We investigated the role of histone 3 lysine 9 trimethylation (H3K9me3), an epigenetic mechanism involved in the repression of synaptic plasticity and memory-related genes, within aging and Alzheimer's disease (AD). Our study reveals that elevated cortical H3K9me3 strongly correlates with cognitive dysfunction in individuals with mild cognitive impairment (MCI) and AD. In old (18 months) and younger (14 months) APPSWE/PS1ΔE9 and 3xTg AD mouse models, inhibiting SUV39H1 methyltransferase with ETP69, substantially reduces cerebral H3K9me3 levels and attenuates amyloid-β burden, tau pathology, and gliosis. Administration of ETP69 further promotes dendritic spine formation, leading to rapid and sustained improvements in cognitive function. Proteomics analysis indicates that a significant proportion of dysregulated proteins in the brains of AD-model mice are reversed by ETP69. These proteins are enriched for synaptic plasticity and learning-related pathways. ETP69 exerts its effects through multiple neuroprotective mechanisms, including regulation of neuroinflammation, induction of both blood and cerebral-infiltrating monocytes involved in cerebral Aβ clearance. Moreover, ETP69 activates brain-derived neurotrophic factor (Bdnf) network, and particularly its downstream effector neurosecretory protein Vgf. These findings support the pharmacological inhibition of H3K9me3-mediated gene silencing to reverse AD-related pathology and cognitive decline. ### Competing Interest Statement KLB is the CEO and shareholder of Fortem Neurosciences, Inc. KLB and MKH are inventors of a related patent Compositions and methods for treating Alzheimer's disease (PCT/US2021/044195). The other authors have no conflicts to disclose.

Figures S8 and S9. Quantification of expression of laminin-411 β1 chain, its binding β1 integrin, Notch family members and other stem cell markers after nanobioconjugate treatment. All markers were significantly suppressed in treated tumors.

The glioblastoma multiforme (GBM) microenvironment is highly immunosuppressive, leading to poor clinical outcomes and median survival of less than 15 months. Information about the nature of immunosuppressive and immunostimulatory cell types in GBM is fragmented, and a better understanding is urgently needed. We have developed a spatial phenotyping application that permits comprehensive characterization of the human GBM immune microenvironment. The Phenocycler-Fusion is a fast spatial biology solution that affords ultrahigh-plex single-cell spatial readouts. We used this solution for deep immune phenotyping of >50 proteins, comprising immune cell lineages, activation states and checkpoints, as well as markers for tumor, vascular and neuronal landscapes of GBM. Via single cell spatial phenotyping, we then isolated spatial signatures within the GBM tissue immune microenvironment. We focused on large numbers of glioma-associated macrophages that were identified via the combinatorial expression of key markers CD68, CD163, STAT1, STAT6, Iba-1 and SIRPα. The macrophages and their biomarker expression profiles displayed significant inter- and intra-tumoral heterogeneity, indicative of the complex biology of GBM. In addition, we profiled other immunosuppressive elements, including Tregs and exhausted T cells. Our data provide a comprehensive account of diverse immune cell populations in GBM and they may uncover systematic differences among GBM patient samples. Our application has enormous potential to further our understanding of the GBM immune microenvironment and is ready to be deployed by other laboratories.

Introduction: Tumor growth, invasion, and escape from immune surveillance largely depend on cancer microenvironment. Laminins are trimeric proteins and essential components of glioblastoma (GBM) microenvironment/extracellular matrix (ECM). In brain glioma samples from 230 patients, we found a correlation between the overexpression of tumor ECM protein laminin-411 (α4β1γ1) and faster tumor recurrence with shorter patient survival. Laminin-411 is produced by endothelial cells, neutrophils, monocytes, platelets, lymphocytes, and glioma cells and can modulate the immune system. Novel nanotechnology approach to block trimeric laminin-411 and activate of brain local immune system with brain delivery of PD-1 checkpoint inhibitor was developed for future translational application. Methods: Nanobioconjugates (NBC) based on poly (β-L-malic acid, P), were synthesized, characterized and used to intravenously treat mice with intracranial syngeneic GL261 or CT-2A GBM. The lead NBCs P/PEG/LLL(40%)/AON(α4β1)(2.0%)/AP-2(2%) and P/PEG/LLL(40%)/AP-2(2%)/αPD-1(0.2%) contained antisense to laminin-411 α4 and β1 chains, or αPD-1 antibody as well as trileucine (LLL) peptide for endosomal escape and AP-2 peptide for BBB crossing and tumor cell targeting. CRISPR/Cas9 constructs were used to knockdown α4 and β1 laminin chains in GBM ex vivo. Flow cytometry and RNA-seq analyses were performed to evaluate treatment. Results: Laminin-411 depletion with CRISPR/Cas9 and multifunctional NBC in vivo treatment equally suppressed GBM growth and significantly prolonged animal survival. The brain privileged immune system was activated upon treatment with a significant increase of CD3+, CD8+ T cells, NK, IFNγ+ NK cells, and M1 macrophages. RNA-sec analyses after treatment with a combination of NBC suppressing laminin-411 and checkpoint PD-1 showed enhanced anti-tumor effect with upregulation of genes coding for apoptotic Caspase 3 and IFNγ, and reduction of proliferation markers EGFR, c-Myc and Ki-67. Conclusion: Study describes novel GBM treatment strategy via NBCs crossing blood-brain barrier and targeting critical ECM and immune components of tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma. Citation Format: Alexander V. Ljubimov, Rameshwar Patil, Hui Ding, Liron Israel, Eggehard Holler, Julia Y. Ljubimova, Tao Sun, Keith L. Black. Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 575.

Video schematically shows the mechanism of glioblastoma treatment by laminin-411 inhibiting nanobioconjugate

Sex differences in cancer incidence are pervasive regardless of age or race. However, the molecular mechanisms underlying these differences remain undefined. Previously, we showed that sex differences in the regulation of retinoblastoma protein (Rb) function has been shown to correlate with differential vulnerability to malignant transformation to mesenchymal glioblastoma (mes-GBM) in male and female cortical astrocytes lacking functional neurofibromin and p53. We hypothesized that these sex differences in susceptibility to transformation may be driven by the differential activities of cyclin dependent kinase inhibitors. We found that p16 and p21, but not p27 exhibited sexually dimorphic activity corresponding to sex differences cell cycle arrest and transformation. In response to serum deprivation and DNA damage, female mes-GBM astrocytes increase p16 and p21 expression respectively, leading to cell cycle arrest. In contrast, male mes-GBM astrocytes continue to proliferate, acquire chromosomal aberrations, and display greater clonogenic cell frequency and in vivo tumorigenesis. Abrogation of p16 and p21 activity in female mes-GBM astrocytes mitigates these phenotypic differences. An analysis of pharmacological CDK inhibition revealed that the sex differences in p16 and p21 activity are correlated with sex differences in response to CDK4/6 and CDK5-CDK2 pathway inhibitors. These data provide new evidence for sex differences in growth regulation and transformation and suggest that these differences can render male and female cells differentially sensitive to targeted therapeutics. Finally, they indicate that sex differences should be considered in the investigation of novel cancer therapeutics in both the lab and clinical setting.

BACKGROUND: Male prevalence across histological subtypes of brain tumors is well known but unexplained. The effects of sex on human disease frequently result from acute actions of circulating sex hormones on cellular physiology and immune function. However, differences in brain tumor rates between males and females are comparable in prepubertal children, and pre and post-menopausal adults, suggesting that alternate mechanisms may underlie this disparity. Here we show that sex-specific differences exist in thresholds for malignant transformation within the astrocyte lineage and that this can contribute to male predominance in the most common brain tumor, glioblastoma (GBM). METHODS: Analysis of three publically accessible databases was utilized to determine whether sex differences are equally present in each of the four molecular subtypes of GBM. Stepwise transformation was modeled for Mesenchymal GBM in separate male and female murine astrocyte cultures by sequential inactivation of neurofibromin (NF1) and p53. Thresholds for transformation were measured in soft agar assays and with in vivo tumorigenesis assays. The molecular basis for differences in thresholds for transformation between male and female astrocytes was investigated in evaluations of stem cell induction, Rb inactivation and p21 activation. RESULTS: Significant male predominance was consistently observed in Proneural and Mesenchymal subtypes of GBM. We modeled Mesenchymal subtype GBM with combined loss of Nf1 and p53 function through cre-mediated knock-out of Nf1 (Nf1-/-) and expression of a dominant negative p53 construct (DNp53). These changes induced a subpopulation of cells with clonogenic, stem cell function in male but not female astrocytes. Male Nf1-/-,DNp53 astrocytes exhibited significantly greater growth compared to female Nf1-/-,DNp53 astrocytes. In addition, male but not female Nf1-/-,DNp53 astrocytes were transformed with either EGF treatment or in vivo implantation. Male Nf1-/-,DNp53 astrocytes exhibited greater in vivo tumorigencitiy regardless of the sex of the recipient mouse. Induced intracranial tumors were diagnosed as grade 4 astrocytomas based on standard histological features. Enhanced stem cell function and tumorigenicity in male Nf1-/-,DNp53 astrocytes was associated with greater inactivation of Rb and less p21 activation compared to female Nf1-/-,DNp53 astrocytes. CONCLUSIONS: Sex differences in cancer, including GBM can involve cell intrinsic sexual dimorphism in tumor suppressor pathways resulting in different thresholds for transformation in males and females. These results suggest that tumor suppressor and oncogene mechanisms should be evaluated in a sex-specific fashion and that screening and treatment of GBM and other cancers may need to be sex-specific. SECONDARY CATEGORY: n/a.

Abstract Background: Clinical studies have shown that brain cancers including Glioblastoma Multiforme (GBM) and several subtypes of medulloblastomas, occur more frequently in males than in females worldwide. The reason for this sex disparity is poorly understood. Sex differences in brain tumor rates exist not only in adults but also in the pediatric population. In children, brain tumor incidence peaks at approximately 4 years of age when the levels of circulating sex hormones are equivalently low in both boys and girls. This suggested to us that cell-intrinsic sex differences may play a role in the oncogenesis of brain tumors. The current study examines the contribution of cell intrinsic sexual dimorphism in oncogenic pathways to the sex differences in brain cancers. Methods: We analyzed 3 publicly accessible gene expression databases containing over 600 GBM patient specimens to determine whether sex differences are present in a molecular subtype specific fashion. We were able to assign sex to each patient based on the Y-chromosome gene expression, and found that male prevalence exists consistently in both proneural and mesenchymal subgroups of GBMs. Mesenchymal GBM is frequently associated with inactivation of two tumor suppressors Neurofibromin 1 (NF1) and TP53. Therefore, to model the impact of sex on the oncogenesis of glioblastoma, we established a step-wise transformation system by using murine astrocytes with ablation of Nf1. The Nf1-/- astrocytes were further engineered to lose p53 function by over-expression of dominant-negative p53 (DNp53). These Nf1-/-;DNp53 astrocytes were either treated with epidermal growth factor (EGF) in vitro, or implanted into immunodeficient mice. Results: Over-expression of DNp53 in Nf1-/- astrocytes enhanced the growth rate in both male and female astrocytes, but with a much greater increase in male cultures. In addition, EGF treatment resulted in in vitro transformation of male but not female, Nf1-/-;DNp53 astrocytes. In line with these results, limiting dilution analyses demonstrated that there is a much higher stem cell frequency in male than in female Nf1-/-;DN-p53 cells. Implants of Nf1-/-;DN-p53 cells into nude mice led to a male predominant pattern of tumor growth. Survival analyses on the nude mice with intracranial implants of the Nf1-/-;DNp53 cells showed a sex-dependent survival pattern with 100% death in mice bearing male Nf1-/-;DNp53 cells vs. 36% death in mice carrying the female counterparts. Conclusions: We established a model system that allows us to model the impact of sex on gliomagenesis. By employing Nf1-/-;DNp53 mouse astrocytes, we were able to demonstrate that male astrocytes are more susceptible to malignant transformation both in vitro and in vivo. Furthermore, our tumor formation data suggest that it is the cell-intrinsic sex differences rather than circulating sex hormones that may determine the sex disparity in glioma rates in patients. Citation Format: Tao Sun, Nicole M. Warrington, Jingqin Luo, Michael Brooks, Sonika Dahiya, Steven C. Snyder, Rajarshi Sengupta, Joshua B. Rubin. Investigating the sexually dimorphic susceptibility to brain cancer in a glioblastoma model system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 71. doi:10.1158/1538-7445.AM2014-71

In order to solve the problem of low space object interception rate by use of common radar search fence,a high interception rate seamless search method is proposed on the basis of periodicity of transit.Space target can be intercepted by search fence at least once at transiting,when the longitude coverage of search fence is greater than or equal to the repeat longitude difference of the target.Based on this characteristic,by optimizing the antenna elevation angel and the dwell time of beam positions,the space and energy distributions of search fence are changed to make the longitude coverage equal to the repeat longitude difference of the space objects in each orbit height,so the high interception rate search is realized with the least time resource expenditure.Numerical simulations have shown that the proposed seamless search method can increase the mean interception rate from 90.93% to 99.94%,compared with the common search method under the condition of the same time resource expenditure.The research in this paper has the high application value in enhancing the search efficiency of space surveillance radars.

Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.

Speculation regarding dysregulation of cAMP metabolism in oncogenesis has existed since the discovery of cAMP more than 50 years ago. Recent data confirm the relevance of disordered cAMP metabolism to the genesis of multiple cancers and suggest that the mechanism might involve altered expression and activity of phosphodiesterases (PDEs). These discoveries coincide with the rapid development and clinical evaluation of PDE inhibitors for non-cancer indications. Thus, the time is ripe to evaluate PDE inhibitors as cancer chemotherapeutics. Here we highlight recent evidence that abnormal regulation of cAMP levels might be a determinant of brain tumorigenesis and that altered PDE expression is one the mechanisms of its dysregulation. Recent preclinical and clinical experience with inhibitors of PDE4 indicates that this might be a promising approach to brain tumor therapy.