bluuebunny/crossref_metadata_2025 huggingface dataset:
TAMI-03. IDENTIFICATION OF NOVEL DRIVERS OF LUNG-TO-BRAIN METASTASIS THROUGH IN VIVO FUNCTIONAL GENOMICS
Authors: Aghaei Nikoo , Lam Fred , Venugopal Chitra , Singh Sheila
Published: December 2020

Brain metastasis, the most common tumor of the central nervous system, occurs in 20-36% of primary cancers. In particular, 40% of patients with non-small cell lung cancer (NSCLC) develop brain metastases, with a dismal survival of approximately 4-11 weeks without treatment, and 16 months with treatment. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Genomic interrogation of LBM using CRISPR technology can inform preventative therapies targeting genetic vulnerabilities in both primary and metastatic tumors. Loss-of-function studies present limitations in metastasis research, as knocking out genes essential for survival in the primary tumor cells can thwart the metastatic cascade prematurely. However, gene overexpression using CRISPR activation (CRISPRa) has the potential for overcoming dependencies of gene essentiality. We theorize that an in vivo genome-wide CRISPRa screen will identify novel genes that, when overexpressed, drive LBM. We have developed a patient-derived orthotopic murine xenograft model of LBM using primary patient-derived NSCLC cell lines (termed LTX cells) from the Swanton Lab TRACERx study. We are now poised to transduce LTX cells with a human genome-wide CRISPRa single guide RNA (sgRNA) library, and to subsequently inject the cells into the lungs of immunocompromised mice. We will then track the process of LBM using bioluminescent and MRI imaging until mice reach endpoint. Sequencing of primary lung tumors and subsequent brain metastases promises to uncover enriched sgRNAs, which may represent novel drivers of primary lung tumor formation and LBM. To the best of our knowledge, this study is the first in vivo genome-wide CRISPRa screen focused on identifying novel drivers of LBM, and can inform future preventative therapies to improve survival outcomes for NSCLC patients.
TAMI-04. TUMOR TREATING FIELDS (TTFIELDS) HINDER GLIOMA CELL MOTILITY THROUGH REGULATION OF MICROTUBULE AND ACTIN DYNAMICS
Authors: Voloshin Tali , Schneiderman Rosa , Volodin Alexandra , Shamir Reuben , Kaynan Noa , Zeevi Einav , Koren Lilach , Klein-Goldberg Anat , Paz Rom , Giladi Moshe , Bomzon Zeev , Weinberg Uri , Palti Yoram
Published: December 2020

The ability of glioma cells to invade adjacent brain tissue remains a major obstacle to therapeutic disease management. Therefore, the development of novel treatment modalities that disrupt glioma cell motility could facilitate greater disease control. Tumor Treating Fields (TTFields), encompassing alternating electric fields within the intermediate frequency range, is an anticancer treatment delivered to the tumor region through transducer arrays placed non-invasively on the skin. This novel loco-regional treatment has demonstrated efficacy and safety and is FDA-approved in patients with glioblastoma and malignant pleural mesothelioma. TTFields are currently being investigated in other solid tumors in ongoing trials, including the phase 3 METIS trial (brain metastases from NSCLC; NCT02831959). Although established as an anti-mitotic treatment, the anti-metastatic potential of TTFields and its effects on cytoskeleton rapid dynamics during cellular motility warrant further investigation. Previous studies have demonstrated that TTFields inhibits metastatic properties of cancer cells. Identification of a unifying mechanism connecting the versatile TTFields-induced molecular responses is required to optimize the therapeutic potential of TTFields. In this study, confocal microscopy, computational tools, and biochemical analyses were utilized to show that TTFields disrupt glioma cellular polarity by interfering with microtubule assembly and directionality. Under TTFields application, changes in microtubule organization resulted in activation of GEF-H1, which led to an increase in active RhoA levels and consequent focal adhesion formation with actin cytoskeleton architectural changes. Furthermore, the optimal TTFields frequency for inhibition of invasion in glioma cells was 300 kHz, which differed from the optimal anti-mitotic frequency leading to glioma cell death of 200 kHz. The inhibitory effect of TTFields on migration was observed at fields intensities of 0.6 V/cm RMS (below the threshold of 1 V/cm RMS previously reported for cytotoxic effects). Together, these data identify discrete TTFields effects that disrupt processes crucial for glioma cell motility.
TAMI-08. A TALE OF TWO TELOMERE MAINTENANCE MECHANISMS: TERT EXPRESSION AND THE ALT PATHWAY INDUCE UNIQUE MRS-DETECTABLE METABOLIC REPROGRAMMING IN LOW-GRADE GLIOMAS
Authors: Viswanath Pavithra , Batsios Georgios , Gillespie Anne Marie , Luchman Hema Artee , Costello Joseph , Pieper Russell , Ronen Sabrina
Published: December 2020

Telomeres are nucleoprotein structures at chromosomal ends that shorten with cell division and constitute a natural barrier to proliferation. In order to proliferate indefinitely, all tumors require a telomere maintenance mechanism (TMM). Telomerase reverse transcriptase (TERT) expression is the TMM in most tumors, including low-grade oligodendrogliomas (LGOGs). In contrast, low-grade astrocytomas (LGAs) use the alternative lengthening of telomeres (ALT) pathway as their TMM. As molecular hallmarks of tumor proliferation, TMMs are attractive tumor biomarkers and therapeutic targets. Non-invasive imaging of TMM status will, therefore, allow assessment of tumor proliferation and treatment response. However, translational methods of imaging TMM status are lacking. Here, we show that TERT expression and the ALT pathway are associated with unique magnetic resonance spectroscopy (MRS)-detectable metabolic reprogramming in LGOGs and LGAs respectively. In genetically-engineered and patient-derived LGOG models, TERT expression is linked to elevated 1H-MRS-detectable NAD(P)/H, glutathione, aspartate and AXP. In contrast, the ALT pathway in LGAs is associated with higher α-ketoglutarate, glutamate, alanine and AXP. Importantly, elevated flux of hyperpolarized [1-13C]-alanine to pyruvate, which depends on α-ketoglutarate, is a non-invasive in vivo imaging biomarker of the ALT pathway in LGAs while elevated flux of hyperpolarized [1-13C]-alanine to lactate, which depends on NADH, is an imaging biomarker of TERT expression in LGOGs. Mechanistically, the ALT pathway in LGAs is linked to higher glutaminase (GLS), a key enzyme for α-ketoglutarate biosynthesis while TERT expression in LGOGs is associated with elevated nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme for NADH biosynthesis. Notably, TERT expression and the ALT pathway are linked to MRS-detectable metabolic reprogramming in LGOG and LGA patient biopsies, emphasizing the clinical validity of our observations. Collectively, we have identified unique metabolic signatures of TMM status that integrate critical oncogenic information with noninvasive imaging modalities that can improve diagnosis and treatment response monitoring for LGOG and LGA patients.
TAMI-09. ENERGY METABOLISM AND THERAPEUTIC T CELL EFFICACY IN THE GLIOBLASTOMA MICROENVIRONMENT
Authors: Logun Meghan , Colonna Maxwell , Edison Arthur , Karumbaiah Lohitash
Published: December 2020

Glioblastoma (GBM), like most cancers, undergo metabolic alterations to primarily utilize aerobic glycolysis in the hypoxic tumor microenvironment (TME). Similarly, activated T cells switch to glycolysis upon antigen recognition to cope with proliferation needs but are not equally equipped to survive in the hypoxic TME. Metabolic reprogramming within GBM TME contributes to therapeutic resistance and tumor progression, but the effects of metabolic alterations on therapeutic T cell survival and efficacy have not been fully elucidated. We hypothesized that hypoxia in GBM/T-cell co-cultures will significantly impair T cell proliferation and function. We conducted in vitro co-culture assays and nuclear magnetic resonance (NMR)–based assessments in hypoxic (1%O2) or normoxic conditions to detect metabolic changes in real-time. Imaging cytometry for cell cycle assessment demonstrated that GSCs were unaffected by hypoxia, but roughly 90% of healthy T cells arrested in G0/G1 along with significant reduction in glycan precursor UDP-GlcNAc presence. Media samples over 96h in normal and hypoxic oxygen conditions from cells in solitary or co-cultures were analyzed using a Bruker Avance III HD spectrometer at 600 MHz for comparison over time using PCA analysis of metabolic intermediate differences. After 16h, there was observable differences in produced metabolites between the T cells cultured alone or co-culture with GSCs, compared to the GSCs alone or media alone controls. Quantifiable changes in glucose, lactate, fumarate, acetate and pyruvate, among others, indicated a large shift in T cell metabolism dependent on oxygen conditions and co-culture interactions, while GSCs are less metabolically responsive to culture conditions. Ongoing experiments will examine precise changes in UDP-GlcNAc and glycosylation precursors in T cells and CAR-T cells via targeted NMR analysis, which we expect will help us understand energy dependent mechanisms of T cell exhaustion and lead to development of novel strategies to sustain T cell function in the hostile TME.
TAMI-18. SINGLE-CELL SEQUENCING AND GENETIC LABELING TO CHARACTERIZE HYPOXIC GBM CELLS IN THEIR MICROENVIRONMENT
Authors: Sattiraju Anirudh , Marallano Valerie , Ramakrishnan Aarthi , Brusco Concetta , Shen Li , Wojcinski Alexandre , Kesari Santosh , Friedel Roland H , Zou Hongyan
Published: December 2020

Glioblastoma (GBM) recurrence, arising from treatment-resistant hypoxic cells, is a major contributor to patient mortality. We have engineered GBM cell lines with a novel genetic reporter system (“HRE-UnaG”), which sensitively labels hypoxic cells with green fluorescence. Using murine GL261 HRE-UnaG GBM cells in intracranial transplants in immunocompetent hosts, we detected hypoxic cells primarily around pseudopalisading structures. Here, we report our new results of single cell RNA-seq sequencing of intracranial GL261-HRE-dUnaG GBM tumors, which revealed in hypoxic UnaG+ GBM cells upregulation of more than 60 genes, including canonical hypoxia response genes such as Slc2a1, Bnip3, Ldha and Vegfa, and downregulation of a smaller number of genes, such as Rgcc, Cdc20 and Hist1h2ap, which regulate cell cycle progression. This novel GBM hypoxia signature was in TCGA patient samples mostly upregulated in recurrent GBM and associated with worse prognosis, especially within proneural and mesenchymal subtypes. Gene set enrichment analysis revealed that the GBM hypoxia signature was positively correlated with hypoxia, glycolysis, TNFα and MTORC1 signaling, and negatively correlated with oxidative phosphorylation, Myc and DNA repair pathways. Subclustering revealed four distinct subpopulations of hypoxic GBM cells, which were distinguished by differential expression of pathway genes for proliferation, angiogenesis, stress response, and type I/II interferon signaling. Intercellular communication between hypoxic GBM cells and the stromal cells were predicted using NicheNET package. To spatio-temporally label in future studies hypoxic GBM cells and their progeny, we have engineered GBM cells with an inducible, hypoxia-sensitive lineage-tracing reporter (HRE-Cre; floxed-tdTomato). In combination with the HRE-UnaG hypoxia reporter, we will be able to reveal the role of “hypoxia memory” in GBM expansion and recurrence after treatment. Identifying key markers of hypoxic cells in respect to treatment resistance and relapse will lead to improvements in GBM detection and new options for therapeutic treatments.
TAMI-22. VARIATION AMONG INTACT TISSUE SAMPLES REVEALS THE CORE TRANSCRIPTIONAL IDENTITIES OF CELL TYPES IN THE GLIOMA MICROENVIRONMENT
Authors: Schupp Patrick , Oldham Michael
Published: December 2020

Adult low-grade gliomas generally progress to glioblastoma, a more aggressive CNS tumor with an extremely poor prognosis. Despite intensive efforts, numerous promising glioma therapies have failed to provide survival benefits. These failures reflect many factors, including intertumoral heterogeneity and immunosuppression by the tumor microenvironment (TME). We propose a novel approach to addresses these challenges through integrative deconvolution of bulk gene expression data generated from more than 5000 human gliomas and 7000 normal human brain samples. Inherent variation in the cellular composition and cellular activities of these samples allowed us to identify highly correlated modules of genes that represent specific cell types and cell states. By comparing gene coexpression modules in glioma vs. normal human brain, we have identified cell type-specific gene expression changes in the glioma TME that are highly reproducible. In contrast to single-cell methods, which sample only a fraction of the tumor tissue and fail to capture major nonmalignant cell-types, our results derive from billions of cells and thousands of individuals and are therefore highly robust. We find that a number of genes encoding cell-surface proteins are specifically up-regulated in immune and vascular cells of the glioma TME. Surprisingly, among those genes up-regulated in glioma vasculature are multiple members of the angiotensin pathway, suggesting non-canonical roles for these proteins in the glioma setting. We propose that these proteins may form a specific ‘zip code’ for glioma within the brain’s vasculature that can be targeted directly or by conjugation with existing drugs. More generally, our analytical approach has revealed reproducible gene expression changes in specific cell types of the glioma TME that provide more stable therapeutic targets than those that are expressed by genetically mutable malignant cells. We have also discovered novel, aberrantly coexpressed genes in microglia, oligodendrocytes, and astrocytes which we are testing in state-of-the-art human brain assembloid systems.
TAMI-24. BEHAVIOR OF GLIOBLASTOMA STEM-LIKE CELLS WITH KNOWN IDH1 STATUS IN CEREBRAL ORGANOIDS
Authors: Singh Sanjay , Munyeshyaka Maxime , Gumin Joy , Yang Jing , Ledbetter Daniel , Hossain Anwar , Kerrigan Brittany Parker , Lang Frederick
Published: December 2020

Glioblastomas (GBM) exhibit high proliferative index, areas of necrosis, high vascularization, and are highly invasive to normal brain tissues. The most common and lethal form of GBMs are primary GBMs, with no prior clinical history. Whereas, secondary GBMs arise from low-grade gliomas and are associated with IDH1 mutation. Pre-clinical studies of GBM largely depend on patient-derived GBM stem-like cells (GSCs) in vitro and in vivo as orthotopic xenografts. Cerebral organoids (COs) derived from induced pluripotent stem cells can serve as allogenic in vitro model systems to study interactions between normal brain and GSCs. COs have been shown to harbor neural stem cells and their differentiated progenies as well as microglia within distinct niches. Here, we co-cultured 45 day-old COs and MDA-GSCs lines representing mesenchymal sub-group (M-MDA-GSC), classical sub-group (C-MDA-GSC), and IDH1 mutant (IDH1R132H-MDA-GSC). MDA-GSCs stably express fluorescent proteins and is used to track GSCs within COs. These GSC bearing COs were fixed, embedded, sectioned, immuno-stained, and imaged by confocal microscope. There was a positive correlation between GSC numbers in allografted niche and invasion into COs as measured from the edge of organoid, M-MDA-GSC (R2=0.99; 0.89μm/cell), C-MDA-GSC (R2=0.92; 0.66μm/cell), and IDH1R132H-MDA-GSC (R2=0.89; 0.5μm/cell). Additionally, M-MDA-GSCs had significantly high percentage of Ki67+ve invasive cells (24%) in comparison to C-MDA-GSCs (5.1%; p=0.0057). As a measure of interaction of MDA-GSC with normal cells, we assessed proximity of IBA1+ve microglia in GSC niche within organoids and show that M-MDA-GSC and IDH1R132H-MDA-GSC highly co-localized with IBA1+ve microglia on day12 of co-culture. In conclusion, our cerebral organoid-based allograft study shows that mesenchymal GSCs (M-MDA-GSC) are most invasive whereas IDH1 mutant GSCs (IDH1R132H-MDA-GSC) are least invasive. C-MDA-GSCs are least proliferative while invading into normal COs. Uniqueness of CO based allograft system is highlighted by observed similarity between M-MDA-GSC and IDH1R132H-MDA-GSC for their potential to attract IBA1+ve microglia.
TAMI-27. RADIATION THERAPY REPROGRAMS THE TUMOR METABOLISM TO PROMOTE SURVIVAL AND IMMUNOSUPPRESSION IN GLIOBLASTOMA
Authors: De Martino Mara , Daviaud Camille , Vanpouille-Box Claire
Published: December 2020

Immune checkpoint blockade has re-stimulated the interest of immunotherapeutic strategies for glioblastoma (GBM). However, PD-1 blockade in recurrent GBM patients failed to meet the primary endpoint of superior survival (NCT02017717) highlighting the need to identify mechanisms of GBM immune evasion. One possibility is the metabolic landscape that cancer cells generate to promote survival and prevent immune responses. Radiation therapy (RT), a standard of care for GBM, is well-documented to reprogram the metabolism of the tumor microenvironment (TME). Therefore, we hypothesize that RT generates a metabolic shift in the TME to support GBM survival and immunosuppression. To address this question, we first defined RT-induced metabolic changes by Seahorse assay. We observed that mitochondrial respiration and ATP production were significantly increased in 10 gray (Gy)-irradiated GL261 cells after 24hr. Metabolomic profiling of 10Gy-irradiated GL261 cells showed a strong increase in metabolic pathways related to nucleotide, amino acids and lipid metabolism. We next ask whether RT was modulating the fatty acid synthase (FASN), a key lipogenic enzyme. Western blot analysis indicated upregulation of FASN expression in irradiated GL261 cells. Interestingly, flow cytometry of orthotopically implanted GL261 tumors not only confirmed FASN upregulation in cancer cells in vivo, but also revealed a marked FASN expression in tissue-resident microglia after 10Gy focal irradiation. Moreover, flow cytometry analysis showed increased expression of the immunosuppressive markers PD-L1 and CSF-1R in macrophages co-cultured with irradiated GL261. Finally, targeting FASN using shRNA or pharmacological inhibition, significantly reduced the proliferation of GL261 cells. Altogether, our data indicate that RT is rewiring the energy supply of GBM to presumably foster its growth and immunosuppression of the TME by promoting lipid biosynthesis. While much work remains to be done to confirm this hypothesis, our data propose FASN as a novel therapeutic target to overcome RT resistance and reduce immunosuppression in GBM.
TAMI-32. TEMPOROSPATIAL INVASION AND GENETIC EVOLUTION FROM INFRATENTORIAL TO SUPRATENTORIAL COMPARTMENT IN DIFFUSE MIDLINE GLIOMA
Authors: Georgescu Maria-Magdalena
Published: December 2020

Diffuse midline gliomas (DMGs) are very aggressive pediatric brain tumors with dismal prognosis due to therapy-resistant tumor growth and invasion. We performed the first integrated histologic/genomic/proteomic analysis of 21 tumor foci from three pontine DMG cases with supratentorial dissemination. Histone H3.3 K27M was the driver mutation, usually at high variant allele fraction due to recurrent chromosome 1q copy number gain, in combination with germline variants in ATM, FANCM and MYCN genes. Both previously reported and novel recurrent copy number variations and somatic pathogenic mutations in chromatin remodeling, DNA damage response and PI3K/MAPK growth pathways were variably detected, either in multiple or isolated foci. Proteomic analysis showed global upregulation of histone H3, lack of K27 tri-methylation, and further impairment of polycomb repressive complex 2 by ASXL1 downregulation. Activation of oncogenic pathways resulted from combined upregulation of N-Myc, SOX2, p65/p50 NF-kB and STAT3 transcription factors, EGFR, FGFR2, PDGFRa/b and MET receptor tyrosine kinases, and downregulation of PHLPP1/2, PTEN and p16/INK4A tumor suppressors. Upregulation of SMAD4, PAI-1, CD44, and c-Src in multiple foci most likely contributed to invasiveness. This integrated comprehensive analysis allowed spatiotemporal modeling of tumor progression and identified two general pathways of supratentorial invasion, and a multitude of migratory subpopulations within the infratentorial compartment. It also delineated common signaling pathways and potential therapeutic targets, revealing an unsuspected activation of a multitude of oncogenic pathways that may explain the resistance of DMG to current therapies.
TAMI-37. SEX DIFFERENCES IN REDOX STATE UNDERLIE GLUTAMINE DEPENDENCY IN MALE GLIOBLASTOMA
Authors: Sponagel Jasmin , Zhang Shanshan , Jones Jill , Chinnaiyan Prakash , Rubin Joshua , Ippolito Joseph
Published: December 2020

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. GBM occurs more commonly in males but female patients survive significantly longer. Understanding the molecular mechanisms underlying this clinical sex disparity could support novel treatment strategies to improve outcomes for GBM patients. In this regard, we found that male and female GBM patient tissues differ in their metabolite profiles and that male GBM exhibit a higher abundance of amino acid metabolites. We confirmed these findings in a murine model of GBM. Furthermore, we found that male GBM cells are more sensitive to amino acid deprivation. This male-specific dependency on amino acids is almost entirely driven by amino acids involved in the synthesis of the antioxidant glutathione. Glutaminase 1 (GLS1) mediates the conversion from glutamine to glutamate, a crucial component of glutathione. We found that male GBM cells are more sensitive to GLS1 inhibition with the clinical inhibitor CB-839. This correlated with significantly increased reactive oxygen species (ROS) in male GBM. We further confirmed sex differences in redox state through pharmacological depletion of glutathione, which resulted in a significant increase in ROS and cell death in male GBM cells. Moreover, assays of glutathione oxidation demonstrated that male GBM cells exist in a chronically oxidized state. Finally, we found that mitochondrial structure and function, including TCA cycle flux, NADH levels, and antioxidant activity, differ between male and female GBM cells. Together, these data suggest that (1) male and female GBM differ in their amino acid requirements, (2) male GBM are more dependent on glutamine to regulate ROS levels, and (3) sex differences in mitochondrial physiology may result in ROS accumulation and increased susceptibility to drugs targeting the redox state in male GBM. Our data reveal novel metabolic targets for GBM and underline the importance of considering sex in metabolic targeting approaches.
TAMI-49. THE ALTERNATIVE SPLICING FACTOR MBNL1 INHIBITS GLIOBLASTOMA TUMOR INITIATION AND PROGRESSION BY REDUCING HYPOXIA-INDUCED STEMNESS
Authors: Voss Dillon , Sloan Anthony , Bar Eli , Bar Eli
Published: December 2020

Muscleblind-like-proteins (MBNL) belong to a family of tissue-specific RNA metabolism-regulators that control pre-messenger RNA-splicing (AS). Inactivation of MBNL causes an adult-to-fetal AS transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer, glioblastoma (GBM), maintains stem-like features (GSC) through hypoxia-induced responses. Accordingly, we hypothesized that the hypoxia-induced responses in GBM might also include MBNL-based AS to promote tumor progression. When cultured in hypoxia, GSCs rapidly export MBNL1 out of the nucleus resulting in significant inhibition of MBNL1 activity. Notably, the hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Using a tetracycline-inducible system, induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression resulting in a significant prolongation of survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, whereby hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may, therefore, yield potent tumor-suppressor activities, uncovering new therapeutic opportunities to counter this devastating disease.
TAMI-50. CHITINASE-3-LIKE-1 PROTEIN BINDING COMPLEXES REGULATE IMMUNE SUPPRESSION IN GLIOBLASTOMA
Authors: Chen Apeng , Jiang Yinan , Li Zhengwei , Zou Han , Santiago Ulises , Xiao Xiangwei , Camacho Carlos , Pollack Ian , Hu Baoli
Published: December 2020

Glioblastoma (GBM), the most common and lethal brain tumor, remains incurable despite intensive multimodal treatment. While immunotherapies have been highly effective in some types of cancer, the disappointing results from clinical trials for GBM immunotherapy represent continued challenges. GBM is highly immunosuppressive and resistant to immunotherapy because of glioma cells escaping from immune surveillance by reprograming the tumor microenvironment (TME). However, understanding the mechanisms of immune evasion by GBM remains elusive. Here, we found that Chitinase-3-like-1 (CHI3L1) is highly expressed in GBM and associated with a poor clinical outcome. CHI3L1, also known as human homolog YKL-40, plays a role in tissue remodeling, inflammation and cancer. Interestingly, we found that genetic knockdown (KD) of Chi3l1 in syngeneic immunocompetent mouse GBM models resulted in increased tumor-infiltrating lymphocytes, tumor size reduction, and improved animal survival. Surprisingly, the parallel loss-of-function experiment revealed that Chi3l1 KD did not repress tumor progression in the orthotopic immunodeficient mice with deficient T and B cells. These results suggest the predominant role of CHI3L1 in regulating the GBM immune TME, rather than in tumor cells per se. Mechanistically, we discovered that Galectin-3 (Gal-3) and Galectin-3 binding protein (Gal-3BP) interact competitively with the same binding motif on CHI3L1, leading to selective migration of protumor M2-like versus antitumor M1-like bone marrow-derived macrophages (BMDMs) and resident microglia (MG). Transcriptomic analysis revealed that pro-inflammatory signature and T cell mediated immunity and cytotoxicity signaling are significantly enriched in tumor associated macrophages/microglia (TAMs) composed of BMDMs and MG, which were isolated from tumors with Chi3l1 KD versus wild type. In vitro validations suggest that CHI3L1-Gal-3, but not CHI3L1-Gal-3BP protein binding complex, activates PI3K/AKT/mTOR signaling to control the TAM switch of immune suppression and immune stimulation. Together, these results shed light on molecular mechanism of GBM immune evasion and potential new immunotherapeutic strategies for GBM treatment.
TAMI-54. THE PRESENCE OF IMMUNE CELL INFILTRATES IN THE TISSUE MICROENVIRONMENT OF HIGH-GRADE GLIOMAS AND THEIR ASSOCIATION WITH OVERALL SURVIVAL
Authors: Rishi Anupam , Mohammadi Homan , Martir Daniela , Welsh Eric , Robinson Timothy , Oliver Daniel , Eschrich Steven , Torres-Roca Javier , Yu Hsiang-Hsuan , Grass George , Ahmed Kamran
Published: December 2020

OBJECTIVE

Tumor-associated microglia and macrophages (TAMs) influence brain tumor biology and promote tumor-proliferating phenotype. Studies have shown these cell types predict outcomes in various tumor sites with limited evidence in high-grade gliomas (HGG). In this study, we assessed the presence of immune cell infiltrate (ICI) and overall survival (OS) in HGGs.

METHODS

A total of 97 consecutively treated patients with primary HGG with complete gene expression profiling were identified from our IRB-approved institutional tissue biorepository. Patients underwent primary surgical resection between 02/2004 and 03/2011. Gene expression levels were assessed by Affymetrix Hu-RSTA assays (Affymetrix; Santa Clara, CA). CIBERSORT estimated the presence of ICI via gene expression deconvolution. Tumor characteristics and the presence of 22 individual ICI subtypes were assessed with respect to OS. Time-to-event analyses were performed with Kaplan-Meier estimates and compared via log-rank test. Associations between the ICI and outcomes were explored using Cox-regression. P< 0.05 (two-tailed) was considered statistically significant.

RESULTS

Median follow-up from primary surgical resection was 12.8 months (range: 0.1-162.8), and median age was 58 years (20-85). Most patients were male (n=63; 65%) and had grade 4 tumors (n=71; 73%). OS differed by grade with 24-month actuarial OS rates of 81% and 34% (P< 0.0001) for grade 3 and 4 gliomas, respectively. The presence of M0 (HR 2.2; 95% CI 1.4-3.6; P=0.001), M1 (HR 1.8; 95%CI 1.1-2.9; P=0.01), and M2 macrophages (HR 1.9; 95%CI 1.2-3.2; P=0.007) predicted OS. No other ICI subtypes were predictive of OS. The presence of M0- and M2-polarized macrophages were more common in grade 4 compared to grade 3 gliomas 46% vs. 11% (P=0.002) and 69% vs. 31% (P=0.0007), respectively.

CONCLUSION

The increased presence of non-polarized or M2 TAMs within the glioma microenvironment was significantly associated with OS in HGG. Their presence may serve as unique stratification and a potential therapeutic target.
TAMI-59. METABOLIC REGULATION OF THE EPIGENOME DRIVES LETHAL INFANTILE EPENDYMOMA
Authors: Michealraj Antony , Kumar Sachin , Kim Leo , Rich Jeremy , Taylor Michael
Published: December 2020

Ependymomas are malignant glial tumors that occur throughout the central nervous system. Of the nine distinct molecular types of ependymoma, Posterior Fossa A(PFA) ependymomas, found in the hindbrain of infants and young children, are the most prevalent type. They rarely harbor recurrent somatic single nucleotide or copy number aberrations, but rather display epigenetic dysregulations. Transcriptional analyses show that PFAs, but not other molecular ependymoma variants, have elevated hypoxic signaling. We hypothesized that the metabolic environment of the developing human fetal hindbrain contributes to PFA ependymomas through the intermediary metabolic mechanism. Hypoxia microenvironment is essential for PFA survival and upholding their epigenetic dysregulation. Hypoxia blocks methyltransferase activity by upregulating EZHIPs expression and restricting SAM abundance. Fine-tuning the abundance of a-KG and acetyl-CoA, hypoxia fuels demethylase, and acetyltransferase activity which collectively resulting in H3K27 hypomethylation and hyperacetylation. Genome-wide essentially screen further underscore that minimal basal level of H3K27me3 is essential for PFA survival and further attenuation of H3K27me3 decreases the fitness of PFAs. PFA Ependymomas have a unique epigenome, suggesting a model in which they thrive in a narrow Goldilocks zone, with deviation to either increased or decreased H3K27me3 levels leading to diminished cellular fitness. PFAs are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation and increase histone demethylation and acetylation at H3K27. PFAs initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3 and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA growth. Microenvironmental regulation of PFA ependymoma epigenome appears to play a major role in tumorigenesis. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.
TMOD-01. DEVELOPING CLINICALLY RELEVANT BRAIN METASTATIC TUMOR MODELS FOR TARGETED STEM CELL THERAPEUTICS
Authors: Kitamura Yohei , Moleirinho Susana , Kanaya Nobuhiko , Franco Arnaldo , Shah Khalid
Published: December 2020

Brain metastasis is associated with increased mortality in patients with cancer, 15-20% of patients will develop brain metastasis during the course of the disease. Characterizing clinically relevant brain metastasis models and exploring different options to deliver drugs across blood brain barrier in such models are fundamental for the development of novel therapies for metastatic brain tumors. We have created imageable breast to brain metastasis tumor models using patient derived brain seeking basal like breast cancer (BLBC) lines. We show a widespread distribution of micro- and macro-metastasis and the close association of tumor cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. These models enables us to investigate the pathophysiology of this little-known phenomenon and test the efficacy of new approach targeting the dormant cancer cells in perivascular niche. Next, we assessed the EGFR and DR4/5 levels in BLBC and show that brain seeking BLBC have higher EGFR and DR5 levels than their primary counterparts. We also show that intracarotid artery injection or intrathecal injection of stem cells engineered to express optical imaging markers home to metastatic brain tumors. These findings provide useful models for testing treatments for metastatic brain tumors and also provide insights into the changes in gene expression in metastatic BLBC and means to deliver therapeutics to metastatic tumors in the brain.
TMOD-02. IN VITRO & IN VIVO CULTURE OF PATIENT DERIVED (PD) CSF-CTCS IN LEPTOMENINGEAL DISEASE (LMD) FROM MELANOMA TO IDENTIFY NOVEL TREATMENT STRATEGIES
Authors: Law Vincent , Evernden Brittany , Puskas John , Caceres Gisela , Ryzhova Elena , Smalley Inna , Tran Nam , Etame Arnold , Magliocco Anthony , Smalley Keiran , Forsyth Peter
Published: December 2020

BACKGROUND

Approx. 5% of melanoma pts develop LMD. There are essentially no models of LMD available for therapeutic development. A significant barrier to the development of effective therapies against LMD has been the inability to culture and expand LMD cells. Here we report our strategies to in vitro & in vivo culturing of CSF-CTCs. As a proof of concept, we assessed response to Ceritinib (Cer), a non-canonical IGF1R inhibitor) in combination with MEK inhibitor.

METHODS

We collected CSF from 11 patients (pts) from various sources (ie: LPs, Ommayas, autopsies). 3 pts CSF were collected at autopsies. PD-CSF-CTCs were expanded in vitro in conditioned media and in vivo using CDX model. scRNAseq analysis was performed to assess expression profiles of PD-CSF-CTCs.

RESULTS AND DISCUSSION

Of the total 61 PD-CSF-CTCs collected from 11 pts (avg: 4.07 CSF collections/patient), we successfully cultured PD-CSF-CTCs from 3 pts (20%) and were able to grow them in vivo from 2 pts (18%). scRNAseq analysis identified MLANA, IGF1R, SOX9 and ErbB3 were among genes highly expressed in our PD-CSF-CTCs. We evaluated the responses of the combination Cer with MEKi (Tra) in vitro and in vivo and found that these agents produced therapeutic effects to both established melanoma cell lines and our PD-CSF-CTCs. For example, in vivo testing showed a median survival (MS): 18, 35, and 27 days in WM164, WM164R and the PD-CSF-CTCs, respectively, in control groups. Whereas treatment with Cer + Tra produced significantly better MS in all three in vivo models and was not reached in WM164, WM164R (p< 0.001 & p< 0.047, respectively) and 38.5 days in PD-CSF-CTCs (p< 0.032).

CONCLUSIONS

Though the sample size is small, this is the first report of the successful in vitro & in vivo culture of CSF-CTCs from pts with LMD.
TMOD-03. PAN-CANCER ANALYSIS OF ORTHOTOPIC PATIENT DERIVED XENOGRAFTS FROM BRAIN METASTASES
Authors: Zeng Yu , Pelton Kristine , Yerrum Smitha , Kao Pei-Lun , Sinai Claire , Tran Tony , Sinha Rileen , Shetty Aniket , Tolstorukov Michael , Jaber Aliya , Freitas Dylan , Pisano William , Verselis Sigitas J , Herbert Zach T , Lin Nancy , Zhao Jean J , Weinstock David M , Chukwueke Ugonma , Aizer Ayal A , Chiocca E Antonio , Bi Wenya Linda , Wen Patrick , Lee Eudocia , Nayak Lakshmi , Meredith David , Santagata Sandro , Chow Kin Hoe , Ligon Keith
Published: December 2020

Brain metastases (BM) are a leading cause of cancer death and prognosis remains poor despite treatment advances at other sites. Models are central to therapeutic development, but few orthotopic patient-derived xenograft (PDX) models of BM exist. To represent diversity across BM types, we established a program to create orthotopic PDX at scale from all BM patients. To date BM were received from 100 patients and PDX attempted by direct brain injection (PDX, n=89) or injection of low passage patient-derived cell lines (PDCLX, n=11). We created 65 successful BM PDX from 13 cancers: 17 lung (55% take), 15 breast (68%), 6 melanoma (75%), 5 CNS lymphoma (83%), 3 gastrointestinal (75%), 2 esophageal (40%), 2 ovarian (67%), 1 sarcoma (100%), 1 laryngeal (100%), 1 prostate (100%), 1 pancreatic (100%), 1 uterine adenosarcoma (100%), and 1 yolk sac tumor (100%). Take rate was similar for models derived from patients with prior chemotherapy-only versus immune/targeted therapy-only (63 vs 58%). Fifteen patients had live tumor and matching PBMCs archived for modeling in vitro immunotherapy responses. Mean time to moribund among different cancer types ranged from 27 days (yolk sac tumor) to 177.5 days (ovarian). BM PDX had a favorable timeline for preclinical study (90% moribund at 180 days). All PDX retained high fidelity to the patient driver SNVs and copy aberrations, even at >P4. No significant differences noted by immunodeficient strain (SCID versus NSG) or injection site (orthotopic versus heterotopic). Explants from BM PDX were able to generate long-term cell lines (60%) or short-term cultures with qualitative concordance of model-to-patient responses to targeted therapy (Osimertinib, EGFRi) and immunotherapy (Pembrolizumab, PD1i). Genomic and clinical data were used to create the DFCI BM PDX cBioPortal for public release and models distribution will be available through the DFCI Center for Patient Derived Models.
TMOD-12. ESTABLISHING A CLINICALLY RELEVANT MODEL OF MESENCHYMAL GLIOBLASTOMA (GBM) TO STUDY RESPONSE TO STANDARD OF CARE TREATMENT AND IMMUNE CHECKPOINT INHIBITION (ICI).
Authors: Clerkin James , Connor Kate , Sweeney Kieron , White Kieron , Shiels Liam , Maher Stephen , Marignol Laure , Prehn Jochen H M , O’Brien David , Byrne Annette
Published: December 2020

GBM is a devastating disease with peak incidence in the seventh decade. Pre-clinical models are essential for studying resistance mechanisms and screening novel therapies. However, historically these models have failed to predict response in humans. Current models seldom incorporate surgical resection, and commonly use young animals whose immune contexture differs from older patients. Here, we have established an orthotopic model employing the syngeneic mesenchymal-NFpp10a-cell line which incorporates surgical resection in aged mice. We further characterise response to ICI and temozolomide monotherapy. NFpp10a and GL261-cell lines were exposed in vitro to irradiation (0Gy/2Gy/5Gy) and response assessed using colony formation assays. NFpp10a formed significantly more colonies at 5Gy compared to GL261 at both day 10 (NFpp10a 5.167 vs GL261 1.4; p=0.0017) and day 14 (NFpp10a 3.5 vs GL261 0; p< 0.0001). Hence, NFpp10a displays increased radioresistance. Next, NFpp10a-luciferin expressing cells were orthotopically implanted into young (6-8weeks;n=16) and aged (18months;n=16) C57BL/6-mice. Weekly bioluminescence imaging (BLI) was performed to monitor growth. Mice undergoing resection showed a median 18.47-fold drop in BLI signal. We demonstrated resection survival advantage in aged mice (Resection:33.5 days vs Non-Resection:18 days, p= 0.0166) and showed young age to be a positive prognostic factor (Young:62 days vs Aged:22 days, p=0.0002). Subsequently, we orthotopically implanted NFpp10a-Luc2 cells into C57BL/6 mice and treated with temozolomide (n=24) or PBS control (n=23), and anti-PD1 (n=24) or IgG (n=23). We observed that temozolomide and anti-PD1 monotherapy had no impact on NFpp10-Luc2 growth (temozolomide-overall:p=0.9001, anti-PD1-overall:p=0.7933) or survival (temozolomide-overall:p=0.3035, anti-PD1-overall:p=0.6328). Overall, we have established an NFpp10-Luc2 mesenchymal-GBM model in aged mice which incorporates surgical resection and accurately displays significant resistance to temozolomide and anti-PD1 monotherapy. We are currently employing this model to study the efficacy of neoadjuvant anti-PD1 therapy. Mechanistic analyses with multiplex-immunohistochemistry, scRNA and whole exome sequencing are planned to interrogate treatment effects on the tumor microenvironment.
TMOD-34. PATIENT-DERIVED XENOGRAFT AND CELL LINE MODELS FACILITATE NOVEL TREATMENT DISCOVERY IN CENTRAL NERVOUS SYSTEM LYMPHOMAS
Authors: Meredith David , Chow Kin Hoe , Kao Pei-Lun , Zeng Yu , Sinha Rileen , Shetty Aniket , Tolstorukov Michael , Tran Tony , Yerrum Smitha , Chukwueke Ugonma , Bi Wenya Linda , Arnaout Omar , Smith Timothy , Ligon Keith , Nayak Lakshmi
Published: December 2020

Lymphomas of the central nervous system (CNSL) are rare tumors with few treatment options; however, recent genomic studies have uncovered several druggable targets. To facilitate novel treatment discovery, we sought to establish patient-derived xenograft (PDX) models and cell lines from patients with newly diagnosed or recurrent CNSL. PDX models were attempted from fresh tumor samples from 18 consented patients for whom rich clinical annotation was available. Orthotopic intracranial injections were performed via stereotactic injection. Cell line generation was attempted from both primary tumors (PDCL) and PDX samples (PDXCL). Characterization included histopathology, targeted exome sequencing, low-pass whole-genome sequencing for copy number evaluation, and expression profiling of matched patient tumors and PDXs. Drug response testing was performed using CellTiterGlo. From 18 samples attempted, 8 PDX models were successfully generated: 2 primary CNS diffuse large B-cell lymphoma (DLBCL), 5 secondary CNS DLBCL, and 1 secondary cutaneous T-cell lymphoma (CTCL). Mean time to moribund was 66 days. All 8 PDXs grew successfully as orthotopic models, and 4 also grew in subrenal capsule. Additionally, 6 PDCL and 2 PDXCL were established (> P3). Models faithfully represented primaries, showing nearly identical histopathology, immunoprofiles, and genomic signatures. To support expanded and rapid preclinical use of these models in drug testing we documented that PDX could be explanted to create short-term cell preparations or long-term cell lines with a 100% and 40% success rate, respectively. Proof of principle testing showed that explanted DLBCL PDX cells and cell lines showed sensitivity to copanlisib and venetoclax, which were not efficacious in the CTCL PDX. These findings showcase a diverse collection of CNSL models demonstrating high fidelity to primary tumors at the genomic and phenotypic levels, emphasizing their utility for preclinical studies and as patient avatars to rapidly determine sensitivities to existing and novel regimens prior to initiation of therapy.
TMOD-35. DEVELOPING RECURRENT GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES
Authors: James Charles , Cheng Shi-Yuan , Horbinski Craig , Sarkaria Jann , Stupp Roger , Ahmed Atique
Published: December 2020

PURPOSE

Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients.

METHODS

Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants.

RESULTS

Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant.

CONCLUSIONS

The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.
ATRT-01. UPREGULATION OF PROTEIN SYNTHESIS AND PROTEASOME DEGRADATION CONFERS SENSITIVITY TO PROTEASOME INHIBITOR BORTEZOMIB IN MYC-ATYPICAL TERATOID/RHABDOID TUMORS
Authors: Tran Minh-Huy , Wu Kuo-Sheng , Chang Che-Chang , Wong Tai-Tong
Published: December 2020

BACKGROUND

Atypical teratoid rhabdoid tumors (ATRTs) are among the most malignant brain tumors in early childhood and remain incurable. Myc-ATRT is driven by the Myc oncogene, which directly controls the intracellular protein synthesis rate. Proteasome inhibitor bortezomib (BTZ) was approved by the Food and Drug Administration as a primary treatment for multiple myeloma. This study aimed to determine whether the upregulation of protein synthesis and proteasome degradation in Myc-ATRTs increases tumor cell sensitivity to BTZ.

METHODS

We performed differential gene expression and gene set enrichment analysis on matched primary and recurrent patient-derived xenograft (PDX) samples from an infant with ATRT. The expressions of proteasome-encoding genes were compared among this paired model as well as between the 24 human ATRT samples and normal brain tissues. The antitumor effect of BTZ was evaluated in three human Myc-ATRT cell lines (PDX-derived tumor cell line Re1-P6, BT-12, and CHLA-266) and in the orthotopic xenograft models of Re1-P6 cell.

RESULTS

Concomitant upregulation of the Myc pathway, protein synthesis, and proteasome degradation were identified in recurrent ATRTs. In ATRTs, the proteasome-encoding genes were highly expressed compared with in normal brain tissues, correlated with the malignancy of tumor cells, and were essential for tumor cell survival. BTZ inhibited proliferation and induced apoptosis through the accumulation of p53 in in vitro drug tests. Furthermore, BTZ inhibited tumor growth and prolonged survival in Myc-ATRT orthotopic xenograft mice.

CONCLUSIONS

Our findings suggest that BTZ may be a promising targeted therapy for Myc-ATRTs.