bluuebunny/crossref_metadata_2025 huggingface dataset:
EXTH-36. PHARMACEUTICAL INHIBITION OF CYCLIN DEPENDENT KINASE 4/6 (CDK4/6) IN GLIOBLASTOMA
Authors: Nuga Oluwademilade , Meng Yuling , Berezovsky Artem , Nelson Kevin , de Carvalho Ana
Published: December 2020

Potent and selective CDK4/6 inhibitors (CDK4/6i) have potential for treating glioblastoma. Although genomic abnormalities affecting CDK4/6-RB1 signaling axis, such as CDK4 or CDK6 amplification and CDKN2A deletion, which are frequent in GBM, have been proposed to predict response of RB1-wildtype GBM to CDK4/6i, there is not enough data validating these biomarkers as sufficient for patient selection. Here we employ a panel of GBM patient derived cancer stem cells (CSC) representing the most frequent somatic genomic alterations in GBM to test their response to CDK4/6i. Twelve CSC lines were treated in quintuplicate for 4–7 days with 0–10 mM Abemaciclib, Ribociclib or vehicle control. Cell viability was measured using CellTiterGlo and dose response curves were analyzed using GRmetrics in R to determine Area Above the Curve (AAC) and IC50, factoring variable growth rates among cell lines. Experiments were repeated 2–4 times. Abemaciclib was more potent in reducing cell viability, based on the RB1-null CSC line measured resistance (mean AAC = 0.3 +/- 0.06), ribociclib was more specific (mean AAC = 0.05 +/-0.01). 4/7 CSCs with CDKN2A homozygous deletion were sensitive (mean AAC > 0.7) while 3/7 were resistant (mean AAC < 0.33) to 4 and 7-day treatments of abemaciclib. One CSC from a newly diagnosed GBM bearing CDK4, MYC and EGFR amplifications was sensitive to both inhibitors (mean AAC = 0.5 abemaciclib and ribociclib), while CSC from the matched recurrent tumor presenting the same driver genomic alterations was significantly more resistant (mean AAC = 0.2 for abemaciclib and ribociclib) (p < 0.05, t-test). Additionally, we exposed a sensitive cell line to conditioned media from a resistant cohort, resulting in significantly reduced proliferation and increased resistance to CDK4/6i (p< 0.05, Dunn). These findings underscore the importance of a utilizing a robust molecular profiling approach in evaluating which patients will benefit from CDK4/6i therapy.
EXTH-38. ORTHOGONAL IN VIVO MODELS WERE THE SOLE PRECLINICAL PREDICTOR OF CLINICAL EFFICACY IN PHASE 1 TRIALS OF TARGETED THERAPIES FOR GLIOBLASTOMA: RESULTS OF A SYSTEMATIC REVIEW
Authors: Gunjur Ashray , Balasubramanian Adithya , Hafeez Umbreen , Menon Siddharth , Cher Lawrence , Parakh Sagun , Gan Hui
Published: December 2020

INTRODUCTION

No drug has improved survival in recurrent glioblastoma despite encouraging activity preclinically. We undertook a systematic review of matched preclinical and Phase 1 trials (P1Ts) of targeted agents to investigate potential preclinical predictors of clinical efficacy.

METHODS

We identified all adult glioblastoma monotherapy P1Ts of targeted agents & preceding preclinical data published between 2006–2019 via structured searches of EMBASE/MEDLINE/PUBMED. For preclinical studies, data regarding in vitro models, in vivo models (species, implantation site, cell-line type) and efficacy (growth inhibition, regression rate, survival) were extracted. For P1T, response rate (RR) data were collected as absolute (%) and categorical (RR< 5% vs. RR≥ 5%) variables. Associations were compared by chi-square/Fisher’s exact test, Kruskal-Wallis or Mann-Whitney U testing as appropriate with 2-sided p-values.

RESULTS

We found 28 P1Ts with median RR 2.9% (range 0.0–33.3%) and mOS 8.0mo (range 4.6–13.0mo). Seven (25%) had ‘minimal’ published pre-clinical data (5 missing entirely; 2 in vitro only); 12 (43%) utilised one cell line in vivo (‘single model’ group); and 9 (32%) used 2+ biologically distinct in vivo models (‘orthogonal’ group). There was strong reliance on U87-based cell lines (14/21 (71%)) in the latter groups. None of the variables tested were associated with RR except for use of ‘orthogonal models’. Compared to the ‘orthogonal’ group, the P1T RR rate was lower in ‘single’ and ‘minimal’ groups (6.8% vs 1.2%, p= 0.043 and 6.8% vs 0.0%, p= 0.026 respectively). The frequency of P1T with a RR > 5% was also higher in the ‘orthogonal’ compared to the same two groups (78% vs 20%, p= 0.042 and 78% vs 17%, p= 0.041).

CONCLUSION

The availability of good quality pre-clinical data, especially the use of orthogonal models in vivo, was significantly associated with P1T response rates and warrants further investigation as a minimal threshold of evidence in future drug development.
EXTH-41. THE microRNA miR-138 MAY BE RESPONSIBLE FOR SOME OF THE EFFECTS OF METABOLIC KETOSIS ON MALIGNANT GLIOMA
Authors: Perryman Richard , Zeng Qingyu , Raha Oishik , O’Neill Kevin , Scheck Adrienne , Syed Nelofer
Published: December 2020

We and others have demonstrated that altering metabolism through the use of a ketogenic diet (KD) has numerous effects in mouse models of malignant glioma including slowed growth, reduced hypoxia and angiogenesis, enhanced survival and potentiation of the effects of radiation and chemotherapy. These effects are recapitulated in vitro when the ketone β-hydroxybutyrate (BHB) is added to mouse and human malignant glioma cells grown under high glucose conditions. BHB has been demonstrated to be an epigenetic modifier, providing a potential mechanism for the pluripotent effects of ketones even in the presence of high glucose. Therefore, we have analyzed alterations in microRNA (miRNA) expression in tumors and cells treated with a KD or BHB, respectively. MicroRNAs are short, single-stranded, noncoding RNAs that posttranscriptionally control gene expression. Each miRNA can affect the expression of many genes and they are implicated in all hallmarks of cancer. We found a number of miRNAs deregulated in tumor tissue from mice fed a KD, particularly mmu-miR-138 which was increased over 30-fold relative to tumors from mice fed a standard diet. MiR-138 is involved in a variety of anti-tumor effects that correlate with our data from KD treated gliomas. We have shown that ketones radiosensitize glioma cells, potentially through a reduction in DNA damage repair. MiR-138 has been shown to reduce the expression of H2AX which plays a key role in sensing and repairing DNA damage. Hypoxia inducible factor-1α (HIF-1α) is also a target of miR-138, and we have demonstrated a reduction in hypoxia, angiogenesis and the expression of HIF-1α in tumors from mice fed a KD. Furthermore, when we genetically engineered tumor cells to express miR-138 and injected them into mice, only necrotic tumor foci were found. Thus, we suggest that miR-138 is an important regulatory mechanism for the anti-tumor actions of metabolic ketosis in glioma.
EXTH-47. PRECISION IMMUNOTHERAPY VACCINES FOR GLIOBLASTOMA USING CANCER IMMUNOGENOMICS AND SELECTIVE GENE ENRICHMENT STRATEGY
Authors: Trivedi Vrunda , Yang Changlin , Yegorov Oleg , Dyson Kyle , Mitchell Duane
Published: December 2020

BACKGROUND

Development of successful immunotherapy targeting antigens in Glioblastoma (GBM) remains a challenge owing to the heterogeneous nature of GBM and the low mutation burden. It is therefore important to identify multiple tumor antigens that can be targeted simultaneously. Our goal is to develop a personalized RNA vaccination approach that employs the use of cancer immunogenomics in identifying tumor immunogenic epitopes and target enrichment strategies that allow for targeting only the tumor specific antigens.

APPROACH

RNAseq and WES was performed for murine GBM tumors KR158 and GL261. Using our Open Reading Frame Antigen Network (O.R.A.N.) algorithm, we identified neoantigens and tumor-associated antigens (TAAs) which includes cancer testis and developmental antigens that are aberrantly over-expressed in the KR158 and GL261 tumors. The genes identified as harboring the target epitopes were subjected to a gene enrichment strategy which included hybridization, capture and amplification of our identified genes from a pool of total tumor cDNA. The precision RNA libraries generated from the enriched cDNA samples were then validated for enrichment of target genes using RNAseq.

RESULTS

We predicted 12 neoantigens and 15 TAAs as immunogenic targets for the KR158 tumor and 192 neoantigens and 37 TAAs for GL261. Pre-capture and amplification, the 27 KR158 tumor genes constituted only 1.14% of the total tumor cDNA pool, however, we were able to enrich the selected genes to up to 95% of the cDNA pool. The IVT RNA library generated from the enriched cDNA templates also consisted of a 90–95% majority of the predicted tumor-specific genes and only 5–10% of the genes were background.

CONCLUSION

We have demonstrated the ability to generate tumor antigen specific personalized RNA libraries for murine GBM tumors. RNA vaccines targeting specifically and simultaneously numerous tumor antigens, will pave way for the future of immunotherapy vaccines without the induction of intolerable autoimmunity.
EXTH-50. ACTIVATION OF THE MITOCHONDRIAL CLPP PROTEASE IS SYNTHETICALLY LETHAL WITH HDAC1/2 INHIBITION IN GLIOBLASTOMA MODEL SYSTEMS
Authors: Schiffgens Salveena , Nguyen Trang , Shu Chang , Elena Bianchetti , Torrini Consuelo , Karpel-Massler Georg , Siegelin Markus
Published: December 2020

Novel therapeutic targets are critical to unravel for recalcitrant malignancies, such as the most common primary brain tumor in adults, glioblastoma (GBM). Heterogeneity remains a hallmark of primary glial brain tumors and therefore targeting several pathways simultaneously is an appropriate approach. Here, we showed that pharmacological activation of the mitochondrial ClpP protease through utilization of the novel imipridone compounds (ONC206 and ONC212) in combination with global (Panobinostat) and selective (romidepsin) HDAC – inhibitors caused synergistic reduction of viability in established and patient-derived xenograft (PDX) cultures of human GBM. This effect occurred independent of TP53 status and was partially mediated by activation of a cell death with apoptotic features accompanied by activation of initiator and effector caspases as well as cleavage of PARP. Consistently, the combination treatment altered the expression of anti-apoptotic and pro-apoptotic Bcl-2 family members, resulting in down-regulation of Bcl-xL and Mcl-1. Knockdown experiments targeting Noxa, BIM, Bcl-xL and Mcl-1 confirmed a functional implication of these proteins in the reduction of cellular viability mediated by the combination treatment. Importantly, knockdown of the ClpP protease significantly rescued the reduction of cellular viability by ClpP activators and the combination treatment, respectively, suggesting critical involvement of this protein. Finally, using a PDX model, we demonstrated that the combination treatment of romidepsin and ONC206 reduced tumor growth more potently than single treatments or vehicle by enhanced reduction of cellular proliferation and pronounced induction of cell death in vivo. Collectively, these observations suggest that the efficacy of HDAC-inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.
EXTH-52. GLIOBLASTOMA-TARGETING AUTOLOGOUS INDUCED NEURAL STEM CELL THERAPY: EVALUATING SAFETY, TOXICITY, PERSISTENCE, AND TRANSPLANT METHODS IN A POST-SURGICAL CANINE MODEL
Authors: Bomba Hunter , Sheets Kevin , Valdivia Alain , Khagi Simon , Ruterbories Laura , Mariani Christopher , Borst Luke , Tokarz Debra , Hingtgen Shawn
Published: December 2020

BACKGROUND

Glioblastoma patient survival statistics have remained unchanged for more than three decades. Despite tumor resection and chemoradiotherapy, recurrence is inevitable. Moreover, the invasive behavior of glioblastoma confounds treatment. To improve patient survival statistics, a targeted therapy that can home to distant tumor foci is desperately needed. Induced neural stem cells (iNSCs) armed with cytotoxic payloads have proven efficacious against human xenograft models of glioblastoma. To further propel iNSCs to human clinical trials, we investigated the safety, toxicity, and persistence of iNSCs in a canine model.

METHODS

Autologous iNSCs generated from the skin of four non-tumor-bearing, purpose-bred, male beagles were engineered to express TRAIL and thymidine kinase (TK). iNSCs were loaded with ferumoxytol to facilitate MRI-tracking. Canines were divided into two cohorts to denote iNSC administration route: scaffold encapsulation or intracerebroventricular (ICV). Two dose levels were investigated: 1′106 iNSCs/kg or 3′106 iNSCs/kg. The scaffold cohort received a single dose of iNSCs while the ICV cohort received three doses of iNSCs via a Rickham reservoir. To activate TK, canines were administered valganciclovir. Canine health was assessed via neurological exams, MRI, and serial blood, urine, and CSF analyses.

RESULTS

No acute injection reactions were observed. Three of four canines exhibited surgery-induced blindness. Urine and CSF analyses were unremarkable. Unexpectedly, blood analyses showed transient neutropenia. Hypodense signal was observed on all MRI sequences through endpoint. Post-mortem histopathology of the spleen, liver, and lung were unremarkable. As expected, brain tissues exhibited gliosis, fibrous thickening, and inflammation. Spinal cords exhibited acute hemorrhaging, attributed to perimortem CSF draws. Surprisingly, significant testicular degeneration was observed; this was confirmed to be caused by valganciclovir. In conclusion, iNSCs exhibit limited toxicity and warrant further exploration.

FUTURE DIRECTIONS

Prospective studies will investigate the efficacy of autologous iNSCs in a spontaneous canine glioma model in preparation for human clinical trials.
EXTH-53. A BRAIN-PENETRANT MICROTUBULE-TARGETING AGENT THAT DISRUPTS HALLMARKS OF GLIOMA TUMORIGENESIS
Authors: Horne Eric , Vicente Ruiz Juan J , Cimino Patrick , Jung Erik , Xu Cong , Diaz Philippe , Hamel Ernest , Kumasaka Debra K , Wagenbach Michael J , Winkler Frank , Wordeman Linda G , Holland Eric , Stella Nephi
Published: December 2020

Most cancer therapeutics developed to date are excluded from the brain and are therefore ineffective in treating glioma, the most devastating type of brain cancer in adults. Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of solid and hematopoietic tumors, and evidence suggests that glioma is sensitive to MTAs; but most MTAs do not cross the blood-brain-barrier. To address this limitation, we developed a brain-penetrant MTA, ST-401, that inhibited the growth of human glioma in culture at nanomolar concentrations. ST-401 bound to the colchicine site, inhibited tubulin assembly and reversibly reduced microtubule (MT) dynamics. Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity does not correlate with any of the compounds tested thus far through this platform but showed weak correlations for two MTA that work through distinct mechanisms: taxol (p=0.46) and vinblastine (p=0.34). Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function. We discovered that ST-401 killed patient-derived (PD) glioma isolates in both mitosis and interphase, and inhibited the formation of tumor microtubes, MT-rich structures that connects glioma cells to a network that is resistant to standard therapies. Pharmacokinetic analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft model demonstrated significant antitumor activity. In an immunocompetent mouse model of platelet-derived growth factor B-driven glioma, ST-401 significantly enhanced the therapeutic efficacies of standard care therapies temozolomide and radiation therapy. Our study identified novel aspects of glioma tumorigenesis that exhibit enhanced sensitivity to MTAs and shows that the brain-penetrant MTA, ST-401, represents a promising chemical scaffold to develop therapeutics for the treatment of patients diagnosed with glioma.
EXTH-62. PRECLINICAL EFFICACY OF THE IMIPRIDONE ONC-206 AGAINST MEDULLOBLASTOMA
Authors: MacDonald Tobey , Malhotra Anshu , Liu Jingbo , Zhang Hongying , Schneiderjan Matthew , Vega Jose Velazquez , Prabhu Varun , Allen Joshua
Published: December 2020

Treatment for medulloblastoma (MB) is typically ineffective for MYC amplified or metastatic SHH, Group 3 and 4 subgroups. Promising preclinical and clinical results have been obtained for adult and pediatric malignant glioma treated with ONC-201, a selective antagonist of DRD2, a G-protein coupled receptor that regulates prosurvival pathways. Herein, we report the activity of ONC-201 and ONC-206, which has increased non-competitive antagonism of DRD2, against MB. We treated three different MB cell types representative of SHH- and Group 3-like cells, with varied levels of DRD2 expression, and consistently observed increased cell death in a dose-dependent manner at lower doses of ONC-206 compared to ONC-201. We also evaluated ClpP as an additional drug target in MB. ClpP is a mitochondrial protease that has been shown to directly bind and be activated by ONC 201, and is highly expressed at the protein level across pediatric MB, malignant glioma and ATRT, but not normal brain. We observed that similar to ONC-201, ONC-206 treatment of MB cells induces the restoration of mitochondrial membrane potential to the non-proliferative state, degradation of the mitochondrial substrate SDHB, reduction in survivin and elevation in ATF4 (integrated stress response). Importantly, ONC-206 treatment induced significant cell death of patient-derived SHH, WNT, and Group 3 tumors ex vivo and Group 4 cells in vitro, while having no observable toxicity in normal brain. ONC-206 treatment of a transgenic mouse model of Shh MB in vivo significantly reduces tumor growth and doubles survival time in a dose-dependent manner following 2 weeks of therapy. Additional in vivo data will be reported in preparation for a planned Phase I study of ONC-206 in children with malignant brain tumors.
EXTH-67. PHARMACOLOGIC INHIBITION OF LYSINE SPECIFIC DEMETHYLASE-1 (LSD1) AS AN ADJUVANT IMMUNE-SENSITIZATION STRATEGY IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)
Authors: Bailey Cavan , Collier Tiara , Henry Verlene , Monje Michelle , Nazarian Javad , Lee Dean , Wang Linghua , Gopalakrishnan Vidya , Becher Oren , Chandra Joya
Published: December 2020

Diffuse midline gliomas (DMG), including brainstem DIPG (diffuse intrinsic pontine glioma), are incurable pediatric brain tumors. Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, highlighting epigenetic aberrations in these tumors. Lysine specific demethylase-1 (LSD1), is an enzyme that acts upon mono and di-methylated histone H3K4/K9, and pharmacological inhibitors of its enzymatic and scaffolding domains are clinically viable. Patient-derived DIPG cell lines, orthotopic mouse models, and pediatric high-grade glioma (pHGG) datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Because an unique immune signature was sen with enzymatic LSD1 inhibitors in DIPG cells in vitro, immune cell cytotoxicity was assessed in DIPG cells pre-treated with LSD1 inhibitors and informatics platforms were used to determine immune infiltration of pHGG. Selective cytotoxicity and an immunogenic gene expression signature was established in DIPG cell lines and in brainstem tumor-bearing mice using clinically-relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit using this LSD1-dependent gene expression signature. On-target binding of catalytic LSD1 inhibitors was confirmed in DIPG and pre-treatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival while CD8 T-cells are negatively prognostic. Catalytic LSD1 inhibitors are non-perturbing to NK cells while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene expression signature in DIPG cells. Treatment of orthotopic xenograft bearing DIPG mouse models with catalytic LSD1 inhibitors and NK cells showed a significant reduction in tumor burden. In summary, we find that LSD1 inhibition using catalytic inhibitors is both selectively cytotoxic and promotes a functional immune gene expression signature that is associated with NK cell killing, representing a therapeutic opportunity for pHGG.
EXTH-70. CYP450 AND METABOLIC PROFILING OF ANTI-CANCER IMIPRIDONE ONC201
Authors: Morrow Sara , Prabhu Varun , Tarapore Rohinton S , Guo Ailan , Fang Yunlan , Nej Magdalena , Stogniew Martin , Allen Joshua
Published: December 2020

Imipridone ONC201 is an investigational agent in phase II clinical trials for high grade gliomas as a single agent once weekly. Systemic and tumor exposure to ONC201 exceeds therapeutic thresholds consistently; however, exposure is variable despite fasted conditions and does not strongly covary with body weight or body surface area. Clinical trials with ONC201 have restricted the use of concomitant medication that induce or inhibit cytochrome P450 (CYP) enzymes; however, several supportive medications for glioma patients such as corticosteroids and anti-seizure medications can affect these enzymes. Studies in pooled human liver microsomes revealed that ONC201 is not an inducer of seven major drug-metabolizing human CYP enzymes: CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. However, ONC201 inhibits multiple CYPs IC50 values ranging from 34.9 to 428.6 μM and is substrate of CYP3A4 and, to lesser extent, other CYPs. Given potential metabolism of ONC201 by these enzymes, we conducted metabolic profiling in human hepatocytes that revealed at least 13 metabolites; however, only one was >10% abundance. The major metabolite, termed ONC207, that lacks a benzyl moiety of ONC201 was assayed by LC-MS-MS in plasma samples obtained from 16 ONC201-treated H3 K27M-mutant glioma patients. Peak plasma concentrations of ONC207 relative to ONC201 was 22% (range: 6%-48%) and its abundance was not correlated with ONC201, suggesting that variable exposure to ONC201 may not be associated with variable metabolism. ONC207 does not inhibit dopamine receptor D2 or cancer cell viability in contrast to the parent compound. In summary, ONC201 is an inhibitor and substrate, but not an inducer, of multiple CYP enzymes. The major metabolite of ONC207 appears to be biologically inactive and is unlikely to explain variable ONC201 exposure or its prolonged biological activity that outlives its systemic presence.
EXTH-72. CONTINUOUS INFUSION STUDIES REVEAL THE POTENCY OF ELACRIDAR TO ACT AS A PHARMACO-ENHANCER FOR TREATMENT OF INTRACRANIAL DISEASES BY INHIBITING ABCB1 AND ABCG2 AT THE BLOOD-BRAIN BARRIER
Authors: van Tellingen Olaf , de Gooijer Mark C , Zuidema Stefanie , Meurs Amber , Çitirikkaya Ceren H , Venekamp Nikkie , Beijnen Jos H
Published: December 2020

The blood-brain barrier (BBB) is a formidable hurdle to successful pharmacotherapy of intracranial diseases. ABCB1 and ABCG2 are efflux transporters that play an important role in the BBB, keeping substances out of the brain. Elacridar and tariquidar are third-generation ABCB1-inhibitors developed for treatment of multidrug-resistant tumors. Later, they were shown to also inhibit ABCG2. We aim to improve pharmacotherapy of brain cancer by concomitant use of potentially effective drugs with elacridar. This study was undertaken to determine the relationship between the plasma concentration of the inhibitor and the brain-to-plasma (B/P) ratio of (model) substrate drugs in order to assess which type of drug may best qualify taking into account clinically achievable plasma levels of the inhibitor. We used Abcg2;Abcb1a/b double knockout (DKO), Abcb1a/b KO, Abcg2 KO and wild-type mice receiving a cocktail of 9 drugs at a fixed low dose plus a range of doses of inhibitor by 3-h intraperitoneal infusion to achieve steady-state conditions. DKO mice are the reference for complete inhibition, while single KO mice allow interrogation of the other transporter when using dual substrate drugs. Complete inhibition of Abcb1 by elacridar requires plasma levels of about 1000 nM. Inhibition of Abcg2 is more difficult. For erlotinib and palbociclib about 1000 nM of elacridar is sufficient, but other more profound substrate drugs (e.g. vemurafenib and afatinib) do not reach the B/P ratios achieved in DKO mice, even at 4000 nM. The improvement in B/P ratio that can be reached differs per substrate. Compounds like palbociclib benefit markedly from elacridar with B/P ratios rising from 0.25 to 7, whereas others (e.g. erlotinib and dasatinib) increase from about 0.10 to only 0.40. Thus, elacridar is an efficient pharmaco-enhancer of ABCB1 substrates and weaker ABCG2 substrates, but is not able to improve brain delivery of drugs that are profound ABCG2 substrates.
EXTH-73. HETEROGENEITY IN CELL SURFACE ANTIGEN AND HLA CLASS I EXPRESSION IN PEDIATRIC BRAIN TUMORS AND ITS IMPACT ON T-CELL IMMUNOTHERAPY
Authors: Houke Haley , Zhu Xiaoyan , Mercer Kimberly S , Stripay Jennifer L , Chiang Jason , Baker Suzanne , Roussel Martine F , Gottschalk Stephen , Krenciute Giedre
Published: December 2020

Immunotherapy with tumor antigen-specific chimeric antigen receptor (CAR) and/or ab (T-cell receptor) TCR T-cells has the potential to improve clinical outcomes of patients with pediatric brain tumors. As a prerequisite for successful T-cell therapies, we must determine which cell surface antigens are expressed and targetable in these tumors, and if HLA Class I is present, which is necessary for ab TCR T-cell recognition. Therefore, in this study we systematically analyzed pediatric patient-derived orthotopic xenograft (PDOX) brain tumor samples for cell surface expression of five known CAR targets: IL13Ra2, HER2, EphA2, B7-H3, and GD2, as well as HLA Class I. We established and validated a flow cytometry-based method of profiling tumor-associated antigens. Fifty-three PDOX samples have been profiled to date, including medulloblastoma, high grade glioma (HGG), diffuse intrinsic pontine glioma (DIPG), atypical teratoid rhabdoid tumor (ATRT), and ependymoma, among others. Our results showed high variability within and between individual samples. B7-H3 was the most consistently expressed, seen in 98% of the samples tested. We validated these results by conventional immunohistochemistry staining for B7-H3 and found comparable RESULTS: HLA Class I was highly expressed on all HGG samples but was undetectable on 47.8% of other brain tumor samples. This suggests that down-regulation of HLA class I is one mechanism by which brain tumors evade conventional T-cells, and that HLA-independent CAR T-cells would be useful therapies. We also compared expression of antigens in fresh patient samples and corresponding PDOX tumors and saw that they were indeed similar. To our knowledge, this is the largest group of pediatric brain tumor PDOX samples methodically analyzed for potential CAR target antigens and HLA Class I. Taken together, our data demonstrate that elimination of tumors by CAR T-cell immunotherapies will require targeting multiple antigens, and our profiling method could inform how to circumvent antigen-negative relapse.
IMMU-04. IMMUNE-RELATED ADVERSE EVENTS STRONGLY PREDICT SUPERIOR OUTCOMES IN BRAIN METASTASES PATIENTS RECEIVING LOCAL TREATMENT AND IMMUNE CHECKPOINT INHIBITORS
Authors: Hulsbergen Alexander , Lak Asad , Lo Yu Tung , Lamba Nayan , Nagtegaal Steven , Job Arun , Verhoeff Joost , Smith Timothy , Broekman Marike , Phillips John
Published: December 2020

INTRODUCTION

In several cancers treated with immune checkpoint inhibitors (ICIs), a remarkable association between the occurrence of immune-related adverse events (irAEs) and superior oncological outcomes has been reported. This effect has hitherto not been reported in the brain. This study aimed to investigate the relation between irAEs and outcomes in brain metastases (BM) patients treated with both local treatment to the brain (LT; i.e. surgery and/or radiation) and ICIs.

METHODS

This study is a retrospective cohort analysis of patients treated for non-small cell lung cancer (NSCLC) BMs in a tertiary institution in Boston, MA. Outcomes of interest were overall survival (OS) and intracranial progression-free survival (IC-PFS), measured from the time of LT. Sensitivity analyses were performed to account for immortal time bias (i.e., patients who live longer receive more cycles of ICIs and thus have more opportunity to develop an irAE).

RESULTS

A total of 184 patients were included; 62 (33.7%) were treated with neurosurgical resection and 122 (66.3%) with upfront brain radiation. irAEs occurred in 62 patients (33.7%). After adjusting for lung-Graded Prognostic Assessment, type of LT, type of ICI, newly diagnosed vs. recurrent BM, BM size and number, targetable mutations, and smoking status, irAEs were strongly associated with better OS (HR 0.33, 95% CI 0.19 – 0.58, p < 0.0001) and IC-PFS (HR 0.41; 95% CI 0.26 – 0.65; p = 0.0001). Landmark analysis including only patients who received more than 3 cycles of ICI (n = 133) demonstrated similar results for OS and IC-PFS, as did sensitivity analysis adjusting for the number of cycles administered (HR range 0.36 – 0.51, all p-values < 0.02).

CONCLUSIONS

After adjusting for known prognostic factors, irAEs strongly predict superior outcomes after LT in NSCLC BM patients. Sensitivity analysis suggests that this is unlikely due to immortal time bias.
IMMU-05. CSF METABOLOMICS AS A PREDICTOR OF SURVIVAL IN PATIENTS WITH BRAIN METASTASIS RECEIVING IMMUNOTHERAPY
Authors: Sarangi Sasmit , Zhang Devin , Dong Anita , Lok Edwin , Wong Eric
Published: December 2020

INTRODUCTION

Checkpoint inhibitors show a positive intracranial response in around 20–50% of patients with metastatic melanoma and non-small cell lung cancer (NSCLC). There is a current paucity of biomarkers to prospectively identify these patients. We hypothesize that CSF would provide useful data on tumor-related metabolites to help stratify responders and non-responders. We focused on adenosine and related nucleotides based on their role in creating an immunosuppressive tumor microenvironment.

METHODS

Fifty-one patients (43 with melanoma and 8 with NSCLC) received immunotherapies. The CSF samples from this cohort were compared to 30 controls and 60 non-immunotherapy-treated patients (13 with melanoma and 47 with NSCLC) using liquid chromatography/tandem mass spectrometry for quantitative assessment of metabolites.

RESULTS

The age and sex distribution were comparable in both cohorts. The most common immunotherapy regimens were ipilimumab, α-interferon and IL-2, while the most common non-immunotherapy regimens were carboplatin/taxol, carboplatin/pemetrexed and pemetrexed alone. CSF AMP, but not adenosine, ADP or ATP, from the immunotherapy cohort was higher in comparison to the non-immunotherapy cohort (P=0.0495). An adenosine/AMP ratio (AAR) was used to quantify relative adenosine production from AMP. AAR of 3.5–7 was associated with lower median survival from diagnosis of brain metastasis in immunotherapy-treated patients (9.1 months, log-rank P=0.0197). AAR below or above this range had significantly higher median survival (27.7 and 24.2 months, respectively). The same AARs could not stratify for survival in non-immunotherapy-treated patients (log-rank P=0.3039). Compared to the non-immunotherapy cohort, the immunotherapy cohort had higher kynurenine, glyceraldehyde 3-phosphate, pyruvate and lactate, as well as succinate, fumarate, malate, oxaloacetate, aconitate and isocitrate (all P< 0.05).

CONCLUSION

AAR of 3.5–7 could indicate an immunosuppressive microenvironment and needs further investigation to identify immunotherapy non-responders. The presence of elevated kynurenine, succinate, fumarate, malate and oxaloacetate suggests glutaminolysis related to immunotherapy. Research supported by the 2019 AACR-Bayer Innovation and Discovery Grant.
IMMU-06. DISCERNING THE DIFFERENT BONE MARROW IMMUNE LANDSCAPES
Authors: Strong Michael , Ahmed Aqila , Calinescu Anda-Alexandra , Zhou Xiaofeng , Robinson Tyler , Rocco Sabrina , Siegel Geoffrey , Clines Gregory , Moore Bethany , Keller Evan , Szerlip Nicholas
Published: December 2020

Roughly 400,000 people have bone metastases in the U.S. with the vast majority of these occurring in the spine. The etiology of bone metastasis still remains to be fully elucidated. This study explored the differences in immune landscape between long bone and spine that may contribute to higher rates of bone metastasis to the spine. Spines and femurs from male C57BL6/J mice (N=10) were processed for flow cytometry and immunophenotyping using Mass Cytometry by Time-Of-Flight (CyTOF). The cells were analyzed with CyTOF using a 33-surface protein marker mouse antibody panel. Spines (N=3) and femurs (N=2) from patients were analyzed with CyTOF using the Maxpar Complete Human T cell Immuno-Oncology Panel Set. There are global differences in the immune cell composition between the long bone and spine microenvironment. Flow cytometry revealed slight increases in the CD45+ and Cd11b+ cell populations in the bone marrow of murine spines compared to murine long bone, which are markers for myeloid-derived suppressor cells (MDSCs). Using CyTOF, significant differences in the immune cell landscape between long bone and spine were observed. In the murine long bone, an increase in monocytes/macrophages, myeloid progenitors, granulocytic MDSCs, granulocytes, and mast cells was observed compared to the spine. In the murine spine an elevation of CD8a+ DC cells, classical monocytes, MDSCs, pDCs, memory T helper cells, and NK T cells was seen. Evaluation of human long bone and spine revealed similar trends with a predominance of myeloid progenitor cells and monocytes in the human vertebra compared to the human long bone marrow. Significant differences in the immune microenvironment exist between the spine and long bone marrow in both murine and human samples. This is the first report of significant differences in immune cell populations between different skeletal locations. However, the functional significance of these differences has yet to be determined.
IMMU-08. MODELING UPFRONT GLIOBLASTOMA SURGICAL RESECTION AND STEROID USE REVEALS IMMUNOSUPPRESSIVE CHANGES AND SUGGESTS THAT PERIPHERAL LYMPHOCYTE COUNTS ARE ASSOCIATED WITH TUMOR VOLUME AND PROGNOSIS
Authors: Otvos Balint , Alban Tyler , Grabowski Matthew , Bayik Defne , Winkelman Robert , Johnson Sarah , Rabljenovic Anja , Vogelbaum Michael , Ahluwalia Manmeet , Fecci Peter , Lathia Justin
Published: December 2020

Glioblastoma (GBM) and its treatment produces systemic immunosuppression, which is being targeted by immunotherapies. However, it remains unclear how surgical resection and steroids specifically in GBM alter the immune system. To further explore this issue, immunocompetent C57Bl/6 mice were intracranially inoculated with syngeneic glioma cells (GL261 and CT-2A) and growth of tumors was evaluated by MRI. Host immune cell populations were analyzed during surgical resection and steroid administration. Mice with surgically resected tumors had a longer median survival compared to mice subjected to tumor biopsies, and had increased bone marrow sequestration of both CD4 and CD8 T cells with corresponding decreased blood lymphocytes. Furthermore, physiologic doses of dexamethasone administered perioperatively decreased tumor edema, but increased the number and proliferative capacity of both marrow and circulating MDSCs while generating no survival benefit. Independent of therapy or dexamethasone, intracranial tumor volume correlated linearly with decreased CD4 and CD8 T cells in peripheral blood, and increased T cell sequestration within the bone marrow. We validated these parameters in steroid-naïve newly diagnosed GBM patients and observed decreased lymphocytes correlated linearly with increased tumor volume. When initial lymphocyte counts in both steroid-naïve and steroid-administered patients were used in univariate and multivariate models predicting progression-free survival and overall survival, decreased initial lymphocyte counts were an independent predictor of decreased progression free survival and decreased overall survival, with steroid use and initial tumor size falling out of significance during stepwise selection. Taken together, tumor volume is linearly correlated with marrow sequestration of lymphoid cells, but both surgery and steroid administration further suppress active immune responses along lymphoid and myeloid lineages. Furthermore, decreasing peripheral lymphocyte counts at diagnosis of GBM indicate an immune system less able to mount responses to the tumor and portent a worse progression free and overall survival.
IMMU-09. CONCURRENT DEXAMETHASONE LIMITS THE CLINICAL BENEFIT OF IMMUNE CHECKPOINT BLOCKADE IN GLIOBLASTOMA
Authors: Iorgulescu Bryan , Gokhale Prafulla , Speranza Maria , Eschle Benjamin , Poitras Michael , Wilkens Margaret , Soroko Kara , Chhoeu Chhayheng , Knott Aine , Gao Yang , Lim-Fat Mary Jane , Baker Gregory , Bonal Dennis , Nguyen Quang-De , Grant Gareth , Ligon Keith , Sorger Peter , Chiocca E Antonio , Anderson Ana , Kirschmeier Paul , Sharpe Arlene , Freeman Gordon , Reardon David
Published: December 2020

BACKGROUND

Dexamethasone, a uniquely potent corticosteroid, is frequently administered to brain tumor patients to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in glioblastoma patients – particularly in the context of immunotherapy.

METHODS

We evaluated the dose-dependent effects of dexamethasone when administered with anti-PD-1 and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 or CT-2A glioblastoma tumors, including analyses of intracranial tumors, draining lymph nodes, and spleen. Clinically, the effect of dexamethasone on survival was additionally evaluated in 181 consecutive IDH-wildtype glioblastoma patients treated with anti-PD-(L)1, with adjustment for relevant prognostic factors.

RESULTS

Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy decreased survival in a dose-dependent fashion and decreased survival following anti-PD-1 plus radiotherapy in both GL261 and immunoresistant CT-2A models. Dexamethasone quantitatively decreased T lymphocytes by reducing the proliferation while increasing apoptosis. Dexamethasone also decreased lymphocyte functional capacity. Myeloid and NK cell populations were also generally reduced. Thus, dexamethasone negatively affects both the adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade revealed worse survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone use – regardless of dose – was the strongest predictor of poor survival (reference no dexamethasone; < 2mg HR 2.28, 95%CI=1.41–3.68, p=0.001; ≥2mg HR 1.97, 95%CI=1.27–3.07, p=0.003).

CONCLUSIONS

Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for glioblastoma patients. Our preclinical analyses also suggest that dexamethasone’s detrimental effects are dose-dependent, suggesting that the lowest possible dose should be used for patients when dexamethasone use is unavoidable. Careful evaluation of dexamethasone use is warranted for neuro-oncology patients undergoing immunotherapy clinical trials.
IMMU-14. ONCOLYTIC ADENOVIRUS DELTA-24-RGD ENGINEERED TO EXPRESS 4-1BBL AS A THERAPEUTIC APPROACH FOR DIPG
Authors: Laspidea Virginia , Puigdelloses Montserrat , Martinez-Velez Naiara , Garcia-Moure Marc , Labiano Sara , Ausejo Iker , Patiño-García Ana , Becher Oren J , Jiang Hong , Gupta Sumit , Fueyo Juan , Gomez-Manzano Candelaria , M Alonso Marta
Published: December 2020

Despite the advances in our understanding of pediatric diffuse midline gliomas (DMG) they remain the leading cause of pediatric death caused by cancer. Our group has demonstrated that the administration of Delta-24-RGD in DIPG models is safe and therapeutically efficacious. An on-going clinical trial with this virus has proven to be safe for these patients. To further improve the anti-tumoral response of the virus, we armed Delta-24-RGD with 4-1BBL (Delta-24-ACT). 41BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. Here, we showed that in vitro Delta-24-ACT can infect and express 4-1BBL in murine and human DIPG cell lines. Importantly, 4-1BBL expression in DIPG cell lines was able to activate lymphocytes and increase their IFN-gamma production. In addition, Delta-24-ACT triggered immunogenic cell death in DIPG cell lines, as shown by the release of DAMPs such as ATP, HMGB1, Hsp90a and calreticulin translocation. Delta-24-ACT administration in orthotopic DIPG models was well tolerated and safe. We confirmed the expression of the ligand within the tumor. Moreover, using flow cytometry and multispectral immunohistochemistry we observed profound changes in the tumor microenvironment with an increase in the T-cell populations and a remodeling of the myeloid component before and after treatment. Functional studies showed no differences in lymphocytes isolated from mice treated splenocytes but uncovered TILS that showed a significantly increased in the production of IFN-gamma. Delta-24-ACT treatment of mice bearing orthotopic DIPG murine tumors resulted in a significant increase in the median survival and led to free of disease long-term survivors. In summary, Delta-24-ACT is a virus that builds in our clinical experience with Delta-24-RGD in DIPG patients going a step further to boost the antitumor effect of viral therapy while maintaining a safe profile.
IMMU-17. SINGLE-CELL RNA-SEQ REVEALS HIGH-GRADE GLIOMA ASSOCIATED MICROGLIA WITH PROINFLAMMATORY AND STEM-LIKE FEATURES SUPPORT TUMOR PROGRESSION
Authors: Liu Hailong , Liu Yongqiang , Franco-Barraza Janusz , Yu Xinguang , Feng Shiyu
Published: December 2020

Poor response of human glioblastoma to current therapies are influenced by tumor microenvironment. Although glioblastoma is recognized by large enrichment of microglia, characterization of diverse cell subsets and their functions remain challenging because of high heterogenicity. Here, we analyzed single-cell transcriptomics to comprehensively map the cell populations and determine the roles of microglia in IDH1/2 wild-type (IDH-wt) glioblastoma progression. Besides finding microglia were significantly enriched in IDH-wt glioblastoma compared to IDH1/2 mutant (IDH-mut) gliomas, we identified a unique high-grade glioma microglia (HGAM) subtype characterized by proinflammatory and stem-like features. In particular, HGAM’s pro-tumoral IL1β secretion is mediated via ApoE-induced activation of NLRP1 inflammasome. HGAM phagocytosed OPC-like malignant cells forming the neoplastic microglia, which presented the stem-like potential giving rise to activated microglia. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation. Additionally, an intricated evaluation of glioma patients revealed that SETD2 mutation/low-expression correlated with adverse prognosis. Further analysis showed that SETD2 -dificient tumor cells presented hypersensitivity to HGAM-derived IL1β via epigenetic dysregulation of PHF6. Also, SETD2 -deficient tumor cells produced TGF-β1 contributing to microglia activation. Finally, targeting the TGF-β1/TβRI signaling impaired HGAM activation and tumor growth. Our studies identify a unique neoplastic microglia subpopulation and establish cellular basis of interactions with tumor cells important for disease progression.
IMMU-20. SINGLE-CELL RNASEQ OF TUMOR INFILTRATING IMMUNE CELLS FROM NEOADJUVANT ANTI-PD1 TREATED GBM PATIENTS REVEALS GLOBAL TRANSCRIPTIONAL CHANGES AND IMMUNOSUPPRESSIVE ADAPTIVE RESPONSES BY MYELOID CELLS
Authors: Lee Alexander , Mochizuki Aaron , Chow Frances , Reynoso Jeremy , Orpilla Joey , Sun Lu , Chavez Carolina , Galvez Mildred , Kienzler Jenny , Yong William , Everson Richard , Nathanson David , Bensinger Steven , Liau Linda , Cloughesy Timothy , Hugo Willy , Prins Robert
Published: December 2020

INTRODUCTION

Neoadjuvant anti-PD1 therapy (neo-aPD1) was previously shown to significantly increase the survival of recurrent glioblastoma patients in a small randomized clinical trial. However, neo-aPD1 alone was not curative so defining the limitations of neo-aPD1 and discovering where other immunotherapies can be used alongside neo-aPD1 is needed.

METHODS

To understand how immune cells in the tumor microenvironment change with neo-aPD1, we used single-cell RNAsequencing to analyze cells from 27 glioma patients (n = 105,143 cells) of which 9 patients had received neo-aPD1 (n = 33,325 cells). Using unsupervised clustering and pseudotime trajectory analysis, we characterized the transcriptional changes within immune cells and how these populations changed with therapy.

RESULTS

We defined the immune landscape of the glioblastoma tumor microenvironment. Compared to no immunotherapy treatment, neo-aPD1 significantly increased the ratio of T cells to myelo-monocytic cells and led to significant increases in the effector and memory T cell populations but no significant changes in myeloid cell composition. Our differential gene expression analysis of the myeloid compartment showed significant increases in interferon-γ-responsive genes and down-regulation of genes associated with M2 macrophages and MDSCs, suggestive that neo-aPD1 influences the transcriptional profile of myeloid cells in the tumor microenvironment. Interestingly, our psuedotime trajectory analysis showed that neo-aPD1 was associated with cells expressing both lymphoid and myeloid-related genes, which we theorized to actually be lymphoid-myeloid cell doublets caused by increased interactions between myeloid and lymphoid cells. These doublets were highly enriched in MHC I and II, macrophage, T cell, and T cell activation and exhaustion genes indicating that neo-aPD1 may result in some adaptive immunosuppressive mechanism by increasing these interactions. This could explain why neo-aPD1 alone is not curative for glioblastoma patients.

CONCLUSIONS

In total, neoadjuvant anti-PD1 therapy enhances effector T cell activity, but may concomitantly induce adaptive resistance mediated by myeloid cells in glioblastoma.