bluuebunny/crossref_metadata_2025 huggingface dataset:
TAMI-28. DIFFERENTIAL MIGRATION MECHANICS AND IMMUNE RESPONSES OF GLIOMA SUBTYPES
Authors: Shamsan Ghaidan , Liu Chao , Braman Brooke , Rathe Susan , Sarver Aaron , Ghaderi Nima , McMahon Mariah , Klank Rebecca , Tschida Barbara , McFarren Joey , Sarkaria Jann , Clark H Brent , Rosenfeld Steven , Largaespada David , Odde David
Published: December 2020

In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p < 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.
TAMI-36. CONNEXIN 43 BLOCKADE INHIBITS PROLIFERATION IN IDH1-MUTANT GLIOMA CELLS
Authors: Melamed Lisa , Lee Christine , Nagashima Hiroaki , Miller Julie , Wakimoto Hiroaki , Cahill Daniel
Published: December 2020

IDH1-mutant gliomas are characteristically sensitive to NAD+ depletion. It is known that NAD+ can move between cells through gap junctions, which provides an opportunity for treatments that prevent NAD+ sharing across tumor cells, effectively decreasing available NAD+. Previous studies have also shown the role of connexin 43 (Cx43) in mediating communication in glioma cell networks and have identified Cx43 as a potential mediator of temozolomide resistance in glioma. We hypothesized that blocking Cx43 would prevent intercellular NAD+ sharing in IDH-mutant gliomas, causing tumor cells to be more vulnerable to metabolic NAD+ depletion via nicotinamide phosphoribosyltransferase (NAMPT) inhibitors and temozolomide (TMZ) treatment. Here, we show that blockade of Cx43 with α-connexin carboxyl-terminal (ACT1) is able to inhibit growth of patient-derived IDH1-mutant tumor cells. ACT1 sensitizes cells to TMZ and inhibits growth of IDH1-mutant gliomas via an NAD-dependent mechanism. We also found that ACT1 can be used in combination with other NAD-depleting drugs, such as NAMPT inhibitors, to enhance its effect and provide a viable therapeutic window. Overall, our results suggest that ACT1 may enhance the efficacy of treatments for IDH1-mutant tumors by blocking metabolic buffering through tumor cell gap junctions.
TAMI-41. PI3K-GAMMA INHIBITION SUPPRESSES ACCUMULATION OF GLIOBLASTOMA-ASSOCIATED MICROGLIA TO RECAPITULATE THE MICROENVIRONMENT OF EXCEPTIONAL RESPONDERS
Authors: Li Jie , Kaneda Megan , Ma Jun , Li Ming , Patel Kunal , Koga Tomoyuki , Sarver Aaron , Furnari Frank , Xu Beibei , Dhawan Sanjay , Ning Jianfang , Zhu Hua , Wu Anhua , You Gan , Jiang Tao , Venteicher Andrew , Glass Christopher , Varner Judith , Chen Clark
Published: December 2020

INTRODUCTION

Exceptional responders refer to the rare subset of patients who derive extraordinary benefits from a therapy compared to most treated patients. Understanding the basis for such responses represents a key pillar in the framework of precision medicine.

OBJECTIVE

Here, we examine the molecular basis underlying exceptional responses in glioblastoma patients who underwent concurrent radiation and temozolomide (TMZ) therapy.

METHODS

mRNA profiles derived from clinically annotated glioblastoma specimens were analyzed by Single sample gene set enrichment (ssGSEA) and Gene Cluster Expression Summary Score (GCESS). Key results were confirmed using cell-based assays and murine glioblastoma models.

RESULTS

Integrated, multi-platform analyses of RNA profiles derived from clinically annotated glioblastoma samples revealed decreased accumulation of microglia/macrophages in the tumor microenvironment of exceptional responders. Further analysis revealed that glioblastoma-associated microglia/macrophages secreted interleukin 11 (IL11) activate a STAT3-MYC signaling axis in glioblastoma cells. This signaling facilitated glioblastoma transition into stem cell states that confer enhanced tumorigenicity as well as resistance to the TMZ. Pharmacologic inhibition or genetic inactivation of a myeloid cell restricted isoform of PI3K, PI3K-gamma, disrupted this signaling axis by suppressing microglia/macrophage accumulation and IL11 secretion in the glioblastoma microenvironment. Mirroring the clinical outcome of exceptional responders, PI3K-gamma inhibition synergistically enhanced the anti-neoplastic effects of TMZ in orthotopic murine glioblastoma models. Moreover, inhibition or genetic inactivation of PI3K-gamma in murine glioblastoma models recapitulated expression profiles observed in clinical specimens isolated from exceptional responders.

CONCLUSIONS

Our results suggest key contributions from tumor-associated microglia/macrophages in exceptional responses and highlight the translational potential for PI3K-gamma inhibition as a glioblastoma therapy.
TAMI-42. H3K27M MUTANT GLIOMAS HIJACK A CONSERVED AND CRITICAL METABOLIC PATHWAY USED BY IDH1 MUTANT GLIOMAS TO MAINTAIN THEIR PREFERRED EPIGENETIC STATE
Authors: Chung Chan , Sweha Stefan , Pratt Drew , Tamrazi Benita , Panwalkar Pooja , Banda Adam , Bayliss Jill , Hawes Debra , Yang Fusheng , Lee Ho-Joon , Shan Mengrou , Cieslik Marcin , Qin Tingting , Werner Christian , Wahl Daniel , Lyssiotis Costas , Yadav Viveka Nand , Koschmann Carl , Chinnaiyan Arul , Blumul Stefan , Judkins Alexander , Venneti Sriram
Published: December 2020

H3K27M-midline gliomas are fatal tumors that mainly harbor H3.3K27M mutations resulting in global H3K27me3 reduction that impacts neuroglial-differentiation. However, the exact mechanisms by which H3.3K27M mutations promote cancer are poorly understood. Metabolic reprogramming is a hallmark of cancer and we hypothesized that H3.3K27M mutations can reprogram metabolism to support uncontrolled growth. We demonstrate that H3.3K27M-mutant cells show elevated levels of critical enzymes related to glycolysis and TCA cycle metabolism including hexokinase-2, isocitrate dehydrogenase (IDH)-1 and glutamate dehydrogenase. H3.3K27M cells also demonstrated enhanced glycolysis, glutamine and TCA-cycle metabolism accompanied by increased alpha-ketoglutarate (α-KG) production. Mutant IDH (mIDH)1/2 converts α-KG to D-2-hydroxyglutarate (D-2HG). D-2HG increases H3K27me3 by inhibiting α-KG’s function to drive H3K27-demethylases. We discovered that H3.3K27M cells use α-KG in an opposing manner to maintain low H3K27me3. Inhibiting enzymes related to α-KG generation including hexokinase-2, glutamate-dehydrogenase and wild type-IDH1 increased global H3K27me3, altered chromatin accessibility at neuroglial-differentiation factors and lowered tumor cell proliferation. In vivo inhibition of glutamine metabolism and/ or wild type-IDH1 using blood-brain barrier penetrant small molecule inhibitors increased overall survival in vivo in two independent H3.3K27M animal models (p< 0.0001). H3K27M and mIDH1 were mutually exclusive in patient tumor samples and D-2HG treatment or forced-mIDH1 expression in H3.3K27M cells increased global H3K27me3 and cell death. Finally H3.3K27M and mIDH1 were synthetic lethal in vitro. Our data suggest that H3.3K27M and mIDH1 hijack a critical and conserved metabolic pathway in opposing manners to regulate global H3K27me3. These results have implications for understanding the pathogenesis of fatal H3K27M-gliomas and for developing therapeutic strategies by disruption of an integrated metabolic/epigenetic-axis.
TAMI-55. THE EVOLUTIONARY ENIGMA OF FATTY ACID DESATURATION IN GLIOBLASTOMA
Authors: Oatman Nicole , Reisz Julie , D’Alessandro Angelo , Dasgupta Biplab
Published: December 2020

Fatty acid desaturation is an enzymatic reaction in which a double bond is introduced into an acyl chain. Of the four functionally distinct desaturase subfamilies, the First Desaturase Family of enzymes introduce the first double bond into a saturated fatty acid, resulting in the synthesis of monounsaturated fatty acids (MUFA). MUFA are essential components of membrane and storage lipids and exert a profound influence on the fluidity of biological membranes. A disequilibrium in saturated to unsaturated fatty acid ratio alters cell growth, differentiation and response to external stimuli, and thus affects a range of pathologies including cancer. The most abundant and key First Desaturase Family enzyme is the delta 9 desaturate called Stearoyl Co-A Desaturase (SCD and SCD5 in humans, and SCD1-4 in mice). SCD desaturates Stearoyl-CoA (C18) and palmitoyl-CoA (C16) to oleoyl-CoA (C18:1) and palmitoyl-CoA (C16:1), respectively. Besides SCD, the only known First Desaturase in mammals with dual function is FADS2 which desaturates palmitate to Sapienate (C16:1, a positional isomer of palmitoleate) in skin cells. A recent study showed that some cancer cells can use FADS2 to bypass the SCD reaction. SCD and SCD5 are by far the most abundant desaturases expressed in the human brain. We made an unexpected discovery that SCD undergoes monoallelic codeletion with PTEN on chromosome 10, and is also highly methylated in glioblastoma (GBM). More surprisingly, all GBM cell lines with SCD codeletion/methylation (that expressed very little SCD protein) are completely resistant to SCD/SCD5 inhibition, yet their phospholipids contained abundant oleic acid. It is unknown if GBMs bypassed SCD, but retained the delta 9 desaturation reaction through a novel enzymatic activity. Our targeted and untargeted metabolomics studies revealed unexpected findings that cannot be explained by conventional wisdom, and may lead to identification of novel lipogenic targets in GBM.
TAMI-56. HYPOXIA-DRIVEN NECROSIS DRIVES DYNAMIC TUMOR PROGRESSION IN GLIOBLASTOMA
Authors: Markwell Steven , Olson Cheryl , Ross James , Nunez-Santana Felix , Brat Daniel
Published: December 2020

The onset of necrosis correlates with aggressive malignant progression in solid tumors. Whether necrosis arises from or promotes accelerated tumor proliferation and progression remains unknown, primarily due to inadequate models systems that fail to induce or capture these dynamic changes as they develop. In glioblastoma (GBM; WHO grade IV), the most malignant primary brain tumor, vascular pathology and central necrosis precedes rapid, radial expansion resulting in patient mortality. While genetic alterations in GBM have been highly characterized, biological adaptations arising from sustained hypoxia/anoxia require further mechanistic investigation. This sustained nutrient deprivation leads to necrosis which dramatically changes the tumor microenvironment (TME). To reveal the dynamic temporal and spatial changes promoting expansive progression, we are generating mouse models that more appropriately capture events found in human gliomas, accounting for unique microenvironmental stressors often lacking in GBM animal models, specifically central necrosis. Combining hypoxia-induced focal necrosis within high grade gliomas with intravital microscopy captures TME restructuring to understand its impact on glioma progression. Our studies use both genetically characterized patient-derived orthotopic GBM xenografts, alongside an immunocompetent RCAS/tv-a model, to determine how antagonizing these processes impacts disease progression and outcomes across multiple GBM subtypes. Complementary to this, we are investigating how increasing hypoxia and necrosis-driven signaling events promotes tumor-associated macrophages (TAMs) influx and reprogramming during glioma progression. Our preliminary data indicate substantial differences pre- and post-necrosis regarding TAM enrichment, immunosuppressive phenotypic conversion and their biological impact, however these mechanisms have not yet been elucidated. Our models capture glioma growth dynamics, reactive neuroinflammation and therapeutic resistance, facilitating innovative pre-clinical interventions to improve patient outcome.
TAMI-61. TUMOR-ASSOCIATED MACROPHAGES ARE INDEPENDENT PROGNOSTIC MARKERS IN MENINGIOMAS
Authors: Liu Fang , Jungwirth Gerhard , Warta Rolf , Unterberg Andreas , Herold-Mende Christel
Published: December 2020

Tumor-associated macrophages (TAMs) play an emerging role in tumor progression by creating an immunosuppressive tumor microenvironment. Therefore, we investigated TAM numbers and their activation (M1/2) in primary (p-) and recurrent (r-) meningiomas and evaluated their impact on survival. Presence of TAMs was analyzed in 195 clinically well-annotated cases (WHO°I n = 43, WHO°II n = 95, WHO°III n = 57; pMGM n = 120, rMGM n = 75). TAMs were quantified by a semiautomated analysis on whole tissue sections stained by multicolor immunofluorescence for CD68 as general TAM marker, and for CD163 and CD204 as M2 marker. CD68+ macrophages positive for either CD163 or CD204 were regarded as M2-polarized TAMs. Furthermore, concentration of 27 immune-relevant cytokines and chemokines from meningioma tissue samples (n = 46) were assessed by Luminex analysis. Median TAM infiltration accounted for 2.47% per total cell count in pMGM and 3.22% in rMGMs. Although no significant WHO°-dependent changes regarding TAM numbers were observed, high numbers of TAMs were associated with shorter progression-free survival (PFS) independent of prognostic confounding variables such as WHO, sex and age. In pMGMs, proportion of M2-polarized TAMs was significantly increased in WHO°II and WHO°III tumors compared to WHO°I (P = 0.001 and P = 0.03, respectively). Luminex data revealed a WHO°-dependent increase of IL-8, IL-10, and IL-12p70. Tissue samples containing higher numbers of TAMs demonstrated significant elevated concentrations of interleukin-1 family members (IL-1β, IL-1Ra), and G-CSF. Furthermore, upregulation of IL-1β and IL-1Ra resulted in shorter overall survival of patients (P = 0.049, P = 0.006, respectively). Taken together, we identified higher numbers of TAMs as an independent prognostic factor for shorter progression-free survival in meningioma patients. This was associated with increased levels of the interleukin-1 family members IL-1β and IL-1Ra and a shorter overall survival, indicating an important function in the tumor microenvironment.
TMOD-05. EXTRACRANIAL TUMORS INFLUENCE INTRACRANIAL RESPONSE TO IMMUNE CHECKPOINT INHIBITORS IN PRE-CLINICAL MODELS OF MELANOMA BRAIN METASTASIS
Authors: Nayyar Naema , Singh Mohini , Stocking Jackson , Brehm Michael , Brastianos Priscilla
Published: December 2020

Melanomas frequently spread to the brain, with up to 75% of patients developing brain metastases. Treatment of metastatic melanoma has been revolutionized by the advent of immune checkpoint inhibitors (ICI) targeting PD-1 and CTLA-4. However, responses to ICI within the immune-specialized brain environment are not well understood owing to the lack of immunocompetent animal models for the investigation of intracranial responses to checkpoint blockade. We studied the effect of ICI in a syngeneic mouse model of melanoma brain metastasis with concurrent intracranial and subcutaneous tumors. Either D3UV3 cells, derived from a UVB irradiated clone of D4M.3A melanoma cell line, or vehicle were subcutaneously injected into the flank of C57BL/6 mice. 3 days later, D3UV3 cells tagged with firefly luciferase were implanted into the striatum using stereotactic injection. Mice were then treated with anti-PD-1 antibody, anti-CTLA-4 antibody, a combination of anti-PD-1 and anti-CTLA-4 antibodies, or isotype controls. We observed striking differences in survival between mice with concurrent intracranial and subcutaneous tumors compared to intracranial tumors alone. Mice with intracranial tumors alone experienced no benefit in survival following monotherapy with anti-PD-1 (p=1) or anti-CTLA-4 (p=0.1) compared to isotype-treated mice, and only a slight benefit with combination treatment (p=0.049). In contrast, mice with concurrent tumors experienced a significant increase in overall survival with anti-CTLA-4 monotherapy (p=0.01) and combination anti-CTLA-4 and anti-PD-1 treatment (p=0.01) compared to isotype-treated mice, although treatment with anti-PD-1 alone did not increase survival (p=0.28). These results indicate that the presence of extracranial disease can modulate intracranial immune responses following checkpoint blockade. We have therefore established a pre-clinical model with concurrent intracranial and extracranial tumors to better recapitulate the clinically observed context of brain metastases. We hope this model will lead to a better understanding of the setting in which ICI is effective for patients with melanoma brain metastases.
TMOD-11. APPLICATION OF A NOVEL TRANSGENIC AND SYNGENEIC MOUSE MODELS OF HUMAN CLASSICAL GLIOBLASTOMA FOR DRUG SCREENING AND PRE-CLINICAL TRIALS
Authors: Singh Olivia , Voisin Mathew , Li Mira , Karimi Shirin , Walia Kanwalpreet , Ali Hafsah , Nassiri Farshad , Suppiah Suganth , Aldape Kenneth , Wei Qingxia , Zadeh Gelareh
Published: December 2020

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with 1 year median survival. The classical subtype of GBM occurs in about 10% of GBM cases and has the worst clinical outcome. The most frequent genetic alteration associated with classical GBM is amplification in the epidermal growth factor receptor (EGFR) and homozygous deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A). We generated a transgenic mouse harboring EGFRvIII, a constitutively active mutant of EGFR, knock in and homozygous deletion in p19 ARF, an alternative spliced form of the CDKN2a gene, restricted to glial cells. Transgenic mice develop tumors as early as 1 month and 73% of mice die by 6 months of age. MRIs reveal that mice develop infiltrative tumor cells throughout the brain and craniospinal junction with some infiltration within the leptomeninges that can result in hydrocephalus. Histology shows that 72% of tumors are high grade gliomas and some tumors demonstrate classical histological features of human GBM such as pseudopallisading necrosis, hyper vascularity and cellular atypia. We generated primary cell lines from transgenic mice that retained EGFRVIII expression and P19 ARF deletion and formed allografts in syngeneic mouse brains. Epigenetic modifications are considered a key mechanism in GBM development. We have therefore evaluated the efficacy of a panel of 42 compounds that target various epigenetic proteins including the readers, writers, and editors of several histone marks available from the Structural Genomics Consortium in primary cell lines derived from our mouse model. The primary cell lines were exposed to epigenetic drugs showing different responses to the same epigenetic modifiers. Key candidate drugs will further be tested pre-clinically in our syngeneic mouse model. In summary, we developed a novel transgenic and syngeneic mouse model for human classical GBM that can be used for drug screening and pre-clinical trials.
TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS
Authors: Vaubel Rachael , Mladek Ann , Zhao Yu , Gupta Shiv K , Kim Minjee , Mohammad Afroz S , Bakken Katrina , He Lihong , Hu Zeng , Burgenske Danielle M , Carlson Brett L , Elmquist William F , Sarkaria Jann
Published: December 2020

Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.
TMOD-16. A NOVEL ADENOVIRAL-PERMISSIVE, IMMUNOCOMPETENT HAMSTER GLIOMA MODEL TO EVALUATE ONCOLYTIC ADENOVIRAL THERAPY
Authors: Phillips Lynette , Gumin Joy , Li Shoudong , Daou Marc , Ledbetter Daniel , Hossain Anwar , Kerrigan Brittany Parker , Gomez-Manzano Candelaria , Fueyo Juan , Lang Frederick
Published: December 2020

Oncolytic adenoviruses, including Delta-24-RGD, target tumors by direct tumor cell oncolysis and by activation of an anti-tumor immune response. Due to the species selectivity of oncolytic adenoviruses, there is currently no single preclinical animal model of glioma that supports viral replication, tumor oncolysis, and virus-mediated immune responses. To address this gap, we took advantage of the Syrian hamster to develop the first glioma model that is both adenovirus replication-permissive and immunocompetent. Hamster glioma stem-like cells (GSCs), transformed by forced expression of hTERT, SV40 large T antigen, and h-RasV12, reproducibly form intracranial tumors in hamsters. In vitro, electron microscopy and cytopathic effect assays demonstrated that hamster GSCs supported viral replication and were susceptible to Delta-24-RGD-mediated cell death. In vivo, hamster GSCs consistently developed into highly proliferative tumors resembling high-grade gliomas. Following intratumoral delivery of Delta-24-RGD, immunohistochemistry for viral proteins demonstrated viral infectivity and replication in hamster gliomas. Flow cytometry revealed increased T cell infiltration in Delta-24-RGD-infected tumors. Delta-24-RGD treatment of tumor-bearing hamsters led to significantly increased survival compared with hamsters treated with PBS. Using this model, we evaluated the effects of corticosteroid-mediated immunosuppression on Delta-24-RGD efficacy. Dexamethasone treatment significantly decreased peripheral blood lymphocytes, decreased tumor-infiltrating lymphocytes, and suppressed the levels of serum anti-adenovirus antibodies. Dexamethasone reduced the number of long-term survivors and decreased the median survival (50 days for Delta-24-RGD + dexamethasone vs undetermined for Delta-24-RGD alone). In summary, we have developed the first adenovirus-permissive, immunocompetent hamster glioma model, addressing a critical need for a model in which to study the role of direct oncolysis in driving immune mediated viral clearance versus driving an antiglioma immune response. Understanding these mechanisms is critical to optimizing the success of oncolytic adenoviral therapy in the clinic.
TMOD-17. ONCOLYTIC POLIOVIRUS AS A PROBE FOR MECHANISMS OF IMMUNE RESISTANCE IN GLIOBLASTOMA
Authors: Yang Yuanfan , Brown Michael , Gromeier Matthias , Bigner Darell
Published: December 2020

Glioblastoma is an immunologically cold tumor that employs multiple resistance mechanisms to escape activation of both innate and adaptive immunity. Transient immune activation and responses induced by immunotherapies in glioblastoma are almost always suppressed by potent intrinsic and extrinsic resistance mechanisms, limiting therapeutic efficacy. PVSRIPO is a recombinant poliovirus/rhinovirus chimera that elicits robust innate immune activation through infecting and lysing glioblastoma cells expressing the human poliovirus receptor (CD155), and through sublethal infection of tumor associated macrophages, setting the stage for lingering viral replication. We have constructed a pathological model using murine CT2ACD155 intracranial tumors stereotactically infused with a single dose of PVSRIPO (5×107 pfu) or mock control to study the response of glioblastoma in an immune competent in vivo tumor microenvironment. Tumor bearing brains were harvested before therapy and on days 2, 4, 6, 9, 12 post therapy for histology. On day 4 post therapy, histopathology showed > 90% shrinkage of intracranial tumor diameter (n= 6/6). Residual tumor is surrounded by a necrotic rim on histology with profound infiltration of F4/80+ macrophages and substantially reduced expression of Ki-67 on IHC compared to controls. Other markers including CD3, CD8 and Cleaved-Caspase 3 showed no difference in tumors of both groups. Interestingly, most residual tumor progressed on day 6 post therapy and caught up with the size of control tumor by day 9. Long-term remission of tumor occurred in only 10% of mice. This model is valuable in studying the mechanisms of intrinsic resistance and the timeline of resistance to develop in order to obtain more durable immunotherapy responses in glioblastoma. Future steps are to track inflammation induced by viral infection by RNAseq, and to find potential therapeutic interventions (including blocking PD1:PD-L1 axis) to mitigate therapy resistance in this preclinical model.
TMOD-25. IN VIVO MODEL OF TREATMENT-RESISTANT GLIOBLASTOMA HIGHLIGHTS SEX DIFFERENCES IN SURVIVAL
Authors: Al-Gizawiy Mona , Wujek Robert , Prah Melissa , Molino Salvatore , Chitambar Christopher , Schmainda Kathleen
Published: December 2020

BACKGROUND

Sex differences independent from acute hormone action are evident in the incidence and tumorigenesis of several cancers, including glioblastoma (GBM). Malignant brain tumors with mesenchymal, proneural, and neural GBM subtypes occur twice as frequently in males than in females, implying a molecular basis for the sex disparities observed. Accordingly, response to therapy and overall survival, may also differ between male and female GBM patients. Further elucidation of these differences would benefit from a robust preclinical testing system, such as the one described here.

METHODS

Adult (U87-MG) GBM cells were exposed to a total radiation dose of 10 Gy, effecting an epithelial to mesenchymal transition. The resultant irradiated U87-10Gy cells were tested for cell viability and allowed to reach confluence prior to stereotactic implantation into the right striatum of male and female athymic rats. In vivo advanced MR imaging at 9.4T was carried out weekly starting two weeks after implantation. Advanced MRI parameters were processed for enhancing tumor ROIs in OsiriX 8.5.1 (lite) with Imaging Biometrics™ Software (IQ-AI, Ltd.).

RESULTS

Tumor take was 90% in males and 60% in females, indicating an ‘incidence’ ratio of 1.5. Overall survival was significantly higher in females (median = 48, range = 40-72 days) than in males (median = 28, range = 27-30 days) (p = 0.024). Immunohistochemical and imaging analyses are pending and will be discussed.

CONCLUSION

The outcomes noted in this pilot study mirror incidence and survival outcomes noted in GBM patients undergoing standard care. Our model of treatment-resistant glioma provides a robust testing system to study underlying sex differences in GBM biology and treatment response in parallel analyses of male and female data.
TMOD-28. NEW APPROACHES FOR ELUCIDATING MEDULLOBLASTOMA DEVELOPMENT VIA HINDBRAIN BLASTOCYST COMPLEMENTATION
Authors: Schmidt Christin , Carlson Annika , Weiss William , Schwer Bjoern
Published: December 2020

Understanding the etiology of brain cancers requires elucidation of developmental origins, genetic drivers, and the tumor microenvironment. This requires reliable in vivo approaches, which are currently lacking. Current in vivo models for pediatric brain tumors rely on generation of xenografts or allografts in immunodeficient mice or generation of transgenic mice. These approaches have severe limitations, including lack of a functional immune system, a restricted developmental time window defined by the cell of origin, or time-consuming workflows for the generation of transgenic mice. We recently developed neural blastocyst complementation (NBC), an organogenesis approach for the forebrain. NBC involves injection of donor mouse embryonic stem cells (ESC) into genetically-engineered blastocysts that are programmed to ablate dorsal telencephalic progenitors. This results in the formation of a donor-cell derived, intact forebrain. Based on this general approach, we are developing an in vivo platform for studies of brain cancer. We will report on our efforts and progress toward the generation of an organogenesis approach for the hindbrain and related studies that aim to define developmental origins and drivers of medulloblastoma.
TMOD-33. THE ROLE OF BAFF-R SIGNALING IN THE GROWTH OF PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA
Authors: Mulazzani Matthias , Zhou Xíaolan , Zhang Wenlong , Straube Andreas , von Baumgarten Louisa
Published: December 2020

Primary CNS lymphoma (PCNSL) is an aggressive brain tumor. Despite improvements in therapeutic algorithms, long-term survival remains rare, illustrating an urgent need for novel therapeutic targets. BAFF-R is a pro-survival receptor expressed on most malignant B cells, including PCNSL. To date, its role in PCNSL growth remains elusive. Here, we created a BAFF-R knockout lymphoma cell line (BAFF-R-KO) using CRISPR-Cas9. In serum-starved conditions, BAFF-R-KO cells exhibit decreased viability in vitro compared to BAFF-R+ cells. Combining an orthotopic mouse model of PCNSL with chronic cranial windows and intravital microscopy, we demonstrate a significant delay in tumor growth in mice inoculated with BAFF-R-KO cells compared to BAFF-R+ PCNSL. Additionally, median survival of BAFF-R-KO mice was significantly prolonged. Altogether, our results indicate a potential of BAFF-R as a novel treatment target for PCNSL.
ATRT-16. MODELLING ATRT THROUGH SWI/SNF COMPLEX DEFICIENCY IN GENETICALLY-ENGINEERED MOUSE MODELS
Authors: Bondoc Andrew , Golbourn Brian , Smith Christian , Huang Annie , Rutka James
Published: December 2020

Atypical Teratoid/Rhabdoid Tumours (ATRT) are highly malignant neoplasms arising primarily in the CNS of children. They are defined by loss of function mutations in smarcb1, a gene serving a vital role in neurogenesis and differentiation. In order to recapitulate ATRT in the mouse, we used a Cre-Lox recombination system to conditionally knockout smarcb1 in specific cell compartments. Loss of smarcb1 in BLBP-expressing cells of the developing brain led to severe neurologic defects. Mice exhibited seizures, ataxia, and median 12-day survival. Histological analysis revealed severe thinning of the cerebral cortex and cerebellum. Temporally-targeted smarcb1 loss in BLBP/Nestin-expressing embryonic compartments did not result in tumour formation. Similarly, BLBP-expressing, smarcb1-deficient neural stem/progenitor cells (NSC/NPCs) were isolated and allografted but did not form tumours. These cells demonstrated decreased proliferation, higher apoptosis, and upregulation of p53, CDKN1A, and CDKN2A. In contrast, ubiquitous smarcb1 loss at the earlier embryonic day 6.5 produced widespread tumorigenicity in the forebrain, hindbrain, skullbase, and spine; each with unique phenotypes, survival, and morphology. We employed a clinically-relevant Nanostring gene-panel screen to stratify tumours into genetically distinct subgroups. Our findings indicate that smarcb1 plays an important role in CNS development. Loss of smarcb1 in NSC/NPCs is lethal, and its developmental context influences cell fate. Targeted smarcb1 loss likely plays a tumorigenic role at an earlier developmental stage than previously determined, in a diverse array of primitive stem cells. These data support the generation of a murine ATRT model capable of producing distinct tumour entities that recapitulate the human disease.
ATRT-17. TARGETING GLUTAMINE METABOLISM LOWERS METHYLATION POTENCIALS IN AT/RT AND SYNERGIZE WITH TAZEMETOSTAT
Authors: Nakata Satoshi , Wang Sabrina , Alt Jesse , Slusher Barbara , Eberhart Charles , Raabe Eric , Rubens Jeffrey
Published: December 2020

Atypical teratoid/rhabdoid tumors (AT/RT) have a single recurring genetic mutation in SMARCB1. This deletion leads to an abnormal SWI/SNF chromatin remodeling complex and the constitutive activation of EZH2. S-adenosyl-L-methionine (SAM) donates a methyl group to EZH2 which then methylates DNA and histones leading to the abnormal gene expression responsible for AT/RT’s aggressive phenotype. We have previously shown that glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) confers a survival advantage in AT/RT. In this study, we identified with ultra-high performance liquid chromatography mass spectrometry that DON treatment lowered the methylation potential in AT/RT (Decreased SAM:SAH ratio, t-test in 5 AT/RT human-derived cell models comparing DON treatment to DMSO control, p<0.05). AT/RT cell lines grown in glutamine deplete media compared to normal growth conditions also had a reduced methylation potential (decreased SAM:SAH, t-test, p<0.05). DON treatment over 5 days decreased histone methylation (as determined by western blot for H3K27me3). Tazemetostat is a small molecule inhibitor that blocks the SAM methyl donor site on EZH2. We find that DON combines synergistically with Tazemetostat to slow AT/RT cell growth (MTS assay, p<0.01 t-test; MUSE viability assay, p<0.01 ANOVA) and enhances cytotoxicity (MUSE Annexin-V, p<0.01 by ANOVA). Synergies were especially pronounced at low concentrations of Tazemetostat which is significant given that Tazemetostat’s efficacy in AT/RT has been limited by poor CNS penetration. These studies identify a novel treatment strategy that has potential to improve survival in AT/RT.
ATRT-19. EPIGENETIC REPROGRAMMING LEADS TO INNATE IMMUNE PATHWAY ACTIVATION IN AT/RT
Authors: Hariharan Seethalakshmi , Kilic Cem , Bowie Michelle , Reitman Zachary , Ashley David
Published: December 2020

BACKGROUND

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive brain tumors affecting early childhood and are characterized by bi-allelic inactivation of the SMARCB1 gene. Though patients benefit from multimodal therapy, there is no improvement in overall survival necessitating exploration of alternative approaches including innate-based immune therapy and epigenetic therapy, which have shown promise in treating adult brain tumors and other cancers. Though reconstitution of SMARCB1 in SMARCB1-deficient cells leads to activation of interferon-stimulated genes, the role of innate immune signaling has not been investigated in AT/RTs.

METHODS

Our data from a panel of AT/RT cell lines indicates loss of expression of key innate signaling components, like RIG-I, MDA-5, cGAS and STING that are required for sensing extracellular dsRNA and dsDNA. These cell lines also do not respond to dsDNA-based or dsRNA-based innate agonists. However, co-treatment of the BT-16 cell line with two epigenetic drugs, panobinostat and 5-azacytidine leads to re-expression of STING and RIG-I. Panobinostat/5-azacytidine co-treatment followed by either genomic DNA (dsDNA agonist) or poly(I:C) (dsRNA agonist) treatment results in induction of innate responses, measured by STAT1 phosphorylation and production of ISG-15 and IFIT-1.

CONCLUSION

Our data suggests that AT/RT cell lines are unresponsive to innate agonists possibly due to the loss of expression of key innate immune components. However, these pathways can be reactivated by epigenetic drugs and further potentiated by dsDNA/dsRNA-based innate agonists. Combined epigenetic reprogramming and innate pathway stimulation may serve as a potential therapy option for treating AT/RT.
ATRT-22. HIGH-THROUGHPUT DRUG SCREENING OF FDA-APPROVED CANCER DRUGS REVEALS POTENTIAL THERAPEUTIC APPROACHES FOR ATYPICAL TERATOID RHABDOID TUMOUR
Authors: Chong Wai Chin , Zhukova Nataliya , Wood Paul , Downie Peter A , Cain Jason E
Published: December 2020

Atypical teratoid/rhabdoid tumors (ATRT), are the most common brain tumor in children under the age of 1 year with an overall survival of ~17%. Like extracranial rhabdoid tumors, ATRT is exclusively characterized by bi-allelic loss of SMARCB1, a critical subunit of the SWI/SNF chromatin remodeling complex, implicating epigenetic deregulation in the pathogenesis of disease. We have previously shown the ability of the histone deacetylase inhibitor, panobinostat, to mimic SMARCB1-mediated SWI/SNF functions in extracranial rhabdoid tumors to inhibit tumor growth by driving multi-lineage differentiation in vitro and in vivo. Whether this also applies to ATRT is unknown. Using a panel of human-derived ATRT cell lines, representing defined molecular subgroups, we have shown that prolonged treatment with panobinostat at nanomolar concentrations results in markedly reduced clonogenicity, and increased senescence, preceded by increased H3K27 acetylation, decreased H3K27 trimethylation and EZH2 expression. To determine potentially synergistic therapies, we performed high-throughput drug screening of 622 compounds already in advanced clinical trials or FDA-approved for other indications, across our panel of ATRT models and identified 30 common compounds, which decrease cell viability by >50%, with no effect on neural stem cell controls and 12 compounds which demonstrated subgroup specificity, highlighting the necessity to consider therapies in the molecular context. In addition to HDACi, consistent with our panobinostat in vitro findings, inhibitors of CDK, survivin and PI3K were the top hits. In vitro and in vivo validation of these compounds alone, and in combination with panobinostat is ongoing.
ATRT-26. META-ANALYSIS OF TREATMENT MODALITIES IN METASTATIC ATYPICAL TERATOID/RHABDOID TUMORS IN CHILDREN
Authors: Underiner Reena M , Eltobgy Mostafa , Stanek Joseph R , Finlay Jonathan L , AbdelBaki Mohamed S
Published: December 2020

BACKGROUND

Metastatic atypical teratoid/rhabdoid tumors (AT/RT) are aggressive central nervous system tumors that present during infancy and are associated with dismal outcomes. Patients receive multimodal treatment including surgical resection, systemic chemotherapy and one or more of intrathecal chemotherapy (IT), marrow-ablative chemotherapy with autologous hematopoietic cell rescue (AuHCR) and radiation therapy (XRT). While data regarding treatment modalities for AT/RT patients exist, no comprehensive data have been published regarding the metastatic patient population.

METHODS

We performed a meta-analysis of 1,578 articles published through September 2018, including 44 studies with a total of 123 subjects. Additionally, seven patients were incorporated through chart review of patients treated at Nationwide Children’s Hospital.

RESULTS

Analysis of 130 patients revealed a 3-year overall survival (OS) of 25%. Age at diagnosis had a significant impact on survival (p=0.0355); 3-year OS for infants < 18 months was 21%; 18–36 months was 26%; and > 36 months was 36%. Location of the primary tumor, metastatic stage and extent of surgical resection did not have significant impact on OS. On univariate analysis, XRT (p<0.0001), IT (p=0.01) and AuHCR (p<0.0001) were found to significantly improve survival. The most substantial effect was noted in patients who received AuHCR (3-year OS of 60% versus 9% in those who did not). On multivariable analysis XRT (p=0.0006), IT (p=0.0124) and AuHCR (p<0.0001) were independently associated with reduced risk of death.
COVD-01. VINBLASTINE MONOTHERAPY INDUCTION FOR LOCALISED CNS GERMINOMA DURING THE COVID-19 PANDEMIC
Authors: Moleron Rafael , Stoneham Sara , Ajithkumar Thankamma , Cross Justin , Nicholson James , Murray Matthew
Published: December 2020

INTRODUCTION

Patients with localised CNS-germinoma have excellent survival. More recently, intensive inpatient chemotherapy (carboPEI=carboplatin/etoposide/ifosfamide in Europe) has been effectively employed to reduce radiotherapy fields and/or dose. Current research priorities focus on reducing treatment burden and long-term sequelae. Of note, outpatient-based single-agent carboplatin chemotherapy is associated with excellent outcomes in metastatic testicular seminoma (an identical pathology) [Alifrangis,EJC,2020]. Recently, successful vinblastine monotherapy was reported in localised CNS-germinoma [Murray,Neurooncol-Adv,2020].

METHODS

Due to the COVID-19 pandemic, adapted UK guidelines for germ-cell-tumour management were distributed, including potential non-standard treatment options that would reduce hospital visits/admissions. A 30-year-old patient presented with a 32mmx30mmx35mm diameter solid+multi-cystic localised pineal CNS lesion, consistent radiologically with a germ-cell-tumour with prominent teratoma component. Investigation revealed negative AFP/HCG markers and biopsy-proven pure germinoma. After appropriate consent, the patient commenced 12-week induction with weekly vinblastine monotherapy (low-grade-glioma dosing [Lassaletta,JCO,2016]), with wk6&12 MRI re-assessment prior to definitive radiotherapy.

RESULTS

Vinblastine was well-tolerated. After initial 4mg/m2 test-dosing (wk1), standard 6mg/m2 was delivered for wk2, but resulted in asymptomatic neutropenia (nadir 0.3x10^9/l) and missed dosing at wk3. Subsequent doses were 4mg/m2, with no further neutropenia. As expected, MRI showed moderate 40% tumour volume reduction by wk12. Surgical resection of the residual presumed teratoma component was undertaken prior to radiotherapy.

CONCLUSION

Patients with CNS-germinoma have excellent outcomes and reduction of treatment-effects remains a priority. The exquisite chemosensitivity of germinoma, excellent results from monotherapy for metastatic testicular disease, and early promise of vinblastine monotherapy lend itself to further exploration for CNS-germinoma.
DDEL-06. DRUG INTERACTION BETWEEN EVEROLIMUS AND CANNABIDIOL IN PEDIATRIC PATIENTS WITH SUBEPENDYMAL GIANT CELL ASTROCYTOMAS: A SINGLE INSTITUTION EXPERIENCE
Authors: Sabus Ashley , Winzent Shelby , Hemenway Molly , Levy Jean Mulcahy , Foreman Nicholas
Published: December 2020

Tuberous sclerosis complex (TSC) is an autosomal recessive genetic disorder associated with clinical manifestations including subependymal giant cell astrocytomas (SEGA) and seizures. The combination of everolimus and Epidiolex, a purified form of cannabidiol, has become an increasingly common treatment regimen in this population. Everolimus is primarily metabolized via CYP3A4, which may be inhibited by cannabidiol. We seek to describe our institution’s experience with this drug interaction. METHODS: Investigators conducted a retrospective review of neuro-oncology patients with TSC and SEGA who were treated concurrently with everolimus and cannabidiol. Data collected included demographics, body surface area, everolimus dose, everolimus troughs, date of cannabidiol initiation, documented symptoms, liver and renal function tests, and reason for discontinuing therapy. RESULTS: Three patients (ages 11–17 years) met inclusion criteria. All patients were stable on everolimus doses ranging from 6.5 to 9.5 mg/m2/day and achieving trough goals of 5–10 ng/mL. Two to four weeks after initiating cannabidiol, everolimus trough concentrations rose 200–860% above goal. One patient reported new-onset involuntary movements, but no other toxicities were noted. Cannabidiol was discontinued in all cases due to caregiver concerns regarding drug interactions. All patients were able to achieve goal trough concentrations on previously stable doses of everolimus after discontinuing cannabidiol. CONCLUSIONS: Cannabidiol appears to modulate everolimus metabolism leading to significantly elevated serum concentrations. Additional research is required to determine the need for empiric dose adjustments upon cannabidiol initiation. Patient counseling, frequent trough monitoring, and surveillance for adverse effects are crucial for optimizing outcomes in patients prescribed this regimen.
DIPG-07. HIGH THROUGHPUT DRUG SCREENING IDENTIFIES POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)
Authors: Upton Dannielle , Valvi Santosh , Liu Jie , Yeung Nicole , George Sandra , Ung Caitlin , Khan Aaminah , Franshaw Laura , Ehteda Anahid , Shen Han , Orienti Isabella , Farruggia Giovanna , Nath Christa , Reynolds Patrick , Tsoli Maria , Ziegler David
Published: December 2020

DIPGs are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12 months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,500 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds- auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine- that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in-vitro. To determine whether the in-vitro efficacy could be replicated in-vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different Fenretinide formulations (LYM-X-Sorb and NanoMicelle) which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.