioma Models Reveals Subtype-Specific Therapeutic Vulnerabilities.

Pediatric high-grade gliomas (pHGG) are lethal, incurable brain tumors frequently driven by clonal mutations in histone genes. They often harbor a range of additional genetic alterations that correlate with different ages, anatomic locations, and tumor subtypes. We developed models representing 16 pHGG subtypes driven by different combinations of alterations targeted to specific brain regions. Tumors developed with varying latencies and cell lines derived from these models engrafted in syngeneic, immunocompetent mice with high penetrance. Targeted drug screening revealed unexpected selective vulnerabilities-H3.3G34R/PDGFRAC235Y to FGFR inhibition, H3.3K27M/PDGFRAWT to PDGFRA inhibition, and H3.3K27M/PDGFRAWT and H3.3K27M/PPM1DΔC/PIK3CAE545K to combined inhibition of MEK and PIK3CA. Moreover, H3.3K27M tumors with PIK3CA, NF1, and FGFR1 mutations were more invasive and harbored distinct additional phenotypes, such as exophytic spread, cranial nerve invasion, and spinal dissemination. Collectively, these models reveal that different partner alterations produce distinct effects on pHGG cellular composition, latency, invasiveness, and treatment sensitivity. SIGNIFICANCE: Histone-mutant pediatric gliomas are a highly heterogeneous tumor entity. Different histone mutations correlate with different ages of onset, survival outcomes, brain regions, and partner alterations. We have developed models of histone-mutant gliomas that reflect this anatomic and genetic heterogeneity and provide evidence of subtype-specific biology and therapeutic targeting. See related commentary by Lubanszky and Hawkins, p. 1516. This article is highlighted in the In This Issue feature, p. 1501.

Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells.

BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

H3K27me3 spreading organizes canonical PRC1 chromatin architecture to regulate developmental programs.

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights: The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions.The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs.Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture.The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

Arsenic Trioxide exerts cytotoxic and radiosensitizing effects in pediatric Medulloblastoma cell lines of SHH Subgroup.

We evaluated the potential effects of ATO in different pediatric SHH-MB cell lines (ONS-76: TP53-wild type; DAOY and UW402: TP53-mutated). MB cell lines molecular subgroup was confirmed and TP53 mutations were validated. Cell viability, clonogenicity and apoptosis were evaluated after ATO treatment at different concentrations (1-16 µM) alone or combined with irradiation doses (0.5, 1, 2 and 4 Gy). Rad51 and Ku86 proteins were evaluated by WB. ATO treatment reduced cell viability for all SHH-MB cell lines. Significant decrease of clonogenic capacity and higher apoptosis rates were also observed after ATO exposure, being cell death more pronounced (>70%) for the SHH-MB TP53-mutated. Combined treatment of ATO with irradiation also reduced colonies formation in UW402 tumor cells, which was independent of DNA damage repair proteins Rad51 and Ku86. In silico analyses suggested that a set of genes from cell cycle and p53 pathways are differentially expressed in SHH tumor subtypes, suggesting that cell lines may respond to therapies according to the gene expression profiles. Herein, we showed ATO cytotoxicity in pediatric SHH cell lines, with marked radiosensitizing effect for the MB-SHH TP53-mutated cells. These results highlight the potential of ATO, alone or in combination with radiotherapy, supporting further clinical investigations.

Molecular characterization of Wnt pathway and function of ß-catenin overexpression in medulloblastoma cell lines.

Medulloblastoma (MB) is the most common malignant childhood brain tumor. MB is currently classified into four molecular subgroups (Wnt, Shh, Group 3, and Group 4). The wingless (Wnt) pathway is responsible for embryonic development and is deregulated in MB. We analyzed the activation of the Wnt pathway in MB cell lines and its correlation with the Shh pathway, with emphasis on the importance of cellular characterization. Transient ß-catenin transfection led to an increase in the ß-catenin gene and protein expression in MB cell lines. Wnt pathway activation resulted in a reduced number of colonies in all cell lines studied and a significant increase in the G2/M cell cycle phase only in ONS-76 cells. Regarding the Shh pathway, transfection caused a reduced expression of the PTCH1 and SMO genes only in the UW473 cells. Further studies are needed to understand the mechanism underlying the molecular events associated with the effects of Wnt activation in MB.

Antitumour activity of AMG 900 alone or in combination with histone deacetylase inhibitor SaHa on medulloblastoma cell lines.

OBJECTIVES: Medulloblastoma (MB) is the most common malignant childhood brain tumour. Aurora kinases are essential for cell division and are primarily active during mitosis. Recently, the combination of aurora kinases inhibitors (iAURK) and histone deacetylase inhibitors (iHDAC) has shown potential antitumour effects and had significant biological effects in preclinical cancer models. In this study, we analysed the effects of the pan-aurora kinases inhibitor AMG 900 alone or in combination with the iHDAC SaHa (Vorinostat) on paediatric MB cell lines (UW402, UW473 and ONS-76). METHODS: Cell proliferation was measured by XTT assay, apoptosis was determined by flow cytometry and clonogenic capacity was studied. qRT-PCR assays were used to determine the mRNA expression in MB cell lines after treatment. Drug combination analyses were made based on Chou-Talalay method. RESULTS: AMG 900 caused the inhibition of cell proliferation, diminution of clonogenic capacity and increased the apoptosis rate in cell lines (P < 0.05). A synergistic effect in the AMG900-SaHa combination was evidenced on the inhibition of cell proliferation in all cell lines, especially in sequential drug treatment. Moreover, the combination of these drugs reached 100% of the inhibition in colony formation (synergistic effect). The treatment with AMG 900 increased the p21 and GDF15 expression, but did not alter the TP53 in one of the cell lines. CONCLUSIONS: These results indicate that AMG 900 may be a promising drug for the adjuvant treatment of MB, mainly when combined with iHDAC.

Chromosomal heterogeneity and instability characterize pediatric medulloblastoma cell lines and affect neoplastic phenotype.

Chromosomal heterogeneity is a hallmark of most tumors and it can drive critical events as growth advantages, survival advantages, progression and karyotypic evolution. Medulloblastoma (MB) is the most common malignant central nervous system tumor in children. This work attempted to investigate chromosomal heterogeneity and instability profiles of two MB pediatric cell lines and their relationship with cell phenotype. We performed GTG-banding and cytokinesis-block micronucleus cytome assays, as well as morphological characterization, cell population doubling time, colony-forming efficiency, and chemo-sensitivity assays in two pediatric MB cell lines (UW402 and UW473). Both MB cells showed a high chromosomal heterogeneity. UW473 cells showed ~2 fold higher both clonal- and non-clonal chromosomal alterations than UW402 cells. Besides, UW473 showed two clonal-groups well-differentiated by ploidy level (<2n> and <4n>) and also presented a significantly higher number of chromosomal instability biomarkers. These results were associated with high morphological heterogeneity and survival advantages for UW473 and proliferation advantages for UW402 cells. Moreover, UW473 was significantly more sensitive to methotrexate, temozolomide and cisplatin while UW402 cells were more sensitive to doxorubicin. These data suggest that distinct different degrees of karyotypic heterogeneity and instability may affect neoplasic phenotype of MB cells. These findings bring new insights into cell and tumor biology.