Chromodomain Helicase DNA-Binding Protein 7 Is Suppressed in the Perinecrotic/Ischemic Microenvironment and Is a Novel Regulator of Glioblastoma Angiogenesis.

Tumorigenic and non-neoplastic tissue injury occurs via the ischemic microenvironment defined by low oxygen, pH, and nutrients due to blood supply malfunction. Ischemic conditions exist within regions of pseudopalisading necrosis, a pathological hallmark of glioblastoma (GBM), the most common primary malignant brain tumor in adults. To recapitulate the physiologic microenvironment found in GBM tumors and tissue injury, we developed an in vitro ischemic model and identified chromodomain helicase DNA-binding protein 7 (CHD7) as a novel ischemia-regulated gene. Point mutations in the CHD7 gene are causal in CHARGE syndrome (a developmental disorder causing coloboma, heart defects, atresia choanae, retardation of growth, and genital and ear anomalies) and interrupt the epigenetic functions of CHD7 in regulating neural stem cell maintenance and development. Using our ischemic system, we observed microenvironment-mediated decreases in CHD7 expression in brain tumor-initiating cells and neural stem cells. Validating our approach, CHD7 was suppressed in the perinecrotic niche of GBM patient and xenograft sections, and an interrogation of patient gene expression datasets determined correlations of low CHD7 with increasing glioma grade and worse patient outcomes. Segregation of GBM by molecular subtype revealed a novel observation that CHD7 expression is elevated in proneural versus mesenchymal GBM. Genetic targeting of CHD7 and subsequent gene ontology analysis of RNA sequencing data indicated angiogenesis as a primary biological function affected by CHD7 expression changes. We validated this finding in tube-formation assays and vessel formation in orthotopic GBM models. Together, our data provide further understanding of molecular responses to ischemia and a novel function of CHD7 in regulating angiogenesis in both neoplastic and non-neoplastic systems. Stem Cells 2019;37:453-462.

Collaborating to support school reintegration following suicide-related crises: Voices from the field.

Adolescent psychiatric hospitalization for suicide-related crises continues to rise. Although previous reviews have identified frameworks for supporting youth as they return to school settings, there is a need to identify and address barriers to collaboration across hospitals and schools. This qualitative study explored school and hospital professional perspectives to inform a pathway toward partnership for improving practices for school reintegration. As part of a larger project that has been developing guidelines for adolescent school reintegration following psychiatric hospitalization for suicide-related crises, the present study explored professional perceptions of (a) school interactions during hospital stays and (b) recommendations for adolescents, families, school professionals, and hospital professionals. We conducted in-depth interviews with 19 school professionals and seven hospital professionals and analyzed transcribed interviews using Applied Thematic Analysis. Communication and collaboration emerged as cross-cutting themes across research questions, with additional themes considered across a continuum of care. Findings inform the ways in which professionals can collaborate to support adolescent recovery, spanning universal approaches implemented in advance of a crisis to approaches enacted during and following psychiatric care. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Mutant tristetraprolin: a potent inhibitor of malignant glioma cell growth.

Malignant gliomas rely on the production of certain critical growth factors including VEGF, interleukin (IL)-6 and IL-8, to fuel rapid tumor growth, angiogenesis, and treatment resistance. Post-transcriptional regulation through adenine and uridine-rich elements of the 3' untranslated region is one mechanism for upregulating these and other growth factors. In glioma cells, we have shown that the post-transcriptional machinery is optimized for growth factor upregulation secondary to overexpression of the mRNA stabilizer, HuR. The negative regulator, tristetraprolin (TTP), on the other hand, may be suppressed because of extensive phosphorylation. Here we test that possibility by analyzing the phenotypic effects of a mutated form of TTP (mt-TTP) in which 8 phosphoserine residues were converted to alanines. We observed a significantly enhanced negative effect on growth factor expression in glioma cells at the post-transcriptional and transcriptional levels. The protein became stabilized and displayed significantly increased antiproliferative effects compared to wild-type TTP. Macroautophagy was induced with both forms of TTP, but inhibition of autophagy did not affect cell viability. We conclude that glioma cells suppress TTP function through phosphorylation of critical serine residues which in turn contributes to growth factor upregulation and tumor progression.

Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase.

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas' disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

Kinomic profiling of glioblastoma cells reveals PLCG1 as a target in restricted glucose.

BACKGROUND: For glioblastoma (GBM) treatments to be effective in vivo, understanding the effects of the tumor microenvironment is imperative. In traditional cell culture conditions, glucose concentrations do not model physiologic levels, nor the diminished concentrations found in tumor niches. We therefore sought to profile the differences in kinase activity in GBM cells cultured in restricted glucose to identify pathways that could be targeted with small molecule inhibitors. METHODS: Using the PamStation12 platform, we examined the ability of GBM lysates from cells cultured in standard or low glucose conditions to phosphorylate 144 tyrosine and 144 serine/threonine peptides that correspond to known protein phosphorylation sites. Potential kinase targets were identified and validated using small molecule kinase inhibitors in GBM spheroid cultures. RESULTS: Using results from two GBM patient-derived xenografts, we determined common changes to peptides derived from Phospholipase C, Gamma 1 (PLCG1) and Raf-1. Using PLC and Raf inhibitors, we found a significantly stronger growth inhibitory effect of the PLC inhibitor U73122 under restricted glucose conditions. In contrast, Raf inhibitors were significantly growth inhibitory regardless of the nutrient level tested. CONCLUSIONS: Together, our data demonstrate that kinase activity is altered in low glucose conditions and that kinomic profiling can assist with the identification of effective strategies to target GBM growth. Our data further suggest the importance of accurately modeling the tumor microenvironment to reproduce cancer cell signaling and develop drug screens for anti-cancer agents.

Reactive species balance via GTP cyclohydrolase I regulates glioblastoma growth and tumor initiating cell maintenance.

Background: Depending on the level, differentiation state, and tumor stage, reactive nitrogen and oxygen species inhibit or increase cancer growth and tumor initiating cell maintenance. The rate-limiting enzyme in a pathway that can regulate reactive species production but has not been thoroughly investigated in glioblastoma (GBM; grade IV astrocytoma) is guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1). We sought to define the role of GCH1 in the regulation of GBM growth and brain tumor initiating cell (BTIC) maintenance. Methods: We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples, and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production, and survival in orthotopic patient-derived xenograft models were determined. Results: GCH1 was expressed in GBMs with elevated but not exclusive RNA and protein levels in BTICs in comparison to non-BTICs. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In converse experiments, GCH1 knockdown with short hairpin RNA led to GBM cell growth inhibition and reduced self-renewal in association with decreased CD44 expression. GCH1 was critical for controlling reactive species balance, including suppressing reactive oxygen species production, which mediated GCH1 cell growth effects. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence, and worse survival. Conclusions: GCH1 expression in established GBMs is pro-tumorigenic, causing increased growth due, in part, to promotion of BTIC maintenance and suppression of reactive oxygen species.

NOS Expression and NO Function in Glioma and Implications for Patient Therapies.

SIGNIFICANCE: Gliomas are central nervous system tumors that primarily occur in the brain and arise from glial cells. Gliomas include the most common malignant brain tumor in adults known as grade IV astrocytoma, or glioblastoma (GBM). GBM is a deadly disease for which the most significant advances in treatment offer an improvement in survival of only ∼2 months. CRITICAL ISSUES: To develop novel treatments and improve patient outcomes, we and others have sought to determine the role of molecular signals in gliomas. Recent Advances: One signaling molecule that mediates important biologies in glioma is the free radical nitric oxide (NO). In glioma cells and the tumor microenvironment, NO is produced by three isoforms of nitric oxide synthase (NOS), NOS1, NOS2, and NOS3. NO and NOS affect glioma growth, invasion, angiogenesis, immunosuppression, differentiation state, and therapeutic resistance. FUTURE DIRECTIONS: These multifaceted effects of NO and NOS on gliomas both in vitro and in vivo suggest the potential of modulating the pathway for antiglioma patient therapies. Antioxid. Redox Signal. 26, 986-999.

Modeling Physiologic Microenvironments in Three-Dimensional Microtumors Maintains Brain Tumor Initiating Cells.

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity, including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy, because BTICs are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using the human-derived biomatrix HuBiogel™ and maintained with physiologically relevant microenvironments. BITC percentages were determined using cell surface marker expression, label retention, and neurosphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient-restricted microtumors significantly increased the percentage of both CD133+ and CFSEhigh cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintained neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

Development of a Sox2 reporter system modeling cellular heterogeneity in glioma.

BACKGROUND: Malignant gliomas are complex systems containing a number of factors that drive tumor initiation and progression, including genetic aberrations that lead to extensive cellular heterogeneity within the neoplastic compartment. Mouse models recapitulate these genetic aberrations, but readily observable heterogeneity remains challenging. METHODS: To interrogate cellular heterogeneity in mouse glioma models, we utilized a replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor/tumor virus A (RCAS-tva) system to generate spontaneous mouse gliomas that contained a Sox2-enhanced green fluorescent protein (EGFP) reporter. Glial fibrillary acidic protein-tva mice were crossed with Sox2-EGFP mice, and tumors were initiated that contained a subpopulation of Sox2-EGFP-high cells enriched for tumor-initiating cell properties such as self-renewal, multilineage differentiation potential, and perivascular localization. RESULTS: Following implantation into recipient mice, Sox2-EGFP-high cells generated tumors containing Sox2-EGFP-high and Sox2-EGFP-low cells. Kinomic analysis of Sox2-EGFP-high cells revealed activation of known glioma signaling pathways that are strongly correlated with patient survival including platelet-derived growth factor receptor beta, phosphoinositide-3 kinase, and vascular endothelial growth factor. Our functional analysis identified active feline sarcoma (Fes) signaling in Sox2-EGFP-high cells. Fes negatively correlated with glioma patient survival and was coexpressed with Sox2-positive cells in glioma xenografts and primary patient-derived tissue. CONCLUSIONS: Our RCAS-tva/Sox2-EGFP model will empower closer examination of cellular heterogeneity and will be useful for identifying novel glioma pathways as well as testing preclinical treatment efficacy.

Method for Efficient Transduction of Cancer Stem Cells.

Ectopic gene expression through introduction of cDNA and gene silencing by RNA interference each facilitate the elucidation of molecular pathways in both normal and pathologic states. As transfection efficiency in some primary and established cells is low, lentivirus based expression systems with high infection rates can improve experimental design. For example, glioblastoma cells and particularly the cancer stem cell (CSC) fraction can be difficult to transfect but are amenable to viral infection. Greater utilization of lentivirus for expression of cDNA and shRNA in CSCs may be limited due to technical challenges, including elimination of pro-differentiating fetal bovine serum (FBS). We therefore generated a subline of 293Ts that can proliferate and efficiently produce virus in CSC media, designated CSC293Ts. We provide detailed protocols for the generation of CSC293Ts and for the production of lentivirus for CSC infection using glioblastoma as a model. Our data demonstrate that serum free media from CSC293Ts consistently produces greater than 80% infection rates without virus concentration. We believe that the detailed protocols provided here can be adapted for multiple cell types for broad utility.