Murine Glioblastoma Model Research Articles

EPCO-22. SINGLE CELL ANALYSIS TO UNDERSTAND SEX DIFFERENCES IN GBM OUTCOME

Abstract Males have a higher incidence and mortality in certain cancers compared to females. The same is true in glioblastoma (GBM) regardless of age, suggesting that cell-intrinsic factors rather than extrinsic factors such as circulating sex hormones are causing these sex differences. Our lab has demonstrated cell-intrinsic sex differences with respect to growth, clonogenicity, and in vivo tumorigenesis using an isogenic murine astrocyte cell culture model of GBM (Nf1 -/-; DNp53). Male astrocytes are more likely to adapt and grow when exposed to irradiation-induced stress compared to female astrocytes, which are more likely to undergo growth arrest after the same exposures. These sex differences in response to treatment can be explained by the different chromatin and transcriptional profiles established from the moment of fertilization that can result in different levels of transcriptional heterogeneity, a well-established cause for resistance to induced stress in GBM. However, so far sex has not been considered when exploring transcriptional heterogeneity. As such, we used 10x Genomics technology to extract single nuclei RNA (snRNA) and ATAC (snATAC) sequencing from the same nuclei and identified different levels of transcriptional heterogeneity in male and female astrocytes cells from our model. We also identified different chromatin profiles of crucial genes, such as Sox2, an important TF for the reprogramming of differentiated GBM into stem-like cells, which can then form tumors in vivo. We expect for the male astrocytes to be more effective in escaping growth arrest and adapting to irradiation-induced stress because of these different transcriptional and chromatin profiles. Our work will identify epigenetic mechanisms by which male cells are more able to adapt to treatment-induced stress compared to female cells and will further advance our understanding of sex differences in treatment-response in GBM and cancer overall, while giving avenues for the development of novel treatments.

EXTH-46. RNA LIPID PARTICLES INDUCE BI-DIRECTIONAL IMMUNITY AGAINST DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is uniformly fatal. Upon diagnosis, DIPG cannot be safely debulked and systemic therapies have unproven benefit. Immunotherapy can overcome these obstacles, but objective responses are rare; and cancer antigens, even when tumor specific (i.e. H3K27M), are usually poorly immunogenic. OBJECTIVE We sought to develop a murine model of diffuse midline glioma (DMG) that better recapitulates human disease. In these models, we tested different iterations of mRNA loaded nanoparticle (NP) vaccines for their therapeutic effect. APPROACH: We implanted murine gliomas (expressing H3K27M mutation) midline into developing neonatal brains. We manufactured different mRNA constructs encoding for foreign proteins (e.g. GFP), H3K27M or wildtype H3K27 and evaluated immunogenicity and anti-tumor efficacy. These mRNA vaccines were layered into lipid particles (RNA-NP) and administered intravenously when animals became acutely symptomatic (similar to when patients are diagnosed). RESULTS Surprisingly, all mRNA constructs tested were sufficient to elicit anti-tumor efficacy against DMGs. This was true for both antigen specific (H3K27M) and non-antigen specific constructs (GFP, H3K27 wildtype). While H3K27M encoding RNA-NPs were superior to H3K27 wildtype encoding RNA-NPs (p=0.057), the adaptive immune effects appear marginal relative to the innate immune responses generated by all RNA-NP constructs. These innate responses are characterized by induction of type I interferon response from plasmacytoid DCs. Interestingly, these effects could not be recapitulated in murine models of adult type glioblastoma (KR158b) which required antigen specificity for induction of anti-tumor efficacy. CONCLUSION These data suggest that pediatric gliomas may be particularly sensitive to innate immunity. We have optimized development of a target H3K27M mRNA construct that balances innate and adaptive bi-directional immune induction. We are completing FDA-IND enabling data to support translation of H3K27M loaded RNA-NPs into human clinical trials.

EPCO-29. KLF5 IS REQUIRED FOR THE SEX-DIFFERENCES ACROSS MULTIPLE TUMORIGENIC PHENOTYPES IN PEDIATRIC GLIOBLASTOMA (GBM)

Abstract High-grade glioblastoma (GBM) is a highly heterogeneous, malignant cancer with no cure to date. A sex-biased incidence and therapeutic response is reported (1) for GBM with a male to female incidence ratio of 1.6:1 and female patients having a 4-6 month survival advantage (2) compared to male patients. In our current work, we sought to determine the molecular basis for the sex-bias in incidence and therapeutic response using an isogenic murine model of GBM with dual loss of neurofibromin 1 (NF1) and P53 (DNP53) in astrocytes. Our model yielded GBM from the murine astrocytes in a sex-biased manner, with male GBM cells exhibiting a highly significant proliferative, migratory, in-vivo tumorigenicity, stem-cell enrichment rate and therapeutic resistance compared to female GBM cells. Using transposon calling cards, we mapped Brd4 enhancer usage across the genome in male, compared to female GBM cells and identified key transcription factors that differentially enriched at male-biased, compared to female-biased Brd4 bound enhancers. Interestingly, female-biased Brd4-bound enhancers were enriched for transcription factors with tumor suppressor roles including P53 and SMAD4, whereas male-biased Brd4-bound enhancers were enriched for transcription factors with oncogenic roles, including Oct4, MYC and KLF5. Genetic or chemical silencing of KLF5 reduced the tumorigenic potential in male GBM with rates of cellular proliferation, survival, migration and stem-cell frequency significantly reduced to levels that resemble female GBM. Using transposon calling cards, we mapped KLF5 genomic localization in male and female GBM cells and identified significant peaks that affiliated with differentially expressed genes between the sexes. Interestingly, genes induced by KLF5 in the male cells were affiliated with poor survival in patients with various cancers, whereas those induced by KLF5 in the female cells were affiliated with favorable prognosis. This signifies the need to explore molecular mechanisms by which KLF5 drives these sex differences in GBM.

The Impact of Chemoradiotherapy on Biophysical Properties of Glioblastoma Utilizing a Novel Murine Model

<h3>Purpose/Objective(s)</h3> Glioblastoma, historically viewed as a single disease entity, is now known to be composed of various subtypes. Here, we present our work evaluating the effects of chemoradiotherapy on cellular biophysical processes using a novel murine model of glioblastoma developed by our group. <h3>Materials/Methods</h3> Primary tumor cell lines were established from a murine glioma model generated from a synthetic DNA transposon system delivered oncogenic drivers SV40-LgTA and either NRASV12 or PDGFB. We have previously shown these cell lines, termed NRAS and PDGF, respectively, recapitulate the mesenchymal and proneural tumor subtypes. Treatment was administered with temozolomide (TMZ) to a maximum of 100µM, single fraction radiation therapy (RT) to a maximum of 10 Gy, or both. Cells were stained with Hoechst dye, imaged with an epifluorescence microscope with 10X objective lens, and counted with an image segmentation algorithm daily for 4 days to estimate cell survival and proliferation. On post-treatment day 4, surviving cells were plated on Matrigel coated 9.3kPa polyacrylamide gel and imaged with an inverted, transmitted light microscope with 10X objective lens every 5 minutes for 4 hours. Single cell mean squared displacement (MSD) was measured from time lapse images via an image segmentation algorithm. The random motility coefficient (µ) was calculated by the relationship MSD(t) = 4µt, where t is time. Data was compared via the Kruskal-Wallis test with Bonferroni correction for multiple comparisons when appropriate. <h3>Results</h3> There was no significant difference in cellular death or proliferation between treatment and control arms for the PDGF cell lines. In the NRAS cell lines, TMZ alone did not induce cell death while RT (P < 0.01) and combined modality therapy (P < 0.01) yielded cellular killing in a dose dependent manner. Furthermore, RT-treated NRAS cells proliferated more slowly than non-treated cells (0.05 vs. 0.20 hr-1, P=0.04). Control NRAS cell lines migrated faster than their PDGF counterparts at baseline (3.82 vs. 0.73 µm2/hr, P<1E-5). Within each cell line, the use of combined modality therapy significantly reduced cellular motility (PDGF: 0.73 vs. 0.21 µm2/hr, P=0.008; NRAS: 0.65 vs. 3.82 µm2/hr, P<1E-5). RT monotherapy additionally reduced motility in NRAS cell lines (1.07 vs. 3.82 µm2/hr, P<1E-5) while no difference was observed in PDGF cells. TMZ monotherapy did not affect motility within either cell line. <h3>Conclusion</h3> We evaluated biophysical properties of a novel murine glioma model utilizing oncogenic drivers overexpressing NRASV12 or PDGFB which we have previously shown recapitulates the mesenchymal and proneural subtypes of human glioblastoma, respectively. Here, we observed phenotypic differences in cellular migration following RT and TMZ regimens which could have subtype-specific ramifications for radiotherapy dose and target volume delineation.

PEGylation of Phosphatidylglycerol/Docosahexaenoic Acid Hexosomes with d-α-Tocopheryl Succinate Poly(ethylene glycol)2000 Induces Morphological Transformation into Vesicles with Prolonged Circulation Times.

Considering the broad therapeutic potential of omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA), here we study the effect of PEGylation of DHA-incorporated hexosomes on their physicochemical characteristics and biodistribution following intravenous injection into mice. Hexosomes were formed from phosphatidylglycerol and DHA with a weight ratio of 3:2. PEGylation was achieved through the incorporation of either d-α-tocopheryl succinate poly(ethylene glycol)2000 (TPGS-mPEG2000) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol)2000 (DSPE-mPEG2000) at a concentration of 1.5 wt %. Nanoparticle tracking analysis, synchrotron small-angle scattering, and cryo-transmission electron microscopy were employed to characterize the nanodispersions. The results show that PEGylated lipids induce a structural transition from an inverse hexagonal (H2) phase inside the nanoparticles (hexosomes) to a lamellar (Lα) phase (vesicles). We also followed the effect of mouse plasma on the nanodispersion size distribution, number, and morphology because changes brought by plasma constituents could regulate the in vivo performance of intravenously injected nanodispersions. For comparative biodistribution studies, fluorescently labeled nanodispersions of equivalent quantum yields were injected intravenously into healthy mice. TPGS-mPEG2000-induced vesicles were most effective in avoiding hepatosplenic clearance at early time points. In an orthotopic xenograft murine model of glioblastoma, TPGS-mPEG2000-induced vesicles also showed improved localization to the brain compared with native hexosomes. We discuss these observations and their implications for the future design of injectable lyotropic nonlamellar liquid crystalline drug delivery nanosystems for therapeutic interventions of brain and liver diseases.

BSCI-15 INVESTIGATING CD8 T CELL EXHAUSTION STATES WITHIN THE TME AND DRAINING LYMPH NODE OF PRIMARY BRAIN TUMORS AND BRAIN METASTASES

Abstract Brain metastases affect nearly 20% of all cancer patients. Likewise, glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Current immunotherapeutic efficacy is hindered by immunosuppression present in the brain tumor microenvironment (TME). T-cells, critical for tumor clearance, take on a functionally exhausted phenotype. Importantly, two exhaustion states, progenitor (Tpe) and terminal (Tte), have been identified in models of chronic infection and cancer. This distinction is particularly relevant, as Tpe can remain responsive to immune checkpoint blockade (ICB), while Tte cannot. To date, the dynamics and characteristics of these exhausted populations in primary tumors and brain metastases remain unclear. Using intracranially implanted murine models of GBM (CT2A) and metastatic melanoma (B16F10), Tpe and Tte were identified by flow cytometry as PD1+SLAMF6+ and PD1+TIM3+, respectively. Functional differences between subsets were evaluated via intracellular staining of IFNγ, TNFα, IL2, CD107a, and Ki67. To determine the role of antigen, we performed adoptive lymphocyte transfers of tumor-specific and non-tumor-specific transgenic T-cells into a TRP2 or OVA overexpressing intracranial CT2A or B16 tumor, respectively. Tte displayed higher cytotoxic molecule expression than Tte, consistent with chronic infection models. Key exhaustion-associated transcription factors were identified in exhaustion subsets, including Tox, Tcf7, T-bet, and Eomes. Tox and Tcf7 expression identified Tpe within tumor-draining lymph nodes, suggesting a potential origin outside of the tumor, and the capacity for rescued function. We observed a decline in the Tpe population over time, with an associated rise in Tte within both tumor types. Notably, the ratio of Tpe to Tte was higher at all time points in B16F10 compared to CT2A. Tte appeared only in tumor-specific T-cells of the TME, further confirming the tumor-antigen dependence of T cell exhaustion. Tpe may arise outside the TME in tumor-specific T-cells. Further study may reveal a means and time window for rescuing T-cell function with ICB for brain tumors.

STING activation promotes robust immune response and NK cell–mediated tumor regression in glioblastoma models

Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The stimulator of interferon genes (STING) DNA sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties. Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils, and natural killer (NK) populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, chimeric antigen receptor T cells, NK therapies, and immune checkpoint blockade.

GBP3 promotes glioblastoma resistance to temozolomide by enhancing DNA damage repair.

Glioblastoma is the most common malignant brain cancer with dismal survival and prognosis. Temozolomide (TMZ) is a first-line chemotherapeutic agent for glioblastoma, but the emergence of drug resistance limits its anti-tumor activity. We previously discovered that the interferon inducible guanylate binding protein 3 (GBP3) is highly elevated and promotes tumorigenicity of glioblastoma. Here, we show that TMZ treatment significantly upregulates the expression of GBP3 and stimulator of interferon genes (STING), both of which increase TMZ-induced DNA damage repair and reduce cell apoptosis of glioblastoma cells. Mechanistically, relying on its N-terminal GTPase domain, GBP3 physically interacts with STING to stabilize STING protein levels, which in turn induces expression of p62 (Sequestosome 1), nuclear factor erythroid 2 like 2 (NFE2L2, NRF2), and O6-methlyguanine-DNA-methyltransferase (MGMT), leading to the resistance to TMZ treatment. Reducing GBP3 levels by RNA interference in glioblastoma cells markedly increases the sensitivity to TMZ treatment in vitro and in murine glioblastoma models. Clinically, GBP3 expression is high and positively correlated with STING, NRF2, p62, and MGMT expression in human glioblastoma tumors, and is associated with poor outcomes. These findings provide novel insight into TMZ resistance and suggest that GBP3 may represent a novel potential target for the treatment of glioblastoma.

Abstract 5329: Pt(IV)-M13, a blood-brain-barrier penetrant macrocyclic peptide-platinum(IV) conjugate, leads to enhanced drug uptake and efficacy in murine glioblastoma models

Abstract INTRODUCTION. Glioblastoma is the most aggressive primary malignant brain tumor and has a median survival of approximately 15 months. Treatment is hindered by the blood-brain barrier (BBB) that restricts entry of the vast majority of cancer therapeutics to the brain. We have designed a peptide-drug conjugate, (Pt(IV)-M13), comprised of M13, a perfluoroaryl-stapled cell-penetrating peptide, covalently linked to Pt(IV) via an amide bond. Upon intracellular reduction the Pt(IV) prodrug releases active cisplatin eliciting DNA damage and cell death. To investigate the properties of Pt(IV)-M13 in detail, we determined in vitro cell killing, platinum brain accumulation, biodistribution, and efficacy in mouse models. METHODS. Pt(IV)-M13 tumor cytotoxicity was assessed in vitro using standard approaches. In vivo platinum levels were measured in the brain by ICP-MS, and BBB penetration assessed through a multicellular 3D in vitro BBB model, and biodistribution to peripheral organs was determined. Toxicity from bi-weekly injections of the Pt(IV)-M13 platinum conjugate was monitored from 5mg/kg to 30mg/kg. Animal survival was determined in a glioblastoma xenograft model. DNA damage was quantified by γH2AX staining. RESULTS. Pt(IV)-M13 possesses in vitro tumor cell killing effects similar to cisplatin and Pt(IV) alone. Pt(IV)-M13 showed increased BBB penetration when compared to cisplatin in our in vitro BBB spheroid model (&amp;gt;20-fold), in brain tissue (7-fold) and glioblastoma tumor-bearing mice (8-fold). Five hours after intravenous injection, biodistribution of platinum delivered by Pt(IV)-M13 was higher than cisplatin in the spleen, but lower in the heart. Bi-weekly injections of Pt(IV)-M13 were well-tolerated in nude mice to at least 15mg/kg, three times the reported cisplatin MTD. Finally, Pt(IV)-M13 treatment increased survival in a murine glioblastoma xenograft model (median survival= 33 days for Pt(IV)-M13 vs. 22.5 days for cisplatin and 24 days Pt(IV) alone. Elevated γH2AX foci were observed in Pt(IV)-M13 treated mice providing direct support for the improved delivery of cisplatin to brain tumors using this approach. CONCLUSION. Pt(IV)-M13 leads to enhanced BBB penetration and cisplatin accumulation in glioblastoma models, with consequent animal survival benefit. Overall, our data support further investigation and development of Pt(IV)-M13 for glioblastoma treatment. Future studies will include additional glioblastoma models, and further optimization of this approach. Citation Format: Jorge-Luis Jimenez-Macias, Yen-Chun Lee, Tomer Finkelberg, Gilles Berger, Michal Nowicki, Choi-Fong Cho, Bogdan Fedeles, Andrei Loas, Brad Penetelute, Sean E. Lawler. Pt(IV)-M13, a blood-brain-barrier penetrant macrocyclic peptide-platinum(IV) conjugate, leads to enhanced drug uptake and efficacy in murine glioblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5329.

Abstract 2057: Utilizing ‘viral mimicry’ as a novel therapeutic approach in conjunction with anti-PD-1 immunotherapy increases immune activation and extends survival in a glioblastoma murine model

Abstract Therapeutic advances of glioblastoma (GBM) in the last two decades have only extended survival by a few months, maintaining a five-year survival rate of less than five percent. While immunotherapy has shown promise for other types of cancer, it has proved less efficacious as a monotherapy in GBM, partly due to a suppressive immune microenvironment. Being able to stimulate anti-tumor immune activity through alternative methods may allow for a more successful immunotherapeutic response. One such method is by disengaging epigenetic maintenance of repetitive element (RE) silencing. It was recently shown that the epigenetic pathway, FBXO44/SUV39H1, could be targeted specifically in cancer cells to release inhibition of REs such as endogenous retroviruses and retrotransposons. This effect resulted in increased activation of antiviral pathways and interferon signaling. We hypothesized using this treatment in conjunction with anti-PD-1 immunotherapy would allow for a synergistic effect against GBM tumor growth and increased survival. We therefore treated mice with intracranially implanted GL261 cells with a combined therapy of anti-PD-1 and SUV39H1 inhibition. One week after treatment started, we used single cell analysis using multiplexed flow cytometry to look at different immune populations. The largest percentage of immune cells comprised macrophages; with multiplex flow analysis, we were able to differentiate activated microglia (the brain resident immune cell) versus bone marrow derived macrophages (BMDMs; macrophages derived from the blood) based on CD49d expression. While no differences were found in overall macrophage numbers, the percentage of microglia was significantly higher than BMDMs. Strikingly, expression of PD-1 was significantly higher on microglia compared to BMDMs, while CD206, a marker of immune suppression, was significantly higher on the latter. T cells were also examined, with overall numbers again, not significantly different between treatment groups. However, those from mice treated with either the SUV39H1 inhibitor alone or in combination with anti-PD-1 had significantly higher levels of the co-stimulatory molecule CD40L on both CD4+ and CD8+ T cells. Lastly, survival analysis revealed a significant increase in survival with combinatorial treatment compared to either therapy alone or no treatment control. These results indicate the efficacy of an alternative treatment method working synergistically with anti-PD-1 immunotherapy to stimulate the immune system and slow GBM progression in a murine model. Citation Format: Katie B. Grausam, Jia Z. Shen, Joshua J. Breunig, Charles Spruck, Stephen L. Shiao. Utilizing ‘viral mimicry’ as a novel therapeutic approach in conjunction with anti-PD-1 immunotherapy increases immune activation and extends survival in a glioblastoma murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2057.

Abstract 3238: GBP3 promotes glioblastoma resistance to temozolomide by enhancing DNA damage repair

Abstract Glioblastoma is the most common malignant brain cancer with dismal survival and prognosis. Temozolomide (TMZ) is a first-line chemotherapeutic agent for glioblastoma, but the emergence of drug resistance limits its anti-tumor activity. We previously discovered that the interferon inducible guanylate binding protein 3 (GBP3) is highly elevated and promotes tumorigenicity of glioblastoma. Here, we show that TMZ treatment significantly upregulates the expression of GBP3 and stimulator of interferon genes (STING), both of which increase TMZ-induced DNA damage repair and reduce cell apoptosis of glioblastoma cells. Mechanistically, relying on its N-terminal GTPase domain, GBP3 physically interacts with STING to stabilize STING protein levels, which in turn induces expression of p62 (Sequestosome 1), nuclear factor erythroid 2 like 2 (NFE2L2, NRF2), and O6-methlyguanine-DNA-methyltransferase (MGMT), leading to the resistance to TMZ treatment. Reducing GBP3 levels by RNA interference in glioblastoma cells markedly increases the sensitivity to TMZ treatment in vitro and in murine glioblastoma models. Clinically, GBP3 expression is high and positively correlated with STING, NRF2, p62, and MGMT expression in human glioblastoma tumors, and is associated with poor outcomes. These findings provide novel insight into TMZ resistance and suggest that GBP3 may represent a novel potential target for the treatment of glioblastoma. Citation Format: Ming Li, Hui Xu. GBP3 promotes glioblastoma resistance to temozolomide by enhancing DNA damage repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3238.

Abstract 4184: RNA-seq analyses reveal remodeling of tumor microenvironment and reversal of glioma resistance to oncolytic viruses by targeting immunometabolism

Abstract Viroimmunotherapy aims to infect cancer cells to elicit anti-tumor immune responses. In clinical trials, glioma treatment with oncolytic viruses induced durable clinical responses in a small fraction of patients. To improve the percentage of responders, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. Thus, we engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus that carries the cDNA of the T-cell activator, OX40L. In this work, we observed that Delta-24-RGDOX triggered a dramatic reshaping of the tumor microenvironment dominated by strong changes in immune processes as indicated by RNA-sequencing via ingenuity pathway analyses in a murine glioblastoma model. Paradoxically, network analyses revealed that Delta-24-RGDOX also induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of the cancer cells to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma bearing mice. Importantly, addition of the IDO inhibitor to Delta-24-RGDOX decreased the activation of the IDO network. IDO inhibition did not affect virus infection or replication in human or murine glioma cells. Flow cytometry assays revealed that the combination therapy increased the frequency of activated CD8+ T cells and decreased the presence of the immunosuppressive cell populations, MDSCs and Tregs. Gene set enrichment analyses confirmed the decrease of MDSCs and Tregs in the combination treated glioma-bearing mice compared to the virus alone. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens, and that this activation was reversed by kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing immunosuppression caused by virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Sagar Sohoni, Sanjay K. Singh, Yisel Rivera-Molina, Hong Jiang, Xuejun Fan, Joy Gumin, Frederick F. Lang, Christopher Alvarez-Breckenridge, Marta M. Alonso, Filipa Godoy-Vitorino, Lijie Zhai, Erik Ladomersky, Kristen L. Lauing, Derek A. Wainwright, Juan Fueyo, Candelaria Gomez-Manzano. RNA-seq analyses reveal remodeling of tumor microenvironment and reversal of glioma resistance to oncolytic viruses by targeting immunometabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4184.

Abstract 4235: PPRX-1701, a deliverable nanoparticle formulation of 6-bromo-acetoxime, blocks tumoral IDO1 expression and shows efficacy in immunocompetent murine glioblastoma models

Abstract Introduction: We previously showed that derivatives of the Chinese traditional medicine indirubin promote survival in murine models of the brain tumor glioblastoma. However, poor drug solubility hampered further development of this approach. Here we introduce PPRX-1701, a 6’-bromoindirubin acetoxime (BiA) containing drug/polymer nanoparticle formulation which can be injected intravenously at relatively high concentrations. Here we examine the efficacy and mechanism of PPRX-1701 in murine glioblastoma models. Methods: In vitro glioblastoma cell proliferation and migration assays were performed using standard approaches. Effects of PPRX-1701 on interferon-γ (IFN γ) signaling were determined by Western blotting. Delivery to murine GBM was assessed using a luminescent reporter assay, and survival studies were performed in immunocompetent murine glioblastoma models. Results: PPRX-1701 effects on glioblastoma cell proliferation and migration were indistinguishable from BiA alone. Our data show that intravenous administration of PPRX-1701 is well-tolerated and can reach intracranial murine glioblastoma as assessed by luminescent reporter assays. PPRX-1701 administration leads to improved survival in the GL261 glioblastoma mouse model (median survival 30 days (control), 47 days (treated), p &amp;lt; 0.0001). Treatment with PPRX-1701 was associated with alterations in the tumor immune microenvironment, with reduced Tregs and pro-tumor macrophages, and increased CD8+ T cells. Further mechanistic studies have shown that PPRX-1701/BiA blocks the expression of multiple immunosuppressive molecules in GBM downstream of interferon-γ (IFNγ) including PD-L1 and indoleamine 2,3-dioxygenase 1 (IDO1) - a key enzyme in the tryptophan-kynurenine-aryl hydrocarbon receptor (Trp-Kyn-AhR) immunosuppressive pathway. BiA promoted more effective T-cell mediated tumor cell killing using patient derived glioblastoma ex vivo co-culture models. Conclusion: This data supports further development of PPRX-1701 as a candidate immunotherapeutic agent for glioblastoma treatment. Ongoing pre-clinical studies are investigating combination with other relevant therapies. Citation Format: Mykola Zdioruk, Michal Nowicki, Bin Wu, Paul Boucher, Yuji Takeda, Weiyi Li, E Antonio Chiocca, Sean E. Lawler. PPRX-1701, a deliverable nanoparticle formulation of 6-bromo-acetoxime, blocks tumoral IDO1 expression and shows efficacy in immunocompetent murine glioblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4235.

Brain Tumor Imaging and Delivery of Sub-5 nm Magnetic Iron Oxide Nanoparticles in an Orthotopic Murine Model of Glioblastoma

Brain Tumor Imaging and Delivery of Sub-5 nm Magnetic Iron Oxide Nanoparticles in an Orthotopic Murine Model of Glioblastoma

HGG-25. Targeting KLF5 reduces tumorigenic phenotype in a murine glioblastoma (GBM) model

Sex differences are evident in the incidence, therapeutic response and survival of patients with various cancers including the most common, aggressive and incurable GBM. We generated a murine GBM model of male and female astrocytes with dual loss of NF1 and P53 that yielded a sex-biased transformation of the astrocytes with male cells being significantly more tumorigenic and therapeutically resistant compared to female cells. In our current work, we aimed to delineate the molecular mechanisms driving this sex-biased tumorigenic phenotype in order to deliver therapies that are more effective to patients with GBM. We examined the inhibition of KLF5 on tumorigenic phenotypes using cellular bio-functional assays. We used barcoded transposon calling cards to determine the genomic localization of KLF5 and the differentially induced gene expression in male versus female GBM cells. Chemical inhibition or shRNA knock-down of KLF5 significantly reduced proliferation, migration, clonogenic stem-cell frequency and survival, but increase apoptosis in male and female GBM cells. Interestingly, male, but not female, GBM cells exhibited an increased migratory phenotype after radiation that inhibition of KLF5 significantly reduced. Moreover, KLF5 inhibition significantly reduced the protein expression of tumorigenic PDGFRB, AKT, ERK and the stem marker Sox-2. Transposon calling cards mapped unique KLF5 genomic localization in male versus female GBM cells with significantly differential gene expression profiles between the two. The top genes induced by KLF5 in male cells were primarily affiliated with poorer prognosis and reduced survival, whereas in female cells they were affiliated with better prognosis and improved survival in patients with cancer. Our findings provide a promising exploratory avenue for KLF5 as a therapeutic target in GBM and warrants further investigation in order to delineate the precise molecular mechanisms driving this antitumor response so that targeted therapy would be more effective taking into consideration the sex-differences in patients with GBM.

JCI‑20679 suppresses the proliferation of glioblastoma stem cells by activating AMPK and decreasing NFATc2 expression levels.

The prognosis of glioblastoma, which is the most frequent type of adult‑onset malignant brain tumor, is extremely poor. Therefore, novel therapeutic strategies are needed. Previous studies report that JCI‑20679, which is synthesized based on the structure of naturally occurring acetogenin, inhibits mitochondrial complex I and suppresses the growth of various types of cancer cells. However, the efficacy of JCI‑20679 on glioblastoma stem cells (GSCs) is unknown. The present study demonstrated that JCI‑20679 inhibited the growth of GSCs derived from a transposon system‑mediated murine glioblastoma model more efficiently compared with the growth of differentiation‑induced adherent cells, as determined by a trypan blue staining dye exclusion test. The inhibition of proliferation was accompanied by the blockade of cell‑cycle entry into the S‑phase, as assessed by a BrdU incorporation assay. JCI‑20679 decreased the mitochondrial membrane potential, suppressed the oxygen consumption rate and increased mitochondrial reactive oxygen species generation, indicating that JCI‑20679 inhibited mitochondrial activity. The mitochondrial inhibition was revealed to increase phosphorylated (phospho)‑AMPKα levels and decrease nuclear factor of activated T‑cells 2 (NFATc2) expression, and was accompanied by a decrease in calcineurin phosphatase activity. Depletion of phospho‑AMPKα by knockdown of AMPKβ recovered the JCI‑20679‑mediated decrease in NFATc2 expression levels, as determined by western blotting and reverse transcription‑quantitative PCR analysis. Overexpression of NFATc2 recovered the JCI‑20679‑mediated suppression of proliferation, as determined by a trypan blue staining dye exclusion test. These results suggest that JCI‑20679 inhibited mitochondrial oxidative phosphorylation, which activated AMPK and reduced NFATc2 expression levels. Moreover, systemic administration of JCI‑20679 extended the event‑free survival rate in a mouse model transplanted with GSCs. Overall, these results suggested that JCI‑20679 is a potential novel therapeutic agent against glioblastoma.

Functional virus-specific memory CD8+ T cells survey glioblastoma

Abstract Glioblastoma multiforme (GBM) is among the most aggressive, treatment resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host, including the brain, and are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8+ T cells expressing tissue resident markers populate the mouse and human glioblastoma microenvironment. Reactivating virus-specific memory T cells through intra-tumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to anti-neoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific memory T cells are a significant part of the glioblastoma immune microenvironment and may be leveraged to promote anti-tumoral immunity. Supported by UMN SPORE Program Project Planning grant (DM, CCC), NCI 1R01CA238439 (DM), Humor to Fight the Tumor Foundation (JN), NCI 5P30CA023108-42 (PR)

All-stage targeted therapy for glioblastoma based on lipid membrane coated cabazitaxel nanocrystals.

Glioblastoma (GBM) is the most aggressive brain tumor with poor prognosis and frequent recurrence. The blood-brain barrier (BBB), blood-brain tumor barrier (BBTB) hinder the entry of therapeutics into the glioma region. Vasculogenic mimicry (VM) formed by invasive glioma cells is also related to recurrence of GBM. VAP is a D-peptide ligand of GRP78 protein overexpressed on BBTB, VM, and glioma cells but not on normal tissues. Besides, p-hydroxybenzoic acid (pHA) can effectively traverse the BBB. Herein we developed an all-stage glioma-targeted cabazitaxel (CBZ) nanocrystal loaded liposome modified with a "Y" shaped targeting ligand composed of pHA and VAP (pV-Lip/cNC). The pure drug nanocrystal core provided high drug loading, while lipid membrane promoted the stability and circulation time. pV-Lip/cNC exhibited excellent glioma homing, barriers crossing, and tumor spheroid penetrating capability in vitro. Treatment of pV-Lip/cNC displayed enhanced CBZ accumulation in glioma and anti-glioma effect with a median survival time (53days) significantly longer than that of cNC loaded liposomes modified with either single ligand (42days for VAP and 45days for pHA) in the murine orthotopic GBM model. These results indicated pV-Lip/cNC could traverse the BBB and BBTB, destruct VM, and finally kill glioma cells to realize all-stage glioma therapy.

P-243 - ImmunoPET imaging combined with focused ultrasound to study brain pharmacokinetic of 89Zr labeled anti-PD-L1 antibodies in a syngeneic murine model of glioblastoma

P-243 - ImmunoPET imaging combined with focused ultrasound to study brain pharmacokinetic of 89Zr labeled anti-PD-L1 antibodies in a syngeneic murine model of glioblastoma

Tumor Treating Fields dually activate STING and AIM2 inflammasomes to induce adjuvant immunity in glioblastoma.

Tumor Treating Fields (TTFields), an approved therapy for glioblastoma (GBM) and malignant mesothelioma, employ noninvasive application of low-intensity, intermediate-frequency, alternating electric fields to disrupt the mitotic spindle, leading to chromosome missegregation and apoptosis. Emerging evidence suggests that TTFields may also induce inflammation. However, the mechanism underlying this property and whether it can be harnessed therapeutically are unclear. Here, we report that TTFields induced focal disruption of the nuclear envelope, leading to cytosolic release of large micronuclei clusters that intensely recruited and activated 2 major DNA sensors — cyclic GMP-AMP synthase (cGAS) and absent in melanoma 2 (AIM2) — and their cognate cGAS/stimulator of interferon genes (STING) and AIM2/caspase 1 inflammasomes to produce proinflammatory cytokines, type 1 interferons (T1IFNs), and T1IFN-responsive genes. In syngeneic murine GBM models, TTFields-treated GBM cells induced antitumor memory immunity and a cure rate of 42% to 66% in a STING- and AIM2-dependent manner. Using single-cell and bulk RNA sequencing of peripheral blood mononuclear cells, we detected robust post-TTFields activation of adaptive immunity in patients with GBM via a T1IFN-based trajectory and identified a gene panel signature of TTFields effects on T cell activation and clonal expansion. Collectively, these studies defined a therapeutic strategy using TTFields as cancer immunotherapy in GBM and potentially other solid tumors.