Preclinical Models Of Glioblastoma Research Articles

Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging

The tumor microenvironment plays a crucial role in determining response to treatment. This involves a series of interconnected changes in the cellular landscape, spatial organization, and extracellular matrix composition. However, assessing these alterations simultaneously is challenging from a spatial perspective, due to the limitations of current high-dimensional imaging techniques and the extent of intratumoral heterogeneity over large lesion areas. In this study, we introduce a spatial proteomic workflow termed Hyperplexed Immunofluorescence Imaging (HIFI) that overcomes these limitations. HIFI allows for the simultaneous analysis of > 45 markers in fragile tissue sections at high magnification, using a cost-effective high-throughput workflow. We integrate HIFI with machine learning feature detection, graph-based network analysis, and cluster-based neighborhood analysis to analyze the microenvironment response to radiation therapy in a preclinical model of glioblastoma, and compare this response to a mouse model of breast-to-brain metastasis. Here we show that glioblastomas undergo extensive spatial reorganization of immune cell populations and structural architecture in response to treatment, while brain metastases show no comparable reorganization. Our integrated spatial analyses reveal highly divergent responses to radiation therapy between brain tumor models, despite equivalent radiotherapy benefit.

Abstract 2663: Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma

Abstract Despite advances in care in many cancer types, patients with glioblastoma (GBM) have a grim prognosis of 15 to 21 months and unchanged standard therapy since 2005. These tumors have a profoundly immunosuppressive tumor immune microenvironment. Immunotherapy, including the checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4, has transformed the treatment of many cancers but has failed in trials of both newly diagnosed and recurrent GBM. Tumor mutational burden (TMB) has been proposed as a predictor of response to ICI treatment, presumably through an increase in neoantigens that can be recognized by cytotoxic CD8+ T cells leading to tumor rejection. GBM typically has a low TMB, a feature speculated as a contributor to poor response to ICI treatment. However, cases with an extremely high TMB (i.e., patients with the biallelic mismatch repair deficiency syndrome) do show response to immunotherapy. To begin to investigate the impact of mutational burden on immune response in GBM, we created a murine syngeneic tumor model using CRISPR-Cas9 to knockout (KO) the mismatch repair protein Msh2, the lynchpin to mismatch recognition, in the SB28 GBM model that has a low TMB (108 mutations) at baseline. Msh2 KO increased the TMB from 2 to 12-fold depending on single cell sorted clone, resulting in between 150 and 200 predicted strong MHC-I binding neoantigens estimated by the NetMHC algorithm. Preliminary studies have demonstrated that in vitro proliferation is similar amongst low TMB and high TMB SB28 clones. Flank implantation of high TMB SB28 clones revealed slower growth compared to the low TMB SB28 tumors. Furthermore, treatment with ICIs did provide a survival advantage in this flank model. However, no survival advantage was noted when the high TMB clones were implanted intracranially either with or without ICI treatment. These results suggested that either there is poor immune cell trafficking to the intracranial tumors, or a robust immune response was causing increased intracranial pressure that is prematurely causing death. Use of dexamethasone starting at day 11 to control cerebral edema has shown some survival improvement with ICI treatment, supporting that an inflammatory response is at least partially responsible for the lack of improved survival in early studies. We are currently evaluating CD4+ and CD8+ T cell clonality, earlier timepoints to evaluate the immune infiltrate and anti-tumor immune response, and alterations in chemokines/cytokines in the tumor microenvironment. By creating high mutation burden clones from a GBM with a low mutational burden, we will be able to interrogate potential mechanisms of heightened immunogenicity that has been reported in other cancers. Citation Format: Kevin Breen, Tuesday Haynes, Masashi Watanabe, Mark Gilbert. Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2663.

Abstract 3222: Acute and long-term responses to treatment with the HDAC inhibitor quisinostat in preclinical models of glioblastoma

Abstract Introduction: High expression of histone deacetylases (HDACs), particularly HDAC1, is strongly associated with poor prognosis for glioblastoma (GBM) patients. Quisinostat is a second-generation, class I-II selective HDAC inhibitor with high specificity for HDAC1 and potent anti-tumor activity. Here, we evaluated GBM PDX tissues and glioma stem cells (GSCs) for cellular, transcriptomic and phenotypic changes after acute and long-term treatment with quisinostat. Methods: Multiple GBM PDX models and PDX-derived GSCs were treated with quisinostat, radiation, or a combination of quisinostat and radiation. Tissue and cells were processed for RNA-seq, qRT-PCR, and immunofluorescence assays to examine alterations in neuronal signaling, cell fate, DNA damage, and markers of stemness and differentiation. Results: Bulk RNA sequencing of tumor tissue revealed that chronic co-treatment with quisinostat and radiation led to increased cellular expression of neuronal signaling and neuronal development pathways compared to the untreated control group. Increase in expression of neuronal signaling genes was also observed upon in vitro treatment of GSCs with quisinostat. Immunocytochemistry and immunofluorescence staining confirmed changes in neuronal signaling and neuronal cell fate proteins after quisinostat treatment. Conclusions: Quisinostat treatment induces a shift in tumor cells towards a neuronal-like cell fate. Considering that GSCs have been shown to establish synaptic junctions with healthy neurons to evoke enhanced excitatory currents within glioma cells and contribute to treatment resistance, further investigation of this shift upon HDAC inhibition is crucial. Ongoing studies are aimed at evaluating the significance of these cellular and molecular changes resulting from quisinostat treatment and whether the quisinostat-induced cell fate changes can be exploited for future combination therapy for GBM. Citation Format: Keely Orndorff, James McNamara, Mariya Stavnichuk, Costanza Lo Cascio, Connor White, Ernesto Luna Melendez, Gozde Uzunalli, Nader Sanai, Shwetal Mehta. Acute and long-term responses to treatment with the HDAC inhibitor quisinostat in preclinical models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3222.

Abstract 4006: IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells

Abstract Introduction: EGFRvIII is a commonly studied tumor-specific antigen in glioblastoma (GBM) and poses an enticing target for immunotherapy. Its varied expression, however, complicates its effectiveness as a Chimeric Antigen Receptor (CAR) T cell target. Given the increasing prominence of Interleukin-12 (IL12) in treating solid tumors, its synergy with EGFRvIII specific CARs in treating preclinical models of GBM is explored. Methods: To simulate tumor heterogeneity, mice were implanted with equal ratios of CT2A and CT2A EGFRvIII+ cells. We developed EGFRvIII-specific third-generation CAR T cells, both with and without IL12 using a MSGV retrovirus backbone and transduced them into murine T cells. Their antitumor effectiveness was gauged by evaluating survival rates, and their capability to enhance and modify the tumor environment was determined using single-cell sequencing. Results: Standalone EGFRvIII CARs were unsuccessful in curing mice with heterogeneous tumors. However, 60% of mice treated with EGFRvIII CARs combined with IL12 were long term survivors. IL12 secreting CARs exhibited prolonged persistence in vivo, and there was no need for prior lymphodepletion before ALT introduction. Our studies reveal that EGFRvIII CARs secreting IL12 can eliminate EGFRvIII negative tumor cells by drawing on the inherent CD8 T cell compartment. Moreover, IL12 prominently impacted microglia among all Antigen Presenting Cells in the tumor environment, amplifying antigen presentation processes and broadening epitope exposure. Conclusion: By leveraging IL12, EGFRvIII CARs can counteract antigen heterogeneity in tumors by recruiting endogenous CD8 T cells and polarizing microglia. These IL12-secreting CARs outlast their traditional counterparts without requiring lymphodepletion. Harnessing CAR-T cells that secrete IL12 represents a novel strategy for overcoming tumor heterogeneity and treating GBM in a clinical context. Citation Format: Aditya Mohan, Steven Shen, Kelly Hotchkiss, Sarah Quackenbush, John Sampson, Peter Fecci, Anoop Patel. IL12-secreting CAR-T cells eliminate the requirement for lymphodepletion and promote epitope spreading via local antigen presenting cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4006.

EGFR amplification and EGFRvIII predict and participate in TAT-Cx43266-283 antitumor response in preclinical glioblastoma models.

Glioblastoma (GBM) commonly displays epidermal growth factor receptor (EGFR) alterations (mainly amplification and EGFRvIII) and TAT-Cx43266-283 is a Src-inhibitory peptide with antitumor properties in preclinical GBM models. Given the link between EGFR and Src, the aim of this study was to explore the role of EGFR in the antitumor effects of TAT-Cx43266-283. The effect of TAT-Cx43266-283, temozolomide (TMZ) and erlotinib (EGFR inhibitor) was studied in patient-derived GBM stem cells (GSCs) and murine neural stem cells (NSCs) with and without EGFR alterations, in vitro and in vivo. EGFR alterations were analyzed by Western blot (WB) and Fluorescence In Situ Hybridization (FISH) in these cells, and compared with Src activity and survival in GBM samples from TCGA. The effect of TAT-Cx43266-283 correlated with EGFR alterations in a set of patient-derived GSCs and was stronger than that exerted by TMZ and erlotinib. In fact, TAT-Cx43266-283 only affected NSCs with EGFR alterations, but not healthy NSCs. EGFR alterations correlated with Src activity and poor survival in GBM patients. Finally, tumors generated from NSCs with EGFR alterations, showed a decrease in growth, invasiveness and vascularization after treatment with TAT-Cx43266-283, which enhanced the survival of immunocompetent mice. Clinically relevant EGFR alterations are predictors of TAT-Cx43266-283 response and part of its mechanism of action, even in TMZ- and erlotinib-resistant GSCs. TAT-Cx43266-283 targets NSCs with GBM-driver mutations, including EGFR alterations, in an immunocompetent GBM model in vivo, suggesting a promising effect on GBM recurrence. Together, this study represents an important step towards the clinical application of TAT-Cx43266-283.

Antitumoral Activity of Cecropia Pachystachya Leaves Extract in Vitro and in Vivo Model of Rat Glioma: Brain and Blood Effects.

The aim of this study was to investigate the antiglioma effect of Cecropia pachystachya Trécul (CEC) leaves extract against C6 and U87 glioblastoma (GB) cells and in a rat preclinical GB model. The CEC extract reduced in vitro cell viability and biomass. In vivo, the extract decreased the tumor volume approximately 62%, without inducing systemic toxicity. The deficit in locomotion and memory and an anxiolytic-like behaviors induced in the GB model were minimized by CEC. The extract decreased the levels of reactive oxygen species, nitrites and thiobarbituric acid reactive substances and increased the activity of antioxidant enzymes in platelets, sera and brains of GB animals. The activity of NTPDases, 5'-nucleotidase and adenosine deaminase (ADA) was evaluated in lymphocytes, platelets and serum. In platelets, ATP and AMP hydrolysis was reduced and hydrolysis of ADP and the activity of ADA were increased in the control, while in CEC-treated animals no alteration in the hydrolysis of ADP was detected. In serum, the reduction in ATP hydrolysis was reversed by CEC. In lymphocytes, the increase in the hydrolysis of ATP, ADP and in the activity of ADA observed in GB model was altered by CEC administration. The observed increase in IL-6 and decrease in IL-10 levels in the serum of GB animals was reversed by CEC. These results demonstrate that CEC extract is a potential complementary treatment to GB, decreasing the tumor size, while modulating aspects of redox and purinergic systems.

Abstract A020: Neural stem cells expressing PTEN-L suppress glioblastoma invasion in vivo

Abstract A020: Neural stem cells expressing PTEN-L suppress glioblastoma invasion <i>in vivo</i>

Lucanthone, a Potential PPT1 Inhibitor, Perturbs Stemness, Reduces Tumor Microtube Formation, and Slows the Growth of Temozolomide-Resistant Gliomas In Vivo.

Glioblastoma (GBM) is the most frequently diagnosed primary central nervous system tumor in adults. Despite the standard of care therapy, which includes surgical resection, temozolomide chemotherapy, radiation and the newly added tumor-treating fields, median survival remains only ∼20 months. Unfortunately, GBM has a ∼100% recurrence rate, but after recurrence there are no Food and Drug Administration-approved therapies to limit tumor growth and enhance patient survival, as these tumors are resistant to temozolomide (TMZ). Recently, our laboratory reported that lucanthone slows GBM by inhibiting autophagic flux through lysosome targeting and decreases the number of Olig2+ glioma stem-like cells (GSC) in vitro and in vivo. We now additionally report that lucanthone efficiently abates stemness in patient-derived GSC and reduces tumor microtube formation in GSC, an emerging hallmark of treatment resistance in GBM. In glioma tumors derived from cells with acquired resistance to TMZ, lucanthone retains the ability to perturb tumor growth, inhibits autophagy by targeting lysosomes, and reduces Olig2 positivity. We also find that lucanthone may act as an inhibitor of palmitoyl protein thioesterase 1. Our results suggest that lucanthone may function as a potential treatment option for GBM tumors that are not amenable to TMZ treatment. SIGNIFICANCE STATEMENT: We report that the antischistosome agent lucanthone impedes tumor growth in a preclinical model of temozolomide-resistant glioblastoma and reduces the numbers of stem-like glioma cells. In addition, it acts as an autophagy inhibitor, and its mechanism of action may be via inhibition of palmitoyl protein thioesterase 1. As there are no defined therapies approved for recurrent, TMZ-resistant tumor, lucanthone could emerge as a treatment for glioblastoma tumors that may not be amenable to TMZ both in the newly diagnosed and recurrent settings.

10172-IM-6 DEVELOPMENT OF CAR-NK CELL THERAPY TARGETING B7H3 AGAINST GLIOBLASTOMA

Abstract There is an urgent need to find new treatments for brain tumors with poor prognoses, such as glioblastoma (GBM). Recently, CAR-T cell therapy, which uses genetically engineered T cells to express chimeric antigen receptors (CAR) has been actively investigated. However, although CAR-T cell therapy has shown efficacy in preclinical GBM models, CAR-T cells have a number of limitations. When adapted for clinical use, it may exhibit undesirable side effects such as graft-versus-host disease or cytokine-releasing syndrome. Furthermore, generating an autologous product for each patient needs time and effort and is restrictive for widespread clinical use. In contrast, cord blood-derived natural killer (NK) cells show a robust safety profile in vivo and are an attractive therapeutic option for cancer treatment. In this study, we focused on developing CAR-NK therapy against GBM and used B7H3 which has recently been studied as a tumor antigen. First, we generated CAR-T cells expressing CAR against B7H3 and confirmed their antitumor effect in vitro and in vivo. Next, we generated CAR-NK cells with similar scFV from code blood. We generated eight donors of CAR-transfected NK cells and investigated the gene transfer and cell growth rate. We tested three donors of CAR-transfected NK cells and found that one of them had robust cytolytic activity against GBM cells in vitro. Overall, this study suggests that cord blood-derived CAR-NK cells targeting B7H3 may be a promising therapy for GBM.

MODL-19. DEVELOPMENT OF A RECURRENT GLIOBLASTOMA MURINE MODEL FOLLOWING SURGICAL RESECTION: INVESTIGATING THE TUMOR-IMMUNE MICROENVIRONMENT TO EVALUATE NOVEL PERIOPERATIVE THERAPIES

Abstract BACKGROUND Most glioblastoma (GBM) patients experience tumor recurrence following standard-of-care treatment which includes tumor resection, radiation, and chemotherapy with concomitant and adjuvant temozolomide. Currently, no murine surgical GBM resection models to evaluate novel therapies exist and understanding of postsurgical tumor microenvironment (TME) has yet to be fully elucidated in pre-clinical GBM models. METHODS GL261 GBM cells were orthotopically implanted in C57BL/6 mice on day 1. On day 14, tumor resection was performed using the NICO Murine Neuroscience System, an automated, intraoperative, pre-clinical surgical resection system designed to minimize damage to surrounding tissue thus preventing the need for animal sacrifice. Standard T2-MRI and Terason uSmart 3200T ultrasound system were used to analyze tumor size following resection and recurrent growth. Immunological and biochemical assays were performed to explore local TME. RESULTS We observed that surgically resected GL261 GBM mice survived 55 + 8 days whereas mice with unresected GBM survived 35 + 7 days following tumor implantation. This new preclinical surgical resection recurrent immunocompetent GBM (Sur-rGBM) model was used for analyzing TME. Our results demonstrated significant changes in TME, such as an increase in astrocytic activation (GFAP+), increased microglia/macrophage (CD11b+, and IBA-1+) infiltration to surrounding tissue, increase in M2 macrophage (TGM2+ and CCL22+) and NK regulatory cells (CDC 26 +), and increased angiogenesis (VEGF) in Sur-rGBM as compared to unresected GBM. We also observed increased proliferation (Ki-67) and upregulation of stem cell markers CD133, Nestin, and Olig2 at the protein level. CONCLUSION Our data demonstrated the upregulation of tumor-promoting activation factors in Sur-rGBM as compared to unresected GBM. Currently, the Sur-rGBM model does not fully resemble the entire treatment regimen offered to GBM patients. Thus, our future aim is to further refine this resection model and assess adjuvant therapies at different time points along disease progression without needing to sacrifice the animal for analysis of tissue.

EXTH-38. VELCRIN MOLECULAR GLUES INDUCE CELL DEATH IN GLIOBLASTOMAS WITH HIGH PDE3A AND SLFN12 EXPRESSION

Abstract Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase, SLFN12, and the phosphodiesterase, PDE3A. Formation of the complex activates SLFN12, which cleaves tRNALeu(TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumor cell lines from the Cancer Cell Line Encyclopedia (CCLE), including glioblastoma cell lines. We therefore aimed to characterize velcrins as novel therapeutic agents in glioblastoma. We measured PDE3A and SLFN12 expression levels in commercially available glioblastoma cells as well as glioblastoma neurospheres from the Center for Patient Derived Models at Dana Farber Cancer Institute. We identified velcrin-sensitive glioblastoma cell lines and neurospheres, and determined that velcrin response is highly correlated with PDE3A expression. We also determined that sensitive cells exhibit a reduction in nascent polypeptide formation upon velcrin treatment, which is rescued by CRISPR-induced knockout of PDE3A. To assess velcrin efficacy in vivo, we measured central nervous system and plasma levels of the velcrin, BAY 2666605, and determined that it freely crosses the blood brain barrier. We also determined that twice daily dosing of BAY 2666605 elicits full tumor regression in an orthotopic glioblastoma xenograft model, with sustained complete responses after treatment withdrawal. Lastly, we demonstrated that the velcrins BAY 2666605 and BAY 2501293 induce tumor regression in biomarker-positive subcutaneous glioblastoma patient-derived xenograft models. In summary, our data demonstrates that velcrins have anti-tumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents.

CTNI-61. MYCOPHENOLATE MOFETIL INHIBITS GLIOBLASTOMA PURINE METABOLISM IN HUMANS: AN INTERIM RESULT OF PHASE 0/1 CLINICAL TRIAL TO OVERCOME TREATMENT RESISTANCE IN NEWLY DIAGNOSED AND RECURRENT GLIOBLASTOMA

Abstract BACKGROUND Mycophenolate mofetil (MMF), a purine synthesis inhibitor, improves radiation and temozolomide efficacy in preclinical glioblastoma models. This phase 0/1 trial (NCT04477200) is the first-in-human study to assess the tolerability of MMF with chemoradiation in glioblastoma, MPA accumulation, and purine synthesis inhibition in tumor. METHODS Adult patients meeting eligibility are given MMF 500-2000 PO BID using TITE-CRM dose assignment. Thirty recurrent glioblastoma patients receive MMF one-week prior to and concurrently with re-irradiation (40.5 Gy), with 28 days of dose-limiting toxicity (DLT) period. Thirty newly-diagnosed glioblastoma patients receive MMF one-week prior to and concurrently with chemoradiation and followed for 28 days for DLT1-period. During the adjuvant temozolomide (days 1-5/28-day cycle) period, MMF is given days 0-5 on a separate dose-escalation (DLT2 period = cycles 1-2). Tissues from phase-0 recurrent glioblastoma patients (N = 8) receiving MMF 500-2000mg BID one-week pre-operatively are analyzed using mass spectrometry. RESULTS Enhancing and non-enhancing tumor from phase 0 subjects yielded above 1 µM active drug metabolite, and the GTP/IMP ratio was decreased by 75% in enhancing tumor in MMF-treated patients compared to untreated controls (p = 0.009), indicating effective target engagement and inhibition of purine synthesis. No DLT has been observed with adjuvant cyclic temozolomide in phase 1 newly-diagnosed group (N = 26), nor in the recurrent-disease group (N = 18) with re-irradiation. In newly diagnosed arm, 3 subjects experienced DLT1 at 2000mg BID; grade-3 hemiparesis and cognitive disturbance, and grade-4 thrombocytopenia. Other main toxicities are mild nausea and fatigue. Interim median overall survival in recurrent phase 1 is 15.6 months, and not reached yet in newly diagnosed phase 1. CONCLUSION MMF combined with chemoradiation has been reasonably well tolerated in glioblastoma patients with preliminary promising evidence of intracranial target engagement of its active metabolite. This study will yield a recommended phase 2 dose and preliminary efficacy estimate for future randomized phase 2/3 trial.

EXTH-81. RE-EXPRESSION OF CXCL9 FOLLOWING ADENO-ASSOCIATED VIRUS (AAV) TRANSGENE DELIVERY TRANSFORMS THE IMMUNOLOGICAL LANDSCAPE OF GLIOBLASTOMA, SENSITIZING TUMORS TO PD-1 IMMUNE CHECKPOINT BLOCKADE (ICB)

Abstract BACKGROUND To date, few glioblastoma (GBM) patients have demonstrated clinical benefit from treatment with anti-PD-1 ICB. GBM is notoriously devoid of lymphocytes, signifying that a possible obstacle for effective ICB response may be the limited ability of cytotoxic T-lymphocytes (CTLs) to home to these tumors. CTLs are recruited via long-range signaling mediated by the diffusion of chemokines present in inflammatory environments. Following a comprehensive chemokine screen of clinical tumor specimens, we identified that CXC motif ligand 9 (CXCL9) is notably absent in GBM. To test if GBM reconstitution with CXCL9 could transform these intractable tumors into ICB-responsive, we leveraged AAV gene therapy to generate durable production of this potent lymphotactic signal. HYPOTHESIS AAV mediated re-expression of CXCL9 will improve lymphotaxis in GBM, sensitizing these tumors to PD-1 ICB. METHODS 3-dimensional whole brain fluorescent imaging was performed to characterize the distribution, tropism, and durability of AAV transgene expression in preclinical GBM models. Single-cell RNA sequencing (scRNAseq) and multiparameter flow cytometry was utilized to immunologically profile tumors in response to single and combination treatment. Survival studies with and without lymphocyte depletion were performed to evaluate immunogenic responsiveness of therapy. RESULTS Intra-tumor delivery of AAV6 yields stable transduction of tumor-reactive astrocytes, producing a localized, peri-tumoral pattern of transgene signal distribution. Combination therapy increases tumor infiltration by lymphocytes. scRNAseq analyses reveal widespread innate and adaptive immune activation in response to each CXCL9 and PD-1 ICB treatment. Infiltrating CD8 T-lymphocytes demonstrate inflammatory signatures associated with increased effector function, including antigen presentation, cytotoxicity, and TCR signaling. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration. CONCLUSIONS This work suggests direct conditioning of the tumor microenvironment by AAV-CXCL9 could potentially overcome a key resistance factor in GBM, sensitizing tumors to immune mediated therapies such as PD-1 ICB.

EXTH-70. UTILIZING ONCOLYTIC VIROTHERAPY AS AN APPROACH TO ENHANCE NEOANTIGEN SPECIFIC IMMUNE RESPONSES IN GLIOBLASTOMA

Abstract Despite the success in treating many solid cancers, immunotherapy has not shown significant efficacy in patients with glioblastoma (GBM). The lack of tumor-infiltrating T cells recognizing tumor antigens displayed with major histocompability complex I (MHC I) is one of the reasons for GBM’s evasion from immunotherapy. Our preclinical GBM models and an ongoing clinical trial with oncolytic herpes simplex virus-1 (oHSV) at our institution (NCT03152318) demonstrated that oHSV treatment leads to an increase influx of CD8+ and CD4+ T cells into tumor. However, it remained unclear whether these T cells are specific to the tumor antigens (neoantigens) or reactive to only viral antigens. To determine whether infiltrating CD8 T cells target tumor antigens, we first performed whole-exome and RNA sequencing of CT2A, a preclinical model of GBM impervious to immunotherapies for identification of neoantigens. We screened immunogenicity of identified neoantigens by ELISPOT assay. TILs from the oHSV-treated (but not from control group) reacted to two of the neoantigens that were identified with our pipeline and this activation was similar in magnitude to the reaction to the immunodominant HSV antigen. FACS analyses of TILs seven days after virotherapy showed that oHSV-treated tumor had higher level of TILs than vehicle control tumors, a finding that mirrored what we observed in our phase 1 clinical trial with oHSV. High dose of oHSV treatment not just induces the infiltration of viral specific T cells, but infiltration of T cells targeting neoantigens that were not presented without OV infection. Phosphoproteomic analysis of bulk tumor revealed activation of key inflammatory pathways, including that of T cells receptor signaling upon virus treatment. We evaluated the efficacy of therapeutic neoantigen vaccination in combination with oncolytic intratumoral virotherapy. Survival analysis showed that combination therapy not just delays the tumor progression but also provides survival advantage compared to monotherapies.

MODL-35. BIOENGINEERED HYDROGEL AND BLOOD BRAIN BARRIER-ON-A-CHIP SYSTEM TO STUDY GLIAL CELL ACTIVATION AND BLOOD BRAIN BARRIER DYSFUNCTION IN GLIOBLASTOMA

Abstract Treatments for glioblastoma (GBM) are limited leading to short patient life expectancies after diagnosis. Current preclinical models of GBM fail to accurately replicate the function of the blood brain barrier (BBB) and the extreme heterogeneity of the GBM tumor hindering the drug development process. Therefore, there is a pressing need for improved model systems to study how different GBM subpopulations interact with glial cells and the BBB. To build such a model, we first focused on designing a 3D extracellular matrix-based hydrogel biomaterial that would support human astrocytes and a functional BBB. The hydrogel chosen for these studies is composed of functionalized hyaluronic acid (HA) and gelatin and has similar mechanical properties to the human brain. Using confocal imaging and qPCR, we have shown that astrocytes encapsulated in this hydrogel have greater branching and express fewer markers of activation compared to astrocytes grown in commonly used 3D biomaterials such as collagen I and Matrigel. Treatment with inflammatory cytokines and GBM cell derived extracellular vesicles (EVs) revealed that astrocytes in this environment display the activation and inflammatory phenotypes observed in vivo in GBM. Next, this hydrogel biomaterial was deployed in a bioengineered BBB-on-a-chip. We have shown that this system supports the formation of 3D microvessel structures with human brain endothelial cells, pericytes, and astrocytes that accurately mimic the function of the human BBB. Studies using this BBB-on-a-chip have shown that invasive glioma stem cells alter BBB permeability influencing the transport of certain therapeutics to the tumor site. We are now beginning to test how various subpopulations of GBM tumor cells behave in this model and how changes to glial cells and the BBB impact drug delivery and efficacy. This study shows that engineered in vitro models can provide critical insight into disease mechanisms and inform treatment design in GBM.

DDDR-13. ST-401, A BRAIN PENETRATING MICROTUBULE TARGETING AGENT THAT KILLS GLIOBLASTOMA IN INTERPHASE AND BY DISRUPTING MITOCHONDRIA FUNCTION

Abstract The development of small molecular entities that can effectively penetrate the blood-brain barrier and kill glioblastoma (GBM) is crucial in Neuro-Oncology. ST-401, a unique microtubule depolymerizer, has shown remarkable potential as a therapeutic agent for GBM in PD-GBM isolates (nanomolar potencies) and in vivo preclinical GBM models (potencies DNA-damaging agents). This study aims to investigate the novel mechanism of action of ST-401 and its impact on GBM cell death. We recently showed that increasing concentrations of ST-401 disrupts mitochondrial function, leading to cell death initially in interphase and then in mitosis. Through single-cell RNA sequencing analysis, significant changes in mRNA related to mitochondrial functions were observed, including ribosomal proteins and OXPHOS genes. The study focuses on the role of mitochondria in GBM tumorigenesis and explores how ST-401 affects mitochondrial OXPHOS function. We identified three GBM cell lines (U251, SNB-19, and SF-539) with increasing vulnerability to ST-401, respectively, as measured by using WST-1, an index of mitochondria OXPHOS. Further investigation using the Seahorse platform demonstrated that SF-539 cells treated with ST-401 exhibited a loss of spare capacitance and significantly reduced mitochondrial function, including basal and maximal OXPHOS response, as well as ATP production. In conclusion, our results emphasize the existence of specific subtypes of GBM that show vulnerability or resistance to the antitumor effects of ST-401. Identifying these subtypes is crucial for advancing precision Neuro-Oncology and facilitating human trials. Our results also suggest that ST-401's inhibition of microtubule function and induction of cell death in interphase are associated with the loss of mitochondrial respiratory function. Given the limited treatment options for brain tumors and the challenges in delivering drugs across the blood-brain barrier, the development of brain-penetrant drugs like ST-401 holds great promise. Funded by NIH (CA244213 to NS and NS106924 to NS).

EXTH-67. LP-184, AN MGMT-AGNOSTIC SMALL MOLECULE, HAS POTENT SYNERGY WITH SPIRONOLACTONE TO EFFECTIVELY INHIBIT ORTHOTOPIC GBM XENOGRAFT TUMORS

Abstract Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to extend survival of patients with newly diagnosed MGMT-methylated glioblastoma (GBM). Blood-brain barrier (BBB) permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT-unmethylated GBM) are needed. Lantern Pharma is advancing LP-184, an acylfulvene prodrug activated by the enzyme PTGR1. In MGMT-expressing TMZ-resistant GBM cultures, LP-184 was found to be ~5000X more potent than TMZ with LP-184 IC50s ranging from 20-200 nM. DNA damage induced by acylfulvenes is primarily repaired via transcription-coupled nucleotide excision repair (TC-NER) mediated by ERCC complexes. ERCC3 is proteolytically degraded in response to the FDA approved BBB permeable diuretic, spironolactone (SP). We found that SP induces rapid ERCC3 degradation by up to 95% in GBM cells and validated that SP-induced ERCC3 loss led to substantial increases in LP-184 responses (DNA damage, tumor cell death) in vitro and in subcutaneous (s.c.) tumor xenografts. Co-treatment of GBM cells with LP-184 and SP resulted in a 3-6 fold decrease in LP-184 IC50s in PDX-derived (M1123 and Mayo39) and cell line (U87) cultures in vitro. Systemic SP sensitized pre-established s.c. and orthotopic (i.c.) GBM xenografts to LP-184 therapy. Pre-established s.c. U87 tumors recurred after initial regression in 5/5 animals following treatment with LP-184 alone whereas tumors showed durable complete regression without recurrence in 4/5 animals following treatment with LP-184 + SP. Pre-established M1123 i.c. xenografts treated with LP-184 + SP were ~3-fold smaller than those treated with LP-184 alone. LP-184 is a promising agent with potential clinical translation in GBM patients. Our results indicate that LP-184 is MGMT-agnostic and effective in TMZ-resistant preclinical GBM models. The first-in-human LP-184 dose escalation Phase 1A trial enrolling solid tumor patients including GBM is planned to launch Q3 2023.

STING activation counters glioblastoma by vascular alteration and immune surveillance

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease. In this study, we used two preclinical models of GBM: orthotopically delivering either GBM stem cells or employing CRISPR-mediated tumorigenesis by adeno-associated virus, to establish immunologically proficient and non-inflamed tumors, respectively. After tumor development, the innate immune system was activated through long-term STING activation by a pharmacological agonist, which reduced tumor progression and prolonged survival. Recruitment and activation of cytotoxic T-cells were detected in the tumors, and T-cell specificity towards the cancer cells was observed. Interestingly, prolonged STING activation altered the tumor vasculature, inducing hypoxia and activation of VEGFR, as measured by a kinome array and VEGF expression. Combination treatment with anti-PD1 did not provide a synergistic effect, indicating that STING activation alone is sufficient to activate immune surveillance and hinder tumor development through vascular disruption. These results guide future studies to refine innate immune activation as a treatment approach for GBM, in combination with anti-VEGF to impede tumor progression and induce an immunological response against the tumor.

Near-Infrared Fluorescence Imaging in Preclinical Models of Glioblastoma.

Cancer is a public health problem requiring ongoing research to improve current treatments and discover novel therapies. More accurate imaging would facilitate such research. Near-infrared fluorescence has been developed as a non-invasive imaging technique capable of visualizing and measuring biological processes at the molecular level in living subjects. In this work, we evaluate the tumor activity in two preclinical glioblastoma models by using fluorochrome (IRDye 800CW) coupled to different molecules: tripeptide Arg-Gly-Asp (RGD), 2-amino-2-deoxy-D-glucose (2-DG), and polyethylene glycol (PEG). These molecules interact with pathological conditions of tumors, including their overexpression of αvβ3 integrins (RGD), elevated glucose uptake (2-DG), and enhanced permeability and retention effect (PEG). IRDye 800CW RGD gave the best in vivo fluorescence signal from the tumor area, which contrasted well with the low fluorescence intensity of healthy tissue. In the ex vivo imaging (dissected tumor), the accumulation of IRDye 800CW RGD could be appreciated at the tumor site. Glioblastoma tumors were presently detected with specificity and sensitivity by utilizing IRDye 800CW RGD, a near-infrared fluorophore combined with a marker of αvβ3 integrin expression. Further research is needed on its capacity to monitor tumor growth in glioblastoma after chemotherapy.

Identification of Excellent Prognosis IDH Wildtype Glioblastomas Using Genomic and Metabolic Profiling

High grade gliomas (HGGs) are aggressive brain tumors with altered cellular metabolism. HGGs can carry mutations in the tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase 1 (IDH1), conferring distinct biology and improved patient prognosis compared to IDH wildtype (wt) tumors. Using metabolomic analyses of tumor tissue, we previously showed that IDH wt and IDH mutant (IDH mut) tumors have unique metabolomic signatures that correlate with different survival outcomes. Among this cohort of 69 HGG samples, we identified two unique patient tumors that metabolically clustered with IDH mut tumors, but lacked both the IDH mutation and its product 2-hydroxyglutarate. We aimed to discover unique mutations in these two tumors that may impart an IDH mutant-like phenotype in the absence of an IDH1 or IDH2 mutation. Whole exome sequencing (WES) was performed on frozen tumor samples from two patients diagnosed as glioblastoma (GBM), IDH wt via Agilent v5 + IncRNA platform. Alignment to the hg38 genome and variant calling were completed using an accelerated implementation of GATK's BWA and MuTect2 algorithms from Sentieon. Variants were filtered based on supporting reads and variant allele thresholds, with synonymous variants and common SNPs removed. High-confidence variants were further filtered by membership in the four KEGG pathways associated with IDH1 and IDH2. Identified variants were corroborated with metabolomics data from the two unique IDH wt tumors compared with classical GBM IDH wt, oligodendrogliomas IDH mut and astrocytomas IDH mut to identify putative drivers of an IDH mutant-like metabolomic phenotype in these unique IDH wt tumors. Despite the lack of an IDH mutation, one patient survived 45.6 months and the other patient remains alive at last follow up 64 months post diagnosis, much longer than the 16-18-month median survival typical of patients with GBM IDH wt. WES of outlier IDH wt tumor samples revealed 65 unique mutations in the queried KEGG pathways, of which 34 had a variant allele frequency > = 0.15. These variants were processed in Gprofiler, confirming expected enrichment of the carboxylic acid metabolic biologic process, a functional gene set consisting of TCA genes, among these variants (p = 0.002, 3.6-fold enrichment). Accordingly, metabolite levels of intermediates of the TCA cycle, including malate and isocitrate were decreased in the outlier tumor samples compared to classic GBMs IDH wt (p<0.001). Presence of genetic alterations in key variants of the carboxylic acid metabolic biologic process (including ME1, GYP4F3, PTGIS, PFKL, PSPH, AKR1A1, HK2, NOS1) correlated with improved overall survival among GBM patients in the TCGA (p = 0.04). Laboratory validation of these findings in preclinical GBM models is ongoing. Disruption of the TCA cycle independent of an IDH mutation is associated with favorable survival in GBM. Pharmacologic inhibition of these pathways may be a promising strategy to improve GBM outcomes.