Epigenetic Reprogramming of Autophagy Drives Mutant IDH1 Glioma Progression and Response to Radiation.

Mutant isocitrate dehydrogenase 1 (mIDH1; IDH1 R132H ) exhibits a gain of function mutation enabling 2-hydroxyglutarate (2HG) production. 2HG inhibits DNA and histone demethylases, inducing epigenetic reprogramming and corresponding changes to the transcriptome. We previously demonstrated 2HG-mediated epigenetic reprogramming enhances DNA-damage response and confers radioresistance in mIDH1 gliomas harboring p53 and ATRX loss of function mutations. In this study, RNA-seq and ChIP-seq data revealed human and mouse mIDH1 glioma neurospheres have downregulated gene ontologies related to mitochondrial metabolism and upregulated autophagy. Further analysis revealed that the decreased mitochondrial metabolism was paralleled by a decrease in glycolysis, rendering autophagy as a source of energy in mIDH1 glioma cells. Analysis of autophagy pathways showed that mIDH1 glioma cells exhibited increased expression of pULK1-S555 and enhanced LC3 I/II conversion, indicating augmented autophagy activity. This dependence is reflected by increased sensitivity of mIDH1 glioma cells to autophagy inhibition. Blocking autophagy selectively impairs the growth of cultured mIDH1 glioma cells but not wild-type IDH1 (wtIDH1) glioma cells. Targeting autophagy by systemic administration of synthetic protein nanoparticles packaged with siRNA targeting Atg7 (SPNP-siRNA-Atg7) sensitized mIDH1 glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory, when used in combination with IR. Our results indicate autophagy as a critical pathway for survival and maintenance of mIDH1 glioma cells, a strategy that has significant potential for future clinical translation. One Sentence Summary: The inhibition of autophagy sensitizes mIDH1 glioma cells to radiation, thus creating a promising therapeutic strategy for mIDH1 glioma patients. Graphical abstract: Our genetically engineered mIDH1 mouse glioma model harbors IDH1 R132H in the context of ATRX and TP53 knockdown. The production of 2-HG elicited an epigenetic reprogramming associated with a disruption in mitochondrial activity and an enhancement of autophagy in mIDH1 glioma cells. Autophagy is a mechanism involved in cell homeostasis related with cell survival under energetic stress and DNA damage protection. Autophagy has been associated with radio resistance. The inhibition of autophagy thus radio sensitizes mIDH1 glioma cells and enhances survival of mIDH1 glioma-bearing mice, representing a novel therapeutic target for this glioma subtype with potential applicability in combined clinical strategies.

KRT17High/CXCL8+ tumor cells display both classical and basal features and regulate myeloid infiltration in the pancreatic cancer microenvironment.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is generally divided in two subtypes, classical and basal. Recently, single cell RNA sequencing has uncovered the co-existence of basal and classical cancer cells, as well as intermediary cancer cells, in individual tumors. The latter remains poorly understood; here, we sought to characterize them using a multimodal approach. EXPERIMENTAL DESIGN: We performed subtyping on a single cell RNA sequencing dataset containing 18 human PDAC samples to identify multiple intermediary subtypes. We generated patient-derived PDAC organoids for functional studies. We compared single cell profiling of matched blood and tumor samples to measure changes in the local and systemic immune microenvironment. We then leveraged longitudinally patient-matched blood to follow individual patients over the course of chemotherapy. RESULTS: We identified a cluster of KRT17-high intermediary cancer cells that uniquely express high levels of CXCL8 and other cytokines. The proportion of KRT17High/CXCL8+ cells in patient tumors correlated with intra-tumoral myeloid abundance, and, interestingly, high pro-tumor peripheral blood granulocytes, implicating local and systemic roles. Patient-derived organoids maintained KRT17High/CXCL8+cells and induced myeloid cell migration in an CXCL8-dependent manner. In our longitudinal studies, plasma CXCL8 decreased following chemotherapy in responsive patients, while CXCL8 persistence portended worse prognosis. CONCLUSIONS: Through single cell analysis of PDAC samples we identified KRT17High/CXCL8+ cancer cells as an intermediary subtype, marked by a unique cytokine profile and capable of influencing myeloid cells in the tumor microenvironment and systemically. The abundance of this cell population should be considered for patient stratification in precision immunotherapy.

H3.3-G34R Mutation-Mediated Epigenetic Reprogramming Leads to Enhanced Efficacy of Immune Stimulatory Gene Therapy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are diffuse and highly aggressive CNS tumors which remain incurable, with a 5-year overall survival of less than 20%. Within glioma, mutations in the genes encoding the histones H3.1 and H3.3 have been discovered to be age-restricted and specific of pHGGs. This work focuses on the study of pHGGs harboring the H3.3-G34R mutation. H3.3-G34R tumors represent the 9-15% of pHGGs, are restricted to the cerebral hemispheres, and are found predominantly in the adolescent population (median 15.0 years). We have utilized a genetically engineered immunocompetent mouse model for this subtype of pHGG generated via the Sleeping Beauty-transposon system. The analysis of H3.3-G34R genetically engineered brain tumors by RNA-Sequencing and ChIP-Sequencing revealed alterations in the molecular landscape associated to H3.3-G34R expression. In particular, the expression of H3.3-G34R modifies the histone marks deposited at the regulatory elements of genes belonging to the JAK/STAT pathway, leading to an increased activation of this pathway. This histone G34R-mediated epigenetic modifications lead to changes in the tumor immune microenvironment of these tumors, towards an immune-permissive phenotype, making these gliomas susceptible to TK/Flt3L immune-stimulatory gene therapy. The application of this therapeutic approach increased median survival of H3.3-G34R tumor bearing animals, while stimulating the development of anti-tumor immune response and immunological memory. Our data suggests that the proposed immune-mediated gene therapy has potential for clinical translation for the treatment of patients harboring H3.3-G34R high grade gliomas.

Analysis of Donor Pancreata Defines the Transcriptomic Signature and Microenvironment of Early Neoplastic Lesions.

The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. SIGNIFICANCE: Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression. See related commentary by Hoffman and Dougan, p. 1288. This article is highlighted in the In This Issue feature, p. 1275.

Arginase 1 is a key driver of immune suppression in pancreatic cancer.

An extensive fibroinflammatory stroma rich in macrophages is a hallmark of pancreatic cancer. In this disease, it is well appreciated that macrophages are immunosuppressive and contribute to the poor response to immunotherapy; however, the mechanisms of immune suppression are complex and not fully understood. Immunosuppressive macrophages are classically defined by the expression of the enzyme Arginase 1 (ARG1), which we demonstrated is potently expressed in pancreatic tumor-associated macrophages from both human patients and mouse models. While routinely used as a polarization marker, ARG1 also catabolizes arginine, an amino acid required for T cell activation and proliferation. To investigate this metabolic function, we used a genetic and a pharmacologic approach to target Arg1 in pancreatic cancer. Genetic inactivation of Arg1 in macrophages, using a dual recombinase genetically engineered mouse model of pancreatic cancer, delayed formation of invasive disease, while increasing CD8+ T cell infiltration. Additionally, Arg1 deletion induced compensatory mechanisms, including Arg1 overexpression in epithelial cells, namely Tuft cells, and Arg2 overexpression in a subset of macrophages. To overcome these compensatory mechanisms, we used a pharmacological approach to inhibit arginase. Treatment of established tumors with the arginase inhibitor CB-1158 exhibited further increased CD8+ T cell infiltration, beyond that seen with the macrophage-specific knockout, and sensitized the tumors to anti-PD1 immune checkpoint blockade. Our data demonstrate that Arg1 drives immune suppression in pancreatic cancer by depleting arginine and inhibiting T cell activation.

Analysis of donor pancreata defines the transcriptomic signature and microenvironment of early pre-neoplastic pancreatic lesions.

The adult healthy human pancreas has been poorly studied given lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathological analysis of the samples revealed PanIN lesions in most individuals irrespective of age. Using a combination of multiplex immunohistochemistry, single cell RNA sequencing, and spatial transcriptomics, we provide the first ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts, and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. Statement of significance: The causes underlying the onset of pancreatic cancer remain largely unknown, hampering early detection and prevention strategies. Here, we show that PanIN are abundant in healthy individuals and present at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell intrinsic factors that restrain, or, conversely, promote, malignant progression.

Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance.

PURPOSE: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). EXPERIMENTAL DESIGN: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. RESULTS: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. CONCLUSIONS: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.

WNT signaling in the tumor microenvironment promotes immunosuppression in murine pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is associated with activation of WNT signaling. Whether this signaling pathway regulates the tumor microenvironment has remained unexplored. Through single-cell RNA sequencing of human pancreatic cancer, we discovered that tumor-infiltrating CD4+ T cells express TCF7, encoding for the transcription factor TCF1. We conditionally inactivated Tcf7 in CD4 expressing T cells in a mouse model of pancreatic cancer and observed changes in the tumor immune microenvironment, including more CD8+ T cells and fewer regulatory T cells, but also compensatory upregulation of PD-L1. We then used a clinically available inhibitor of Porcupine, a key component of WNT signaling, and observed similar reprogramming of the immune response. WNT signaling inhibition has limited therapeutic window due to toxicity, and PD-L1 blockade has been ineffective in PDA. Here, we show that combination targeting reduces pancreatic cancer growth in an experimental model and might benefit the treatment of pancreatic cancer.

H3.3-G34 mutations impair DNA repair and promote cGAS/STING-mediated immune responses in pediatric high-grade glioma models.

Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation led to the downregulation of DNA repair pathways. This resulted in enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG led to the accumulation of extrachromosomal DNA, which activated the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier-permeable PARP inhibitor pamiparib and the cell-cycle checkpoint CHK1/2 inhibitor AZD7762), and these combinations resulted in long-term survival for approximately 50% of the mice. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induced immune-mediated therapeutic efficacy in G34-mutant pHGG.

Using Single Cell Transcriptomics to Elucidate the Myeloid Compartment in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a 5-year survival rate of 10%. A hallmark feature of this disease is its abundant microenvironment which creates a highly immunosuppressive milieu. This is, in large part, mediated by an abundant infiltration of myeloid cells in the PDAC tumor microenvironment. Consequently, therapies that modulate myeloid function may augment the efficacy of standard of care for PDAC. Unfortunately, there is limited understanding about the various subsets of myeloid cells in PDAC, particularly in human studies. This review highlights the application of single-cell RNA sequencing to define the myeloid compartment in human PDAC and elucidate the crosstalk between myeloid cells and the other components of the tumor immune microenvironment.

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

A novel miR1983-TLR7-IFNß circuit licenses NK cells to kill glioma cells, and is under the control of galectin-1.

Although pharmacological stimulation of TLRs has anti-tumor effects, it has not been determined whether endogenous stimulation of TLRs can lead to tumor rejection. Herein, we demonstrate the existence of an innate anti-glioma NK-mediated circuit initiated by glioma-released miR-1983 within exosomes, and which is under the regulation of galectin-1 (Gal-1). We demonstrate that miR-1983 is an endogenous TLR7 ligand that activates TLR7 in pDCs and cDCs through a 5'-UGUUU-3' motif at its 3' end. TLR7 activation and downstream signaling through MyD88-IRF5/IRF7 stimulates secretion of IFN-ß. IFN-ß then stimulates NK cells resulting in the eradication of gliomas. We propose that successful immunotherapy for glioma could exploit this endogenous innate immune circuit to activate TLR7 signaling and stimulate powerful anti-glioma NK activity, at least 10-14 days before the activation of anti-tumor adaptive immunity.

Genetic Alterations in Gliomas Remodel the Tumor Immune Microenvironment and Impact Immune-Mediated Therapies.

High grade gliomas are malignant brain tumors that arise in the central nervous system, in patients of all ages. Currently, the standard of care, entailing surgery and chemo radiation, exhibits a survival rate of 14-17 months. Thus, there is an urgent need to develop new therapeutic strategies for these malignant brain tumors. Currently, immunotherapies represent an appealing approach to treat malignant gliomas, as the pre-clinical data has been encouraging. However, the translation of the discoveries from the bench to the bedside has not been as successful as with other types of cancer, and no long-lasting clinical benefits have been observed for glioma patients treated with immune-mediated therapies so far. This review aims to discuss our current knowledge about gliomas, their molecular particularities and the impact on the tumor immune microenvironment. Also, we discuss several murine models used to study these therapies pre-clinically and how the model selection can impact the outcomes of the approaches to be tested. Finally, we present different immunotherapy strategies being employed in clinical trials for glioma and the newest developments intended to harness the immune system against these incurable brain tumors.

Targeting gliomas with STAT3-silencing nanoparticles.

Glioblastoma is an aggressive brain tumor with poor prognosis. The brain is protected by the blood-brain barrier, which precludes transport of chemotherapeutics. We developed nanoparticles that achieve delivery of small-interfering RNA against Stat3 after systemic administration. Nanoparticles combined with radiation inhibited tumor progression and elicited anti-glioblastoma immunity in mice.

Inhibition of 2-hydroxyglutarate elicits metabolic reprogramming and mutant IDH1 glioma immunity in mice.

Mutant isocitrate dehydrogenase 1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into 2 molecular subgroups: 1p/19q codeletion/TERT-promoter mutations or inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work focuses on glioma subtypes harboring mIDH1, TP53, and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of D-2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma-bearing mice. Also, D-2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to D-2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in WT-IDH gliomas. Thus, we combined D-2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint blockade and observed complete tumor regression in 60% of mIDH1 glioma-bearing mice. This combination strategy reduced T cell exhaustion and favored the generation of memory CD8+ T cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data support the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.

Systemic brain tumor delivery of synthetic protein nanoparticles for glioblastoma therapy.

Glioblastoma (GBM), the most aggressive form of brain cancer, has witnessed very little clinical progress over the last decades, in part, due to the absence of effective drug delivery strategies. Intravenous injection is the least invasive drug delivery route to the brain, but has been severely limited by the blood-brain barrier (BBB). Inspired by the capacity of natural proteins and viral particulates to cross the BBB, we engineered a synthetic protein nanoparticle (SPNP) based on polymerized human serum albumin (HSA) equipped with the cell-penetrating peptide iRGD. SPNPs containing siRNA against Signal Transducer and Activation of Transcription 3 factor (STAT3i) result in in vitro and in vivo downregulation of STAT3, a central hub associated with GBM progression. When combined with the standard of care, ionized radiation, STAT3i SPNPs result in tumor regression and long-term survival in 87.5% of GBM-bearing mice and prime the immune system to develop anti-GBM immunological memory.

An Optimized Protocol for In Vivo Analysis of Tumor Cell Division in a Sleeping Beauty-Mediated Mouse Glioma Model.

Malignant gliomas are the most common and aggressive primary brain tumor in adults, and high mitotic rates are associated with their malignancy. Gliomas were modeled in mice using the Sleeping Beauty system to encode genetic lesions recapitulating the human disease. The presented workflow allows the study of the proliferation of glioma cells in vivo, enabling the identification of different phases of the cell cycle, with the advantage that 5-ethynyl-2'-deoxyuridine staining does not involve denaturation steps and samples do not require histological processing. For complete details on the use and execution of this protocol, please refer to Núñez et al. (2019).

Immunotherapy for gliomas: shedding light on progress in preclinical and clinical development.

INTRODUCTION: Gliomas are infiltrating brain tumors associated with high morbidity and mortality. Current standard of care includes radiation, chemotherapy, and surgical resection. Today, survival rates for malignant glioma patients remain dismal and unchanged for decades. The glioma microenvironment is highly immunosuppressive and consequently this has motivated the development of immunotherapies for counteracting this condition, enabling the immune cells within the tumor microenvironment to react against this tumor. AREAS COVERED: The authors discuss immunotherapeutic strategies for glioma in phase-I/II clinical trials and illuminate their mechanisms of action, limitations, and key challenges. They also examine promising approaches under preclinical development. EXPERT OPINION: In the last decade there has been an expansion in immune-mediated anti-cancer therapies. In the glioma field, sophisticated strategies have been successfully implemented in preclinical models. Unfortunately, clinical trials have not yet yielded consistent results for glioma patients. This could be attributed to our limited understanding of the complex immune cell infiltration and its interaction with the tumor cells, the selected time for treatment, the combination with other therapies and the route of administration of the agent. Applying these modalities to treat malignant glioma is challenging, but many new alternatives are emerging to by-pass these hurdles.

Synthetic High-density Lipoprotein Nanodiscs for Personalized Immunotherapy Against Gliomas.

PURPOSE: Gliomas are brain tumors with dismal prognoses. The standard-of-care treatments for gliomas include surgical resection, radiation, and temozolomide administration; however, they have been ineffective in providing significant increases in median survival. Antigen-specific cancer vaccines and immune checkpoint blockade may provide promising immunotherapeutic approaches for gliomas. EXPERIMENTAL DESIGN: We have developed immunotherapy delivery vehicles based on synthetic high-density lipoprotein (sHDL) loaded with CpG, a Toll-like receptor-9 agonist, and tumor-specific neoantigens to target gliomas and elicit immune-mediated tumor regression. RESULTS: We demonstrate that vaccination with neoantigen peptide-sHDL/CpG cocktail in combination with anti-PD-L1 immune checkpoint blocker elicits robust neoantigen-specific T-cell responses against GL261 cells and eliminated established orthotopic GL261 glioma in 33% of mice. Mice remained tumor free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of immunologic memory. Moreover, in a genetically engineered murine model of orthotopic mutant IDH1 (mIDH1) glioma, sHDL vaccination with mIDH1 neoantigen eliminated glioma in 30% of animals and significantly extended the animal survival, demonstrating the versatility of our approach in multiple glioma models. CONCLUSIONS: Overall, our strategy provides a general roadmap for combination immunotherapy against gliomas and other cancer types.