bims-malgli Biomed News
on Biology of malignant gliomas
Issue of 2022‒05‒15
sixteen papers selected by
Oltea Sampetrean
Keio University

  1. Trends Pharmacol Sci. 2022 May 07. pii: S0165-6147(22)00082-7. [Epub ahead of print]
      Glioblastoma (GBM) is the most common and highly lethal form of primary brain tumor in adults. The median survival of GBM patients is approximately 14-16 months despite multimodal therapies. Emerging evidence has substantiated the critical role of symbiotic interactions between GBM cells and noncancerous immune cells (e.g., myeloid cells and T cells) in regulating tumor progression and therapy resistance. Approaches to target the tumor-immune symbiosis have emerged as a promising therapeutic strategy for GBM. Here, we review the recent developments for pharmacological targeting of the GBM-immune symbiosis and highlight the role of such strategies to improve the effectiveness of immunotherapies in GBM.
    Keywords:  glioblastoma; immunotherapy; macrophages; microglia, MDSCs; symbiosis
  2. FASEB J. 2022 May;36 Suppl 1
      Glioblastoma (GB), is the most aggressive and most commonly diagnosed primary adult neoplasm in the CNS. As GB grows rapidly, it outstrips its oxygen supply, resulting in pockets of hypoxia throughout the developing tumor. Hypoxic glioma cells recruit microglia and macrophages to support angiogenesis to resolve hypoxia and promote an anti-inflammatory tumor milieu. It was previously shown in peripheral cancers that Neuropilin-1 (Nrp1) on macrophages governed entry into hypoxic niches, wherein these cells supported angiogenesis and bolstered immune evasion. We have previously shown myeloid specific Nrp1 deletion or pharmacological antagonism reduces angiogenesis and restores antitumor immunity. Herein, we demonstrate that Nrp1 deletion from myeloid cells reduces infiltration into hypoxic areas of developing gliomas. This results in an increase in tumor hypoxia and myeloid cell abundance. Additionally, we reveal that myeloid cells adopt an M2-like phenotype in hypoxic areas, but that this phenotype is abolished in areas of hypoxia when Nrp1 is deleted, supporting multiple functions of this protein in myeloid cells. Finally, we verified that pharmacological antagonism of the Nrp1 b1 domain is sufficient to increase tumor hypoxia and GAM accumulation in developing gliomas. These data further support efforts co-targeting Nrp1 and hypoxia-associated survival pathways independent of glioma driver mutations.
  3. Cancers (Basel). 2022 Apr 28. pii: 2207. [Epub ahead of print]14(9):
      BACKGROUND: Glioblastoma (GBM) is the most common and deadliest malignant primary brain tumor, contributing significant morbidity and mortality among patients. As current standard-of-care demonstrates limited success, the development of new efficacious GBM therapeutics is urgently needed. Major challenges in advancing GBM chemotherapy include poor bioavailability, lack of tumor selectivity leading to undesired side effects, poor permeability across the blood-brain barrier (BBB), and extensive intratumoral heterogeneity.METHODS: We have previously identified a small, soluble peptide (BTP-7) that is able to cross the BBB and target the human GBM extracellular matrix (ECM). Here, we covalently attached BTP-7 to an insoluble anti-cancer drug, camptothecin (CPT).
    RESULTS: We demonstrate that conjugation of BTP-7 to CPT improves drug solubility in aqueous solution, retains drug efficacy against patient-derived GBM stem cells (GSC), enhances BBB permeability, and enables therapeutic targeting to intracranial GBM, leading to higher toxicity in GBM cells compared to normal brain tissues, and ultimately prolongs survival in mice bearing intracranial patient-derived GBM xenograft.
    CONCLUSION: BTP-7 is a new modality that opens the door to possibilities for GBM-targeted therapeutic approaches.
    Keywords:  blood–brain barrier; brevican; chemotherapy; drug targeting; glioblastoma; peptide; precision medicine
  4. FASEB J. 2022 May;36 Suppl 1
      Glioblastoma multiforme (GBM), the most common malignant brain tumor, is associated with upregulation of a number of G-protein coupled receptors. TCGA analysis of glioblastoma reveals that Gα12 mRNA levels are elevated in 28% of GBM patient tumors, the highest rate for all tumor types surveyed. There are concomitant alterations in expression of a subset of GPCRs that couple to Gα12 . Gα12 signals through activation of RhoA and its downstream effectors. Our earlier studies revealed that gene programs induced through the RhoA-regulated transcriptional co-activators YAP and MRTF-A contribute to glioma stem cell properties and their in vivo growth as orthotopic (brain) tumors (Yu et al., Oncogene 2018). To determine if Gα12 signaling pathways transduce the effects of known and orphan GPCRs activated in the tumor environment we knocked down Gα12 using shRNA in two human GBM cancer stem cell lines, GSC-23 and HK-281. Gα12 mRNA levels decreased by 60-80% without compensatory upregulation of mRNA for the homologous Gα13 . Loss of Gα12 significantly attenuated the stem cell properties of GSC-23 cells as determined by limiting dilution assays and also reduced mRNA levels for multiple canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. Tumor growth of GSC-23 control and Gα12 knockdown (KD) cells was examined following orthotopic injection into mouse brain. Surprisingly, Gα12 deletion had no evident effect on tumor size nor did it prolong mouse survival. On the other hand, histopathological analyses revealed striking differences at the tumor border, indicative of diminished invasive properties and this was confirmed by immunohistochemical staining for the human nucleoli antigen. RNA sequencing and DESeq2 analysis in WT and KD tumors identified a number of genes encoding extra and intracellular proteins related to cell adhesion and migration dynamics which were differentially expressed in Gα12 KDs. Thrombospondin-1 (Thbs-1) was one of the most repressed genes in the Gα12 knockdown tumors and it was previously identified as a YAP-dependent gene in these cells. Expression of Thbs-1 and Gα12 is highly correlated in the more aggressive mesenchymal-types of GBM by Gliovis and IvyGAP tools, and clinical reports show elevated Thbs-1 as a marker of poor prognosis in GBM patients. To assess the effect of Gα12 and Thbs-1 on glioblastoma stem cell migration we performed in vitro Transwell-based assays. These studies demonstrated that both Gα12 and its transcriptional target Thbs-1 are required for agonist-stimulated GSC-23 cell migration and invasion. We also demonstrated through chemogenetic activation of Gα12 by DREADD expression that Gα12 signaling in GSC-23 cells is sufficient to drive Thbs-1 expression and migration. Our findings indicate that Gα12 is a focal point for GPCR control of transcriptional programs for stemness and invasiveness in glioma stem cells derived tumors and suggest targeting this site as a therapeutic intervention to disrupt tumor cell invasion.
  5. FASEB J. 2022 May;36 Suppl 1
      Glioblastomas (GBMs) are primary brain malignancies with poor overall survival; however, disease sequelae following primary treatment is highly variable in nature-leading to variation in therapy response and overall survival. Recent discoveries characterizing the underlying GBM microenvironment evidence dynamic responses in both residential and recruited immune cell populations which may in fact drive these clinical observations. To validate these finding and evaluate the potential role of immune cells in differential survival, we analyzed recurrent GBM RNAseq data from the Chinese Glioma Genome Atlas and subsequently performed unsupervised clustering through k-means to define 3 sub-clusters of recurrent GBM with differential overall survival (p = 0.019). Subsequently, to evaluate the role of immune cells in this paradigm, we applied Weighted Gene Correlation Network analysis to our findings which indicate modulation of immune response as a key driver of this differential clinical pattern. Translating these findings to actionable clinical utilization in neuropathology is however challenged by poor signal-to-noise ratios during immuno-staining and complex arborization of microglial processes. We thus present a convolutional neural network workflow using a UNET framework in python which can mitigate these challenges and successfully annotate diagnostic pathology slides from real glioblastoma patients treated at Ohio State with 80% accuracy-with attribution of lowered accuracy due to annotation error. Using this approach, we plan to subsequently annotate additional histology slides and utilize the morphologic variation within our segmented microglial population to discern post-surgical response patterns in glioblastoma patients.
  6. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. GBM has an ominous prognosis with a survival rate of 14-15 months after diagnosis and despite worldwide initiatives to optimize therapeutic approaches, GBM is still among the most challenging diseases to treat and the fastest to relapse in clinical oncology. The inevitable treatment resistance and relapse are mainly attributed to the presence of glioma stem cells (GSCs), which have the ability to self-renew, indefinitely proliferate and to differentiate into different cell types. We hypothesize that HDAC7, as an epigenetic regulator, is playing a crucial mechanistic role in GBM and constitutes a novel druggable target for this universally fatal disease. By uncovering the HDAC7 interactome in the GSC nucleus, we provide mechanistic insights into the role of HDAC7 in regulating cancer stemness and possible unprecedented targeting strategies.METHODOLOGY: We quantified the HDAC7 expression in tissue samples from 80 GBM patients admitted at Rhode Island Hospital using Custom Nanostring Panels and correlated the HDAC7 expression to overall patient survival. Also, we compared HDAC7 mRNA expression in GBM to the non-tumor samples from TCGA data using GlioVis webtool. Next, we performed siRNA knock-down (KD) of HDAC7 in GSCs followed by RNA-seq and transcriptomic, gene enrichment and stemness enrichment analysis. We used mass spectrometry (Mod Spec) in si-HDAC7 KD versus Control to quantify the potential enzymatic de-acetylation properties of HDAC7 on the global post-translational modifications of histones. Finally, to unravel the protein partners for HDAC7 in the GSC nucleus, we performed Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME).
    RESULTS: Survival analysis of GBM patients showed significant decrease in survival for patients with high HDAC7 mRNA expression. In addition, GlioVis webtool revealed that HDAC7 expression is significantly higher in GBM tumors relative to non-tumor samples. Differential gene expression analysis of RNASeq performed on HDAC7 siRNA KD GSCs versus control, revealed 4963 differentially expressed genes with the Gene Ontology enrichment showing significant enrichment for cell cycle and cell division suppression in HDAC7 KD GSCs. Additionally, 653 genes that were downregulated in HDAC7 KD GSCS were found to play significant role in stemness processes of embryonic stem cells and embryonal carcinoma stem cells. Mass spectrometry on GSCs following HDAC7 siRNA KD, revealed non-significant de-acetylation changes on global histone marks suggesting that HDAC7 has minimal or no histone deacetylation activity. Finally, RIME revealed novel protein partners in the proximity of 2.5 A° to HDAC7 in the GSCs nucleus. The newly discovered protein complex of HDAC7 suggests that HDAC7 regulates heterochromatin formation and spreading, which mechanistically control transcriptional programs of GSCs.
    CONCLUSION: Our data suggest that HDAC7 plays a pivotal role in GBM. It regulates expression of transcripts that define cancer stemness, while inhibition of HDAC7 results in global increase in heterochromatin and transcriptional repression in GSCs. This study supports HDAC7 as a novel druggable target for GBM in a preclinical setting.
  7. Front Oncol. 2022 ;12 841418
      Background: Despite the availability of various therapy options and being a widely focused research area, the prognosis of glioblastoma (GBM) still remains very poor due to therapy resistance, genetic heterogeneity and a diffuse infiltration pattern. The recently described non-apoptotic form of cell death ferroptosis may, however, offer novel opportunities for targeted therapies. Hence, the aim of this study was to investigate the potential role of ferroptosis in GBM, including the impact of treatment on the expression of the two ferroptosis-associated players glutathione-peroxidase 4 (GPX4) and acyl-CoA-synthetase long-chain family number 4 (ACSL4). Furthermore, the change in expression of the recently identified ferroptosis suppressor protein 1 (FSP1) and aldehyde dehydrogenase (ALDH) 1A3 was investigated.Methods: Immunohistochemistry was performed on sample pairs of primary and relapse GBM of 24 patients who had received standard adjuvant treatment with radiochemotherapy. To identify cell types generally prone to undergo ferroptosis, co-stainings of ferroptosis susceptibility genes in combination with cell-type specific markers including glial fibrillary acidic protein (GFAP) for tumor cells and astrocytes, as well as the ionized calcium-binding adapter molecule 1 (Iba1) for microglial cells were performed, supplemented by double stains combining GPX4 and ACSL4.
    Results: While the expression of GPX4 decreased significantly during tumor relapse, ACSL4 showed a significant increase. These results were confirmed by analyses of data sets of the Cancer Genome Atlas. These profound changes indicate an increased susceptibility of relapsed tumors towards oxidative stress and associated ferroptosis, a cell death modality characterized by unrestrained lipid peroxidation. Moreover, ALDH1A3 and FSP1 expression also increased in the relapses with significant results for ALDH1A3, whereas for FSP1, statistical significance was not reached. Results obtained from double staining imply that ferroptosis occurs more likely in GBM tumor cells than in microglial cells.
    Conclusion: Our study implies that ferroptosis takes place in GBM tumor cells. Moreover, we show that recurrent tumors have a higher vulnerability to ferroptosis. These results affirm that utilizing ferroptosis processes might be a possible novel therapy option, especially in the situation of recurrent GBM.
    Keywords:  cell death; ferroptosis; glioblastoma; glioma; immunohistochemistry; protein expression; relapse; therapy resistance
  8. FASEB J. 2022 May;36 Suppl 1
      Glioblastoma (GBM) is a malignant brain cancer that results in patient death within two years following diagnosis. The GBM tumor microenvironment has an important impact on the formation, progression, and drug resistance of this lethal disease. The GBM tumor microenvironment is composed of a variety of cell types that can support tumor growth like microglia. Microglia are immune cells that can switch from a classical, tumoricidal M1 phenotype to an alternative, tumor promoting M2 phenotype which encourages tumor growth. However, there are limited studies about the specific effect(s) that microglia have on GBM thus requiring further research into how these cells affect GBM tumors. Given the importance of the tumor microenvironment to GBM tumor progression, targeting key signaling pathways in tumor-associated cells that are involved in sustaining GBM tumors could be an effective way to reduce tumor progression. One signaling pathway involved in causing different cancers is the nuclear factor-kappa B (NF-κB) pathway, which has been implicated in M1 to M2 phenotype polarization. Therefore, this study focuses on characterization of the p65fl/fl /CX3 CR1creER/+ mouse model, which is an inducible p65 knockout mouse model for studying how inhibition of canonical NF-κB signaling in microglia affects GBM tumors. After tamoxifen is administered to the mouse, p65, a transcription factor of the canonical NF-κB pathway, should be deleted in microglia due to the C-X3 -C chemokine receptor 1 (CX3 CR1) promoter. Tamoxifen binds to the estrogen receptor on the cre recombinase (creER) so that the cre recombinase can recombine the floxed p65 (p65fl/fl ) gene in microglia. Characterization of this particular mouse model is necessary to determine if the floxed p65 gene is effectively deleted in microglia from these mice. Liquid chromatography-mass spectrometry analysis has shown that the orally administered tamoxifen was present and quantifiable in the brains of tamoxifen-treated mice to induce recombination. Based on data from PCR and western blots, tamoxifen given to the p65 knockout mice induced partial deletion of the floxed p65 gene in isolated microglia compared to p65 knockout mice given the vehicle control. Preliminary flow cytometry data suggested that p65 deletion in microglia occurred in p65fl/fl /CX3 CR1creER/+ mice that received GBM implantations and tamoxifen treatment compared to a vehicle control group. Overall, the data from this study suggested that the floxed p65 gene is partially deleted in microglia of the tamoxifen-treated p65 knockout mice compared to the vehicle control mice.
  9. FASEB J. 2022 May;36 Suppl 1
      Epigenetically directed cancer therapeutics, such as targeting the histone demethylase LSD1, are an emerging approach to the treatment of glioblastoma (GBM), with dozens of candidates in preclinical development. To date, knockdown (KD) of LSD1 exerts effects on proliferation, but an evaluation and comparison of pharmacological LSD1 inhibitors in GBM is lacking. Therefore, the present study evaluated the effect of LSD1 KD compared with pharmacological inhibition and studied four LSD1 inhibitors in patient derived GSCs in vitroand in vivo for therapeutic efficacy and genes associated with resistance. Changes in gene expression were observed upon KD of LSD1 and compared with pharmacological inhibition of LSD1 in GBM cells. Four LSD1 inhibitors were assessed in a panel of nine GSC lines for their effects on cell viability and colony formation. The radioresistant GSC line, GSC 20, was used to establish an orthotopic xenograft model to evaluate the in vivo activity of LSD1 inhibition. In parallel, we identified five genes associated with resistance to LSD1 inhibition using RNA-seq comparing non-transformed and isogenic resistant GBM lines. Lastly, we probed for the presence of this gene set in our in vitro and in vivo GSC experiments with LSD1 inhibitors and in GBM patient exome data. In GBM cell lines, we identified an upregulation of genes associated with cell death, regulation of cell proliferation, and regulation of intracellular signaling upon both LSD1 KD and LSD1 inhibition using tranylcypromine treatment. Additional, more selective, pharmacological inhibitors of LSD1 (GSK-LSD1, RN-1 and SP-2577) in vitro had differential responses in the GSCs that were more varied, and responses were dependent on the specific LSD1 inhibitor and GSC line. Importantly, the response to LSD1 inhibition in vitro was not dependent on the GSC radiosensitivity or molecular subtype indicating unique determinants of sensitivity. When assessing in vivoefficacy, the GSC tumor-bearing mice treated with GSK-LSD1 had a strong reduction in tumor burden that occurred seven weeks into treatment. However, tumor regrowth occurred and overall survival was not significantly better in LSD1 inhibitor treated mice. To understand mechanisms associated with tumor regrowth, we mined our existing data on selectivity and sensitivity to LSD1 blockade and identified five genes potentially associated with resistance to LSD1 inhibition, including HKDC1, RAB3IL1, RAB39B, FTH1, and FAM213A. These genes were found to be upregulated in treatment resistant GSCs and in the brain tissue of GSK-LSD1 treated mice after they succumbed to tumor burden. Finally, the resistant-associated genes were present in exome sequencing data from GBM patients where they showed an inverse relationship with LSD1 expression. Future studies will focus on designing combination therapies with LSD1 inhibitors that will enhance their effects and overcome resistance associated with treatment by targeting pathways associated with the genes we have uncovered.
  10. Clin Cancer Res. 2022 May 12. pii: clincanres.2563.2021. [Epub ahead of print]
      PURPOSE: Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for glioblastomas (GBMs). Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy.EXPERIMENTAL DESIGN: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knock-out (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival.
    RESULTS: Combination of CRISPR screen results with outcomes from taxane-treated breast cancer patients led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells and in multiple intracranial glioma xenografts models. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α.
    CONCLUSION: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.
  11. Neoplasia. 2022 May 09. pii: S1476-5586(22)00028-8. [Epub ahead of print]30 100801
      High-grade (WHO grades III-IV) glioma remains one of the most lethal human cancers. Adoptive transfer of tumor-targeting chimeric antigen receptor (CAR)-redirected T cells for high-grade glioma has revealed promising indications of anti-tumor activity, but objective clinical responses remain elusive for most patients. A significant challenge to effective immunotherapy is the highly heterogeneous structure of these tumors, including large variations in the magnitudes and distributions of target antigen expression, observed both within individual tumors and between patients. To obtain a more detailed understanding of immunotherapy target antigens within patient tumors, we immunochemically mapped at single cell resolution three clinically-relevant targets, IL13Rα2, HER2 and EGFR, on tumor samples drawn from a 43-patient cohort. We observed that within individual tumor samples, expression of these antigens was neither random nor uniform, but rather that they mapped into local neighborhoods - phenotypically similar cells within regions of cellular tumor - reflecting not well understood properties of tumor cells and their milieu. Notably, tumor cell neighborhoods of high antigen expression were not arranged independently within regions. For example, in cellular tumor regions, neighborhoods of high IL13Rα2 and HER2 expression appeared to be reciprocal to those of EGFR, while in areas of pseudopalisading necrosis, expression of IL13Rα2 and HER2, but not EGFR, appeared to reflect the radial organization of tumor cells around hypoxic cores. Other structural features affecting expression of immunotherapy target antigens remain to be elucidated. This structured but heterogeneous organization of antigen expression in high grade glioma is highly permissive for antigen escape, and combinatorial antigen targeting is a commonly suggested potential mitigating strategy. Deeper understanding of antigen expression within and between patient tumors will enhance optimization of combination immunotherapies, the most immediate clinical application of the observations presented here being the importance of including (wild-type) EGFR as a target antigen.
    Keywords:  CAR T cells; EGFR; Glioblastoma; HER2; IL13Rα2; antigen escape; immunotherapy; spatial organization of glioblastoma; tumor heterogeneity
  12. Commun Biol. 2022 May 10. 5(1): 436
      Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas.
  13. Clin Cancer Res. 2022 May 12. pii: clincanres.0171.2022. [Epub ahead of print]
      PURPOSE: Dual timepoint FET-PET acquisition (10 and 60 minutes after FET injection) improves the definition of glioblastoma location and shape. Here we evaluated the safety and efficacy of simultaneous integrated boost (SIB) planned using dual FET-PET for postoperative glioblastoma treatment.EXPERIMENTAL DESIGN: In this prospective pilot study (March 2017-December 2020), 17 patients qualified for FET-PET-based SIB intensity-modulated radiotherapy after resection. The prescribed dose was 78 and 60 Gy (2.6 and 2.0 Gy per fraction, respectively) for the FET-PET- and MR-based target volumes. Eleven patients had FET-PET within nine months to precisely define biological responses. Progression-free survival (PFS), overall survival (OS), toxicities, and radiation necrosis were evaluated. Six patients (35%) had tumors with MGMT promoter methylation.
    RESULTS: The one- and two-year OS and PFS rates were 73% and 43% and 53% and 13%, respectively. The median OS and PFS were 24 (95%CI 9-26) and 12 (95%CI 6-18) months, respectively. Two patients developed uncontrolled seizures during radiotherapy and could not receive treatment per protocol. In patients treated per protocol, 7/15 presented with new or increased neurological deficits in the first month after irradiation. Radiation necrosis was diagnosed by MRI three months after SIB in five patients and later in another two patients. In two patients, the tumor was larger in FET-PET images after six months.
    CONCLUSIONS: Survival outcomes using our novel dose escalation concept (total 78 Gy) were promising, even within the MGMTunmethylated subgroup. Excessive neurotoxicity was not observed, but radionecrosis was common and must be considered in future trials.
  14. FASEB J. 2022 May;36 Suppl 1
      This project is to develop therapies to bypass challenges to effective and continuous drug delivery to the brain, for the treatment of glioblastoma multiforme (GBM). Currently, individuals diagnosed with GBM have a short life expectancy of 12-14 months. Our approach has the potential to deliver one single dose of gene therapy directly to the GBM tumor environment and block the production of cancer-driving genes. Epidermal growth factor receptor (EGFR) is dysregulated in 57% of all GBM. Our approach uses an adeno-associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre-mRNA transcript. We have examined the 'pre-mRNA structurome' of EGFR to evaluate the accessibility of targetable regions. To advance our therapeutic strategy, we have analyzed the secondary structure of the EGFR transcript using selective 2' hydroxyl acylation and primer extension followed by mutational profiling (SHAPE-MaP). SHAPE-MaP reactivity profiles were generated revealing the structure of splicing and cryptic polyadenylation signal (PAS) elements within the targeted region. We identified enhancer binding motifs surrounding the 5' splice site and hidden elements of a cryptic polyadenylation signal. Based on these structural profiles, we generated RNA therapies that interact with structural elements to unravel the hidden polyadenylation signal with the potential to activate expression of the short therapeutic isoform. In this project, we cloned these therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. Currently, we are evaluating in vitro, the therapeutic RNA interaction with the target sequence of the EGFR pre-mRNA transcript.
  15. Neuro Oncol. 2022 May 12. pii: noac123. [Epub ahead of print]
      BACKGROUND: Children ≤36 months with Diffuse Intrinsic Pontine Glioma (DIPG) have increased long-term survival (LTS, overall survival (OS) ≥24 months). Understanding distinguishing characteristics in this population is critical to improving outcomes.METHODS: Patients ≤36 months at diagnosis enrolled on the International DIPG Registry (IDIPGR) with central imaging confirmation were included. Presentation, clinical course, imaging, pathology and molecular findings were analyzed.
    RESULTS: Among 1183 patients in IDIPGR, 40 were eligible (median age: 29 months). Median OS was 15 months. Twelve patients (30%) were LTS, 3 (7.5%) very long-term survivors ≥ 5 years. Among 8 untreated patients, median OS was 2 months. Patients enrolled in the registry but excluded from our study by central radiology review or tissue diagnosis had median OS of 7 months. All but 1 LTS received radiation. Among 32 treated patients, 1-, 2-, 3-, and 5-year OS rates were 68.8%, 31.2%, 15.6% and 12.5%, respectively. LTS had longer duration of presenting symptoms (p=0.018). No imaging features were predictive of outcome. Tissue and genomic data were available in 18 (45%) and 10 patients, respectively. Among 9 with known H3K27M status, 6 had a mutation.
    CONCLUSIONS: Children ≤36 months demonstrated significantly more LTS, with an improved median OS of 15 months; 92% of LTS received radiation. Median OS in untreated children was 2 months, compared to 17 months for treated children. LTS had longer duration of symptoms. Excluded patients demonstrated a lower OS, contradicting the hypothesis that children ≤36 months with DIPG show improved outcomes due to misdiagnosis.
    Keywords:  DIPG; International DIPG Registry; outcomes; survival; young
  16. Nature. 2022 May 11.
      Animals constantly receive various sensory stimuli, such as odours, sounds, light and touch, from the surrounding environment. These sensory inputs are essential for animals to search for food and avoid predators, but they also affect their physiological status, and may cause diseases such as cancer. Malignant gliomas-the most lethal form of brain tumour1-are known to intimately communicate with neurons at the cellular level2,3. However, it remains unclear whether external sensory stimuli can directly affect the development of malignant glioma under normal living conditions. Here we show that olfaction can directly regulate gliomagenesis. In an autochthonous mouse model that recapitulates adult gliomagenesis4-6 originating in oligodendrocyte precursor cells (OPCs), gliomas preferentially emerge in the olfactory bulb-the first relay of brain olfactory circuitry. Manipulating the activity of olfactory receptor neurons (ORNs) affects the development of glioma. Mechanistically, olfaction excites mitral and tufted (M/T) cells, which receive sensory information from ORNs and release insulin-like growth factor 1 (IGF1) in an activity-dependent manner. Specific knockout of Igf1 in M/T cells suppresses gliomagenesis. In addition, knocking out the IGF1 receptor in pre-cancerous mutant OPCs abolishes the ORN-activity-dependent mitogenic effects. Our findings establish a link between sensory experience and gliomagenesis through their corresponding sensory neuronal circuits.