bluuebunny/crossref_metadata_2025 huggingface dataset:
CNSC-33. MOLECULAR AND FUNCTIONAL DRIVERS OF ACTIVITY DEPENDENT GLIOBLASTOMA PROLIFERATION
Authors: Negussie Mikias , Krishna Saritha , Wei Ying , Aabedi Alexander , Ambati Vardhaan , Daniel Andy , Lu Katie , Hervey-Jumper Shawn
Published: November 2023

Glioblastoma, the most common primary brain tumor in adults, is a major cause of neurological morbidity and mortality with no effective therapies. Its proliferation and invasion are regulated by direct and paracrine-mediated neuronal activity. In this study, we uncover molecular targets and protein-protein signaling driving activity dependent proliferation. To identify candidate molecular pathways, we used high-density electrode arrays in vivo to record human local field potentials. Single-cell RNA sequencing identified Netrin-G1 (NTNG1) and thrombospondin-1 (TSP1) elevation in glioblastoma clonal population that proliferates in response to the presence of neurons. The amino acid sequence of NTNG1 and the TSR1 domain of TSP1 were utilized to predict the 3D interaction surface of NTNG1 and TSP1. Compared to structure predictions of NTNG1 and its known binding partner NGL1, NTNG1 and TSP1 complex predictions showed agreement between machine learning predicted models and lower predicted aligned error in interdomain regions of the complex, suggesting higher confidence in the relative position of NTNG1-TSP1 as compared to NTNG1-NGL1. We performed 45 protein structure predictions and two molecular dynamics simulations of mutant and WT TSP1 in complex with NTNG1. Each simulation box consisted of >263,000 atoms, each run for 50 million 2 femtosecond timesteps for a total simulation time of 200 ns. These calculations confirmed a stable complex in the WT condition and significant conformational change in the R46A condition, emphasizing the importance of this residue in mediating NTNG1-TSP1 binding. Computational affinity models between NTNG1 and TSP1 were validated by pull-down assays and immunofluorescence labelling using cerebral organoid and human glioma-neuronal coculture models. Finally, we design small molecule and fusion-protein inhibitors disrupting this interaction. Here we advance the development of targeted precision-medicine therapies to treat glioblastoma proliferation through activity-dependent mechanisms.
CNSC-34. BAF60C/SMARCD3 MODULATES CEREBELLUM DEVELOPMENT AND MEDULLOBLASTOMA METASTASIS
Authors: Zou Han , Patel, Yash , Sneiderman Chaim , Talluru Anjali , Zheng Allen , Pearce Thomas , Agnihotri Sameer , Kohanbash Gary , Pollack Ian , Hu Baoli
Published: November 2023

Medulloblastoma (MB), the most common type of embryonal tumor in children, is a fast-growing and heterogeneous brain tumor arising in the cerebellum. The molecular characterization of MB reveals four major subgroups (WNT, SHH, Group 3, and Group 4) and Group 3 is the most aggressive and malignant, characterized by frequent metastasis at diagnosis and the worst prognosis. We have recently found that BAF60C/SMARCD3 is highly expressed in Group 3 MB and associated with poor prognosis. Mechanistically, BAF60C/SMARCD3 hijacks a neurodevelopmental epigenetic program mediated by Disabled1 (DAB1)-Reelin signaling to promote metastatic dissemination in MB. Using single-cell RNA sequencing data of developing mouse and human cerebellum, we found that BAF60C/SMARCD3 expression in Purkinje cells (PCs) is upregulated during embryonic and early postnatal stages, but downregulated during the late postnatal stage of cerebellar development. To examine the effect of BAF60C/SMARCD3 depletion on PC migration and positioning during embryonic and postnatal stages of cerebellar development, we generated targeted knockout mice by crossing the PC-specific Cre mouse (L7Cre-2 and FoxP2-Cre) with the Smarcd3flox/flox mouse. We found that BAF60C/SMARCD3 depletion significantly influences embryo development in FoxP2-Cre; Smarcd3flox/flox but not in L7Cre-2; Smarcd3flox/flox mice, further supporting the expression of BAF60C/SMARCD3 in early stage (Foxp2 +) but not in the late stage (L7/Pcp2+) during cerebellum development. To determine whether BAF60C/SMARCD3 contributes to MB development, we injected lentivirus-carrying constitutively active MYCS62D with/without BAF60C/SMARCD3 in C57BL/6J mice. Interestingly, we found that MYCS62D with BAF60C/SMARCD3 can induce a spontaneous Group3-like MB with strong metastatic dissemination compared to MYCS62D without BAF60C/SMARCD3. These results demonstrate that BAF60C/SMARCD3 plays a pivotal role in PC biology and cerebellar development, and tumor cells hijack BAF60C/SMARCD3-Reelin signaling to promote tumor metastasis in MB.
STEM-04. FATE-BASED CONVERSION FOR IN SITU VACCINATION IN GLIOBLASTOMA
Authors: Tran David , Liu Tianyi , Jin Dan , Le Son , Chen Dongjiang
Published: November 2023

BACKGROUND

Although immune checkpoint inhibitors have shown high benefit for other solid tumors, their efficacy is limited in GBM due in large part to the “cold” tumor microenvironment (TME) of the GBM tumor. New approaches are necessary to overcome these challenges. We propose a shift in the scientific and treatment paradigm of cancer immunotherapy, calling for tumor cells to take on a central, active role in initiating their own immunity right in the TME.

METHODS

Using AI, we identified cell fate determinants (CFDs) to convert GBM cells directly into induced dendritic cells (iDC) inside the TME. CFDs were delivered using a viral vector and conversion assessed by immunophenotyping, scRNA-seq, and functional assays for classical DC properties.

RESULTS

A four-CFD subnetwork anchored by PU.1 was sufficient to convert mouse GBM cells to iDCs (CD45+MHCII+CD80/86+MHCI+). iDCs are growth arrested, exhibit 3-fold higher phagocytic activity and upregulate the canonical antigen processing and presenting machineries by 10-40-fold, resulting in 100-fold greater efficiency at processing ovalbumin and cross-presenting SIINFEKL to naive OTI-CD8+ cytotoxic T lymphocytes (CTL). In addition, iDCs efficiently cross-present exogenous antigens to naïve CD8+ CTLs and elicit >30-fold higher activation and cytotoxicity in tumor-specific T cells compared to native GBM cells, confirming their DC-like properties. Lastly, intratumoral GBM-DC conversion in a syngeneic orthotopic GBM model resulted in significant reduction in tumor burden and increase in survival compared to controls (Log-rank, p = 0.01) with synergistic efficacy with ICIs and classical DC-based tumor vaccination. We have also successfully converted human GBM cells to iDCs with similar properties using a 2-CFD combination also anchored by PU.1.

CONCLUSIONS

GBM-derived iDCs acquired DC-like functions and were capable of eliciting robust intratumor immune activation, thereby overcoming many current limitations of immunotherapy in GBM. Our goal is to develop a low cost, off-the-shelf gene therapy-based fate conversion tumor immunotherapy.
STEM-08. OVERCOMING GLIOBLASTOMA STEM CELL RESISTANCE TO RADIATION THROUGH TARGETING THIOREDOXIN REDUCTASE 1
Authors: Jamali Fatemeh , Lan Katherine , Sabri Siham , Abdulkarim Bassam
Published: November 2023

Glioblastoma, the most aggressive primary tumor of the central nervous system, presents challenges due to its resistance to standard treatment, including radiation therapy (RT). Glioblastoma stem cells (GSCs), known for their chemo-radioresistance, employ adaptive mechanisms to counteract therapy-induced reactive oxygen species (ROS) levels. This includes modulating the activity of thioredoxin (Trx), a key player in the redox system. Furthermore, GSCs exhibit enhanced DNA repair capacity by upregulating DNA repair proteins, such as RAD51 to survive following ROS-induced DNA damage in response to radiation. Our TCGA analysis revealed elevated expression of thioredoxin reductase 1 (TrxR1) in patients diagnosed with GBM and a significant positive correlation between TrxR1 and RAD51. We hypothesized that GSCs might enhance their antioxidant capacity following RT to suppress RT-induced ROS elevation. Hence, combining RT with TrxR1 inhibitors might overcome GSCs radioresistance while reducing RAD51 expression. Auranofin (Au), a drug FDA-approved for rheumatoid arthritis inhibits TrxR1 activity with subsequent ROS increase. We assessed Au cytotoxicity on GSCs with and without ionizing radiation using Alamar Blue and neutrosphere formation assays and ROS assay for quantitative analysis of ROS levels. We evaluated the molecular mechanisms associated with Au and IR-induced cell death in GSCs using western blotting analysis. Our findings suggest that resistance of GSCs to IR was accompanied with increased expression of TrxR1 and RAD51. On the other hand, Au alone significantly decreased GSC viability and neurosphere formation and sensitized GSCs to IR within the nanomolar range (significantly lower than the corresponding IC50s). Au alone and combined with IR increased ROS, while expression of both RAD51 and TrxR1 was decreased. Our study provides insights into repurposing the TrxR1 inhibitor, Au and its combination with RT as a novel therapeutic strategy to overcome GSC radioresistance and ultimately improve the dismal outcome of patients diagnosed with GBM.
STEM-09. EXPLORING THE ROLE OF TP53 IN REDOX REGULATION AND RESPONSE OF GLIOBLASTOMA STEM CELLS TO ROS-INDUCING AGENTS
Authors: Jamali Fatemeh , April Vincent , Sabri Siham , Abdulkarim Bassam
Published: November 2023

Glioblastoma, the most malignant primary tumor of the central nervous system in adults presents a major therapeutic challenge due to the presence of chemo-radioresistant glioblastoma stem cells (GSCs). These GSCs adapt the thioredoxin (Trx) and glutathione (GSH) antioxidant systems to counteract treatment-induced reactive oxygen species (ROS) and promote their survival. The tumor suppressor gene wild-type (wt)TP53 regulates the expression of pro-oxidant or antioxidant genes in response to chemoradiotherapy based on p53 DNA-binding activity in a reducing microenvironment. Given the importance of p53 as a redox-sensitive protein, we hypothesized that p53 might protect GSCs against ROS-inducing agents. Auranofin (Au), a FDA-approved gold(I)-containing compound inhibits thioredoxin reductase 1 (TrxR1), a key antioxidant protein. We investigated the effects of Au on different GSCs including wtp53 GSC line and its counterpart p53-knockdown. We assessed viability, neurosphere formation, ROS levels and expression of p53 and TrXR1 to evaluate the cytotoxicity of Au, alone and in combination with L-buthionine sulfoximine (L-BSO), a GSH-depleting agent. We conducted TCGA analysis to explore the correlation between TrxR1 and p53. Our findings revealed a positive significant correlation between p53 and TrxR1 expression, consistent with lower levels of TrxR1 in p53-knockdown GSCs compared to their wt-p53 counterparts. P53-knockdown GSCs displayed a higher basal level of ROS, which further increased upon treatment with Au. P53-knockdown significantly increased the sensitivity of cells to Au, with pronounced effects for Au/L-BSO combination. Our findings reveal that expression levels of wtp53 affect the role of p53 in TrxR1-redox regulation and sensitivity to ROS-inducing agents in GSCs.
STEM-11. A GLIOBLASTOMA SINGLE-CELL META-ATLAS IDENTIFIES NOVEL NEUROVASCULAR PROGENITOR IN TUMORS
Authors: Fazzari Elisa , Azizad Daria , Li Matthew , Ge Weihong , Bayley Nicholas , Tse Christopher , Tum Andrew , Raleigh David , Patel Kunal , Crouch Elizabeth , Nathanson David , Bhaduri Aparna
Published: November 2023

Glioblastomas (GBM) are defined by cell type heterogeneity that underlies treatment resistance and brain invasion. Recent studies have identified reactivation of neurodevelopmental cells and gene expression programs in GBM. Here, we leverage a neurodevelopmental perspective to identify novel gene signatures in GBM. To do so, we assembled a glioblastoma meta-atlas comprised of 73,376 single-cell transcriptomes from 7 published studies. With these data, we developed a novel module generation strategy to mitigate batch effects, enabled analysis of signatures in each tumors, and emphasized modules observed across multiple tumors. Using this approach, we validated known tumor cell types and axes of variation, and also identified novel stemness programs that were uniquely expressed across distinct progenitor populations. Within progenitor subtypes, we identified a neurovascular progenitor (NVP) of cells expressing a mixed vascular signature including genes characteristic of mural and endothelial identity as well as transcriptional programs reminiscent of neural stem cells. Although tumor associated or glioma stem cell derived vasculature has been described as important for tumor formation and progression, the NVP appears to be stem-like. To our knowledge, this population has not been described in GBM. We validated the existence of this NVP with immunofluorescence and fluorescent activated cell sorting from patient tumors followed by single-cell RNA-sequencing. Using a novel organoid transplantation model, we employed single-cell lineage barcoding to identify the progeny of patient-derived NVP cells, determining that they result in various cell types with diverse morphologies. Exploration of data from more traditional model systems including gliomaspheres and mouse GBM models indicate preservation of this NVP identity across systems. We hypothesize that NVP cells may play important roles in tumor plasticity, and thus may be a valuable therapeutic target to explore.
STEM-18. HYPOXIA-REGULATED EXPRESSION OF CD44 AND OSTEOPONTIN CAN CHANGE THE PHENOTYPE OF GLIOMA STEM-LIKE CELLS FROM HIGHLY INVASIVE TO LESS INVASIVE/PROLIFERATIVE TUMORS IN GLIOBLASTOMA
Authors: Nishikawa Masahiro , Inoue Akihiro , Ohnishi Takanori , Ozaki Saya , Kanemura Yonehiro , Suehiro Satoshi , Kohno Shohei , Ohue Shiro , Shigekawa Seiji , Watanabe Hideaki , Kunieda Takeharu
Published: November 2023

BACKGROUND

The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive and highly migratory glioma stem-like cells(GSCs) in tumors. Upon GBM recurrence or progression, the highly invasive phenotype of GSCs changes to a less-motile, proliferative phenotype, thus generating tumor mass. Elucidating the molecular mechanism underlying this phenotypic transition could lead to the identification of effective molecular targets for treating GBM.

METHODS

We examined the effects of hypoxia (1% O2: severe or 5% O2: moderate) on the expression and functions of hypoxia-inducible factor(HIF), CD44 and osteopontin (OPN) to clarify the role of these molecules in GBM tumor invasion and proliferation. We also investigated the anti-tumor effects of CD44 knockdown in vitro and in vivo using a GSC-transplantation mouse model.

RESULTS

Severe Hypoxia upregulates CD44 expression via activation of HIF-1α, inducing GSCs to assume a highly invasive phenotype. In contrast, moderate hypoxia upregulates OPN expression via activation of HIF-2α. OPN inhibits CD44-promoted GSC migration and invasion and stimulates GSC proliferation, resulting in GSCs assuming a less-invasive, highly proliferative phenotype. CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo, and the mice survived significantly longer than control mice.

CONCLUSION

The highly invasive phenotype of GSCs can be reversed by switching from severe to moderate hypoxia, leading to less-invasive and proliferative tumors. CD44 and OPN play a central role in regulating GSC invasion and proliferation by inducing a phenotypic transition, suggesting that these molecules could be effective targets for treating GBM.
STEM-23. TARGETING SOX2 IN GLIOBLASTOMA CELLS REVEALS HETEROGENEITY IN SOX2 DEPENDENCY
Authors: Dalmo Erika , Rosén Gabriela , Niklasson Mia , Bergström Tobias , Miletic Hrvoje , Lindskog Cecilia , Forsberg-Nilsson Karin , Nelander Sven , Uhrbom Lene , Swartling Fredrik , Westermark Bengt
Published: November 2023

The malignant primary brain tumor glioblastoma (GBM) has a dismal prognosis and current treatment options are insufficient to cure patients. The stem cell transcription factor SOX2 has been indicated as vital for GBM stem cell maintenance in vitro and GBM tumor formation in vivo, rendering it a putative therapeutic target for GBM. However, a few studies, including previous work from our lab, suggest a dichotomy in SOX2 dependence between different patients. To elucidate whether SOX2 independent GBMs exist and if so, how they are distinguished, we have investigated how altered SOX2 expression affects a panel of primary human GBM lines cultured under stem cell conditions. We found that elevated SOX2 expression inhibited proliferation in a dose-dependent manner in three out of four GBM cell lines. Global gene expression analysis showed that cells representing proliferation-inhibited and refractory cell lines end up on different ends of the developmental- to injury response GBM stem cell axis, were resistance to SOX2 overexpression was connected to an injury response expression profile. CRISPR/Cas9 mediated SOX2 knockout revealed a SOX2 independence in the refractory cell line, where cells lacking SOX2 could be propagated both in vitro and in vivo, implicating SOX2 as a non-essential gene for injury response-driven GBM stem cells. In current work, SOX2-dependent cell lines are engineered to express inducible exogenous SOX2 with concomitant endogenous SOX2 knockout, providing a system where SOX2 can be turned off at any time point. This system will be used in further studies elucidating the nature of SOX2 dependence and independence in GBM stem cells.
STEM-24. WDR5 REPRESENTS A THERAPEUTICALLY EXPLOITABLE TARGET FOR CANCER STEM CELLS IN GLIOBLASTOMA
Authors: Mitchell Kelly , Sprowls Samuel , Shakya Sajina , Arora Sonali , Silver Daniel , Goins Christopher , Wallace Lisa , Roversi Gustavo , Schafer Rachel , Kay Kristen , Miller Tyler , Lauko Adam , Bassett John , Kashyap Anjali , D'Amato Kass J , Mulkearns-Hubert Erin , Johnson Sadie , Alvarado Joseph , Rich Jeremy , Paddison Patrick , Patel Anoop , Stauffer Shaun , Hubert Christopher , Lathia Justin
Published: November 2023

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). In this study, we perform an epigenetic-focused functional genomics screen in GBM organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche. Our niche-specific screens identified WDR5, an H3K4 histone methyltransferase responsible for activating specific gene expression, as indispensable for GBM CSC growth and survival. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, required for stem cell maintenance and including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment resulted in dose-dependent silencing of stem cell reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. Our results unveiled the role of WDR5 in maintaining the CSC state in GBM and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. This conceptual and experimental framework can be applied to many cancers, and can unmask unique microenvironmental biology and rationally designed combination therapies.
CCRG-06. PP2A DEFICIENCY INDUCES CELLULAR SENESCENCE AND SENSITIZES P53 MUTANT TUMORS TO SENOLYTIC THERAPY
Authors: Mondal Isha , Das Oishika , Lu Rongze Olivia , Ho Winson
Published: November 2023

Loss of p53 can promote tumor progression by preventing induction of senescence, a critical tumor-suppressive mechanism that limits tumor proliferation. Promotion of senescence in a p53-independent manner can be a novel strategy to overcome treatment resistance in p53-mutant brain tumors. Protein-Phosphatase-2A (PP2A), a ubiquitous serine/threonine phosphatase, regulates various cellular processes including cell-cycle progression. We hypothesize that PP2A-deficiency can induce tumor senescence independent of p53-mutational-status and sensitize tumors to senolytic therapy. CRISPR-KO of PP2A was generated in multiple p53-mutant tumor cell lines: D425 (human medulloblastoma), SB28 and GL261 (murine glioma). Hallmarks of senescence such as b-galactosidase, p21, and p16 were assessed via flow cytometry, RT-PCR and Western-Blot. Secretion of senescence-associated secretory phenotype (SASP), such as IL8, was assessed through ELISA. b-galactosidase, p21, p16 and IL8 were dramatically increased in PP2A-KO compared to WT at baseline. The effect was further enhanced when combined with radiation therapy (RT), a known inducer of senescence, demonstrating PP2A-deficiency synergizes with RT and significantly increases cellular senescence. Expression of CD1d, a MHC1-like molecule associated with senescence and required for invariant NKT cell-activation (iNKT), was dramatically increased in PP2A-KO in comparison to WT via flow-cytometry. In-vitro, sensitivity to WT and PP2A-KO cells to senolytic treatments, such as ABT-737 and iNKT-cell therapy, was tested using an XCelligence-assay system and flow-cytometry-based cytotoxicity assay. PP2A-deficient cells were sensitized to ABT-737, a BCL-xL inhibitor that preferentially targets and kills senescent cells. Co-culture revealed increased cytotoxicity of iNKT-cells against PP2A-deficient tumors compared to WT. In vivo, D425 WT or PP2A-KO cells were injected intracranially into nude NU/J mice, radiated 2 days post-injection, and monitored till endpoint. At baseline, PP2A-KO mice survived longer than WT, and those with PP2A-KO and RT survived the longest. In conclusion, we report that PP2A-deficiency in tumor cells can induce p53-independent senescence and sensitize brain tumors to senolytic therapy.
CSIG-03. SHH PATHWAY ACTIVATION IN DEDIFFERENTIATION DURING TUMOUR PROGRESSION IN MPNST
Authors: Yakubov Rebeca , Bunda Severa , Zadeh Gelareh
Published: November 2023

BACKGROUND

Malignant peripheral nerve sheath tumours (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previous work conducted in our lab used unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumours to uncover two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1 pathway activation). Further, single nuclear RNA-sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively.

PURPOSE & HYPOTHESIS

To examine the expression of transcription factors (TWIST1, SOX9, SNAI2, OTX2, PAX3, and PAX6) known to play canonical roles in the early neural crest cell specification in MPNST-G1 cells. We speculate that MPNST-G1 cells will display overexpression of these transcription factors compared to MPNST-G2 cells and that Sonidegib (SMO inhibitor) will revert dedifferentiation by decreasing activation of the SHH pathway.

METHODS

Dedifferentiation transcription factor expression and the effects of SMO activation and inhibition in MPNST-G1 and MPNST-G2 cells were analyzed using RT-PCR and western blotting. Alamar blue and Trypan blue assays were used to determine the effect of SMO inhibition on proliferation.

RESULTS

Compared to MPNST-G2 cells, MPNST-G1 cells displayed elevated expression of dedifferentiation transcription factors, SMO inhibition was able to reverse these effects. Conversely SMO activation induced the expression of these factors and induced an increase in proliferation.

CONCLUSIONS

The SHH pathway activation promotes the expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options for these lethal cancers.
CSIG-04. INTEGRATED MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES A DIAGNOSTICALLY INFORMED THERAPEUTIC APPROACH FOR MALIGNANT GLIOMA
Authors: Fernandez Elizabeth , Mai Wilson , Song Kai , Bayley Nicholas , Kim Jiyoon , Zhu Henan , Liau Linda , Li Gang , Yong William , Souers Andrew , Li Jingyi Jessica , Graeber Thomas , Cloughesy Timothy , Nathanson David
Published: November 2023

Molecular profiling often fails to predict therapeutic outcomes in cancer. This failure is, in part, due to myriad of genetic alterations and the plasticity of cancer signaling networks. Functional profiling, which ascertains signaling dynamics, has emerged as an alternative method to anticipate drug responses. Here we performed combined molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in glioma patient samples and derivative models. We identified that standard of care treatment rewires the apoptotic machinery in a genotype specific manner, revealing targetable vulnerabilities in gliomas containing specific molecular features (e.g., TP53 WT). However, integration of BH3 profiling uncovered that high mitochondria priming was also required to induce tumor apoptosis. Accordingly, a composite molecular and functional signature best predicted responses of diverse intracranial glioma models to standard of care combined with a new clinical drug targeting the intrinsic apoptotic machinery. This work demonstrates how complementary functional and molecular data can robustly predict therapy-induced cell death.
CSIG-06. CROSSTALK BETWEEN ONCOGENIC SIGNALING PATHWAYS AS DRIVERS OF THERAPY RESISTANCE AGAINST SMALL MOLECULE INHIBITORS IN GLIOBLASTOMA
Authors: Hoosemans Linde , van Heumen Mike , Verduin Maikel , Hoeben Ann , Vooijs Marc
Published: November 2023

Patients with glioblastoma multiforme (GBM) have a median survival of 15 months. Despite an intensive treatment schedule with resection, radio- and chemotherapy, recurrence is inevitable. An important challenge to overcome GBM treatment resistance involves intratumoral heterogeneity, characterized by molecularly, phenotypically, and clinically distinctive GBM subtypes. Different small molecule inhibitors have been designed to inhibit signaling proteins in oncogenic driver pathways, including the EGFR, PDGFR, MET, CDK4, and PI3K/Akt pathway, in GBM. However, these small molecule inhibitors have been unsuccessful in improving patient outcome. Here, we investigate if crosstalk and redundancy in signaling pathways is responsible for single-agent resistance using patient-derived glioblastoma 2D stem cell cultures (pdGBM). Eight brain-penetrable and clinically approved small molecule inhibitors (SMI) were used to target key GBM driver genes. We measured cell viability in triplicate with a CellTiter-Glo assay. The half-maximal inhibitory concentration (IC50) for each inhibitor was defined for six molecularly distinct pdGBM 2D cell cultures. Although inhibition of downstream effector proteins reduced cell viability more effectively (IC50 70nM-1µM), compared to inhibiting upstream membrane-bound tyrosine kinase receptors (IC50 1-15µM), remaining cell proliferation was seen in all pdGBM using the different SMIs as single agent. To identify resistance mechanisms, we investigated changes in phosphokinase activity after SMI monotherapy in the different pdGBM 2D cell cultures. Based upon these results, we could identify combinations of SMIs targeting compensatory pathways that drive treatment resistance. In conclusion, this study presents a mechanistically driven selection of tumor tailored combination treatment to overcome resistance to SMI monotherapy in pdGBM. The efficacy of these novel SMI combinations will be challenged next, moving from pdGBM 2D stem cell cultures towards pdGBM organoids.
CSIG-07. HYPERCELLULAR NODULES IN OLIGODENDROGLIOMA REPRESENT POCKETS OF PROLIFERATIVE STEM-LIKE TUMOR CELLS
Authors: Liu Mengyi , Abramson Karen , Hocke Emily , Hilz Stephanie , Costello Joseph , McLendon Roger , Ashley David , Gregory Simon , Lopez Giselle
Published: November 2023

INTRODUCTION

Oligodendrogliomas are a progressive, infiltrative glioma. By histology, a subset of oligodendrogliomas demonstrate hypercellular nodules (HCN), which often have increased anaplasia compared to surrounding tumor. However, the signals driving anaplasia are unknown.

METHODS

We first performed a GeoMX 89-protein Digital Spatial Profiling (DSP, Nanostring) panel on a cohort of ten oligodendrogliomas with HCN., followed by spatial whole RNA transcriptome analysis (10x Genomics Visium) on six oligodendrogliomas. For the latter, following unsupervised clustering, we performed Gene Ontology (GO) enrichment analysis and Ingenuity Pathway Analysis (IPA, Qiagen) on the differentially expressed genes between HCN and adjacent tumor. We performed paired single nucleus RNA sequencing for deconvolution of the spatial transcriptomic data.

RESULTS AND CONCLUSIONS

By protein DSP, we identified a marker of stemness (OLIG2) and a proliferation marker (Ki-67) as present at increased levels (p< 0.05) within HCN. We subsequently performed spatial whole transcriptome analysis. On unsupervised clustering of differentially expressed genes, the HCN spots cluster away from the remaining tumor spots, supporting a distinct niche. GO and IPA both identified increased activity in several central nervous system developmental pathways, providing further evidence for the “precursor-like” state of cells in HCN. We then annotated paired single nucleus RNA sequencing results to align tumor cells with astrocytic-like, oligodendroglial-like, and stem-like cell states. Using this to deconvolute the spatial transcriptomic data, we found that the vast majority of HCN are enriched for either stem-like or OC-like/stem-like tumor cells. Taken together, the findings support HCN as niches for the most precursor-like, proliferative oligodendroglial cells.
CSIG-14. THE IMPACT OF CORTICOTROPHIN RELEASING HORMONE STRESS NEURO-HORMONES ON THE VIABILITY AND PROLIFERATION OF BRAIN TUMOURS
Authors: Monteiro Ana Rita , Swinny Jerome
Published: November 2023

The majority of brain tumour sub-types are of glial origin. These include high-grade gliomas (HGGs), which lack effective targeted therapies and are thus invariably lethal. We have shown that stress leads to proliferation of brain glia. Such stress-induced glial cell division could provide opportunities for detrimental genetic mutations which increase the risk of oncogenesis. Identifying stress-induced glial plasticity could uncover molecular targets that directly influence glioma viability. Corticotropin-releasing hormone (CRH) stress neurohormones, including urocortin I-III (UCN I-III) are key mediators of stress signalling in the brain including neuroplastic processes. The aims of this project were to compare the expression CRH peptides and receptors in different types of brain tumours and their impact on cells viability and proliferation. Five different brain tumour cell lines were used (two biopsy-derived-in-house-established HGGs – UP007 and SEBTA023 – and three commercially available Medulloblastomas, MBs – UW402, CHLA-01-MED and CHLA-01R-MED). The gene expression profiles of the CRH family were accessed by qRT-PCR (n = 5) and the protein expression by Immunocytochemistry and flow cytometry (FC, n = 3). The effects of targeted drug modulation were assessed through viability (MTS, n = 3) and proliferation assays (Cyquant NF cell proliferation, n = 3). Statistical significance of qRT-PCRs and FC were calculated through a Kruskal-Wallis test, and for viability and proliferation assays through a two-way ANOVA approach. Values of P < 0.05 were accepted as significant. The qRT-PCRs revealed a higher expression of stress neuro-hormones mRNA in MBs compared to HGGs. Immunocytochemistry confirmed the protein expression of CRH and downstream receptors in all cell lines, while FC revealed a higher expression of CRH-Rs in HGGs than MBs. The pharmacological modulation of CRH-Rs induced contrasting effects on cellular viability and proliferation, suggesting patient-specific CRH-CRH-R pathway. This raises the prospect of repurposing available CRH-R ligands for targeted treatment of susceptible tumour types.
CSIG-16. PTEN LOSS IN GLIOMA CELL LINES FACILITATES IMMUNE EVASION BY UPREGULATING EXTRACELLULAR VESICLE BIOGENESIS AND PD-L1 CARGO
Authors: Sanchez Julio , Pierpont Timothy , Argueta-Zamora Dariana , Wilson Kristin , August Avery , Cerione Richard
Published: November 2023

Phosphatase and tensin homologue (PTEN) is deleted or mutated in over 50% of all glioblastoma (GBM) cases. Recently, PTEN loss has been implicated as a prognostic biomarker for patients likely to be non-responders treated with immune checkpoint inhibitor (ICI) therapy. Indeed, a growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1) and the development of an immunosuppressive tumor immune microenvironment (TIME), likely the result of an altered secretome. This raised the question of whether the loss of PTEN impacts the biogenesis and function of extracellular vesicles (EVs), given their functional contributions to immunosuppression and tumor growth. Here we show that glioma cell lines with no or reduced PTEN expression produce elevated numbers of EVs, as measured through nanoparticle tracking analysis. Pharmacological inhibition of PI3K reversed this observation, indicating that this phenomenon occurs in a PI3K-dependent manner. Moreover, we show through flow cytometry and immunoblot analysis that the ability for the inflammatory cytokine interferon-γ (IFN-γ) to upregulate PD-L1 at the cell surface and as EV-associated cargo is markedly increased in cells lacking PTEN. Lastly, we show that in response to IFN-γ, EVs derived from glioma cells lacking PTEN exhibit an enhanced ability to inhibit TCR signaling in a Jurkat reporter system. Taken together, these findings shed new light on how the loss of a major tumor suppressor, PTEN, can alter EV biogenesis and function and contribute to an immunosuppressive extracellular milieu. Future studies will be directed toward exploring whether loss of PTEN function alters additional EV-associated immuno-modulatory proteins relevant to other immune cell populations, toward gaining a further understanding of how EVs shape the “cold” TIME of GBM and the potential therapeutic benefit of combining PI3K inhibitors with ICIs.
CSIG-19. CANCER STEM CELL MEDIATED F11 DRIVES HYPER-THROMBOTIC ACTIVITY IN GLIOBLASTOMA
Authors: Sloan Anthony , Sloan Andrew , Khorana Alok , Horbinski Craig , Cameron Scott , Gupta Anirban Sen , Lathia Justin
Published: November 2023

Cancer-related thrombosis is the second leading cause of death for cancer patients and glioblastoma (GBM), the most common primary malignant brain tumor, is associated with approximately a 30% risk of venous thromboembolism, deep vein thrombosis, or pulmonary embolisms. While this remains a major clinical issue, the interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, including GBM. GBM is a tumor which contains a high degree of cellular heterogeneity and has populations of therapeutically resistant cancer stem cells (CSCs) that drive tumor growth. Histologically CSCs are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcome in many cancers and while platelets are known to promote progression of other tumors. Our previous work demonstrated that CSCs co-opt and activate platelets to promote CSC growth by intrinsically producing thrombin via a cell intrinsic functional coagulation cascade. These findings provided further evidence that CSCs readily execute a highly liver-specific gene expression program that is mechanistically linked to GBM growth. We now demonstrate thrombin and platelet expression in GBM patient specimen creates a hyper-thrombotic GBM tumor-microenvironment. Further, genetic targeting of the CSC intrinsic coagulation cascade, via knockdown of coagulation factor 11 (F11), reduces CSC growth and tumor formation while also decreasing the ability for CSCs to activate platelets, subsequently decreasing the hyper-thrombotic state seen in GBM. Our observations suggest that CSCs execute a cell intrinsic coagulation mechanism to contribute to the hyper-thrombotic tumor-microenvironment seen in GBM and may shed light onto mechanisms of cancer associated thrombosis in not only GBM, but also other malignancies.
CSIG-20. SYSTEMATIC REVIEW OF MOLECULAR TARGETED THERAPIES FOR ADULT-TYPE DIFFUSE GLIOMA: AN ANALYSIS OF CLINICAL AND LABORATORY STUDIES
Authors: Muzyka Logan , Goff Nicolas , Choudhary Nikita , Koltz Michael
Published: November 2023

Gliomas are becoming a frequent topic of investigation for molecular-targeted therapies. However, the current state of research in this area, particularly following the 2021 WHO guideline change, remains undefined. This systematic review sought to characterize the current state of molecular target therapy research for adult-type diffuse glioma to inform scientific progress. A systematic review was conducted in accordance with PRISMA guidelines. Studies meeting inclusion criteria were queried for study design, tumor type, molecular target, molecular pathway, and details pertaining to the molecular targeted therapy– namely the modality, dose, duration of treatment. 350 studies met inclusion criteria: 52 of these were clinical studies, 190 were laboratory studies investigating existing molecular therapies, and 108 were laboratory studies investigating new molecular targets. 119 ongoing clinical trials are also underway, per a detailed query of clinicaltrials.gov. GBM was the predominant tumor studied, with few studies mentioning IDH-mutant astrocytoma or oligodendroglioma. The most common molecular targets in published clinical studies and clinical trials were protein kinase pathways, followed by microenvironmental targets, then immunotherapy and cell cycle/apoptosis pathways. The most common molecular targets in laboratory studies were protein kinase pathways, followed by cell cycle/apoptosis, microenvironmental targets, then immunotherapy pathways, with the wnt/β-catenin pathway arising as a novel target. This systematic review examined the current evidence on molecular targeted therapy for adult-type diffuse glioma and discusses its implications for clinical practice and future research. Published research encompasses clinical studies, laboratory testing of existing therapies, and identification of novel targets, with GBM as the focal point. Ongoing clinical trials are numerous in this area of research as well and follow a similar pattern in tumor type and targeted pathways as published clinical studies. The most common molecular targets were protein kinase pathways, with some variation in the prevalence of other pathways depending on study type.
CSIG-23. PRIMARY CILIA DISRUPTION ALTERS THE TRANSCRITOME OF HUMAN GLIOBLASTOMA CELLS AND SUGGESTS NOVEL THERAPEUTIC STRATEGIES
Authors: Oishi Masahiro , Agyeman-Andoh Afua , Jung Mi-Yeon , Siddegowda Yeshavanth Banasavadi , Burton Eric , Brown Desmond
Published: November 2023

BACKGROUND

Glioblastoma is the most common malignant primary brain tumor. Despite standard-of-care therapy with maximal safe resection, concomitant chemoradiation followed by adjuvant chemotherapy, median survival is 18 months, and 5-year survival is 6.8%. The genetic complexity, ability to suppress host immune surveillance, and the blood-brain-barrier all contribute to the uniform fatality of the disease. Primary cilia are microtubule-based organelles that protrudes from the apical surface of all mammalian cells. They regulate several canonical signal transduction cascades important for development and pathogenesis. Glioblastoma cells are ciliated although the tumor downregulate genes associated with cilia formation and function. Contemporary studies suggest that the regulation of ciliogenesis is an important prognostic consideration. In this study, human glioblastoma cells were modified through stable lentiviral transduction to downregulate KIF3A (a gene required for ciliogenesis), or AURKA (a component of the ciliary disassembly complex). We then performed deep transcriptomic sequencing to determine how the overall transcriptome of human glioblastoma cells change in response to cilia-targeted genetic manipulation.

RESULTS

High-resolution confocal microscopy demonstrated that cilia were absent in KIF3A knockdown cells but abundant following loss of AURKA. Functional loss of primary cilia was confirmed with Gli2A RT-PCR following treatment with SAG (SHH pathway activator). There appeared to be rearrangement of the transcriptome with ciliary loss. Ingenuity Pathway Analysis demonstrated that ciliary loss resulted in the activation of pathways of glioblastoma and other cancer signaling pathways, tumor immune microenvironment pathways, cytokine signaling, and actin cytoskeletal rearrangement among several others.

CONCLUSION

Loss of primary cilia in glioblastomas activate several pro-cancer and immunosuppressive pathways. This suggests that targeting of primary cilia may lead to simultaneous loss of multiple glioblastoma phenotypes that contribute to the tumor’s evolutionary advantage. This may result in a novel paradigm of ‘ciliotherapeutics’ for glioblastomas and other cancers.
CSIG-24. LINEAR GRADING COEFFICIENT REVEALS EMBRYONIC DEVELOPMENT GENE SIGNATURE AS TREATMENT-INDEPENDENT PROGNOSTIC FACTOR IN IDH-MUTANT ASTROCYTOMA
Authors: Ghisai Santoesha , Hoogstrate Youri , Tesileanu Mircea , van Hijfte Levi , Vallentgoed Wies , de Heer Iris , Preusser Matthias , Gorlia Thierry , Sanson Marc , van den Bent Martin , French Pim
Published: November 2023

BACKGROUND

Since there is variation in prognosis within grade II/III and grade IV IDH-mutant astrocytomas, we sought to identify features that may aid further tumor grading.

METHODS

RNAseq was performed on IDH-mutant astrocytomas from the CATNON trial and overlaid with matching genome-wide methylation data. This dataset was extended by multi-omics data of IDH-mutant astrocytomas from the TCGA and GLASS-NL studies. We defined a linear grading coefficient (LGC) based on calibrated DNA methylation-based probability scores and performed differential gene expression (DGE) regression analysis on the LGC. Statistical analyses were intersected across the CATNON and TCGA cohorts ("discovery set") and validated on GLASS-NL. Single-nucleus RNA-sequencing (snRNAseq) data of high-grade IDH-mutant astrocytomas were utilized to explore the source of expression signals. Immunofluorescence was used to confirm findings on protein level.

RESULTS

DGE regression analysis revealed that higher-grade IDH-mutant tumors (high LGC) exhibited mRNA upregulation of key embryonic developmental transcription factors (HOX[A/C/D], NKX, PAX, and TBX). Although the vast majority of CpG sites in LGC high tumors were hypomethylated, homeobox genes were strongly hypermethylated in our discovery set. HOX gene clusters showed gene-ordered (differential) expression where e.g. HOXD10 >D9 >D8 >D4 >D1 and HOXA3 >A7 >A9 >A13 suggesting derepression of enhancer sequences up/downstream of the gene cluster. Our expression-based embryonic development signature was positively correlated with, but not restricted to, the G-CIMP-low subtype and homozygous CDKN2AB deletion. The signature was prognostic (log-rank test) on both the discovery (CATNON: p< 0.0005, TCGA: p< 0.0005) and validation set (GLASS-NL: p< 0.005). As our signature was developed on molecular data from primary resections, its prognostic value is treatment-independent. SnRNAseq on high-grade IDH-mutant astrocytomas indicated expression of embryonic development genes from tumor cells, not restricted to the stemness program.

CONCLUSION

Our study highlights upregulation of embryonic development genes as potential molecular mechanism associated with malignancy of IDH-mutant astrocytomas.
CSIG-25. OVERCOMING RESISTANCE TO THERAPIES TARGETING THE MAPK PATHWAY IN GLIOBLASTOMA
Authors: Bunda Severa , Aldape Kenneth , Zadeh Gelareh
Published: November 2023

Capicua (CIC) is a transcriptional repressor that inhibits expression of genes induced by receptor tyrosine kinase (RTK) activation. RTK/Ras/ERK signaling is one of the most tumorigenic pathways in cancer. In fact, the aggressive nature of the most common and lethal brain tumor, glioblastoma (GBM), has been attributed to hyperactivation of RTK. While CIC is mutated in other tumor types, we found that CIC has a tumor-suppressive function in GBM through an alternative mechanism. We show that Ras/ERK promotes degradation of CIC, resulting in de-repression of CIC targets and amplification of the tumorigenic Ras/ERK signal. Importantly, we show that sustained MEK/ERK inhibition fails to restore CIC protein levels desensitizing from upstream RTK/MEK/ERK inhibition. Our findings suggest the lack of efficacy of MEK/ERK inhibitors in GBM in the clinical context is due to the loss of CIC-mediated repression, hence exposing CIC as a potential therapeutic target in combination with MEK/ERK inhibitors. To uncover targetable pathways that stabilize CIC and sensitize towards MEK/ERK inhibitors in GBM we need to understand how CIC works and causes gene repression. We identified a novel CIC binding partner, Yin Yang 1 (YY1), a context dependent transcription factor important in GBM. We found that YY1/CIC binding co-regulates repression of genes downstream of RTK/Ras/ERK activation important in tumorigenesis. Importantly we uncovered a targetable pathway that leads to stabilization of CIC/YY1 on its target DNA which sensitizes towards MEK/ERK inhibition. These data provide insight into more effective therapeutic avenues to stabilize the CIC/YY1 co-repressor complex and re-sensitize towards RTK/Ras/MEK/ERK inhibition in GBM and likely in other tumors characterized with hyperactive Ras/ERK signaling and CIC loss.
CSIG-31. JAK/STAT PATHWAY CO-OPERATES WITH CIC-FUSIONS TO DRIVE CIC- REARRANGED SARCOMAS
Authors: Kaloti Ramneet , Bunda Severa , Zadeh Gelareh
Published: November 2023

BACKGROUND

CIC-rearranged sarcoma (CRS) is a rare disease driven by a specific fusion protein involving the CIC gene. The native CIC protein is a transcriptional repressor of the (RTK)/Ras/ERK signaling pathway, which is one of the most tumorigenic pathways in cancer. The most common rearrangement is with the double homeobox 4 (DUX4) transcription factor (CIC-DUX4), and others, such as CIC-NUTM1 fusions, have been identified in a subset of pediatric primitive neuroectodermal tumors. However, the molecular mechanisms by which CIC-fusions drive CRS remain unknown.

HYPOTHESIS

Preliminary data shows that CIC-DUX4/NUTM1 fusions activate JAK (cytokine receptors) and its downstream effector STAT1/3 (signal transducers and transcription factors). We hypothesize that the JAK/STAT1/3 signal transduction pathway cooperates with CIC-fusions to induce the expression of oncogenic transcription factors ETV1/4/5 and drive these sarcomas.

METHODS AND RESULTS

JAK1 is a potential target for CIC-fusions - We show high levels of JAK1/STAT1/3 activation in patient-derived CRS cell lines (NCC-SCC-89/C) as compared to other sarcoma cell lines without the fusion. JAK1 inhibition using Solicitinub and Ruxolitinib reduced phosphorylation of STAT1/3 as well as protein and mRNA expression of ETV1/4/5 and cell proliferation. JAK1/STAT1/3 inhibition in patient-derived CIC-DUX4 xenograft models- To evaluate the pre-clinical effect of targeting the JAK1/STAT1/3 pathway, A CRS cell line (NCC-SCC-89/C) were grafted into NSG mice. Ruxolitinib treatment significantly reduced tumor volume, STAT activation, and ETV1/4/5.

CONCLUSION

We show that the JAK1/STAT1/3 pathway plays a critical role in cooperating with CIC-fusions to drive CRS, providing insight into potential therapeutic avenues for these aggressive tumors.