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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

High-grade gliomas (HGG) are the most common malignant primary brain tumors in adults, with a median survival of ~18 months. The standard of care (SOC) is maximal safe surgical resection, and radiation therapy with concurrent and adjuvant temozolomide. This protocol remains unchanged since 2005, even though HGG median survival has marginally improved.Gene therapy was developed as a promising approach to treat HGG. Here we review completed and ongoing clinical trials employing viral and non-viral vectors for adult and pediatric HGG, as well as the key supporting preclinical data.These therapies have proven safe, and pre- and post-treatment tissue analyses demonstrated tumor cell lysis, increased immune cell infiltration, and increased systemic immune function. Although viral therapy in clinical trials has not yet significantly extended survival of HGG, promising strategies are being tested. Oncolytic HSV vectors have shown promising results both for adult and pediatric HGG. A recently published study demonstrated that HG47Δ improved survival in recurrent HGG. Likewise, PVSRIPO has shown survival improvement compared to historical controls. It is likely that further analysis of these trials will stimulate the development of new administration protocols, and new therapeutic combinations which will improve HGG prognosis.

Supplementary Figures and Figure Legends

Table S2. Liquid Chromatography and Mass Spectrometry Measurement of 2-hydroxyglutarate in the Conditioned Media of mIDH1 GCCs untreated, treated with vehicle (DMSO), or mIDH1 inhibitor (AGI-5198 or DS1001b)

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

Supplementary Figure 5: Low dose Rapamycin does not enhance efficacy of Ad-Flt3L + Ad-TK/GCV

Supplementary Figure 2: Effect of Rapamycin on GL26 and RG2 cells

Supplementary materials and methods

Supplementary Figure 1: Flow analysis showing Annexin V and PI stains

The success of immunotherapeutic approaches targeting glioblastoma multiforme (GBM) demands a robust antiglioma T-cell cytotoxic and memory response. Recent evidence suggests that rapamycin regulates T-cell differentiation. Herein, we tested whether administration of rapamycin could enhance the efficacy of immunotherapy utilizing Fms-like tyrosine kinase 3 ligand (Ad-Flt3L) and thymidine kinase/ganciclovir (Ad-TK/GCV). Using the refractory rat RG2 glioma model, we demonstrate that administration of rapamycin with Ad-Flt3L + Ad-TK/GCV immunotherapy enhanced the cytotoxic activity of antitumor CD8+ T cells. Rats treated with rapamycin + Ad-Flt3L + Ad-TK/GCV exhibited massive reduction in the tumor volume and extended survival. Rapamycin administration also prolonged the survival of Ad-Flt3L + Ad-TK/GCV–treated GL26 tumor–bearing mice, associated with an increase in the frequency of tumor-specific and IFNγ+ CD8+ T cells. More importantly, rapamycin administration, even for a short interval, elicited a potent long-lasting central memory CD8+ T-cell response. The enhanced memory response translated to an increased frequency of tumor-specific CD8+ T cells within the tumor and IFNγ release, providing the mice with long-term survival advantage in response to tumor rechallenge. Our data, therefore, point to rapamycin as an attractive adjuvant to be used in combination with immunotherapy in a phase I clinical trial for GBM. Mol Cancer Ther; 13(12); 3024–36. ©2014 AACR.

Supplementary Table 1: Rat and mouse specific antibodies

Supplementary Figure 3: Phenotype of Flt3L derived murine BMDCs

Supplementary Methods

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Plasmid map of ACVR1 G328V Sleeping Beauty plasmid

Supplementary Figures 1-4 from Engineering the Brain Tumor Microenvironment Enhances the Efficacy of Dendritic Cell Vaccination: Implications for Clinical Trial Design

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.

Summplementary Materials and Methods

PDF file - 220K, Correlation between survival and circulating levels of HMGB1.

Purpose: Glioblastoma multiforme is the most common primary brain cancer in adults. Chemotherapy with temozolomide (TMZ) significantly prolongs the survival of patients with glioblastoma multiforme. However, the three-year survival is still approximately 5%. Herein, we combined intratumoral administration of an adenoviral vector expressing Flt3L (Ad-Flt3L) with systemic temozolomide to assess its impact on therapeutic efficacy.Experimental Design: Wild-type or immunodeficient mice bearing intracranial glioblastoma multiforme or metastatic melanoma were treated with an intratumoral injection of Ad-Flt3L alone or in combination with the conditionally cytotoxic enzyme thymidine kinase (Ad-TK), followed by systemic administration of ganciclovir and temozolomide. We monitored survival and measured the tumor-infiltrating immune cells.Results: Although treatment with temozolomide alone led to a small improvement in median survival, when used in combination with gene therapy-mediated immunotherapy, it significantly increased the survival of tumor-bearing mice. The antitumor effect was further enhanced by concomitant intratumoral administration of Ad-TK, leading to 50% to 70% long-term survival in all tumor models. Although temozolomide reduced the content of T cells in the tumor, this did not affect the therapeutic efficacy. The antitumor effect of Ad-Flt3L+Ad-TK+TMZ required an intact immune system because the treatment failed when administered to knock out mice that lacked lymphocytes or dendritic cells.Conclusions: Our results challenge the notion that chemotherapy leads to a state of immune-suppression which impairs the ability of the immune system to mount an effective antitumor response. Our work indicates that temozolomide does not inhibit antitumor immunity and supports its clinical implementation in combination with immune-mediated therapies. Clin Cancer Res; 20(6); 1555–65. ©2014 AACR.

PDF file - 884K, Efficacy of TMZ in combination with Ad-TK and Ad-Flt3L in intracranial astrocytoma and melanoma models.

Table of antibodies used

Supplementary Data from Release of HMGB1 in Response to Proapoptotic Glioma Killing Strategies: Efficacy and Neurotoxicity

PDF file - 1820K, Effect of TMZ on the number of circulating lymphocytes.

PDF file - 2644K, Effect of TMZ in combination with immunotherapy on the content of lymphocytes in the spleen.

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Supplementary Data from Antiglioma Immunological Memory in Response to Conditional Cytotoxic/Immune-Stimulatory Gene Therapy: Humoral and Cellular Immunity Lead to Tumor Regression

PDF file - 2705K, Efficacy of TMZ in combination with Ad-Flt3L in intracranial astrocytoma and melanoma models.

Purpose:Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy.Experimental Design:We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen–specific T cells.Results:Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFβ signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS).Conclusions:This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors.

PDF file - 1495K, Effect of TMZ on the number of T cells in spleen and bone marrow.

Purpose: Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Clinical trials for GBM using dendritic cell (DC) vaccination resulted in antitumor immune responses. Herein, we tested the hypothesis that combining in situ (intratumoral) Ad-Flt3L/Ad-TK–mediated gene therapy with DC vaccination would increase therapeutic efficacy and antitumor immunity.Experimental Design: We first assessed the immunogenicity of tumor lysates generated by Ad-TK (+GCV), temozolomide (TMZ), or freeze/thawing cycles (FTC) in a syngeneic brain tumor model. We also assessed phenotypic markers, cytokine release, and phagocytosis of bone marrow–derived DCs generated by fms-like tyrosine kinase 3 ligand (Flt3L) + IL-6 or by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4. Inhibition of tumor progression and production of anti-GBM antibodies was assessed following vaccination with (i) tumor cell lysates, (ii) DCs generated with either Flt3L/IL-6 or GM-CSF/IL-4 loaded with either Ad-TK/GCV-, TMZ-, or FTC-generated tumor lysates, or (iii) DCs in combination with in situ Ad-Flt3L/Ad-TK gene therapy.Results: DCs loaded with tumor cell lysates generated with either Ad-TK/GCV or TMZ led to increased levels of phagocytosis, therapeutic efficacy, and humoral immune response. In situ immunogene therapy in combination with DC vaccination led to brain tumor regression and long-term survival in about 90% of animals, a significant increase when compared with either therapy alone.Conclusions: Our results indicate that modifying the tumor microenvironment using intratumoral Ad-Flt3L/Ad-TK–mediated gene therapy potentiates therapeutic efficacy and antitumor immunity induced by DC vaccination. These data support novel phase I clinical trials to assess the safety and efficacy of this combined approach. Clin Cancer Res; 17(14); 4705–18. ©2011 AACR.

Supplementary Table 1 Supplementary Figures 1-4

PDF file - 5039K, HMGB1 translocation in vitro in citrine-GL26-Cherry-HMGB1 cells.

Supplementary Figure S7. Anti-CD3ε immunolabeled brain tissue sections from mouse A1 (top image) and mouse N2 (bottom image) shown in Figure 5I.

BACKGROUNDHigh-grade gliomas are fatal with universally poor prognosis. Initiation of effective cancer immune responses requires functional immune cells, especially afferent antigen presenting cells, which are absent from the brain parenchyma.1, 2To address this requirement, two adenoviral vectors expressing HSV1-TK and Flt3L were combined to target human gliomas. This first in human trial assessed safety, cytotoxicity, and immune recruitment to the tumor.METHODSEighteen treatment-naïve adults with high-grade glioma received injections of HSV1-TK- and Flt3L-expressing adenoviral vectors to the tumor bed after maximal safe resection, at six escalating doses (total 1.1×1010-2×1011vp), followed by two 14-day courses of valacyclovir, and standard upfront chemoradiation. Key inclusion criteria were: age 18-75, KPS ≥70, and treatment-naïve high-grade glioma amenable to gross total resection. Patients were consented pre-operatively. Enrollment occurred intraoperatively, upon pathology confirmation.RESULTSThe treatment was well tolerated without dose-limiting toxicity in patients with glioblastoma (n=17) (3 of the gliosarcoma variant), or anaplastic ependymoma (n=1). The maximal-tolerated dose was not reached. The median overall-survival was 21.3 months (95%CI: 11.1, 26.1). Tissues from subsequent resections from 8 subjects showed elevated markers for CD3+ and CD8+ T cells indicating potential successful anti-glioma immunity recruitment. Multiplex immunohistochemistry on two patients indicated a change in the intratumoral redistribution of CD8+ T cells and microglia/macrophages.CONCLUSIONSUse of two adenoviral vectors expressing HSV1-TK and Flt3L is safe and well tolerated in newly diagnosed high-grade glioma patients. Promising multiplex immunocytochemical evidence of immune infiltration warrants further efficacy studies, possibly in combination with blockade of the inhibitory tumor microenvironment.(Funded in part by Phase One Foundation, Los Angeles, CA, The Board of Governors at Cedars-Sinai Medical Center, and The Rogel Cancer Center at The University of Michigan; clinicaltrials.gov:NCT01811992)

Glioblastoma (GBM) is the most aggressive and immunosuppressive form of brain tumor, and its treatment remains a large, unmet medical need. Recently, we introduced oncostreams to refer to dynamic multicellular neuropathological structures that facilitate glioma cell growth and invasion into the normal brain. Moreover, we have shown that targeting Col1α1 within gliomas eradicates oncostreams and prolongs median survival in two genetic murine models. However, the signaling that collagen utilizes to maintain the glioma tumor microenvironment is still unknown. Our RNA-seq data show that collagen receptor DDR1 is overexpressed in genetically engineered glioma mouse models (GEMMs) including NPA (NRAS/shp53/shATRX), NPD (NRAS/shp53/PDGFβ), and NPAI (NRAS/shp53/shATRX/IDH1R132H) compared to healthy mouse brain tissue. We discovered that pharmacological inhibition of DDR1 radio-sensitized gliomas in vitro and dismantled oncostreams ex vivo, imaged with time-lapse confocal microscopy. GEMMs of DDR1 knockdown (NRAS/shp53/shATRX/shDDR1) using the Sleeping Beauty transposase system significantly increased median survival. This suggests that gliomas employ DDR1 mediated mechanisms to promote the immunosuppressive TME and thus stimulate tumor growth. Inhibition of DDR1 within gliomas enhanced intratumoral infiltration of CD45+, and CD3+ immune cells at the tumor core and invasive tumor border and prolonged median survival in GEMMs of glioma. We postulate that glioma DDR1 blocks immune-surveillance by enhancing collagen fiber alignment, which we assessed using collagen-specific second-harmonic generation microscopy. In human datasets such as CGGA and TCGA, DDR1 expression negatively correlates with PTPRC (CD45) gene expression. Furthermore, our results show that DDR1 inhibition suppresses oncostream formation, impairs glioma cell proliferation (PCNA+) and remodeled the tumor microenvironment by lowering Iba1+ glioma-associated microglia. We propose that DDR1 inhibition within glioma cells reprograms the TME to an immune-stimulatory state with enhanced radio-sensitivity. Targeting the DDR1 collagen receptor is a novel and highly promising avenue for GBM therapeutics. Citation Format: Syed M. Faisal, Andrea Comba, Maria L. Varela, Anna E. Argento, Emily Brumley, Molly E. West, Santiago Haase, Anzar A. Mujeeb, Clifford Abel, Marcus N. Barissi, Jarred E. Clewner, Brooklyn Stack, Grace A. Abbud, Maria G. Castro, Pedro R. Lowenstein. Targeting discoidin domain receptor 1 (DDR1) reverses glioma immune suppression by remodeling collagen fiber architecture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5835.

Recent advances in organ on a chip blood brain barriers (BBB) to study brain metastasis have revealed a critical understanding about the metastatic cascade. Most BBB models utilize collagen as brain parenchyma. While the exact role of the tumor microenvironment (TME) in brain metastasis is poorly understood, it has been observed to influence tumor growth. Therefore, the contents of this space must mimic the human brain composition. The extracellular matrix (ECM) occupies 10–20% of the brain volume to form its microenvironment (BME) and contains large quantities of hyaluronan/hyaluronic acid (HyA). Therefore, although collagen is widely used to mimic BME in vitro we propose that HyA is a more appropriate substrate. In this study using our published blood brain barrier niche on a chip (BBN), we use HyA matrix to evaluate brain metastasis. We compared cancer cell phenotypes, and TME remodeling to prior work using type I collagen. The BBN device has two chambers and a 5 µm microporous membrane sandwiched between them. Human brain microvascular endothelial cells (hCMECs) are seeded over the membrane in the upper chamber. The bottom chamber contains a brain stroma and normal human astrocytes (NHA)s. Cancer cells are added into top chamber and allowed to extravasate into the brain niche. We studied the phenotypic differences of human MDA-MB-231 breast cancer cells and their brain-seeking subclone MDA-MB-231-BRs in between 1:3 HyA:media mixture, and 2% collagen diluted in media for 9 days. Both migratory and phenotypic characteristics of cancer cell lines were measured by volume, shape, and positions in the brain stroma relative to the endothelial barrier. We analyzed sphericity and the distance from the barrier to the final positions on days 2 and 9. Comparing with cells extravasated between the collagen and 1:3 mixtures, all parameters showed marked differences, especially in day 9. In 1:3 samples, more cells were in a single plane near barrier than in the 2% collagen which might be result due to biophysical differences. To study the biophysical variations, matrix responses to oscillatory shear were measured using a rheometer. Briefly, the samples were subjected to a frequency sweep with a maximum strain of 1%. Both storage modulus (G’)/stiffness and loss modulus (G”) were measured in the linear viscoelastic region. We evaluated the stiffness of 1:1, 1:3 HyA:media mixtures, undiluted HyA and 2% collagen. Undiluted HyA was the stiffest, around 60 Pa, followed by 1:1 and 1:3 mixtures, about 5 Pa and 3.5 Pa, respectively. 2% collagen was the softest among the samples, much weaker than 0.01 Pa, shown liquid-like behaviors.In conclusion, due to biological and biophysical differences between matrices, marked variations were observed in cancer cell phenotypes. The data gathered can be used to cover the knowledge gaps about brain metastasis and may indicate the need to consider the efficacy of using the BME mimicking HyA for more realistic findings. Citation Format: Ehsanul Hoque Apu, Huing li, Victor Zhao, Habib Serhan, Aki Morikawa, Pedro R. Lowenstein, Maria G. Castro, Sofia D. Merajver, Christopher Ryan Oliver. Comparison of brain extracellular matrices in a blood brain barrier organ on a chip model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1322.

Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. Establishment of glioma-specific methylation patterns by mIDH1 reprogramming drives oncogenic features of cancer metabolism, stemness and therapeutic resistance. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1. Treatment of glioma cells with mIDH1 specific inhibitor AGI-5198, upregulated gene networks involved in replication stress. Specifically, we found that the expression of ZMYND8, a regulator of DNA damage response was decreased in three patient-derived glioma cell cultures (GCC) after treatment with AGI-5198. ZMYND8 functions as a chromatin reader to modulate enhancer activity and recruit DNA repair machinery. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 knockout (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. The accumulation of DNA damage in ZMYND8 KO mIDH1 GCCs promoted the phosphorylation of cell cycle checkpoint proteins Chk1 and Chk2. In addition, the recruitment of ZMYND8 to sites of DNA damage has been shown to be PARP- dependent. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma.Translational Relevance:Our understanding of radioresistance mechanisms in patient-derived glioma cell cultures (GCC) that endogenously express mIDH1-R132H are limited. We have uncovered a novel gene target Zinc Finger MYND-Type Containing 8 (ZMYND8) that is downregulated following treatment of human mIDH1 GCCs with mIDH1 specific inhibitors. We demonstrate that suppression of ZMYND8 expression by shRNA knockdown or genetic knockout reduces the cellular viability of mIDH1 GCCs to ionizing radiation (IR). Our findings reveal an epigenetic vulnerability of mIDH1 GCCs to ZMYND8 knockout (KO) which results in impaired resolution of IR-induced DNA damage and induction of cell cycle arrest. Additionally, ZMYND8 KO mIDH1 GCCs display increased radiosensitivity to inhibition of epigenetic regulators BRD4, HDAC, and PARP which could be mediated by enhanced replicative stress.

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’s poor pharmacokinetic properties, and unfavorable bioavailability have 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.

Background Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. Methods Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected. Results Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial. Conclusions H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.