bluuebunny/crossref_metadata_2025 huggingface dataset:
SCIDOT-39. K16ApoE ENHANCES Aβ-ASSOCIATED 11C-PiB DEPOSITION AND PET SIGNAL IN APP/PS1 TRANSGENIC MICE
Authors: Brown Desmond , Sarkar Gobinda , Decklever Teresa , Curran Geoffry , Sarkar Ameet , Schmeichel Ann , Swaminathan Suresh , Kandimala Karunya , Jenkins Robert , Burns Terry , Lowe Val
Published: November 2019

OBJECTIVE

The K16ApoE peptide enhances delivery of multiple agents across the blood-brain barrier (BBB). Transgenic mouse models are central to elucidating the underlying pathophysiology of Alzheimer’s Disease (AD) and provide a system for evaluating novel therapeutic strategies. PET imaging plays a central clinical role in diagnosing human cases of AD but has had variable performance in mouse models. We investigated the role of K16ApoE to enhance delivery of a radiolabeled PET imaging tracer, 11C-PiB and assess whether this corresponds to improved PET sensitivity in APP/PS1 transgenic mice.

METHODS

Brain-delivery of 11C-PiB was accomplished by successive injections of K16ApoE and 11C-PiB. Distribution of 11C-PiB to the brain and heart was quantified via dynamic PET/CT imaging and digital autoradiography.

RESULTS

K16ApoE increased the brain uptake of 11C-PiB in both wild-type (WT) and APP/PS1 mice. Administration of K16ApoE increased the PET standard uptake value of 11C-PiB at 5 minutes in WT mice from 1.132 to 2.963 (p=0.006) and in APP/PS1 mice from 0.842 to 3.268 (p=0.016). Enhancement peaked at 5 minutes. Binding was reversible with similarly increased kinetics in both WT and APP/PS1 mice. The absolute values were higher in APP/PS1 mice suggesting increased retention. The increased retention in APP/PS1 mice was consistent with specific binding to A-beta plaques as unlabeled PiB reduced 11C-PiB signal retention.

CONCLUSION

K16ApoE mediates enhancement of 11C-PiB signal in APP/PS1 mice brains with increased PET sensitivity. There is increased uptake kinetics in both WT and APP/PS1 mice with specific A-beta plaque binding in the latter. This enhanced delivery of the PET tracer has implications for development and testing of new hypotheses and the efficacy of novel therapeutic paradigms. K16ApoE has potential for improving delivery of several agents across the BBB. This has implication for delivery of diagnostic and therapeutic agents for neurodegenerative and neuro-oncologic diseases.
CSIG-13. PROTEIN KINASE CI DRIVES BOTH TUMOR INVASION AND PROLIFERATION AND IS A COMPELLING TARGET FOR THE TREATMENT OF GLIOBLASTOMA
Authors: Kenchappa Rajappa , Luu Amanda , West Rita , Mladek Ann , Sarkaria Jann , Liu Yi , Murray Nicole , Fields Alan , Rosenfeld Steven
Published: November 2019

In glioblastoma (GBM), invasion and proliferation are reciprocally related—e.g., targeting one stimulates the other. Thus, effective therapy for GBM requires finding targets that drive both invasion and proliferation and which are druggable, ideally with FDA approved therapeutics. We find that the atypical protein kinase C isoform, PKCi meets these criteria. Using a human GBM tissue microarray, we find that PKCi protein expression fits a bimodal distribution, with >35% of GBMs showing high levels of PKCi expression. Genetic deletion of Prkci, the gene encoding mouse PKCi, impairs tumor invasion and proliferation both in vitro and in vivo, and significantly prolongs survival in an immunocompetent PDGF-driven orthotopic mouse model of GBM. PKCi can be selectively targeted with Auranofin, a small molecule PKCi inhibitor that has been FDA approved for treatment of inflammatory diseases. Like genetic deletion of Prkci Auranofin treatment significantly prolongs survival in our murine GBM model, and does so to a similar degree. Interestingly, analysis of a panel of human and murine GBM cells reveals that sensitivity to Auranofin inhibition of transformed growth directly correlates with expression of PKCi, indicating that PKCi expression profiling could serve as a predictive biomarker for responsiveness to Auranofin. However, genetically deleting or pharmacologically inactivating PKCi does not permanently arrest tumor growth, implying that GBM cells acquire mechanisms that allow them to compensate for the loss of this important signaling molecule. We are currently examining potential “escape” mechanisms through ‘omics-based approaches, which may enable us to enhance the therapeutic potential of Auranofin. Taken together, our results demonstrate that PKCi is a compelling therapeutic target for treatment of GBM that deserves further investigation.
RARE-51. RITUXIMAB, METHOTREXATE, BCNU, ETOPOSIDE, AND PREDNISONE (RMBVP) FOR THE TREATMENT OF RELAPSED/RECURRENT PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA: A RETROSPECTIVE SINGLE CENTER STUDY
Authors: Reiss Samantha , Yerram Prakirthi , Modelevsky Lisa , Grommes Christian
Published: November 2019

BACKGROUND

Primary Central Nervous System Lymphoma (PCNSL) is an aggressive rare non-Hodgkin lymphoma. Prognosis is particularly poor for relapsed/recurrent (R/R) patients with no established treatment modality. Methotrexate/BCNU/Teniposide/Prednisone with or without rituximab has efficacy in newly diagnosed PCNSL. Here, we report efficacy and toxicity of RMBVP for the treatment of R/R PCNSL.

METHODS

This retrospective study at MSKCC included PCNSLs treated with MBVP for R/R disease between 5/2009 and 5/2019. Methotrexate (3.5 g/m2; day 1 and 15), etoposide (100 mg/m2; day 2), BCNU (100 mg/m2; day 3), prednisone (60 mg/m2/day; day 1–5) and rituximab (500 mg/m2; day 0 and 14) were given in 28-day cycles. Granulocyte colony-stimulating factor support was given for 5 days in between doses of chemotherapy.

RESULTS

Thirty patients received MBVP; 27 (90%) received RMBVP with a median of 2 cycles given (0.5–5). Median age was 66 years (23–81); median KPS was 70 (30–90); 14 (46.7%) were women. Median prior lines of therapy were 2 (1–5); all received prior methotrexate. Median time from last treatment was 1 month (0–88.3; 21 (70%) < 12 months). Of twenty-nine evaluable patients for response, 23 (76.7%) had a response (9 complete response (CR) (30%), 5 CR-unconfirmed (16.7%), 8 partial response (26.7%), 1 stable disease (3%)). At a median follow up of 14.2 months, median progression free survival (PFS) was 15.6 months and median overall survival was not reached. Median PFS was poorer for patients with < 12 months since last chemotherapy, 10.1 vs 60.6 months. Rates of serious (grade 3 or 4) neutropenia, anemia, thrombocytopenia and transaminitis were 20%, 20%, 16.7%, and 16.7%, respectively. One patient experienced grade 3 nephrotoxicity. No treatment related mortalities occurred.

CONCLUSIONS

RMBVP is a tolerable treatment option for R/R PCNSL, particularly in those with recurrent disease.
CBMT-13. 3DEP SYSTEM TO TEST THE ELECTRICAL PROPERTIES OF DIFFERENT CELL LINES AS PREDICTIVE MARKERS OF OPTIMAL TUMOR TREATING FIELDS (TTFIELDS) FREQUENCY AND SENSITIVITY
Authors: Giladi Moshe , Zeevi Einav , Gotlib Karnit , Wenger Cornelia , Naveh Ariel , Bomzon Ze’ev , Kirson Eilon , Weinberg Uri , Kinzel Adrian , Palti Yoram
Published: November 2019

BACKGROUND

Tumor Treating Fields (TTFields; approved anti-neoplastic treatment modality) are delivered via application of low intensity, intermediate frequency, alternating electrical fields. The electrical properties of cells (eg, permittivity and conductivity) determine the optimal TTFields frequency that would elicit the greatest cell count reduction. Currently, no predictive markers exist to determine TTFields response and optimal frequency for individual patient application. The study goal was to evaluate the correlation between electrical properties of cells and TTFields’s optimal frequency and sensitivity. The 3DEPTM reader (LabTech) determines cellular electrical properties, including permittivity and conductivity, by using dielectrophoresis (DEP) force. DEP is a physical effect that generates a force on polarizable particles, such as cells, subjected to non-uniform electric fields.

METHODS

Utilizing the 3DEP reader, baseline electrical properties (permittivity and conductivity) of 17 cell lines from different tumor-types were determined. Curves were analyzed using 2-way ANOVA. The optimal TTFields frequency of each cell line was determined by evaluating TTFields cytotoxicity at various frequencies using the inovitroTM system. Electrical properties of each cell line were compared with the optimal TTFields frequency and sensitivity.

RESULTS

Significant differences (P< 0.001) were demonstrated between the lower frequency range of the 3DEP curves that correspond to cellular membrane capacitance at TTFields optimal frequencies of 150 kHz (9 cell lines) and 200 kHz (8 cell lines). Also, membrane capacitance was a good predictor of TTFields sensitivity based on curve differences within the low-frequency range.

CONCLUSIONS

These results demonstrate that cell membrane capacitance correlates with TTFields optimal frequency and sensitivity. Based on these data, there is a strong rational to further explore the potential of measuring the electrical properties of cells as predictive markers to help determine the optimal TTFields frequency for individual patient application and to identify ideal treatment-responders to TTFields.
CBMT-16. EGFRvIII EXPRESSION CONFERS CHEMOSENSITIVITY BY INCREASING DNA MISMATCH REPAIR PROTEIN EXPRESSION AND REPLICATION STRESS
Authors: Struve Nina , Binder Zev A , Stead Lucy F , Brend Tim , Bagley Stephen J , Faulkner Claire , Ott Leonie , Müller-Goebel Justus , Weik Anna-Sophie , Hoffer Konstantin , Rieckmann Thorsten , Christin Parplys Ann , Burmester Jasmin , Henze Marvin , Morrissette Jennifer J D , Schüller Ulrich , Petersen Cordula , Rothkamm Kai , Kurian Kathreena M , Short Susan C , Kriegs Malte
Published: November 2019

MGMT promoter methylation is the only accepted biomarker with prognostic role in GBM but its routine implementation is limited partly response to TMZ is heterogeneous, but also due to lack of effective alternative treatment options. Therefore, additional biomarkers are needed to enable better prediction of survival and to improve individualized treatment of GBM patients. A potential new biomarker is the epidermal growth factor receptor variant III (EGFRvIII). This constitutively activated deletion variant is present in approximately one third of all IDH wildtype GBM, but its relevance to treatment response is poorly understood. The aim of the present study was to analyze the impact of endogenous EGFRvIII expression on chemosensitivity and the mechanisms underlying any differential treatment response. EGFRvIII expression was associated with prolonged median overall survival but only for GBM patients with MGMT promoter methylated tumors. In line with this, we observed increased TMZ sensitivity of EGFRvIII+ and MGMT promoter methylated cells, which translated into improved survival in xenograft experiments. The increased TMZ sensitivity was associated with an elevated DNA damage induction accompanied by an increased expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cell lines and EGFRvIII+ GBM patient samples. Subsequently, only a moderate reduction in MMR protein expression resulted in a dramatic TMZ resistance, suggesting that EGFRvIII expression specifically sensitized MGMT deficient cells to TMZ treatment by upregulating MMR. Furthermore, EGFRvIII expression in GBM cell lines was accompanied by increased DNA damage, replication fork slowing, stalling and enhanced origin firing, implying replication stress. Targeting of EGFRvIII-dependent replication stress by irinotecan led to hypersensitivity of EGFRvIII+ cells. Taken together this study illustrates that EGFRvIII-induced upregulation of MMR and replication stress increases chemosensitivity thereby highlighting the vulnerability of EGFRvIII+ GBM to available treatments. These important data may also guide the development of new and more effective personalized strategies.
CBMT-22. REVERSING THE WARBURG EFFECT BY TARGETING NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT), THE RATE-LIMITING ENZYME OF NAD+ SALVAGE PATHWAY INCREASES, CHEMOSENSITIVITY TO TEMOZOLOMIDE IN GLIOMA CELLS
Authors: Sharma Pratibha , Senapedis William , Sampath Deepa , Puduvalli Vinay
Published: November 2019

BACKGROUND

Glioma cells metabolize glucose primarily through incomplete, non-oxidative glycolysis even in the presence of oxygen (the Warburg effect) which provides a constant supply of substrate for proliferation and generates an acidic environment that favors invasion and immunosuppression. NAMPT, the rate-limiting enzyme of the NAD+ salvage pathway, regulates key metabolic processes preferentially used in glioma energy metabolism. Inhibition of NAMPT could have global impact on glioma cell metabolism and could be a promising strategy for glioma therapy. This study examined the effect of NAMPT inhibition using KPT-9274 and FK866 (NAMPTi) on glycolysis and glioma cell metabolism.

METHODS

Metabolic changes related to NAMPT inhibition were examined in glioma cell lines and patient derived glioma stem-like cells (GSC). Mass spectrometry was used for metabolic profiles and the generated data was annotated by comparison against an extensive metabolite library to identify relevant metabolites. The Agilent Seahorse assay was utilized to determine treatment-related changes in glycolysis.

RESULTS

NAMPT inhibition caused NAD, NADH, NADP, NADPH, and ATP depletion in glioma cells. Global metabolomics results showed an accumulation of GMP, IMP, AMP and myo-inositol indicating reprogramming of glioma cell metabolism for de novo purine biosynthesis. Additionally, an accumulation of glucose- and fructose-1,6 bisphosphate in NAMPTi treated cells indicated dysfunctional glycolysis. This was confirmed by the Agilent glycolysis stress test by which NAMPTi-treated glioma cells showed a reduction in glycolysis, glycolytic capacity and reserves. NAMPTi treated cells showed downregulation of LDHA due to NAD depletion. Further, downregulation of LDHA enhanced the chemosensitivity of glioma cells to temozolomide.

CONCLUSIONS

NAMPTi cause profound disruption of glycolysis, reduced glycolytic capacity and glycolytic reserve in gliomas and GSCs irrespective of O[6]-methylguanine-DNA methyltransferase promoter methylation or IDH1 status. Targeting NAMPT to disrupt the Warburg effect is a novel strategy for developing effective therapies against glioma to overcome heterogeneity.
CBMT-23. NON-CANONICAL FUNCTIONS OF TERT IN GLIOBLASTOMA
Authors: Ferdosi Shayesteh , Taylor Brett , Bollam Saumya , Gokhale Vijay , Hurley Laurence , Dhruv Harshil , Berens Michael
Published: November 2019

Telomerase is an enzyme with a catalytic subunit, telomerase reverse transcriptase (TERT), that is in charge of telomere elongation in the nucleus. Promotor region of TERT is commonly mutated across cancers, especially in glioblastoma (GBM) with over 80% frequency. In the absence of any effective molecular targeting therapy for GBM, elucidating oncogenic signaling of TERT could open new avenues in GBM treatment. Canonically, mutations of TERT, which result in TERT upregulation, maintain telomere length in the nucleus and promote indefinite proliferation of cancer cells. However, a non-canonical function of TERT in the mitochondria has recently been suggested. We screened GBM cell models against a novel small molecule inhibitor (RG1534, Reglagene Inc.) that interferes with the functionality of a mutated hTERT promoter. RG1534 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. More interestingly, RG1534 treatment leads to rapid apoptosis induction in glioma cell lines that does not correlate with the time course of the telomere shortening effect. We further validated this rapid apoptosis behavior in glioma cell lines using siRNA and CRISPR/Cas-9 mediated hTERT knockdown. We also measured the protein expression of TERT in subcellular fractions of glioma cell lines and demonstrated the presence of higher TERT expression in mitochondrial extract compared to the nucleus. Finally, using MitoSOX dye we assessed ROS generation in glioma cells in response to an oxidant with or without TERT expresssion. In summary, our results demonstrate that non-canonical functions of TERT may play critical role in glioma pathobiology and require more detail investigation.
CBMT-30. TARGETING NICOTINAMIDE ADENINE DINUCLEOTIDE BIOSYNTHESIS IN CNS TUMORS
Authors: Lindsay Ken , Brenner Charles , Hockenbery David , Paddison Patrick
Published: November 2019

Nicotinamide adenine dinucleotide (NAD) metabolic pathways have been shown to be critical targets in many cancers, including brain tumors. However, inhibiting nicotinamide metabolism has had limited efficacy in the clinic to date due to on-target side effects. To identify new metabolic vulnerabilities in human Glioblastoma, we performed a chemical library screen using 250 inhibitors of metabolic enzymes in Glioblastoma stem-like cells (GSCs) (0131 and 0827) and control hNPC-CB660 cells. Among the screen hits, we identified FK866, an inhibitor of the NAD salvage enzyme NAMPT, as having GSC-selective cytotoxicity. Taking an advantage of an alternative NAD precursor salvage pathway, we show that cytotoxicity with inhibitors of the NAMPT salvage enzyme for neural stem cells was abrogated by media addition of nicotinamide riboside (NR), while certain glioblastoma stem-like cells (GSCs) remained exquisitely sensitive. Examining our GSC isolates, we observed that hypersensitivity correlated with low/absent expression of NMRK1, a kinase required for utilization of NR. We find that 5–20% of most tumor types (CNS, non-CNS) exhibit comparably low NMRK1 mRNA expression. In looking for associations with oncogenic drivers, we noted significant negative correlation with MYCN and MYC expression for multiple tumors. We have additional data suggesting that MYC/MYCN induces the NMRK1low state via transcriptional repression. These results suggest that NAMPT inhibitors, already in clinical trials, can be administered with NR to widen the therapeutic index for NMRK1low tumors. This combined theranostic approach could protect NMRK1-proficient normal cells without compromising treatment efficacy in NMRK1low tumor cells.
CBMT-33. VISUALIZING THE METABOLISM OF GLIOBLASTOMA PATIENT-DERIVED ORTHOTOPIC XENOGRAFTS BY MASS SPECTROMETRY IMAGING
Authors: Upendra Rao Jyotsna , Gibson Katherine , Hamm Gregory , Wright Alan , Fala Maria , Mair Richard , Tsyben Anastasia , Goodwin Richard , Brindle Kevin
Published: November 2019

INTRODUCTION. Glioblastoma multiforme (GBM) has a dismal prognosis, which has been attributed to extensive inter- and intra-tumoural heterogeneity. To investigate the metabolic heterogeneity of GBM, we employed desorption electrospray ionization (DESI) imaging on snap frozen samples of GBM patient-derived orthotopic xenografts (PDOX) in rat brains, following infusion of [U-13C]glucose. METHODS. Athymic nude rats (n=3) were infused with [U-13C]glucose (0.4 mg/g body weight bolus, 0.012 mg/g/min at 300 µL/h infusion [1]) for 40, 80 and 120 min, followed by cardiac puncture to measure blood [U-13C]glucose levels using 13C NMR spectroscopy. A second set of rats (n=2) were implanted intracranially with patient-derived GBM cells. The presence of tumour was confirmed by T2-weighted MRI. One animal was infused with [U-13C]glucose for 2 h, while another with [12C]glucose. The brains were snap frozen in liquid nitrogen followed by analysis with DESI imaging in negative ion mode (35 µm resolution). RESULTS. After a 2 h infusion, 43% of the circulating glucose was 13C labelled. Unsupervised spatial clustering of the MSI data resulted in segmentation of normal brain from tumour. Levels of [12C and 13C]glucose were high in the peri-tumoural region and low within the tumour. [12C and 13C]lactate showed the opposite distribution, indicating that the rate of glucose utilization within the tumour exceeded the rate of delivery. The peri-necrotic and peri-tumoural areas had higher levels of glutamate compared to the rest of the tumour. CONCLUSIONS. The results demonstrate that the PDOX is metabolically heterogeneous and differs from surrounding brain. While the tumour was predominately glycolytic, the presence of glutamate in peri-necrotic and peri-tumoral regions indicates the presence of increased TCA cycle activity. Comparison of MSI data with histological staining of brain sections should yield additional information about the relationship between the tumour and its microenvironment.
CBMT-34. MODULATING DIETARY TRYPTOPHAN OR GUT MICROBIOTA LEVELS DOES NOT IMPROVE THE EFFICACY OF COMBINED TREATMENT WITH RADIATION, ANTI-PD-1 mAb, AND AN IDO1 ENZYME INHIBITOR IN A MODEL OF GLIOBLASTOMA
Authors: Ladomersky Erik , Zhai Lijie , Lauing Kristen , Qian Jun , Bell April , Otto-Meyer Sebastian , Savoor Rohan , Wainwright Derek
Published: November 2019

INTRODUCTION

Immunotherapy has failed to improve glioblastoma (GBM) patient survival in all phase III clinical trials to-date. We recently discovered that a combination immunotherapeutic approach that simultaneously administers radiation (RT), anti-PD-1 mAb, and an IDO1 enzyme inhibitor resulted in long-term survival of ~40% of mice intracranially-engrafted (i.c.) with syngeneic GBM (Ladomersky et al., 2018; CCR). In the same study we discovered that the survival benefit depended on the inhibitor of IDO1 in non-GBM cells. Our current study further interrogates the role of non-GBM cell IDO1 and its role in the brain tumor microenvironment.

METHODS

Mice were engrafted with 2×105 GL261 cells and treated at 14 days post-intracranial injection (i.c.) with RT and/or anti-PD-1 mAb, followed by sampling tissue at 1- and 7-days post treatment. Questioning whether dietary supplementation of Trp would overcome the immunosuppressive activity of the Trp metabolic enzyme, IDO1, mice were placed on ad libitum dietary formulations containing 0–500% normal daily intake. Since Trp is only taken in through the diet, and since bacteria also metabolize Trp, we questioned the role of the gut microbiome on immunotherapy of GBM by injecting mice with an antibiotic cocktail beginning 4 weeks prior to i.c.-injection.

RESULTS

Both RT and anti-PD-1 treatment increase IDO1 expression in the GBM-draining cervical lymph nodes at day 1 post-ic. (P< 0.001). Mice receiving normal (100%) dietary Trp, but no other supplementation levels, achieve a long-term survival benefit (P< 0.001). Unexpectedly, the depletion of gut microbiota has no effect on therapeutic efficacy (P=0.4808).

CONCLUSION

These data suggest that although both systemic and tissue-selective alterations in Trp metabolism occur after RT and anti-PD-1 therapy, modulating dietary Trp levels or gut microbiota composition does not alter treatment efficacy against GBM.
CBMT-35. METABOLIC REWIRING BY ONC201/TIC10 AND 2-DEOXYGLUCOSE HAS SYNERGISTIC ANTI-GLIOBLASTOMA ACTIVITY
Authors: Pruss Maximilian , Dwucet Annika , Hlavac Michal , Kast Richard , Westhoff Mike-Andrew , Wirtz Rainer , Halatsch Marc-Eric , Siegelin Markus , Karpel-Massler Georg
Published: November 2019

BACKGROUND

Metabolic dysregulation is a common feature of cancers such as primary brain malignancies. In this work we examined whether a rewiring of the metabolome by a multi-targeting approach would induce enhanced anti-neoplastic activity in glioblastoma.

METHODS

Preclinical testing of a combined treatment with ONC201/TIC10 and 2-Deoxyglucose was performed in established and primary cultured glioblastoma cells. Extracellular flux analysis was used to determine real-time effects on OXPHOS (OCR) and glycolysis (ECAR). Expression of respiratory chain complexes was analysed by Western blotting. Biological effects on tumor formation were tested in patient-derived model systems on the chorion allantoic membrane (CAM). Protein array analyses were performed to determine effects on phospho protein kinase expression.

RESULTS

Treatment with ONC201/TIC10 leads to impaired mitochondrial respiration and a dose-dependent increase of glycolysis. ONC201/TIC10 combined with 2-Deoxyglucose, induces a state of energy deprivation characterized by a significant decrease in ATP levels. On the molecular level, pAMPK α1 was significantly up-regulated while a hypo-phosphorylation signature was noted including mTOR signaling, src family kinases and receptor tyrosine kinases such as EGFR and PDGFR-β. As a result, synergistic anti-proliferative and anti-migratory effects were observed among established and primary cultured glioblastoma cells. In addition, tumor formation on the CAM was significantly impaired following the combination treatment.

CONCLUSIONS

Simultaneous treatment with ONC201/TIC10 and 2-Deoxyglucose causes a reprogramming of the metabolic circuitry and results in a synergistic anti-glioblastoma activity. Since both agents were tested in clinical trials with good tolerability, and they both penetrate the blood-brain barrier, further clinical evaluation of this therapeutic strategy seems promising.
CBMT-42. A GENOME-WIDE CRISPR-Cas9 SCREEN FOR GENES REGULATING QUIESCENT-LIKE STATES IN GLIOBLASTOMA
Authors: Feldman Heather , Mihalas Anca , Plaisier Christopher , Patel Anoop , Paddison Patrick
Published: November 2019

Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a p27-mVenus reporter construct, which is stabilized when cells enter a G0-like state. By assaying p27 reporteractivity, we were able to identify sgRNAs enriched in p27hipopulations and, which upon retest, trigger a G0-like arrest in GSCs. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that downregulation of KAT5 in vitro and in vivo dramatically increases the pool of cells in G0-like states in GSC cultures and GSC-induced tumors. Using single cell RNA-sequencing, we show that this cell state is characterized by gene expression signatures similar to those found in non-dividing subpopulations of GBM tumors and quiescent neural stem cells. In addition, we perform in-depth molecular and phenotypic characterization of these induced G0-like states, including epigenetic and metabolic profiles. These suggest a key role for KAT5 in regulating genes related to protein synthesis. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors and may provide novel therapeutic opportunities.
CBMT-43. INTEGRATED LIPIDOMIC AND MOLECULAR ANALYSIS REVEALS A SUBSET OF GLIOBLASTOMA VULNERABLE TO FERROPTOSIS
Authors: Morrow Danielle , Bayley Nicholas , Williams Kevin , Muranaka Hayato , Prins Robert , Liau Linda , Cloughesy Timothy , Bensinger Steven , Nathanson David
Published: November 2019

Cancers, including the universally lethal glioblastoma (GBM), have reprogrammed lipid metabolism to fuel tumor growth. However, the molecular alterations responsible for aberrant lipid metabolism, and the potential for identifying new therapeutic opportunities are not fully understood. To systematically investigate the GBM lipidome, we performed integrated transcriptomic, genomic and shotgun lipidomic analysis of a library of molecularly diverse patient-derived GBM cells (n=30). Using this comprehensive approach, we discovered two GBM sub-groups defined by their combined molecular and lipidomic profile. Polyunsaturated fatty acids (PUFAs) were among the most significant lipids that distinguished these two groups of GBM tumors. Intriguingly, this lipid metabolic phenotype was associated with heightened sensitivity to ferroptosis – a newly discovered form of regulated cell death. As PUFA oxidation is a critical feature of ferroptosis, our findings suggest a novel association between specific molecular signatures of GBM, lipid metabolism and ferroptosis. This relationship may present a new therapeutic opportunity to target ferroptosis in a molecularly-defined subset of GBMs.
CBMT-44. COMPREHENSIVE CHARACTERIZATION OF THE INTRINSIC APOPTOTIC MACHINERY REVEALS THE MOLECULAR BLOCKS RESPONSIBLE FOR RESISTANCE TO CELL DEATH IN GLIOBLASTOMA
Authors: Fernandez Elizabeth , Mai Wilson , Bayley Nicholas , Liau Linda , Cloughesy Timothy , Letai Anthony , Nathanson David
Published: November 2019

Conventional therapies (e.g., temozolomide (TMZ), Irradiation (IR)) transiently halt tumor growth of glioblastoma (GBM) but fail to induce cell death through apoptosis. Consequently, the inability to kill GBM tumor cells ultimately leads to disease progression and a poor patient survival. The precise molecular mechanisms by which GBM are refractory to apoptosis remain enigmatic. We preformed BH3 profiling to functionally characterize the intrinsic apoptotic machinery and define the molecular ‘blocks’ that obstruct GBM apoptosis under both basal and treatment states. Using a molecularly diverse panel of freshly purified patient tumors, patient-derived neurospheres and patient-derived orthotopic xenografts, we identified that nearly all GBMs have two anti-apoptotic blocks, BCL-xL and MCL-1, which are essential for GBM survival in an untreated state. TMZ or IR (TMZ/IR) disabled the MCL-1 block in a subset of GBMs, leaving tumors exclusively dependent on BCL-xL for survival. Mechanistic studies revealed that TMZ/IR treatment induced p53-dependent expression of the pro-apoptotic protein, PUMA, which subsequently bound to and neutralized MCL-1. Consequently, pharmacological inhibition of BCL-xL in combination with TMZ/IR initiated intrinsic apoptosis and was synergistically lethal in p53 wild-type GBM. These studies identify the existence of two anti-apoptotic proteins that are critical for GBM survival, which can be therapeutically exploited in a molecularly defined subset of GBMs for tumor eradication.
CBMT-49. OXALOACETATE ALTERS GLUCOSE METABOLISM IN GLIOBLASTOMA: 13C ISOTOPOMER STUDY
Authors: Ijare Omkar , Conway David , Cash Alan , Baskin David , Pichumani Kumar
Published: November 2019

Anhydrous enol-oxaloacetate (AEO) has demonstrated the ability to enhance neuronal cell bioenergetics and activate brain mitochondrial biogenesis. Since oxaloacetate has demonstrated positive effects on brain bioenergetics in neurodegenerative diseases we have begun to investigate whether AEO may also have a positive effect on the altered cellular metabolism found in cancer cells, particularly Glioblastoma multiforme. The “Warburg effect” describes an abnormal metabolic state in cancer, distinct from normal tissue, in which energy is generated through enhanced conversion of pyruvate to lactate even in the presence of oxygen during glycolysis. Oxaloacetate (OAA) is a key anaplerotic substrate that is required to maintain TCA cycle flux. The role of oxaloacetate supplementation on the energy metabolism is not known in cancer cells. Goal of this study is to investigate the changes in metabolic fluxes in glucose metabolism with and without the presence of OAA in patient-derived GBM cells. We use GC-MS based 13C isotopomer analysis for this study. GBM cells are grown in 15mM glucose containing DMEM medium supplemented with 2mM oxaloacetate for 10 days. 6 hours prior to harvesting, [U-13C]glucose is introduced to the medium. 13C isotopomer analysis of GC-MS data showed that OAA supplementation for 10 days drastically decreased Warburg glycolysis by reducing 13C labeling (M+3) by 19.7% and 48.8% in pyruvate and lactate pools respectively in comparison with cells not treated with OAA. M+3 13C labeled pyruvate entered TCA cycle via acetyl-CoA, where we also observed reduced levels of M+2 13C labeled citrate (20.5%) and glutamate (23.9%) isotopomers. Pyruvate can also enter TCA cycle via pyruvate carboxylation pathway and this activity was also found to be slightly decreased in the OAA treated cells. All the differences were statistically significant. These results indicate that OAA can be used to alter bioenergetics of GBM cells, specifically glucose oxidation.
CSIG-01. NA-K-CL COTRANSPORTER PROTEIN IN THE PATHOGENESIS OF LOW-GRADE GLIOMAS
Authors: Luo Lanxin , Guan Xiudong , Begum Gulnaz , Ding Dawei , Kohanbash Gary , Sun Baoshan , Sun Dandan
Published: November 2019

Low-grade gliomas (LGGs) present a high incidence of epilepsy, infiltrative growth, resistance to therapy, and high risk of transforming into high-grade gliomas (HGGs). Temozolomide (TMZ) is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk LGGs. Na+-K+-2Cl- co-transporter 1 (NKCC1) regulates cell volume and intracellular Cl- concentration which promotes glioma cell migration, resistance to TMZ, and tumor-associated epilepsy. Our new bioinformatic analysis of TCGA and CGGA datasets shows that LGGs expressed higher SLC12A2 gene mRNA (encoding NKCC1 protein) than HGGs, which was confirmed at NKCC1 protein levels. In syngeneic mouse glioma models with intracranial transplantation of two different mouse glioma cell lines (GL26 and SB28), we detected upregulated NKCC1 protein expression in glioma tumor cells as well as in peri-tumor reactive astrocytes in response to TMZ monotherapy. This provided a rationale for us to test the efficacy of combining NKCC1 pharmacological blockade with TMZ on improving therapeutic outcomes. 5-day combination therapy of TMZ with potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated TMZ-mediated apoptosis, and decreased peri-tumor reactive astrocyte formation, which collectively led to suppressed tumor growth and prolonged survival of tumor-bearing mice. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in regulating the pathogenesis of LGGs and its blockade presents therapeutic potentials for reducing TMZ-mediated resistance.
CSIG-02. TARGETING CONSTITUTIVE ACTIVATION OF THE EGFR-STAT1 AXIS DECREASES PROLIFERATION OF MENINGIOMA TUMOUR CELLS
Authors: Ferluga Sara , Baiz Daniele , Ercolano Emanuela , Sharma Vikram , Dunn Jemma , Kurian Kathreena M , Hilton David , Oliver Hanemann Clemens
Published: November 2019

Meningiomas are the most frequent primary brain tumours of the central nervous system. The standard of treatment is surgery and radiotherapy, but the lack of knowledge of their tumorigenesis contributes to delay the development of additional therapeutic options. We found STAT1 widely overexpressed in meningioma tumours and in patient-derived meningioma cells but not in the corresponding healthy controls. The protein showed a constitutive phosphorylation on both phosphosites (Y701 and S727), which was not dependent on the JAK/STAT pathway. STAT1 knocked down resulted in a significant reduction of cellular proliferation, showed as a decrease in Ki67-positive cells and Cyclin D1, and deactivation of AKT and ERK 1/2. By studying STAT1 binding partners we isolated a complex composed by STAT1, STAT2, PRMT5 and MEP50. As PRMT5 is known to interact with EGFR, we tested the tyrosine kinase and found that EGFR was constitutively active in meningioma and was responsible for the aberrant phosphorylation of STAT1 on both phosphosites. We tested different drugs inhibiting of EGFR phosphorylation, Canertinib was most effective. It caused a significant reduction in meningioma cells proliferation and a reduction of overall levels of Cyclin D1, phospho- AKT and phospho-ERK 1/2. Hence, STAT1 constitutive phosphorylation, initiated by EGFR activation, is responsible for inducing a positive feedback loop causing its own overexpression and consequently an increased proliferation of the tumour cells. These findings underline a pivotal role of the EGFR and STAT1 axis in meningioma and provides the rationale for further studies aiming to identify novel and effective therapeutic options.
CSIG-06. DEPATUXIZUMAB MAFODOTIN (ABT-414)-INDUCED GLIOBLASTOMA CELL DEATH REQUIRES EGFR OVEREXPRESSION, BUT NOT PHOSPHORYLATION
Authors: von Achenbach Caroline , Blot Vincent , Weiss William , Weller Michael
Published: November 2019

Glioblastomas commonly (40%) exhibit epidermal growth factor receptor (EGFR) amplification, half of these tumors carry the EGFRvIII deletion variant characterized by an in-frame deletion of exons 2–7, resulting in constitutive EGFR activation. Although EGFR tyrosine kinase inhibitors had only modest effects in glioblastoma, novel therapeutic agents targeting amplified EGFR or EGFRvIII continue to be developed. Depatuxizumab mafodotin (ABT-414) is an antibody drug conjugate consisting of the monoclonal antibody 806 and, as its toxic payload, monomethyl auristatin F, designed to target EGFR-overexpressing tumor cells. Since long-term glioma cell lines and patient-derived glioma-initiating cell lines appeared to express too little EGFR in vitro to be sensitive to ABT-414, we generated glioma sublines overexpressing EGFR or EGFRvIII to explore the determinants of ABT-414-induced glioma cell death. Overexpression of EGFRvIII induces sensitization to ABT-414 more readily than overexpression of EGFR. There is no bystander killing of cells devoid of EGFR or EGFRvIII expression. Surprisingly, either exposure to EGF or to EGFR tyrosin kinase inhibitors reduce EGFR protein levels and are thus no strategies to promote ABT-414-induced cell killing. Furthermore, glioma cells overexpressing kinase-dead EGFR or EGFRvIII retain binding of mAb 806 and sensitivity to ABT-414, allowing to dissociate EGFR phosphorylation from the emergence of the “active” EGFR conformation required for ABT-414 binding. The combination of EGFR-targeting antibody drug conjugates with EGFR tyrosine kinase inhibitors carries a high risk of failure. Promoting mere EGFR expression rather than phosphorylation should result in glioblastoma cell sensitization to ABT-414 or related agents.
CSIG-09. PROTEOMIC ANALYSIS OF MENINGIOMA CELL-DERIVED EXTRACELLULAR VESICLES: FIRST OF A KIND
Authors: Ricklefs Franz , Fuh Manka , Maire Cecile , Holz Mareike , Kolbe Katharina , Westphal Manfred , Schlüter Hartmut , Lamszus Katrin
Published: November 2019

BACKGROUND

Extracellular vesicles (EVs) play an important role in cell-cell communication in different types of tumors, carrying multiple layers of biological functional molecules, including proteins, RNA, DNA and lipids. Their implication as biomarkers in tumor disease is under current investigation. We previously showed that EVs in glioblastoma reflect the tumor subtype and that glioblastoma patients have elevated circulating particle counts. Regarding to meningioma, it is not known to what extent these usually benign tumors secrete EVs and how these EVs reflect the tumor. Here we report the first study that analyzed meningioma cell-derived EVs.

METHODS

Meningioma tissue, short-term cell cultures and cell culture-derived EVs (menEVs), (n=4) were analyzed by global mass-spectromety, immunoblotting and imaging flow cytometry and compared to EVs from glioblastoma short-term cell cultures (gEVs), (n=4). Plasma EVs from meningioma patients (n = 12) were analyzed for their tetraspanin marker expression (CD9, CD81 and CD63). EVs were further analyzed by nanoparticle analysis (NTA) and electron microscopy.

RESULTS

menEVs were 110-140nm in size and exhibited vesicular structures by electron microscopy. We identified 269 proteins in menEVs through mass spectometry. 45 proteins were upregulated in menEVs compared to short-term cell culture and original tumor tissue. 99 proteins were exclusively found in menEVs compared to gEVs, with osteopontin being the top highly expressed protein within the mEV fraction. Both meningioma and glioblastoma patients have elevated circulating plasma EV counts (p< 0.01), as measured by NTA.

CONCLUSION

The increase in circulating plasma EVs in meningioma patients suggests that tumor cell-derived EVs augment the pool of circulating EVs and could be utilized to obtain information on the tumor by liquid biopsy. Osteopontin is known to be expressed at high levels in meningiomas and its association with menEVs may facilitate isolation of circulating meningioma-specific EVs for analysis.
CSIG-20. EFFECT OF TUMOR-TREATING FIELDS (TTFIELDS) ON EGFR PHOSPHORYLATION IN GBM CELL LINES
Authors: Luisa D’Angelo Maria , Piffaretti Deborah , Burgio Floriana , Chiappini Alessio , Marchi Francesco , Reinert Michael
Published: November 2019

Glioblastoma (GBM) is the most common high grade and most devastating brain tumor among adults. About 50% of GBM express EGFR (epidermal growth factor receptor) and from these another 50% the mutated form EGFRvIII which is associated with a more aggressive disease and poorer prognosis. We have previously shown that expression of different status of EGFR in GBM cell lines drives the 5-ALA induced fluorescence downstream by influencing the rate-limiting enzyme heme oxygenase-1 (HO-1) probably via PI3K/Akt signalling. We demonstrated that 5-ALA-induced protoporphyrin IX (PpIX) fluorescence is pharmacologically influenced by adding different drugs such as deferoxamine (DFO) and tin-protoporphyrin (SnPP), which are an iron chelator of Fe2+ and an inhibitor of HO-1 respectively, or genistein that promotes PpIX accumulation via functional repression of ABCG2 (ABC transporter G2). Our aim is to increase these pharmacological effects on PpIX fluorescence using tumor-treating fields (TTFields). TTFields, a new therapeutic technology for treating newly diagnosed or recurrent GBM, is able to suppress the growth of cancer cells destabilising microtubule elongation and increasing membrane permeability. Interestingly, TTFields, like as other destabilising drugs and compounds, is able to inhibit the phosphorylation of EGFR and subsequent downregulation of EGFR-induced signalling acting for example on the mechanism that regulate the HO-1 activity. Here, we investigate the effects of TTFields on glioma cells, with different EGFR status and consequently different PpIX fluorescence. Exposure to TTFields during or after pharmacological treatments may represent a novel strategy to block or diminish the phosphorylation of EGFR to ameliorate the visualization of PpIX fluorescence in patients where it is not enough to ensure a safe and precise removal of the tumor bulk. In fact, if a combination of TTFields and drug treatment should give the desired results, this strategy could be applied on patients before being subjected to surgical resection.
CSIG-26. IS INTRINSIC APOPTOSIS THE SIGNALING PATHWAY ACTIVATED BY TUMOR-TREATING FIELDS FOR GLIOBLASTOMA?
Authors: Carlson Kristen , Tuszynski Jack , Bomzon Ze’ev
Published: November 2019

Increasingly, tumor-treating fields (TTFields, 2 V/cm, 200 kHz) are accepted as the fourth treatment modality for glioblastoma. Evidence shows that substituting non-steroidal inflammation control (celecoxib) for dexamethasone increases overall survival from 4.8 to 11.0 months, and more recently, up to 60 months. Toward explaining TTFields mechanism of action (MoA), our numerical simulations indicate that TTFields disrupt functionality of microtubules, which in turn trigger the intrinsic apoptotic pathway independent of cell cycle checkpoints. We present the theory and empirical evidence. 1) TTFields act similarly to chemotherapeutic ‘spindle poisons’ by interfering with microtubule (MT) polymerization, increasing free tubulin by 20% in relative terms; 2) Finite element modeling shows TTFields amplify electric field strength, in accord with empirical results, a) along the MT when aligned with the cell axis, where field strength exceeds 10–16 N required to disrupt motor protein transit, and b) 15x at MT ends when orthogonal to cell axis; 3) Either through producing excess free tubulin, which may block voltage-dependent anion channels, or direct effects on the mitochondrial inner and outer membranes, TTFields inhibit expression of pro-survival protein Bcl-2; 4) Decreased Bcl-2 expression activates the intrinsic apoptotic pathway in a novel cell-cycle-checkpoint and caspase-independent manner; 5) Patients using low (< 4.1 mg/day) vs. high (>4.1 mg/day) dexamethasone doses experienced an average 8.7 vs. 3.2 months OS and up to 60 months; 6) Numerous studies in both brain and other tissues show that dexamethasone a) promotes extrinsic, immune-system apoptosis and b) inhibits intrinsic, Bcl-2/Bax mediated apoptosis; 7) Downstream effects of intrinsic apoptosis are remarkably similar to empirically-observed effects of TTFields on tumor cells. Research supported by Novocure Ltd. Dept of Neurosurgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston MA USA. carlsokw@bidmc.harvard.edu. Dept of Physics, University of Alberta, Edmonton, Canada, Novocure Ltd., Haifa, Israel.
CSIG-29. STRUCTURAL AND FUNCTIONAL STUDIES OF PID1, A NOVEL GROWTH SUPPRESSOR IN BRAIN TUMORS
Authors: Ren Xiuhai , Hirsh Emi , Mahdi Min , Ito Fumiyaki , Chen Xiaojiang , Shackleford Gregory , Erdreich-Epstein Anat
Published: November 2019

BACKGROUND

Brain cancers, including medulloblastomas and gliomas, are a major cause of death in children and adults. We reported that PID1 (Phosphotyrosine Interaction Domain containing 1) is a growth suppressor in medulloblastomas and gliomas (PMID: 24300787). PID1 also enhances the anti-tumor effects of chemotherapy (PMID: 28400607). We are now seeking to better understand the structure and mechanism of PID1, in order to utilize this knowledge to develop innovative PID1-based therapies.

METHODS

PID1 mutants were expressed in E. coli as MBP fusion proteins, purified and used for MALS analysis. Additional epitope tagged PID1 constructs (HA, V5, tGFP, eGFP) were generated, expressed in mammalian cells and analyzed by western blots, immunoprecipitation, and functional assays.

RESULTS

We carried out screening and testing of multiple mutants of purified PID1 and characterized them. Experiments in cell culture supported presence of similar findings in mammalian cells. Colony assays in glioma and medulloblastoma cell lines identified a region in PID1 that confers the most robust growth-inhibitory effect. Experiments are underway to further refine the boundaries and characteristics of this growth-inhibitory region.

CONCLUSIONS

This project, which is focused on better understanding of the structure and function of PID1, is uncovering novel aspects of its molecular function. Insights gained from this work will guide studies to develop innovative PID1-based therapies for gliomas and medulloblastomas.