bluuebunny/crossref_metadata_2025 huggingface dataset:
EXTH-13. COMBINED TARGETING OF TELOMERASE REVERSE TRANSCRIPTASE AND METABOLISM ABROGATES GLIOBLASTOMA PROLIFERATION IN VIVO
Authors: Batsios Georgios , Tran Meryssa , Gillepspie Anne Marie , Taglang Celine , Viswanath Pavithra
Published: November 2022

Telomerase reverse transcriptase (TERT) is essential for tumor immortality. Since TERT is silenced in normal cells, it is an attractive therapeutic target and the TERT inhibitor 6-thio-2’-deoxyguanosine (6-thio-dG) is in clinical trials for gliomas. However, there is a lag period following TERT inhibition before tumor shrinkage is observed. Novel strategies that can be combined with TERT inhibition to enhance anti-tumor activity are sorely needed. We previously demonstrated that TERT increases expression of the alanine transporter ASCT2 in gliomas, an effect that could be non-invasively monitored using hyperpolarized 13C imaging of alanine metabolism (Viswanath et al., Nature Communications, 2021). The goals of the current study were to determine whether combined inhibition of TERT and ASCT2 inhibits GBM growth and to assess the ability of hyperpolarized [1-13C]-alanine to monitor treatment response. Our results indicated that, while both the TERT inhibitor 6-thio-dG and the ASCT2 inhibitor V-9302 individually inhibited viability, the combination of 6-thio-dG and V-9302 maximally inhibited cell viability in the patient-derived GBM1 and GBM6 models. In line with the effect on cell viability, while both compounds reduced lactate production from hyperpolarized [1-13C]-alanine, the combination of 6-thio-dG and V-9302 resulted in the highest inhibition of lactate production in both models. Importantly, treatment of rats bearing orthotopic GBM6 tumors with a combination of 6-thio-dG and V-9302 caused tumor shrinkage, an effect that could be visualized by magnetic resonance imaging by day 21 after treatment. Furthermore, lactate production from hyperpolarized [1-13C]-alanine was significantly reduced at day 7 after treatment with 6-thio-dG and V-9302, when anatomical alterations were absent. Collectively, our results indicate that simultaneously targeting TERT and ASCT2 provides a novel therapeutic opportunity for GBMs and that hyperpolarized [1-13C]-alanine serves as a companion agent for imaging early response to therapy. Our findings pave the way for precision therapy and response assessment for GBM patients.
EXTH-16. THE ORAL IRON-MIMETIC GALLIUM MALTOLATE SUPPRESSES TREATMENT-RESISTANT GLIOBLASTOMA – IN VIVO VALIDATION
Authors: Al-Gizawiy Mona , Wujek Robert , Prah Melissa , Connelly Jennifer , Chitambar Christopher , Schmainda Kathleen
Published: November 2022

BACKGROUND

We previously reported on our in vitro and in vivo studies using the metallocompound gallium maltolate (GaM) to target glioblastoma (GBM). We demonstrated a profound cytotoxic effect in vitrov and in vivo. Additionally, our preliminary in vivo data suggested a substantial inhibitory effect on tumor growth, as well as a significant extension of disease-specific survival (OS). Here, we further validate the inhibitory effects of GaM in vivo.

METHODS

In vitro irradiated adult GBM U87-MG cells were stereotactically implanted into the right striatum of male and female athymic rats. Advanced MR imaging at 9.4T was carried out weekly starting two weeks after implantation. Daily oral GaM (50 mg/kg) or vehicle were provided on tumor confirmation. Longitudinal advanced MRI parameters were processed for enhancing tumor ROIs in OsiriX 8.5.1 (lite) with Imaging Biometrics Software (Imaging Biometrics LLC,). Statistical analyses included Kaplan-Meier survival plots, linear mixed model (LMM) comparisons, and t-statistic for slopes comparison (as indicator of tumor growth rate).

RESULTS

Median OS for the male pilot cohort (5 control, 5 GaM) was 28 and 51 days, respectively; 31 and 59 days for the male replication cohort (1 control, 3 GaM); 37 and 48 days for the female replication cohort (6 control, 7 GaM). GaM-treated xenograft tumors grew significantly slower than control tumors with sex and time dependent growth rates. No significant differences were seen between male and female cohorts in both control and treatment groups at all timepoints. Hence, subsequent analyses were performed on pooled data. Median OS was 30.5 days and 48 days for control and GaM groups, respectively (p < .001). A strong correlation between treatment and survival (Χ2 = 6.238187; p = .0125) and tumor growth suppression (LMM; p < .0001) was evident.

CONCLUSION

We have successfully validated the inhibitory effects of GaM on GBM growth in vivo.
EXTH-19. OPTIMIZING MDM2 INHIBITION FOR THE TREATMENT OF GLIOBLASTOMA
Authors: Rendo Veronica , Lee Eudocia , Khuu Nicholas , Pelton Kristine , Lapinskas Emily , Wen Patrick Y , Ligon Keith , Beroukhim Rameen
Published: November 2022

Over 60% of glioblastomas retain wild-type p53, resulting in potential susceptibility to MDM2 inhibitors. These molecules disrupt the interaction between p53 and its negative regulator MDM2, allowing downstream pathway signaling and cell fate decisions including growth inhibition and death resulting from cellular stress. We have analyzed clinical samples from 10 patients newly diagnosed with glioblastoma multiforme (GBM) and treated with the MDM2 inhibitor KRT-232. We detected upregulation of CDKN1A transcript (a target of p53) in all p53 wild-type tumors (8 of 10), but not in p53-mutant tumors (2 of 10). In patient-derived cell lines, treatment with KRT-232 at the clinically detected concentration was only sufficient to stall tumor growth. Cell death via apoptosis can be achieved when MDM2 inhibition is combined with the chemotherapeutic agent temozolomide. As the effects and resistance mechanisms of MDM2 inhibition remain poorly understood in GBM, we have additionally performed genomic and transcriptomic analyses in patient-derived cell lines to better characterize sensitive tumors and identify putative biomarkers of drug response. Dose response curves and growth assays showed that tumors with inactivating p53 mutations are highly resistant to treatment, but those that retain wild-type p53 exhibit various degrees of drug sensitivity. This suggests that other factors, in addition to p53 mutational status, mediate response to MDM2 inhibition in gliomas. Transcriptional analyses of patient samples following drug treatment suggest that cell division, chromatin reorganization, cell differentiation state and immune response programs become deregulated under MDM2 inhibition and in the absence of p53-inactivating mutations.
EXTH-26. MICROGLIA-SPECIFIC DISRUPTION OF SIALIC ACID-SIGLEC-9/E INTERACTIONS: A NOVEL IMMUNOTHERAPY AGAINST GLIOBLASTOMA?
Authors: Schmassmann Philip , Roux Julien , Tatari Nazanin , Martins Tomas A , Ritz Marie-Françoise , Shekarian Tala , Laeubli Heinz , Hutter Gregor
Published: November 2022

Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as microglia (MG) or monocyte-derived cells (MdCs). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in innate-centered immunotherapy against GBM. TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Using a CT-2A orthotopic GBM mouse model with MG-specific spatio-temporal deletion of Siglece (Sall1CreERT2 x Sigleceflox) , we observed high MG-proliferation upon Siglece knockout (Ki-67+ MG 14.8% in Crenegvs. 34.9% in Creposp < 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Crenegvs. 12.3% in Crepos, p < 0.001). By extending the Siglece knockout to the MdC compartment (Cx3cr1CreERT2x Sigleceflox) we observed a significantly prolonged survival in the Crepospopulation (21d in Crenegvs. 27d post-tumor injection in Crepos, p = 0.018), which could be further promoted by combining Siglece knockout with CD47 blockade (11% long-term remission in Crepos+ anti-CD47). Proteomics analysis revealed increased antigen processing and presentation capabilities of SigleceKOMdCs which was confirmed by ex-vivo OT-1 cross-presentation assays. This increased T cell priming upon MdC SigleceKOwas further boosted by addition of anti-PD1 antibody to the SigleceKO+ anti-CD47 combination. Resulting in 23% of animals experiencing long-term remission in the triple treatment arm, even after tumor re-challenge. Genetic targeting of sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), resulted in increased GBM-cell phagocytosis by MG and MdCs and less exhausted tumor-infiltrating CD8+ T cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003). In a translational approach, we are currently testing anti-Siglec-9 treatment regimens in patient GBM explants, cultured for 5 days in perfusion bioreactors.
EXTH-35. ANTI-TUMOR AROMATHERAPY FOR MALIGNANT BRAIN TUMORS USING VOLATILE COMPONENTS OF HIBA ESSENTIAL OIL
Authors: Saito Norihiko , Hirai Nozomi , Nagata Takuya , Kushida Naoki , Sato Sho , Hiramoto Yu , Hayashi Morito , Iwabuchi Satoshi
Published: November 2022

INTRODUCTION

Glioblastoma is a brain tumor with a very poor prognosis that is highly resistant to treatment. Hiba (Thujopsis dolabrata) has been used as a building material because of its insect repellent effect and refreshing fragrance. In this study, we analyzed the antitumor effects of thujopsen, which is contained in the essential oil of hiba, and investigated its antitumor aroma therapy.Methods and

RESULTS

We investigated the anti-tumor aroma effect of hiba essential oil on glioblastoma cells (U251, T98G), gastric cancer cells (MKN45), and breast cancer cells (MCF7). The anti-tumor effect of hiba essential oil varied depending on the carcinoma, with strong inhibition of cell proliferation observed in MKN45, U251, and T98G cells. Hinokitiol in hiba essential oil has been reported to have antitumor effects, and we isolated and identified a new antitumor factor, thujopsene, by liquid chromatography analysis. In tumor cell proliferation assays, thujopsen inhibited tumor cell proliferation more rapidly and potently than hinokitiol. Comparing the reach of the two compounds, hinokitiol showed a broader anti-tumor effect than thujopsen. The expression of caspase-3 and caspase-9 was increased by thujopsen, and DARTS analysis identified PKM2 as an intracellular binding protein of thujopsen.

DISCUSSION

The present study revealed that thujopsen induces apoptosis via caspase-3 and caspase-9. Furthermore, unlike hinokitiol, thujopsen was suggested to exhibit antitumor effects mediated by PKM2.

CONCLUSION

Thujopsen is a low molecular weight compound and easily evaporates into the air, it is expected to be applied to a new glioblastoma therapy using "aroma".
EXTH-40. DIPGS SUPPRESS THE EFFICACY OF T CELL THERAPIES THROUGH DYSFUNCTIONAL IMMUNE SYNAPSE FORMATION
Authors: Ibanez Jorge , Houke Haley , Elayan Abdul , Ocasio-Adorno Jennifer , Hebbar Nikhil , Velasquez Paulina , Baker Suzanne , Krenciute Giedre
Published: November 2022

Diffuse intrinsic pontine gliomas (DIPGs) are highly lethal pediatric brain tumors. Thus, there is an urgent need for novel therapeutics. While chimeric antigen receptor (CAR) T-cell therapy has the potential to meet this need and early phase clinical studies with CAR T cells have shown safety and some efficacy, additional studies are needed to understand the lack of efficacy of CAR T cells against DIPGs. The goal of this study was to identify CAR T cell efficacy limiting factors in DIPG setting. To study that, we focused on targeting GRP78, an endoplasmic reticulum chaperone protein that is broadly expressed on the cell surface of DIPGs. We generated GRP78-specific CAR T cells and compared their effector function against U87 glioma and the patient-derived DIPG007 cell line. Despite that GRP78-CAR T cells recognize and kill U87 and DIPG007 tumor cells in vitro, they were only effective against U87 in vivo. In addition, in vitro cytokine production and T cell proliferation were heavily diminished in DIPG007, compared to U87. For full activation, T cells require a cell-to-cell interaction and the formation of a highly organized structure called immune synapse (IS). Experiments using confocal microscopy showed that the IS quality, measured as lytic granule accumulation and cytoskeleton organization at the IS, was impaired in DIPG007 when compared to U87. This dysfunctional IS correlated with poor T cell activation (calcium flux) as determined by live cell imaging. We observed the same dysfunctional IS formation regardless of CAR T cell specificity and targeted antigen expression level, indicating that the suppressive effect is DIPG tumor mediated. In summary, we demonstrate that DIPGs suppress CAR T cell effector function through dysfunctional IS formation. We are now testing genetic engineering approaches directed at improving IS formation to overcome the suppressive effects of DIPGs.
EXTH-43. XPO1 INHIBITOR SELINEXOR SENSITIVITY IN GLIOBLASTOMA: POTENTIAL ROLE OF MGMT ACTIVATION THROUGH PKA-CREB PATHWAY
Authors: Suzuki Yuta , Gupta Shiv , Sarkaria Jann , Kitange Gaspar
Published: November 2022

INTRODUCTION

Glioblastoma (GBM) is the most common and aggressive form of adult brain tumor and is an incurable disease due to primary and secondary resistance to therapy. We recently examined the candidate genes modulating temozolomide (TMZ) resistance using genome-wide shRNA screening in TMZ-resistant U251 (U251TMZ) model. We identified a total of 134 TMZ-resistant candidate genes, including Karyopherin A1 (KPNA1) and Exportin 1 (XPO1), the genes critical for nucleocytoplasmic (NC)-transport. Interestingly, XPO1 inhibitor Selinexor is an FDA-approved agent for refractory multiple myeloma, and there is an ongoing interest in evaluating Selinexor for GBM therapy. Therefore, we hypothesized that Selinexor is a promising drug for overcoming TMZ resistance. METHODS and

RESULTS

We conducted a series of experiments in vitro using Selinexor with and without TMZ. Selinexor and TMZ alone didn’t significantly block the growth of U251TMZ cells. In contrast, Selinexor significantly re-sensitized resistant U251TMZ cells to TMZ. Interestingly, MGMT-methylated GBM39 cells were sensitive to Selinexor alone, TMZ alone, and to combined Selinexor/TMZ treatment. Contrarily, MGMT-unmethylated GBM43 cells were only sensitive to combined Selinexor/TMZ treatment. These findings suggested that similar to TMZ, Selinexor sensitivity may be influenced by MGMT expression status, with MGMT expressing cells showing resistance. In line with this notion, re-expression of exogeneous MGMT blocked sensitivity of MGMT-methylated U251 cells to Selinexor and TMZ, but such inhibition was not observed in U251 expressing vector alone. Interestingly, the concentration-dependent experiments demonstrated that Selinexor can induce MGMT expression in unmethylated T98G, GBM6, GBM14, and GBM43 cells. Selinexor-induced MGMT expression was accompanied with increased phosphorylation of CREB protein.

CONCLUSION

These results suggest that Selinexor sensitivity alone and with TMZ may be limited to MGMT-methylated GBM cells and may increase MGMT expression in unmethylated cells through activation of PKA-CREB pathway. Future studies will evaluate how MGMT may impact the sensitivity of GBM cells to Selinexor.
EXTH-48. INHIBITION OF SREBP-1 IS SYNTHETICALLY LETHAL WITH BCL-XL/BCL-2 INHIBITION IN GLIOBLASTOMA IN VITRO
Authors: Tosin Davide , Schlotter Dominik , Hajosch Annika , Hlavac Michal , Westhoff Mike-Andrew , Siegelin Markus , Wirtz Christian Rainer , Karpel-Massler Georg
Published: November 2022

OBJECTIVE

A cellular homeostasis that is shifted away from apoptosis and a reprogramming of the lipid metabolism are both, features that are frequently encountered in glioblastoma. This study aimed at investigating whether interference with the lipid metabolism is synthetically lethal with inhibition of anti-apoptotic Bcl-2 family proteins in glioblastoma in vitro.

METHODS

Established (U251) and primary-cultured glioblastoma cells (PC38, PC40 and PC128) as well as glioblastoma stem-like cells (SC38 and SC40) were treated with the Bcl-xL/Bcl-2 inhibitor ABT-263 (navitoclax) and/or the SREBP-1 inhibitor Fatostatin. MTT-assays were performed to assess effects of the combination therapy on the cell viability. Isobolograms were calculated to characterize the drug-drug interaction. Spheroids were used to determine anti-proliferative effects in a 3-dimensional setting. Staining with annexin V/propidium iodide and flowcytometric analysis were performed to assess pro-apoptotic effects. For molecular analyses, Western blots and specific knock-down experiments with siRNA were performed.

RESULTS

Combined targeting of SREBP-1 and Bcl-xL/Bcl-2 led to a synergistic inhibitory effect on the cellular viability of established, primary-cultured and glioblastoma stem-like cells as well as spheroids. This effect was shown to be at least in part mediated by enhanced apoptosis and to occur in a caspase-dependent manner. On the molecular level, treatment with increasing concentrations of Fatostatin led to a downregulation of Mcl-1.

CONCLUSION

Our study indicates that combined inhibition of Bcl-xL/Bcl-2 and interference with the lipid metabolism targeting SREBP-1 synergistically induces caspase-dependent apoptosis in glioblastoma cells. This effect can also be observed in more complex 3-dimensional glioblastoma cell formations. Further studies will focus on deciphering the drug-induced alterations of the metabolic pathways that are responsible for the synergistic effect of this therapeutic strategy.
EXTH-49. A NOVEL THERAPEUTIC ANTIBODY TARGETING DIFFUSE MIDLINE GLIOMA
Authors: Balakrishnan Illango , Madhavan Krishna , Pierce Angela , Venkataraman Sujatha , Vibhakar Rajeev
Published: November 2022

There is an unmet need to identify novel targeted therapies for H3K27M mutated Diffuse Midline Gliomas. (DMG) Using genetic overexpression and deletion of H3K27M in model cells we identified high expression of CD99 mRNA in H3K27M-mutant expressing DIPGs compared to other normal brain counterparts. A cell surface antigen screen confirmed that CD99 was highly expressed in DMG compared to normal tissues. We then developed a novel chimeric CD99 antibody based on a human IgG4 scaffold and tested the anti-tumor efficacy of this antibody in vitro and in vivo DMG xenografts. Our new CD99 antibody (10D1) significantly reduced DIPG tumor cell proliferation in vitro. In vivo intravenous administration of the antibody in orthotopic DMG tumor bearing mice showed complete clearance of tumor with prolonged animal survival establishing anti-tumor efficacy of 10D1 and demonstrating its ability in crossing the blood-brain-pons barrier. Loco-regional administration of 10D1, intratumorally or to the lateral ventricle, showed similar anti-tumor effects at reduced antibody concentrations while limiting systemic effects on normal T cells. 10D1 combined with radiation to further prolong the survival of orthotopic xenograft-bearing animals. In conclusion, we have identified a novel therapeutic for DMG which is now in further development.
EXTH-51. HSV-PTENΑ TREATMENT TARGETS GLIOMA STEM CELLS TO REDUCE STEMNESS AND SENSITIZES GLIOBLASTOMA TO IRRADIATION
Authors: Mullarkey Matthew P , Sahu Upasana , Hong Bangxing , Kaur Balveen
Published: November 2022

Glioblastoma is one of the most lethal and treatment-resistant tumors of the central nervous system. Despite maximal surgical and medical therapy, survival remains dismal with a median of 21 months. Despite advances in treatment, this has only led to modest survival benefit. A significant challenge in treatment of glioblastoma is targeting glioma stem cells (GSCs) which are a source of therapy resistance. Oncolytic viral (OV) therapy is a promising therapy for solid tumors that preferentially targets tumor cells for lysis and an anti-tumor immune response while sparing normal cells. Among all OVs, oncolytic Herpes Simplex Virus (oHSV) is substantially ahead in the clinic, with an oHSV T-VEC approved by the FDA for metastatic melanoma treatment and G47∆ which received conditional approval for the treatment of GBM in Japan. In prior work, our group has demonstrated that PTENα expression by an oHSV (HSV-P10) results in improved long-term survivors in intracranial tumor-bearing mice compared to HSVQ treatment. We aim to elucidate the mechanism of improved therapeutic efficacy of HSV-P10 against GBM and evaluate if HSV-P10 may overcome radio-resistance. RNA sequencing and GSEA analysis of primary human GBM cells infected with control HSVQ or HSV-P10 reveals that while HSVQ virus infection leads to an increase in genes regulating IL6-STAT3 pathway, pivotal in maintaining stemness properties, HSV-P10 infection leads to a reduction. HSV-P10 reduces CD133+/CD44+ stem cells, induces DNA damage and sensitizes the GBM cells to irradiation. Our findings reveal a novel mechanism induced by HSV-P10 in combination with irradiation where HSV-P10 modulates IL6-STAT3 signaling downregulating genes associated with stemness (Nestin, Sox2). HSV-P10 infection in combination with irradiation reduces GSC tumor sphere formation in vitro and sensitizes GBMs to radiotherapy in an intracranial mouse xenograft model. Our findings uncover a possible mechanism to overcome GSC-mediated therapy resistance to improve the therapeutic efficacy for GBM.
EXTH-61. RPP25L IS A NOVEL DEPENDENCY IN GLIOBLASTOMAS WITH LOW RPP25 EXPRESSION
Authors: Aquilanti Elisa , Chen Rebecca , Hui Keliana , Kageler Lauren , Prensner John , Wen Patrick Y , Meyerson Matthew , Huang Franklin
Published: November 2022

Genetic redundancy is a property whereby two or more genes encode for the same biological function, and inactivation of one redundant gene has little effect on the overall phenotype. In recent years, genetic redundancy has been exploited as a cancer therapeutic strategy, as individual paralogs of essential genes are occasionally lost in cancers because of genomic deletions. Inactivation of the non-deleted paralog can therefore be selectively toxic to tumor cells. To identify cancer cells that may become reliant on the non-methylated paralog of essential genes, we performed an in-silico analysis of the correlation between DNA methylation and genetic dependency across large scale CRISPR knockout screens that revealed novel paralog dependencies. Through this analysis, we identified glioblastoma cell lines with hypermethylation of the RPP25 promoter and with sensitivity to loss of RPP25L. RPP25 is a known structural component of RNAse P, a key enzyme involved in tRNA maturation. While the biological function of RPP25L is unknown, it has significant sequence homology to RPP25. We used CRISPR editing and CRISPR interference to inactivate RPP25L in a panel of glioblastoma cell lines and demonstrated that cell lines that do not express RPP25 (SF295, GB1, LN18) exhibit a rapid reduction in viability upon RPP25L loss, whereas cell lines with retained RPP25 expression or re-expressed RPP25 are not affected. RPP25L dependency is associated with a marked reduction in nascent polypeptide formation as determined by click chemistry. Lastly, we validated RPP25L dependency in glioblastoma patient-derived neurospheres with loss of RPP25 expression. These results indicate that RPP25L is a promising novel therapeutic target in glioblastomas with hypermethylation of the RPP25 promoter.
EXTH-67. PRECLINICAL ASSESSMENT OF THE EFFICACY OF MPT0B640, AN ORALLY BIOAVAILABLE SMALL MOLECULE HSP90 INHIBITOR, AGAINST GLIOMAS
Authors: Sharma Pratibha , Xu Jihong , Chen Xiaohua , Huo Longfei , Ju Zhenlin , Li Ziyi , Ban Kechen , Seah Ethan , Sampath Deepa , Puduvalli Vinay
Published: November 2022

BACKGROUND

Heat-shock protein 90 (HSP90), an evolutionary conserved protein, is a key member of the epichaperome complex involved in maintenance of protein homeostasis during non-stress and stress conditions. It is overexpressed in gliomas and supports tumor cell survival and resistance. Hsp90 inhibition can impact several pathways essential for glioma cell survival and overcome tumor heterogeneity. Here, we examined the activity and toxicity profile of MPT0B640, an orally bioavailable Hsp90 inhibitor, against gliomas and its ability to cross the blood-brain barrier (BBB).

METHODS

Using genetically heterogeneous glioma stem-like cell (GSC) lines, we determined the effects of MPT0B640 on cell proliferation (Cell Titre Glo assay), apoptosis induction and cell cycle changes (flowcytometry; AnnexinV/PI staining), and alterations in protein expressions (western blot and reverse-phase protein array, RPPA assays). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed in non-tumor bearing mice to assess BBB permeability and antitumor effects of MPT0B640.

RESULTS

MPT0B640 inhibited proliferation of both adherent (LN229, A172, U251-HF) and patient-derived GSCs (GSC262, GSC811, GSC11, GSC23, GSC20 and GSC272) in a dose- and time-dependent fashion with an IC50 range of 50-1000 nM. MPT0B640-treated cells underwent apoptosis and/or cell cycle arrest with downregulation of pro-survival signal kinases, phospho-Akt, p-ERK1/2 and p-S6. PK/PD studies showed increased HSP70 expression (a marker of Hsp90 inhibition) and detectable levels of drug in brain specimens from non-tumor bearing mice after a single dose of MPT0B640, indicating BBB permeability and target inhibition of the agent. Results of ongoing animal studies assessing drug toxicity and animal survival with MPT0B640 will be presented.

CONCLUSIONS

MPT0B640 is a potent HSP90 inhibitor that has potent antitumor activity against genetically heterogeneous gliomas in vitro, effectively crosses the intact BBB and demonstrates target inhibition warranting further investigation for treatment of gliomas.
EXTH-68. ONCOMAGNETIC TREATMENT SELECTIVELY KILLS GLIOMA CANCER CELLS BY INDUCING OXIDATIVE STRESS AND DNA DAMAGE
Authors: Hambarde Shashank , Pandey Arvind , Baskin David , Helekar Santosh
Published: November 2022

A new noninvasive therapeutic device developed in our laboratory called the Oncomagnetic device provides a novel approach to glioblastoma (GBM) treatment. It involves repeated stimulation with oscillating magnetic fields (sOMF) produced by spinning permanent magnets. It is technologically and mechanistically distinct from the Optune® device approved by the FDA for the treatment of newly diagnosed and recurrent GBM. Use of this device in one end-stage GBM patient reversed the progression of his recurrent tumor causing >30% reduction in its contrast-enhanced volume within 4 weeks of treatment. Mice with implanted mouse glioma cells in their brains also showed marked reduction in tumor size, increased survival (p< 0.05, n = 10) and higher DNA damage (g-H2AX foci) after sOMF treatment with a whole-body stimulation method developed by us. Normal mice exposed to sOMF for 4 months had no adverse effects on the brain and other organs. In-vitro, sOMF markedly increased reactive oxygen species (ROS) levels in cancer cells leading to the selective death of these cells, while sparing normal neurons and astrocytes. Detection of g-H2AX and 53BP1 foci showed that sOMF caused significant DNA damage in GBM cells and diffuse intrinsic pontine glioma (DIPG) cells but not in normal astroglial SVGp12 cells. Furthermore, sOMF exposure for just 2 h resulted in >40% loss of surviving GBM and DIPG cell colonies detected by clonogenic cell survival assay, similar to that produced by 2 Gy radiation dose. This loss was rescued by the antioxidant Trolox. These results indicate that sOMF stimulation has high anticancer potency comparable to low dose radiation therapy at the cellular level with an underlying mechanism of action that is substantially different from that proposed for Optune® TTF.
EXTH-76. PRMT5 INHIBITION SENSITIZES GLIOBLASTOMA NEUROSPHERES TO TEMOZOLOMIDE
Authors: Rivas Sarah , Rivera-Caraballo Kimberly , Murphy Sara , Otani Yoshihiro , Shelbourn Allison , Ampie Leo , Maric Dragan , Walbridge Stuart , Shah Ashish , Yan Yuanqing , Yoo Ji young , Heiss John , Kaur Balveen , Banasavadi-Siddegowda Yeshavanath
Published: November 2022

INTRODUCTION

The median survival of Glioblastoma (GBM) patients is less than two years with the standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) chemotherapy. Protein Arginine Methyltransferase 5 (PRMT5), which regulates cellular functions by symmetrically di-methylating arginine residues, is overexpressed in GBM. Inhibiting PRMT5 induces apoptosis in differentiated and senescence in stem-like GBM tumor cells. We inhibited PRMT5 in GBM neurospheres to determine if PRMT5 inhibition would enhance TMZ’s antitumor effect.

METHODS

We depleted PRMT5 activity, in vitro, using target-specific siRNA or LLY-283 and combined these with TMZ treatment. We evaluated the antitumor effect of this combination using cell viability assay, cell cycle analysis, apoptosis assay, and western blot.

RESULTS

TMZ reduced the viability of GBMNS with PRMT5 knockdown significantly more than the viability of PRMT5 intact GBMNS. The combination of TMZ and PRMT5 knockdown elevated the expression of cleaved caspase 3 and caspase3/7 indicating that PRMT5 knockdown enhanced the apoptotic effects of TMZ. Cell cycle analysis showed that depleting PRMT5 abrogated TMZ-induced G2/M cell cycle arrest. Further, treatment of PRMT5-depleted GBMNS with TMZ increased ɣ-H2AX expression compared PRMT5 intact GBMNS treated with TMZ, suggesting that PRMT5 depletion enhanced TMZ-induced DNA damage. PRMT5 knockdown also inhibited the symmetric di-methylation of RUVBL1 that is required for homologous recombination repair of TMZ treatment-related DNA damage.

CONCLUSION

Overall, PRMT5 inhibition sensitized GBMNS to TMZ and enhanced TMZ-related DNA damage and cytotoxicity. These findings support further development of this potential therapeutic combination.
EXTH-77. TARGETING CD73 IN GBM SENSITIZES TUMORS TO ONCOLYTIC VIRUS THERAPY
Authors: Murphy Sara , Mullarkey Matthew P , Sahu Upasana , Hong Bangxing , Lewis Cole , Diaz Miguel , Wenzel Pamela , Kamal Abu Hena Mostafa , Putluri Nagireddy , Park Junhyoung , Kaipparettu Benny , Kaur Balveen
Published: November 2022

Glioblastoma Multiforme (GBM) is the most aggressive malignant primary brain tumor and has abysmal 5-year overall survival. With the approval of oHSV Imlygic by FDA for metastatic melanoma and more recently, conditional approval of G47Δ, marketed by Daiichi Sankyo for GBM treatment in Japan, oncolytic viral therapy has emerged as a promising biological approach to treat solid tumors. Our laboratory has previously shown that oHSV expressing long isoform of PTEN (PTENα), HSV-P10, has faster kinetics of virus replication associated with enhanced killing of the glioma cells compared to control HSV. Additionally, HSVP-10 is known to increase mitochondrial membrane potential and cellular ATP production while stimulating anti-tumor immune responses in vivo. RNA sequencing of primary GBM cells infected with HSV-P10 shows altered metabolic pathways relative to control HSV infected cells. 13C metabolic flux analysis in uninfected and control or HSV-P10 infected primary GBM cells reveals that while HSV-P10 infection shuttles most of the glutamine towards citrate by reductive carboxylation of α-ketoglutarate resulting in faster replication of HSV-P10, simultaneously it increased glucose utilization and shuttling towards TCA cycle. This corroborates with enhanced mitochondrial activity leading to increased oxidative phosphorylation and increased cellular and extracellular ATP (eATP). eATP binds to purinergic receptors on tumor and immune cells and boosts anti-tumor immunity. However, the use of extracellular ATP in cancer therapy is limited owing to its short half-life and rapid hydrolysis by ectoenzymes CD39 and CD73 into immune-suppressing adenosine. CD73 carries out the conversion of AMP to adenosine, making it a key regulator of this pathway. Using mice with CD73 knocked out globally (CD73KO), we show that combination of CD73 inhibition with HSV-P10 imparts significant survival benefit compared to WT mice treated with HSV-P10. Our findings will further the understanding of oHSV therapy and the role of the ATP/adenosine in the tumor microenvironment.
EXTH-78. CRISPR/CAS9 KNOCKOUT SCREENS UNRAVEL EPIGENETIC REGULATORS AS DRUGGABLE DEPENDENCIES FOR SONIC HEDGEHOG MEDULLOBLASTOMA
Authors: Tsiami Foteini , Piccioni Federica , Root David , Bandopadhayhay Pratiti , Segal Rosalind , Tabatabai Ghazaleh , Merk Daniel
Published: November 2022

Sonic hedgehog medulloblastoma (SHH-MB) is a malignant, highly heterogeneous brain tumor entity, accounting for 30% of all MBs in the pediatric population and is characterized by aberrant activation of the canonical SHH signaling pathway. Although current therapies targeting Smoothened (Smo) have proven a promising treatment approach for SHH-MB patients, pre-existing or acquired resistance impedes its clinical efficacy. Therefore, novel targeted approaches that overcome mechanisms of resistance are urgently needed. Here, we performed a genome-wide CRISPR/Cas9 knockout screen in a murine and a human SHH-MB cell line, SMB21 and DAOY, respectively, in order to decipher tumor-specific genetic essentialities. Our data demonstrate that SMB21 cells highly depend on key mediators of the SHH pathway, such as Gli2) for their proliferation, as opposed to DAOY cells, suggesting that the latter does not represent a faithful model of SHH-MB. Among other dependencies for SHH-MB are members of the epigenetic machinery such as Dnmt1) and Smarca5). Pharmacologically, we show that DNMT1 inhibition is efficacious at clinically relevant concentrations against Smo inhibitor- sensitive, as well as resistant SHH-MB cell lines. By performing RNA sequencing of SMB21 cells, we identified early and late alterations in global gene expression induced by DNMT1 inhibition, including decreased expression of positive regulators of SHH signaling. An additional knockout drug screen in SMB21 cells unraveled synthetic lethal interactors for DNMT1 inhibitors, as validated in vitro) by drug combination treatments. Further global DNA methylation profiling in SMB cells will help to define the molecular basis of sensitivity to DNMT1 inhibitors in SHH-MB. Last but not least, genetic ablation of epigenetic regulators and combinatorial treatments using DNMT1 inhibition will be investigated in in vivo) established mouse models of SHH-MB. Summarizing, our data indicate the potential of inhibiting epigenetic regulators as novel therapeutic avenues in SHH-MB sensitive, as well as resistant to Smo inhibition.
EXTH-85. TARGETING CLAUDIN 6 WITH CAR T CELL THERAPY FOR ATYPICAL TERATOID/RHABDOID TUMOR
Authors: Madsen Peter , Stern Allison , Griffin Crystal , Oehm Petra , Rengstl Benjamin , Flemming Carina , Resnick Adam C , Storm Philip B , Foster Jessica
Published: November 2022

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive brain tumor that predominantly affects young children and has an average 5-year survival under 50%. Novel, targeted therapies are desperately needed. Claudin 6 (CLDN6) is a tight junction protein present during development and expressed in up to 70% of ATRT specimens but not in normal tissue, making it a promising immunotherapeutic target. CLDN6-targeted chimeric antigen receptor (CAR) T cells in combination with a CAR T cell–amplifying mRNA vaccine have demonstrated antitumor activity against other CLDN6-expressing cancers in pre-clinical and phase I adult trial (NCT04503278; Haanen J et al AACR, 2022). To assess the effectiveness of CLDN6-targeted CAR T cells against ATRT, we utilized a second-generation mRNA CAR with a 4-1BB costimulatory domain and single-chain variable fragment against CLDN6 (Reinhard et al, 2020). Patient-derived ATRT specimens were assessed by RNAseq for CLDN6 expression (mean FPKM= 11.4) and by immunohistochemistry (positive staining in 53% of specimens). Tumor-derived cell lines were validated for CLDN6 expression by flow cytometry. Co-culture of CLDN6-directed mRNA CAR T cells with ATRT cell line 7316-2187 resulted in tumor-specific cytotoxicity compared to CD19-directed control CAR T cells (92% versus 15% at 10:1, p< 0.0001; 86% versus 0% at 5:1, p< 0.0001). Similar results were seen with ATRT cell line 7316-2141 (75% versus 7% at 10:1, p< 0.0001; 53% versus 0% at 5:1, p< 0.0001). Both CLDN6- and CD19-directed CAR T cells showed no cytotoxicity against CLDN6-negative cell line 7316-4149. Patient-derived xenograft models were also created through intracranial injection of multiple ATRT patient cell lines, and ongoing work will evaluate locoregional administration of CLDN6-directed CAR T cells in orthotopic xenograft models to test in vivo efficacy. This work highlights the potential for targeting CLDN6 via CAR T cell therapy in patients with ATRT as a novel therapeutic strategy for these devastating tumors.
EXTH-87. DECITABINE POTENTIATES MENINGIOMA IMMUNOTHERAPY TARGETING NY-ESO-1
Authors: Sun Matthew , Orpilla Joey , Contreras Erick , Imbroane Marisa , Treger Janet , Wang Anthony , Ko MyungJun , Vitte Jeremie , Giovannini Marco , Liau Linda M , Prins Robert , Everson Richard G
Published: November 2022

INTRODUCTION

No immunotherapy exists for meningiomas. NY-ESO-1 is a potential target because in meningiomas it’s the most frequently expressed Cancer-Testis-antigen. Decitabine is a hypomethylating agent known to upregulate CT-antigens. We tested the effects of decitabine on NY-ESO-1 expression in primary and immortalized meningioma cultures, and its impact on the efficacy of NY-ESO-1 T-cell-receptor-transduced T-cells(TCRs), to understand its potential role in meningiomas immunotherapy.

METHODS

We established primary meningiomas cultures from fresh tumor specimens and quantified NY-ESO-1 expression using Western blot and qRT-PCR. Cultures were pre-treated with 1uM of decitabine for at least 48 hours before assays. In vitro cytolysis was measured using the xCelligence Real-Time Cell Analyzer System in meningioma co-cultures with MHC-Class-I(HLA-A*0201)–restricted NY-ESO-1(157 – 165)-specific TCRs.

RESULTS

Nine primary and three immortalized meningioma cultures(Grade I – III) were evaluated for baseline NY-ESO-1 expression. High expression was defined as > 100-fold overexpression compared to glioma(U251) cultures. Decitabine treatment significantly increased NY-ESO-1 expression in 6 primary and 2 immortalized(SF1335, grade 1; IOMM-LEE, grade 3) cultures with low baseline expression(median increase over 100 fold; p > 0.001); whereas it slightly decreased NY-ESO-1 expression in 3 primary and 1 immortalized culture(CH157, WHO grade 3) with high baseline expression(median decrease 20%;p< 0.001). Decitabine pre-treatment of meningioma before co-culture with NY-ESO-1 TCRs at a ratio of 1:1, increased tumor cytolysis for SF1335, from 20% to 40%, and for IOMM-LEE, from 0% to 85%, measured at 10 hours. NY-ESO-1 expression increased 5000-fold vs 10-fold for IOMM-LEE vs SF1335, respectively. Decitabine treatment also increased MHC-Class-I expression for IOMM-LEE but not SF1335. Decitabine pre-treatment did not change cytolysis of CH157 at 65%(10 hrs).

CONCLUSIONS

Decitabine upregulates NY-ESO-1 expression in meningiomas with low baseline expression, and it increases cytolytic effects of NY-ESO-1 TCRs proportional to NY-ESO-1 and MHC-Class-I molecule upregulation. Decitabine may be a clinically feasible adjunct to meningioma immunotherapy.
EXTH-95. UNCOVERING THERAPEUTIC VULNERABILITIES IN MISMATCH REPAIR-DEFICIENT GLIOMAS
Authors: Pal Sangita , Boynton Adam , Johnston Ryan , Currimjee Naomi , Qian Kenin , Touat Mehdi , Bellamy Charlotte , Ho Patricia , Root David , Persky Nicole , Goodale Amy , Berstler Jim , Miller Lisa , Guletsky Alex , Cortes-Cros Marta , Ligon Keith , Beroukhim Rameen , Bandopadhayay Pratiti
Published: November 2022

Approximately 25% of recurrent gliomas exhibit hypermutation, which is most often acquired in chemotherapy sensitive gliomas post-treatment with standard of care alkylating agent temozolomide, Intriguingly, nearly all of these recurrent hypermutant tumors harbor deficiencies in mismatch repair (MMR) genes. Unlike other MMR-deficient cancers, hypermutant gliomas do not exhibit detectable microsatellite instability (MSI) at the population level, and do not share similar dependencies. Thus, strategies to therapeutically target MMR-deficient and hypermutant gliomas are urgently needed, and likely to impact many patients. To evaluate vulnerabilities associated with MMR-deficient gliomas, we have generated isogenic MMR-deficient models by systematic ablation of core MMR genes MSH2, MSH6, MLH1, and PMS2, in multiple patient-derived glioma cell lines using a robust all-in-one CRISPR-Cas9 system. We characterized these isogenic MMR-deficient glioma lines in comparison to respective MMR-proficient control line by performing differential gene expression analysis and subsequent gene set enrichment analyses. Our analyses reveal in an unbiased fashion an enrichment of several hallmark gene sets including cell cycle control and DNA repair in the MMR-deficient cell lines. We performed a CRISPR-Cas9 knockout screen in these MMR deficient and hypermutant models and identified candidate genes involved in DNA repair, cell cycle, RNA metabolism and other pathways as preferential dependencies in the MMR-deficient glioma cells. We also screened multiple MMR-deficient isogenic models against the high-throughput drug repurposing (REPO) chemical library and our preliminary results reveal a number of compounds from a variety of drug classes that selectively kill the MMR-deficient glioma cells, indicating that the loss of MMR confers differential dependencies to these small molecule inhibitors. The candidate drugs and genes identified from these high-throughput assays are currently being subjected to further validation, and they possibly will help us identify novel therapeutic strategies or improve existing therapeutic strategies to target recurrent MMR-deficient gliomas.
EXTH-97. BONE MARROW-DERIVED ENGINEERED MYELOID CELLS ENHANCE THE TRAFFICKING AND ACTIVATION OF CYTOTOXIC T CELLS IN THE GLIOMA MICROENVIRONMENT
Authors: Canella Alessandro , Nazzaro Matthew , Schmitt Claire , Rajendran Sakthi , Haffey Abigail , Nigita Giovanni , Thomas Diana , Rajappa Prajwal
Published: November 2022

INTRODUCTION

Gliomas are the most prevalent type of brain tumors in the adolescent and young adult population (AYA) and one of the leading causes of cancer- related death. Two-third of glioma AYA patients are affected by low-grade gliomas (LGGs), but yet there are no specific treatments. Therefore, a significant percentage of LGG patients experience tumor recurrence and malignant progression to high-grade glioma (HGG) and the immune microenvironment plays a critical role in mediating mechanisms of tumor progression and treatment resistance. Specifically, tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) have been shown to restrict cytotoxic effector trafficking and activation. To that end, first-line immunotherapies have limited efficacy in improving outcomes for HGGs.

HYPOTHESIS

We investigated the preclinical efficacy of a novel immunotherapeutic approach for the treatment of LGGs in AYA mice to modulate and reprogram the immunosuppressive tumor microenvironment (TME) and enhance the trafficking of activated effector T cells.

METHODS

Immunocompetent RCAS n/tv-a LGG bearing animals were treated at day 28 with a single retroorbital dose of bone-marrow derived myeloid cells, engineered for the release of Interlekin-2 (GEMys-IL2). The treatment was well tolerated and no sign of toxicity was noted.

RESULTS

Our results demonstrated that GEMys-IL2 efficiently crossed the BBB, infiltrate the glioma microenvironment, and activate infiltrating immune cells while altering the TME underscored by an increased CD8+ T cell population. Of note, the RCAS/t-va immunocompetent LGG to HGG progression mouse model treated with a single dose of GEMys-IL2 demonstrated a transient arrest of tumor burden within 14 days post treatment, which was associated with a significant advantage in overall survival.

CONCLUSIONS

These results highlight the pre-clinical efficacy and anti-tumoral therapeutic activity of GEMYys-IL2 on CD8+ T cell trafficking and activation to the TME in low-grade glioma in vivo, thus supporting its potential as a novel cell-mediated innate immunotherapy.
EXTH-102. PLASMA AND CEREBROSPINAL FLUID (CSF) PHARMACOKINETICS (PK) OF MIRDAMETINIB IN A NON-HUMAN PRIMATE (NHP) MODEL
Authors: McCully Cynthia Lester , Shearer Todd , Gross Andrea , Langseth Abraham , Peer Cody , Killoran Kristin , Garcia Rafael Cruz , Figg William , Widemann Brigitte
Published: November 2022

Mirdametinib is a MEK inhibitor with reported CSF penetration in pre-clinical models. Murine studies reported ERK phosphorylation inhibition in brain tissue at 1.15 nM (0.73 ng/mL) and tumor cell lines at 0.33–0.59 nM (0.16-0.28 ng/mL). A phase II clinical trial evaluating mirdametinib for neurofibromatosis type 1-related plexiform neurofibromas reported a 42% partial response rate and a mean plasma exposure (AUC0-12h) of 443 h*ng/mL. This study determined the plasma and CSF pharmacokinetic (PK) profile of mirdametinib in a non-human primate (NHP) model where CSF penetration serves as a proxy for CNS penetration.

METHODS

Four NHP received mirdametinib PO q.d, 0.50 mg/kg, in serial studies as a single dose (Single-Dose), three doses (Multiple-Dose), or IV, 0.20 mg/kg, followed by paired plasma and CSF sample collections through 96 hours. Mirdametinib was quantified by LC-MS/MS. PK parameters were calculated via noncompartmental methods. Plasma protein binding was determined by rapid equilibrium dialysis.

RESULTS

Mean □ standard deviation values reported. Three subjects were evaluable for both plasma (total and unbound) and CSF drug concentrations. Plasma – AUClast: Single-Dose 500.3 □ 253.4 and IV 552.3 □ 43 h*ng/mL; AUCtau: Multiple-Dose 456.3 □ 120.6 h*ng/mL. CSF Cmax (ng/mL): Single-Dose 0.43 □ 0.18, IV 2.0 □ 1.8, Multiple-Dose 0.56 □ 0.18. CSF Penetration (%): Single-Dose 1.3 □ 0.45, IV-1.6 □ 0.95 (AUCcsf-last:AUCplasma-last); Multiple-Dose 1.3 □ 0.56 (AUCcsf-tau:AUCplasma-tau). CSF/unbound plasma ratios ranged from 2.5-3.0 Plasma free fraction (%): 0.52 □ 0.1.

CONCLUSION

Notable mirdametinib CSF penetration was demonstrated in this NHP model. A higher CSF to unbound plasma ratio suggests that the efflux of mirdametinib from the CSF was delayed. The NHP CSF Cmax approached the ERK phosphorylation inhibition concentrations previously reported. The ability of this NHP model to predict human PK parameters was demonstrated via the comparable NHP to patient plasma exposure reported in the Phase II clinical trial.