The determinants of immune checkpoint blockade (ICB) response in glioblastoma (GBM) with wild-type isocitrate dehydrogenase remain poorly understood. Here we profiled 181 ICB-treated GBM cases using bulk DNA sequencing, bulk RNA sequencing and single-nucleus RNA sequencing to investigate the genomic features associated with ICB outcomes. Baseline tumor transcriptional subtype was predictive of overall survival following ICB, with mesenchymal (MES) GBM associated with improved outcomes to ICB but not standard chemoradiation. Non-MES-associated genetic lesions, including those in PDGFRA and CDKN2A, were associated with worse survival following ICB but not standard therapy. Tumor mutational burden was not predictive of outcomes. Survival was associated with pre-ICB enrichment for MES-like malignant cells, marked by high human leukocyte antigen class I expression and greater T cell infiltration. Paired tumor analyses linked ICB exposure to outgrowth of subclones harboring lesions associated with non-MES subtypes, supporting MES-to-non-MES transition as a common trajectory of acquired resistance to ICB, distinct from standard chemoradiation.

Although glioblastoma (GBM) harbors multiple genetic abnormalities leading to cell cycle deregulation, a functional mitotic checkpoint is essential to prevent mitotic catastrophe and tumor cell death. Here, we identify the RNA-binding protein HNRNPH1 as a key post-transcriptional modulator of G2/M checkpoint-associated genes in GBM. HNRNPH1 is overexpressed in malignant cells, especially in the neural- and oligodendrocyte-progenitor-like state, and its expression levels are higher in non-hypoxic regions of the tumor. Knocking out HNRNPH1 causes aberrant splicing and downregulation of several genes involved in cell division. These molecular alterations are associated with G2/M cell cycle arrest, reduced cell proliferation, abnormal cell morphology, and increased nuclear fragmentation. Silencing HNRNPH1 in vivo inhibits the tumor growth of patient-derived GBM cell-originated intracranial xenografts and has significant survival benefits. Together, our results show the critical importance of HNRNPH1 in cell cycle progression and tumor growth, potentially impacting the development of novel strategies to treat GBM.

PURPOSE:Integrating external control data into clinical trial designs and analyses has the potential to accelerate drug development processes. We reanalyzed the three experimental arms of the Individual Screening Trial of Innovative Glioblastoma Therapy (INSIGhT), a randomized phase II platform trial in newly diagnosed O6-methylguanine-DNA methyltransferase-unmethylated glioblastoma (ClinicalTrials.gov identifier: NCT02977780). To evaluate the validity of using external data sets, we compared treatment effect estimates based on internal INSIGhT control data and matched external control data. METHODS:The three experimental arms of INSIGhT (abemaciclib [n = 72], neratinib [n = 80], and CC-115 [n = 12]) did not improve survival compared with internal controls (standard chemoradiation [n = 70]). We derived external control patient-level data from multiple real-world and clinical trial data sets. We applied propensity score matching and Cox proportional hazards models to estimate treatment effects with external controls. Additionally, using this glioblastoma (GBM) data collection, we specified simulation scenarios to evaluate trial designs that integrate external controls. RESULTS:After matching to external controls, no survival benefit was observed for patients receiving abemaciclib (hazard ratio [HR], 1.00 [95% CI, 0.75 to 1.34]), neratinib (HR, 0.93 [95% CI, 0.70 to 1.24]), or CC-115 (HR, 0.88 [95% CI, 0.41 to 1.88]). Simulations, together with the INSIGhT data and a collection of GBM data sets, allowed us to examine efficiencies and risks of clinical trial designs that leverage external control data. CONCLUSION:The use of carefully matched external controls, to replace or augment the internal controls of INSIGhT, produced treatment effect estimates that were similar to previously published analyses. Single-arm trial designs and hybrid randomized designs incorporating propensity score-matched external control data evaluated treatment effects in the early-phase testing of experimental therapies in newly diagnosed GBM. The validity of this approach and risks of bias depended on the availability of comprehensive and accurate data on all potential confounders, in the absence of unmeasured confounding.

Abstract Introduction. Intratumoral injection of the oncolytic herpes simplex virus (oHSV) CAN-3110 remodels the immunosuppressive microenvironment of recurrent glioblastoma. The immune infiltration triggered by CAN-3110 treatment is correlated with prolonged survival, especially in HSV1-seropositive individuals (Ling et al., 2023). However, it is still unclear if the immune response induced by oHSV therapy is mainly anti-virus or anti-tumor. We aim to dissect this distinction using ex vivo patient-derived glioma organoid (pGBO)-immune co-culture models to study the impact of oHSV treatment on anti-tumor immunity. Methods. pGBOs were generated from surgically resected glioma specimens and characterized by highly multiplexed cyclic immunofluorescence (CycIF), via (up to date) 29 markers. Viral infection kinetics were assessed in these pGBOs using the GFP-expressing oHSV, rQNestin34.5v.1. Following pGBM characterization, we established a co-culture model of several pGBOs clones with rQNestin34.5v.1 and peripheral blood mononuclear cells (PBMCs) from either healthy donors or the pGBO patients. This system was used to assess tumor-immune interaction characteristics and kinetics under viral and non-viral conditions. Results. Histological and immunofluorescent analyses showed that pGBOs retain tissue microstructures, such as vascular architecture - characterized by endothelial walls containing erythrocytes - for several weeks in culture, even after freezing and thawing the models. The cellular diversity in pGBOs mirrors the heterogeneity found in in vivo, comprising neural stem-like, glial, and residual innate immune cell populations. These distinct cell types exhibited metabolic activity, epithelial-to-mesenchymal transition (EMT), and proliferation. Over a three-month culture period, the architecture and phenotype changed to more aggressively glioma, with increased glioma cell expansion and loss of vasculature. Notably, the underlying cellular heterogeneity remained throughout the process. Co-culture of pGBOs with allogeneic PBMCs triggered infiltration of CD3+, CD8+, and CD4+ lymphocytes under viral and non-viral conditions. Staining for cleaved caspase-3 and granzyme B showed active tumor cell killing by granzyme+ CD8 T cells, which appears unrestricted to infected glioma cells. Further, CD20+ lymphocytes were detected in both infected and uninfected pGBOs. Conclusion. We characterized pGBOs as a physiologically valid ex vivo model, observing both vasculature and cellular composition as found in in situ gliomas. Immune-mediated tumor cell killing was detected, both with and without virus treatment in PBMC x pGBO co-culture, using imaging and molecular readouts. This model enables investigation of how oncolytic HSV infection modulates immune response and provides a tractable system to dissect anti-tumor vs. anti-virus immune responses. Citation Format: C. Zoe Linke, Ethan Chen, Jennifer Gantchev, Aanchalika Chauhan, Christopher Jannotta, Keith L. Ligon, Andres Santos, Nathalie Agar, E Antonio Chiocca, Alexander L. Ling. Dissecting oncolytic virus anti-virus vs anti-tumor immunity in glioma: Insights from a patient derived organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4326.

Abstract Volumetric tumor segmentation improves patient monitoring vs. standard radiological assessment and is often needed for extracting radiomic features. Despite this, widespread implementation of such analyses into clinical and scientific studies is hindered by the time-intensive nature of scan curation and segmentation, and by the significant computational expertise required to process large imaging datasets. To overcome these barriers, we created a python package (astril - https://github.com/Alexander-Ling/astril) that enables fully automated pre-processing, segmentation, and quantification of radiology images using simple command line arguments, starting from unprocessed DICOM directories. astril also supports the training and application of new segmentation algorithms.The first pipeline implemented in astril is for segmenting recurrent glioblastoma (GBM) MRI images, enabling users to automatically pre-process (verify integrity, parse metadata, select optimal series, de-identify, co-align, skull strip, and normalize) and segment (tumor, peritumoral edema, and necrosis) volumes, starting from raw DICOM directories and ending with tabulated volumetric statistics. The built-in CNN segmentation algorithm was trained on manually segmented images from a recurrent GBM patient cohort, enabling the algorithm to correctly handle artefacts such as resection cavities, scar tissue, and low-enhancing tumor. Automated segmentation volumes with astril are highly correlated with manual segmentations and are associated with patient clinical outcomes.This provides a robust platform for rapid, standardized, and automated processing of radiology imaging libraries, and it enables simplified training and distribution of new segmentation algorithms. Citation Format: Alexander L. Ling, C. Zoe Linke, Christopher M. Jannotta, Data Science Teamlab, E. Antonio Chiocca. astril: Automated segmentation toolkit for radiology image libraries [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2770.

Solid tumors are sustained by profoundly immunosuppressive tumor microenvironments (TMEs) that underlie resistance to immunotherapy. Engineered oncolytic viruses and cytokine-based gene therapies can reprogram the TME, converting 'cold' tumors into immune-responsive states and amplifying antitumor immunity. Several agents have achieved regulatory approval, and clinical studies demonstrate that even limited dosing can induce durable changes in immune infiltration and cytokine signaling. Yet consistent and lasting clinical responses remain elusive. Here, we synthesize translational insights from recent trials and highlight emerging strategies to overcome barriers and enhance the therapeutic impact of these TME-modulating biologics.

RNA medicine is a promisingly expanding field in modern health care, but its use in genetically complex diseases, like cancer, has been challenging, mainly due to their reliance on multiple abnormal pathways. Here, we describe a microRNA-based platform that exploits previously unrecognized features of microRNA processing. Leveraging a microprocessor-dependent, cleave-activation strategy, this design allows us to expand biological impact by simultaneously up- and down-regulating desired microRNAs, while using them as structural enablers for other short, noncoding RNAs, such as aptamers. We demonstrate its biological potential in a glioblastoma model, where the simultaneous bidirectional modulation of five among the most deregulated microRNAs results in critical mass interference against the tumor. In parallel, microRNA-mediated chaperoning of an anti-p50 aptamer within the platform allows us to selectively block the nuclear factor κB pathway, a difficult-to-drug target. This work highlights the potential of chimeric microRNA clusters as an emerging therapeutic concept for cancer and other similarly multifactorial diseases.

Glioblastoma remains the most aggressive primary malignant brain tumor in adults, with survival largely unchanged despite advances in molecular diagnostics and supportive care. Therapeutic failure reflects fundamental biological and anatomical barriers, including intratumoral heterogeneity, an immunosuppressive tumor microenvironment, and restricted drug delivery across the blood-brain barrier. In this Review, we summarize the current standard of care and critically examine emerging strategies aimed at overcoming these constraints, including locoregional delivery technologies, immunotherapy, biomarker-defined precision approaches, and adaptive clinical trial designs. We highlight key translational and clinical studies shaping the field and discuss principles for developing more effective, integrated therapeutic paradigms.

Oncolytic virotherapy represents a promising yet under-explored approach for precision cancer treatment, particularly when tailored to tumor-specific molecular profiles. Patients with high-grade isocitrate dehydrogenase (IDH) mutant astrocytomas have limited treatment options and poor prognoses. Here, we investigate the therapeutic efficacy of rQNestin34.5 v.2 (CAN-3110), an engineered oncolytic herpes simplex virus 1 (oHSV-1), in IDH1-R132H-mutant diffuse gliomas. We demonstrate that the IDH1-R132H mutation enhances glioma susceptibility to viral infection through upregulation of Nectin-1, the main HSV-1 entry receptor. Concurrently, IDH1-R132H-driven DNA hypermethylation suppresses interferon (IFN) signaling, creating a permissive microenvironment that facilitates viral replication and tumor cell apoptosis. In immunocompetent murine glioma models, intratumoral administration of rQNestin34.5 v.2 induces robust antitumor immune activation, including increased immune infiltration and systemic IFN-γ release. However, elevated expression of poliovirus receptor (PVR) and the immune checkpoint T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on tumor-infiltrating leukocytes suggests a potential resistance mechanism to virotherapy. Combining rQNestin34.5 v.2 with TIGIT blockade enhances therapeutic efficacy compared to monotherapy, identifying IDH1-R132H as a potential predictive biomarker for oncolytic virotherapy response.

Abstract Background: Recurrent glioblastoma (rGBM) remains refractory to immunotherapy due to sparse and suppressed T cell infiltration. We recently reported that survival correlated with immune activation signatures in rGBM patients receiving the oncolytic HSV-1 (oHSV) rQNestin34.5v.2 (CAN-3110). Here, we provide in-situ and molecular evidence that a single oncolytic virus injection can induce durable, tumor-reactive T cell immunity in rGBM. Methods: We integrated highly multiplexed spatial proteomics (CODEX), spatial transcriptomics (Xenium) with custom probes for viral, immune and TCR targets, and bulk TCR-sequencing on paired pre- and post-treatment specimens from a phase 1 clinical trial (NCT03152318). Results: T cell densities strongly increased after treatment, with deep infiltration into viable tumor regions persisting up to two years after a single intratumoral oHSV injection. Cytotoxic GZMB+ T cells were located in close proximity with cleaved-caspase 3+ apoptotic tumor cells, and shorter T cell-tumor distance correlated with longer-progression free survival, demonstrating ongoing anti-tumor immunity. Spatial transcriptomics identified CD8+ T cells states expressing early TCR activation (NR4A1, CD69) and tissue residency (ZNF683 [HOBIT], ITGAE [CD103]) programs enriched in the tumor bed while stem-like T cells localized within lymphoid aggregates. Bulk and spatial TCR analyses revealed in-situ expansion of pre-existing tumoral T cell clones whose amplification correlated with survival. Expanded clones featured tissue resident phenotypes and were positioned closer to tumor cells than non-expanded T cells. Viral remnants were limited to necrotic regions and did not co-localize with T cells, suggesting persistent tumor recognition rather than viral antigen. Conclusion: These results provide in-situ evidence that a single intratumoral oncolytic virus injection can amplify pre-existing T cells clones and induce sustained T cell mediated tumor cytotoxicity even after virus clearance. This suggests that oncolytic virotherapy is as potent T cell activating strategy in rGBM. Citation Format: Maxime Meylan, Ye Tian, Lijian Wu, Alexander L. Ling, Daniel Kovarsky, Graham L. Barlow, Linh D. Nguyen, Jason Pyrdol, Lucas Westphal, Michel Julius, Nicolas L. Gonzalez Castro, Sydney D. Dumont, Andres Santos, Itay Tirosh, Mario L. Suva, E Antonio Chiocca, Kai W. Wucherpfennig. Pre-existing T cells drive durable anti-tumor immunity after oncolytic virus therapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7743.

FDA-approved oncolytic herpes simplex virus-1 (oHSV) therapy has emerged as a promising viro-immunotherapy for solid tumors. However, tumor- and tumor microenvironment (TME)-associated adaptations following viral treatment, such as feedback immune suppression, neoangiogenesis, and enhanced tumor aggressiveness, often hinder complete tumor eradication. A deeper understanding of the molecular mechanisms underlying resistance to oHSV is crucial to enhancing its clinical impact. We recently discovered that oHSV induces Insulin-like growth factor 2 (IGF2) secretion, shaping an immunosuppressive TME. Similarly, radiotherapy (RTx) activates the IGF1/IGF1R and YAP1 signaling pathways, further promoting therapeutic resistance. In this study, we investigated how oHSV-induced Insulin-like growth factor 1 receptor (IGF1R) signaling drives feedback pro-survival and proliferative pathways in tumor cells and evaluated the therapeutic potential of combining IGF1R blockade with oHSV and RTx. We first demonstrated that oHSV activates IGF1R signaling in vitro and in vivo, promoting tumor proliferation. While IGF1R-targeted monotherapies have shown limited cytotoxicity, its combination with oHSV led to a modest but significant increase in cytotoxicity across tested breast cancer (BC) and primary glioblastoma (GBM) cells in vitro and in vivo xenograft models. Furthermore, we observed that co-treatment with oHSV and RTx robustly activated both IGF1R and YAP1 in resistant cells, revealing the IGF1R/YAP1 axis as a key mediator of resistance to dual oHSV and RTx therapy. Notably, the triple combination of oHSV, RTx, and IGF1R blockade yielded synergistic anti-tumor effects, abolished YAP1 expression and nuclear localization, and significantly enhanced survival in orthotopic BC and GBM models. Collectively, these findings identify the IGF1R/YAP1 axis as a critical driver of resistance to oHSV and RTx and provide a strong rationale for the clinical evaluation of this triple-combination strategy to enhance therapeutic efficacy in patients with BC and GBM.

Background Oncolytic herpes simplex virus (oHSV) therapy is a live virus-based immunotherapy that lyses tumor cells which release antigens and activate antitumor immunity. oHSV therapy has been shown to increase ATP production and release of extracellular ATP (eATP). In the extracellular tumor microenvironment, eATP functions as an immune-activating damage-associated molecular pattern but is hydrolyzed to extracellular adenosine (eADO), which can be immune-suppressive. eADO is generated by the sequential action of ectoenzymes CD39 and CD73 (NT5E). Here, we examined the role of immunosuppressive eADO signaling in regulating antitumor immune efficacy of oHSV.Methods We evaluated changes in eADO signaling in vitro and in patient specimens after virotherapy. A genetic CD73 knock-out mouse model and blocking antibodies were used to assess the impact of CD73 on virotherapy in two different solid tumor models. Single-cell RNA sequencing was employed to assess changes in immune cell infiltration and communication. Flow cytometric immunophenotyping and immunofluorescent imaging were utilized to confirm single-cell sequencing predicted changes in tumor microenvironment.Results Transcriptomic analysis of patient tumors pre-virotherapy and post-virotherapy with CAN-3110 revealed increased expression of the adenosine receptor gene ADORA2B after treatment. High NT5E gene expression, as well as gene signatures suggestive of adenosine signaling, correlated with a significantly worse prognosis for patients with solid tumors. Single-cell sequencing of immune cells recruited to tumor-bearing brain hemispheres in CD73 knockout mice revealed an increase in macrophage-mediated antigen presentation and CD4+ T cell cross-communication. Intracranial tumor-bearing CD73 knock-out mice treated with oHSV showed significant therapeutic improvement as the result of oHSV compared with wild-type mice. Combination of virotherapy with CD73 antibody blockade also resulted in enhanced antitumor efficacy.Conclusions Here, we identify that immunosuppressive eADO signaling in the TME is a major barrier to oHSV therapy and CD73 blockade prevents tumor immune escape. The combination of oHSV with CD73 blockade supports the development of an antitumor immune memory response in solid tumors. This study supports clinical development of this combination strategy.

A recent first-in-human clinical trial demonstrated that survival in glioblastoma (GBM) patients following rQNestin34.5v.2 oncolytic virus treatment was associated with immune activation signatures. This study was registered at ClinicalTrials.gov (NCT03152318). Here, we provide in situ evidence of ongoing T cell-mediated cytotoxicity against tumor cells at late time points following single treatment, with deep and persistent T cell infiltration into tumor regions. Shorter distances between cleaved caspase-3+ tumor cells and granzyme B+ T cells were associated with longer progression-free survival following treatment. Pre-existing tumor-infiltrating T cells expanded locally upon treatment, correlating with longer overall patient survival. T cells with an early activation program closely interacted with tumor cells and were strongly enriched upon treatment. Viral remnants were restricted to necrotic regions, while T cells infiltrated deeply into live tumor regions. These data demonstrate that single oncolytic virus treatment can expand pre-existing T cell clones and trigger persistent T cell-mediated immunity against GBM.

Abstract Introduction Glioblastoma (GBM) is an aggressive primary brain cancer with minimal response to current therapies. An immunosuppressive tumor microenvironment limits immunotherapeutic efficacy. Astrocytes are the most abundant glial cells in the central nervous system and play important roles in locally regulating immune responses. However, in the context of GBM little is known about mechanisms regulating astrocytes or their roles in the modulating anti-tumor immune responses. Methods We used single-cell and bulk RNA sequencing of human GBM specimens and murine preclinical models, multiplexed immunofluorescence, in vivo cell-specific genetic perturbations via CRISPR and in vitro mouse and human experimental astrocyte and GBM models to address this gap in knowledge. Results We identified an astrocyte subset which limits T cell anti-tumor responses by inducing T cell apoptosis via TRAIL, a death receptor ligand. Further, we identified IL-11 produced by GBM cells as a driver of STAT3 signaling in this astrocyte subset and a regulator of TRAIL expression. Astrocyte STAT3 signaling and TRAIL expression were correlated with more aggressive tumor progression and decreased survival in GBM patients. Following in vivo genetic inactivation of TRAIL or of the IL-11 receptor in astrocytes we observed extended survival in GBM mouse models, alongside enhanced T cell and macrophage responses. Finally, we engineered an oncolytic HSV-1 virus to express a TRAIL-blocking single-chain antibody in the tumor microenvironment. TRAIL blockade enhanced therapeutic effect of oncolytic viruses, extended survival and enhanced tumor-specific immunity in preclinical models of GBM. Conclusion In summary, we establish that IL-11—STAT3 signaling drives astrocytes to suppress GBM-specific immune responses by inducing TRAIL-dependent T cell apoptosis, and engineered a therapeutic strategy leveraging oncolytic viruses as delivery vectors to target this mechanism of astrocyte-driven immunosuppression in the tumor microenvironment. Funding Source This work was supported by grants NS102807, ES02530, ES029136, AI126880 from the NIH; RG4111A1 and JF2161-A-5 from the NMSS; RSG-14-198-01-LIB from the American Cancer Society; and PA-1604-08459 from the International Progressive MS Alliance. This work was further supported by a scholarship from the German Academic Exchange Service (DAAD). Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Purpose: Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor, with a median survival of only 15 months. Effective therapeutic advances remain limited, with few clinical trial patients exhibiting robust responses to experimental therapies. This has led to efforts to identify molecular biomarker signatures capable of stratifying patient responses either prognostically (pre-treatment) or diagnostically (post-treatment). This study aims to assess the robustness and reproducibility of published transcriptional GBM biomarker signatures for predicting clinical outcomes in GBM trials. Methods: We analyzed published RNA-seq data from 4 clinical trials involving oncolytic virotherapy and immune checkpoint inhibitors, as well as multiple cohorts of patients treated via the Stupp protocol. The performance of reported immune gene signatures was evaluated across all datasets using both grouped and continuous survival modeling. Intra-patient signature variation between different biopsy sites and timepoints was assessed using longitudinal, multi-site sampling collected by the Break Through Cancer Accelerating GBM Therapies TeamLab. LASSO, multivariate Cox regression, and risk scoring with FDR adjustment were used to identify novel signatures with cross-cohort utility. Key Findings: No single published signature consistently predicted survival across all analyzed trials. A post-treatment ssGSEA antitumor cytokine signature that was associated with survival in the CAN-3110 OV trial (R = 0.74, p < 0.01) was also significantly associated with improved survival in an adjuvant anti-PD1 trial (R = 0.75, p = 0.03) and neoadjuvant anti-PD1 trial (R = 0.30, p = 0.05), but not in standard-of-care controls. PAM clustering of MCP immune signatures, as reported for the DNX-2401 OV+Anti-PD1 trial, also stratified post-treatment survival in the CAN-3110 OV trial (p < 0.01), with the coldest TME subtype consistently predicting worse survival. However, the prognostic value of the most immune enriched TME subtype was inconsistent and did not extend to the Anti-PD1 trials. Furthermore, multi-site, longitudinal biopsy samples revealed marked heterogeneity in biomarker signatures between biopsies from individual patients, even when assessed at the same timepoint. We’ve identified several candidate transcriptomic signatures that show promise for generalizable prognostic value in GBM patient cohorts and are characterizing their spatiotemporal variability. Conclusions: While no individual immune signature is universally predictive in GBM immunotherapy, certain post-treatment cytokine signatures and TME stratifications are significant in multiple immunotherapy contexts, though not in standard-of-care patient cohorts. Marked spatiotemporal heterogeneity in these signatures underscores the need for composite prognostic markers resilient to sampling variance. Citation Format: Christopher M. Jannotta, C Zoe Linke, Charles A. Whittaker, Vikas Patil, Accelerating GBM Therapies TeamLab, Farshad Nassiri, Gelareh Zadeh, E Antonio Chiocca, Alexander L. Ling. Assessing biomarker reproducibility for glioblastoma patient response stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1055.

2006 Background: Glioblastoma (GBM) remains uniformly lethal despite standard therapy. An immunosuppressive tumor microenvironment (TME) and limited T cell infiltration pose major barriers to immunotherapy. Personalized neoantigen vaccines offer a strategy to prime tumor-specific T cell responses by targeting patient-specific mutations, driving effector cells into the CNS. Here, we report results from a phase I trial of a personalized neoantigen peptide vaccine combined with pembrolizumab in newly diagnosed GBM. Methods: Patients with newly diagnosed GBM who were not on dexamethasone following gross total resection were enrolled across four cohorts. Up to 20 synthetic long peptides targeting personal neoantigens were selected per patient, and admixed with the adjuvant poly-ICLC (NeoVax). Following radiotherapy, NeoVax was administered subcutaneously as five priming doses followed by two booster doses. MGMT -unmethylated patients (Cohorts 1A-C) received NeoVax plus pembrolizumab without temozolomide (TMZ) and differed by time of pembrolizumab initiation relative to NeoVax priming; MGMT -methylated patients (Cohort 1D) received standard TMZ and pembrolizumab was initiated after NeoVax priming. Immunogenicity was assessed by ex vivo and in vitro IFN-γ ELISpot assays. Single-nucleus/single-cell RNA sequencing and TCR sequencing (snRNA-seq/scTCR-seq) was performed on paired tumor specimens. Results: Of 39 enrolled patients, 37 initiated NeoVax including 35 who completed priming and 2 with ongoing priming. Treatment was well-tolerated with no serious AEs. Median overall survival was 36.9 months for MGMT -methylated patients and 19.0 months for MGMT -unmethylated patients, compared to 25.3 and 16.7 months for propensity score-matched historical controls, respectively. Ex vivo and in vitro neoantigen-specific T cell responses were detected comparably across all cohorts and overall in 65% and 97% of vaccinated patients, respectively. Among all patients, ex vivo immune responders demonstrated improved overall survival compared to non-responders (HR 0.26, 95% CI 0.09-0.77, P = 0.015). Vaccine-reactive clonotypes, defined by in vitro expansion to vaccine peptides, were identified in post-vaccination tumors. snRNA-seq/scTCR-seq revealed increased intratumoral vaccine-specific T effector populations following vaccination. Conclusions: NeoVax generates durable T cell responses that traffic to GBM tumors, with circulating responses associated with improved survival. Ongoing studies are evaluating peripheral clonotype dynamics, remodeling of malignant cell states and the TME, and the spatial distribution of vaccine-reactive clonotypes, which may guide future combinatorial strategies in GBM. Clinical trial information: NCT02287428 .

Abstract Glioblastoma (GBM) is a WHO grade IV astrocytoma characterized by diffuse infiltration, rapid proliferation, and resistance to conventional therapies, resulting in dismal patient outcomes. Oncolytic virotherapy, particularly utilizing genetically engineered oncolytic herpes simplex virus-1 (oHSV), has demonstrated tumor-selective cytolysis and immunogenic cell death. Despite promising preclinical data, clinical efficacy remains limited due to poorly defined intrinsic resistance mechanisms within the tumor microenvironment (TME) of GBM. Our study identified microRNA-155 (miR-155) as a critical modulator of oHSV therapeutic resistance. We found elevated miR-155 expression in GBM compared to low-grade gliomas, which correlates with a poor prognosis and reduced response to OV therapy. These findings were further supported by The Cancer Genome Atlas (TCGA) data. Ectopic overexpression of miR-155 significantly attenuated oHSV replication and cytopathic effects. Transcriptomic profiling and pathway enrichment analyses revealed that miR-155 upregulates metallothioneins MT1E and MT1F, key regulators of intracellular copper sequestration and antiviral defense. Mechanistically, miR-155 disrupts copper homeostasis via the MT1 axis, impairing viral replication and therapeutic efficacy. Functional ablation of miR-155 via CRISPR/Cas9-mediated knockout or Locked Nucleic Acid (LNA)-based inhibition restored copper homeostasis, oHSV replication kinetics, and enhanced oncolysis in vitro and in orthotopic GBM xenograft models, resulting in prolonged survival. These data suggest that the miR-155/MT1E-MT1F/copper signaling axis represents a novel barrier to oHSV therapy. This work elucidates a previously uncharacterized antiviral resistance pathway in GBM and validates miR-155 inhibition as a rational therapeutic strategy to potentiate oHSV efficacy. Our findings provide a mechanistic framework for overcoming innate resistance and advancing the clinical translation of virotherapy in neuro-oncology. This abstract was originally written by the authors and then edited and revised with generative artificial intelligence (AI). Citation Format: Tae Jin Lee, Min Hye Noh, Citu Citu, Alexandra A. Miller, Jiyeon Kim, Grace Nguyen, Minxin Huang, Amanda S. Kouaho, Stephanie M. Bean, Dohyoung Lee, Lily Nguyen, E. Antonio Chiocca, Zhongming Zhao, Ji Young Yoo. Targeting microRNA-mediated copper homeostasis dysregulation to enhance intratumoral susceptibility to oncolytic herpes simplex virus-1 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2060.