GBM Therapy Research Articles

Abstract PR009: The functional genomic landscape of recurrent glioblastoma

Abstract Glioblastoma (GBM) is a highly fatal brain cancer where mortality is predominantly caused by disease recurrence and lack of second-line therapies. Here, utilizing genome-wide CRISPR-Cas9 screening, we uncover treatment-based conditional genetic interactions modulating sensitivity to combined radiation therapy (RT) and temozolomide (TMZ), the standard of care treatment administered to over 70% of glioblastoma patients. To investigate therapy-induced changes in the functional landscape of glioblastoma stem cells (GSCs) without confounding patient-to-patient differences, we systematically mapped genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent GSCs. This comparison highlights a remodelling of genes and pathways regulating tumor cell survival at disease relapse, arming recurrent GSCs with newly acquired genetic drivers and further loss of tumor suppressors. Among these genes, we identify PTP4A2, encoding protein tyrosine phosphatase 4A2, as a major regulator of stemness and tumorigenicity in recurrent GSCs. Genetic perturbation or pharmacological inhibition of PTP4A2 influences the phosphorylation status and expression of interacting proteins belonging to axonal guidance pathways, as phosphoproteomic studies showed specific enrichment in axon guidance regulator Roundabout Guidance Receptor 1 (ROBO1) in recurrent GSCs. We therefore designed and developed efficacious Camelid biotinylated single-domain antibody (bio-sdAb) and tetrameric complexes targeting human ROBO1 (MKRo-20). Subsequently, we orthotopically engrafted recurrent GSCs into immunocompromised mice and administered local treatments of tetrameric MKRo-20. All mice treated with tetrameric MKRo-20 were rendered tumor free, suggesting that modulation of ROBO1 with bio-sdAbs presents a new immunotherapeutic strategy to target recurrent glioblastoma. Our work reveals a context-specific dependence on axonal guidance through a PTP4A2-ROBO axis in recurrent glioblastoma, highlighting new avenues of therapeutic intervention for second-line therapy in GBM. This abstract is also being presented as PO025. Citation Format: Chirayu Chokshi, David Tieu, Kevin Brown, Chitra Venugopal, Lina Liu, Laura Kuhlman, Katherine Chan, Amy Tong, Neil Savage, Dillon McKenna, Nikoo Aghaei, Minomi Subapanditha, Joseph M Salamoun, Martin Rossotti, Peter Wipf, Elizabeth Sharlow, John S. Lazo, Thomas Kislinger, Kevin Henry, Yu Lu, Jason Moffat, Sheila K. Singh. The functional genomic landscape of recurrent glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR009.

Abstract PO089: Comparison of Two oHSV Vectors for the Treatment of Glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common human brain cancer. Despite a well-established standard of care, the 5-year mortality rate of GBM patients is 95%, highlighting the need for innovative therapeutic interventions. A variety of oncolytic viruses, including those derived from herpes simplex virus (oHSV), have been designed for GBM therapy, but early-phase clinical trials have reported few complete responses without evidence of durable anti-tumor immunity. Potential reasons for the lack of efficacy are limited vector potency (i.e., virulence) and the presence of a highly immunosuppressive tumor microenvironment (TME) comprised of few activated lymphocytes, large numbers of immunosuppressive myeloid cells (macrophages, myeloid derived suppressor cells [MDSCs], microglia), and an agglomerate of immunosuppressive cytokines (IL-10, VEGF, MIF, etc.). Herein we explore these obstacles by comparing the anti-tumor activity of two different oHSV designs and by arming oHSV with an immunomodulatory payload designed to encourage the development of anti-tumor immunity. We first compared the activities of an HSV-1 KOS strain derivative designated KG4:T124 or colloquially, 2A5B, and an F strain derivative designated rQNestin34.5v.1 (a similar oHSV, rQNestin34.5v.2, is currently in a phase I clinical trial for GBM). We evaluated viral entry, replication capacity, and cytotoxicity using both murine and human glioma cell lines in vitro. Viral entry was relatively similar between both vectors, but rQNestin34.5v.1 replicated more effectively and generally induced greater viral mediated cytotoxicity. In syngeneic mice, rQNestin34.5v.1 reduced orthotopic GL261N4 tumor burden compared to 2A5B, but neither virus impeded disease progression in the more pernicious CT2A model. Evaluation of either the GL261N4 or CT2A TME revealed that both viruses induced a high influx of tumor associated macrophages, a transient polymorphonuclear (PMN) cell population, but did not alter the frequency of monocytic MDSCs. As a strategy to enhance oHSV efficacy, we sought to differentiate the highly immunosuppressive MDSC population into pro-inflammatory macrophages using oHSV armed with IL-12, a cytokine that has been shown to provide this activity. We show that oHSV:IL-12 induced a significant increase in tumor-associated macrophages and increased the survival of animals bearing either tumor type. Although we observed a low percentage of complete responses with the presence of anti-tumor CTLs demonstrated by tumor rechallenge experiments, IL-12 alone was not sufficient to significantly improve overall long-term animal survival, suggesting that additional arming genes should be considered in combination with IL-12. Citation Format: Joseph W. Jackson, Bonnie L. Hall, Lisa Bailey, Christophe Quéva, E. Antonio Chiocca, Justus B. Cohen, Joseph C. Glorioso. Comparison of Two oHSV Vectors for the Treatment of Glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO089.

Phase II Trial of Proton Therapy vs. Photon IMRT for GBM: Secondary Analysis Comparison of Progression Free Survival between RANO vs. Clinical Assessment

BackgroundThis secondary image analysis of a randomized trial of proton radiotherapy (PT) versus photon intensity-modulated radiotherapy (IMRT) compares tumor progression based on clinical radiological assessment versus Response Assessment in Neuro-Oncology (RANO).MethodsEligible patients were enrolled in the randomized trial and had MR imaging at baseline and follow-up beyond 12 weeks from completion of radiotherapy. “Clinical progression” was based on a clinical radiology report of progression and/or change in treatment for progression.ResultsOf 90 enrolled patients, 66 were evaluable. Median clinical progression-free survival (PFS) was 10.8 (range: 9.4–14.7) months; 10.8 months IMRT versus 11.2 months PT (P = .14). Median RANO-PFS was 8.2 (range: 6.9, 12): 8.9 months IMRT versus 6.6 months PT (P = .24). RANO-PFS was significantly shorter than clinical PFS overall (P = .001) and for both the IMRT (P = .01) and PT (P = .04) groups. There were 31 (46.3%) discrepant cases of which 17 had RANO progression more than a month prior to clinical progression, and 14 had progression by RANO but not clinical criteria.ConclusionsBased on this secondary analysis of a trial of PT versus IMRT for glioblastoma, while no difference in PFS was noted relative to treatment technique, RANO criteria identified progression more often and earlier than clinical assessment. This highlights the disconnect between measures of tumor response in clinical trials versus clinical practice. With growing efforts to utilize real-world data and personalized treatment with timely adaptation, there is a growing need to improve the consistency of determining tumor progression within clinical trials and clinical practice.

The Multifaceted Glioblastoma: From Genomic Alterations to Metabolic Adaptations.

Glioblastoma multiforme (GBM) develops on glial cells and is the most common as well as the deadliest form of brain cancer. As in other cancers, distinct combinations of genetic alterations in GBM subtypes induce a diversity of metabolic phenotypes, which explains the variability of GBM sensitivity to current therapies targeting its reprogrammed metabolism. Therefore, it is becoming imperative for cancer researchers to account for the temporal and spatial heterogeneity within this cancer type before making generalized conclusions about a particular treatment's efficacy. Standard therapies for GBM have shown little success as the disease is almost always lethal; however, researchers are making progress and learning how to combine therapeutic strategies most effectively. GBMs can be classified initially into two subsets consisting of primary and secondary GBMs, and this categorization stems from cancer development. GBM is the highest grade of gliomas, which includes glioma I (low proliferative potential), glioma II (low proliferative potential with some capacity for infiltration and recurrence), glioma III (evidence of malignancy), and glioma IV (GBM) (malignant with features of necrosis and microvascular proliferation). Secondary GBM develops from a low-grade glioma to an advanced-stage cancer, while primary GBM provides no signs of progression and is identified as an advanced-stage glioma from the onset. The differences in prognosis and histology correlated with each classification are generally negligible, but the demographics of individuals affected and the accompanying genetic/metabolic properties show distinct differentiation [3].

IMMU-17. CAR T CELLS TARGETING THE INTEGRIN ALPHAVBETA3 EXHIBIT ROBUST ANTI-TUMOR RESPONSES AGAINST DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND GLIOBLASTOMA (GBM)

Effective therapies for DIPG and GBM are lacking. CD19 chimeric antigen receptor (CAR) T cells are highly effective in patients with refractory B-cell malignancies. We aim to develop novel CARs for high-grade gliomas. The integrin complex alphavbeta3 was selected as a CAR-T cell target due to its expression on gliomas and their vasculature, yet with minimal expression throughout normal tissues, vessels and organs. Indeed, a majority of DIPG and GBM cell lines express surface α vβ 3. Second-generation CAR-T cells expressing an anti-α vβ 3 scFv and either a CD28 or 4-1BB co-stimulatory domain and CD3zeta were constructed. Transduced healthy, donor-derived T cells exhibited high level CAR expression, efficient expansion, and representative populations of memory subsets including central, effector, and stem cell-like memory CAR-T cells. α vβ 3.28z and α vβ 3.BBz CAR-T cells exhibited antigen-specific in vitro cytotoxicity and cytokine production against DIPG and GBM cell lines. Both CARs mediated rapid and robust anti-tumor responses in NSG mice bearing orthotopic DIPG or GBM tumors. 5/13 α vβ 3.28z and 0/14 α vβ 3.BBz treated animals died without detectable disease within 2 weeks of infusion suggesting different toxicity profiles and is consistent with faster CAR-T cell expansion in CD28-versus 4-1BB-containing CD19 CAR-T cells seen clinically. Our results demonstrate that α vβ 3.BBz CAR-T cell therapy may be both highly effective and safe in DIPG and GBM patients. Due to the restricted nature of α vβ 3 expression in normal tissues, the robust responses seen in tumor-bearing mice, and the slower kinetics of α vβ 3.BBz CAR-T cell expansion, a first-in-human clinical trial is being planned.

A Combination of BRD4 and HDAC3 Inhibitors Synergistically Suppresses Glioma Stem Cell Growth by Blocking GLI1/IL6/STAT3 Signaling Axis.

Glioma stem cells (GSC) are essential for tumor maintenance, invasiveness, and recurrence. Using a global epigenetic screening with an shRNA library, we identified HDAC3 as an essential factor for GSC stemness. Here, we demonstrated that GSCs poorly respond to an HDAC3 inhibitor, RGFP966 (HDAC3i), owing to the production of IL6 and STAT3 activation. To enhance GSC sensitivity to HDAC3i, we explored whether cotreatment with a BRD4 inhibitor, JQ1 (BRD4i), in GSCs produced a better antitumor effect. BRD4i synergistically inhibits GSC growth in association with HDAC3i. HDAC3 inhibition upregulated the acetylation of H3K27, which allowed the recruitment of BRD4 to the GLI1 gene promoter and induced its expression. GLI1, a transcription factor, turned on the expression of IL6, which led to the activation of STAT3 signaling pathways. However, BRD4i inhibited transcription of the GLI1 gene, thereby blocking the GLI1/IL6/STAT3 pathway. In vivo, the HDAC3i/BRD4i combination caused stronger tumor growth suppression than either drug alone. Thus, HDAC3i/BRD4i might provide promising therapies for GBM.

NTRK Fusions and TRK Inhibitors: Potential Targeted Therapies for Adult Glioblastoma.

IntroductionGlioblastoma multiforme (GBM) is the most common primary central nervous (CNS) system malignancy with a poor prognosis. The standard treatment for GBM is neurosurgical resection, followed by radiochemotherapy and adjuvant temozolomide chemotherapy. Predictive biomarkers, such as methylation of the promoter region of the O6-methylguanine DNA methyltransferase (MGMT) gene, can successfully distinguish subgroups with different prognosis after temozolomide chemotherapy. Based on multiomics studies, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), BRAF V600E mutation, neurotrophic tyrosine receptor kinase (NTRK) fusions and other potential therapy targets have been found.MethodsWe have reviewed the preclinical and clinical evidence for NTRK fusions and TRK inhibitors therapy in cancers with NTRK fusions in pan-cancer and gliomas.ResultsSeveral NTRK1/2/3 fusions have been reported in GBM and preclinical studies have proven that NTRK fusions are potential driver mutations in some high-grade gliomas. Tropomyosin receptor kinase (TRK) inhibitors have shown efficacy as targeted therapies for extracranial tumors with NTRK fusions in recent clinical trials, with potential CNS tolerability and activity. However, whether NTRK gene fusions can affect survival status, the efficacy and resistance of TRK inhibitors in GBMs are lacking high-level evidences.ConclusionsFor GBM patients, NTRK fusions and TRK inhibitors are potential target therapy strategy but remain biological mechanism and clinical significance unclarified. More clinical data and future clinical trials are needed to provide more evidence that supports targeted therapy for GBM with NTRK fusions.

IMMU-43. ZIKA VIRUS TO TREAT GLIOMA: TURNING COLD TUMORS HOT

Abstract GBM is a hallmark example of an immunotherapy-resistant tumor. Previously, we discovered that Zika virus (ZIKV) has a natural honing capacity to specifically kill GBM stem cells (GSCs). Using two syngeneic glioma models, GL261 and CT2A, we found that in vivo intratumoral ZIKV treatment reduces tumor size and extends median survival far beyond that expected for its anti-GSC effects; additionally, approximately 50% of mice were long-term survivors. We now report that ZIKV treatment is associated with an increase in tumor-specific CD8+ T cells and activated myeloid cells expressing MHC-II and NOS2. Treatment with CD8-depleting antibody abrogates the survival advantage conferred by ZIKV, demonstrating that CD8+ T cells are required for ZIKV efficacy. Remarkably, ZIKV treatment also confers protection against tumor re-challenge 1.5 years after the original treatment, and this too is dependent on CD8+ T cells. Lastly, in a bilateral tumor implantation model, intratumoral ZIKV treatment of one tumor increases overall survival, suggesting ZIKV is efficacious because it generates a global anti-tumor response. Building on the observation that ZIKV treatment increases CD8+ T cells in the tumor bed, we combined ZIKV with α-PD1 immunotherapy. We found survival was dramatically increased compared to either treatment alone. Finally, to improve safety of potential ZIKV treatment for patients, we generated an immune-sensitized virus. This strain remains efficacious as a single agent and in combination with immunotherapy in vivo. Overall, our results suggest ZIKV treatment addresses two key problems in our current treatment of GBM: it specifically targets highly resistant GSC and initiates a potent anti-tumor immune response against the remaining tumor. Our work reveals the mechanistic basis of ZIKV efficacy and demonstrates the potential for ZIKV as a therapy for GBM.

CTNI-28. A PROSPECTIVE, OBSERVATIONAL STUDY EVALUATING THE SAFETY AND QUALITY OF LIFE OF TUMOR TREATING FIELDS IN CHINESE GLIOBLASTOMA PATIENTS

Abstract OBJECTIVES Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median survival of 15 months. Adding Tumor Treating Fields (TTFields) to first-line therapy in GBM demonstrated improved survival in the EF-14 trial. This prospective study was designed to investigate the safety and effect on QoL of TTFields in Chinese patients with GBM. METHODS A total of 44 patients (29 newly diagnosed GBM, 15 recurrent GBM, median age 51.0 [24.0 - 81.0] years) who underwent TTFields following surgery, concurrent chemoradiotherapy were enrolled into the study. The primary endpoint was the incidence of TTFields-related skin adverse events; the secondary endpoint included quality of life (QoL), treatment adherence. RESULTS Of the 44 patients followed up for at least 3 months, 24 (54.5%) had skin adverse reactions, all of which were grade 1–2. Median time to skin AE was 1.2 months. The common skin AE were dermatitis (53.8%), ulcer (19.2%), infection (19.2%), and most of them could be resolved by symptomatic treatment (topical corticosteroid/antibiotics). In terms of QoL, noticeable improvement in overall health and clinical manifestations such as fatigue, nausea and vomiting were observed. The overall average treatment compliance was 91%: compliance (mean±SD), in newly diagnosed patients, 0.89±0.14, median 0.91; in recurrent patients, 0.86±0.13, median 0.93. CONCLUSIONS The incidence of skin adverse reactions (which could be effectively alleviated with treatment) in GBM patients treated with TTFields was acceptable. From our short follow up, TTFields appear to improve QoL and compliance in Chinese patients comparable to published data.

IMMU-13. EFFICACY OF CXCR6 BLOCKADE AS A POTENTIATOR OF ANTI-PD-1 THERAPY FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive primary tumor of the brain with a dismal prognosis for patients. Despite the standard of care treatment, median survival is 12–15 months. The blockade of inhibitory checkpoints such as PD-1 and CTLA-4 has become the mainstay immunotherapy to treat solid tumors but it lacks efficacy in treating GBM patients. Emergence of alternative checkpoints on T cells as a mechanism of acquired resistance is considered one of the major hurdles for the success of anti-PD-1 therapy in GBM. Using an orthotopic mouse model of GBM, we have seen that cytotoxic T cells infiltrating the tumor show a preponderance of the chemokine receptor CXCR6 on exhausted cells. Furthermore, ablating CXCR6 along with anti-PD-1 therapy greatly improved anti-tumor immune response. Whereas PBS treated and CXCR6 KO mice had no long-term survivors 40 days post-tumor implantation, 90% of anti-PD-1 treated CXCR6 KO mice were long-term survivors, compared with 12% among anti-PD-1 treated wildtype mice. This supports our hypothesis that blockade of CXCR6 licenses anti-PD-1 blockade by alleviating acquired resistance to anti-PD-1 therapy. We have observed CXCR6 expression on exhausted T cells of GBM patients, making it a promising target for dual therapy with anti-PD-1 in clinical trials.

SURG-13. LASER INTERSTITIAL THERMAL THERAPY FOR RECURRENT GLIOBLASTOMA: A REVIEW OF SURVIVAL OUTCOMES COMPARED TO A MATCHED SURGICAL COHORT

Abstract INTRODUCTION Glioblastoma (GBM) is uniformly associated with a poor prognosis and inevitable recurrence. Management of recurrent GBM remains unclear, with repeat surgery often employed with varying degrees of success. We evaluated the efficacy of Laser Interstitial Thermal Therapy (LITT) for recurrent GBM when compared to a carefully matched cohort of patients treated with repeat surgical resection. METHODS A retrospective single-institution database was used to identify patients who underwent LITT or surgical resection of recurrent GBM between 2014-2019. LITT patients were matched with surgical resection patients according to baseline demographics, comorbidities, tumor location, and eloquence. Subgroup analysis matching similar patients for tumor volume was also completed. Overall survival (OS) and progression-free survival (PFS) were the primary endpoints. RESULTS A LITT cohort of 20 patients was matched to 50 similar patients who underwent repeat surgical resection. Baseline characteristics were similar between both cohorts apart from tumor volume, which was larger in the surgical cohort (17.5 cc vs. 4.7 cc, p< 0.01). On long-term follow-up, there was no difference in OS (HR, 0.72; 95%CI, 0.36-1.45) or PFS (HR, 0.67; 95%CI, 0.29-1.53) between the LITT and surgical cohorts when controlling for tumor volume. Subgroup analysis of 23 LITT patients matched according to tumor volume with 23 surgical patients with similar clinical characteristics also found no difference in OS (HR, 0.66; 95%CI, 0.33-1.30) or PFS (HR, 0.58; 95%CI, 0.90-1.05) between the cohorts. LITT patients had shorter length of stays (1 vs. 4 days, p< 0.001) and a higher rate of home discharge (84% vs. 67%, p=0.172) compared to the surgical cohort. CONCLUSION After matching for demographic, clinical, and tumor characteristics, there was no difference in outcomes between patients undergoing LITT compared to surgical resection for recurrent GBM. LITT patients had similar survival outcomes yet shorter hospital stays and more favorable dispositions, potentially mitigating post-treatment complications.

TAMI-01. NEOADJUVANT PD-1 ANTIBODY BLOCKADE REMODELS THE IMMUNE MICROENVIRONMENT OF METASTATIC BRAIN TUMORS

Abstract Brain metastases (BM) commonly arise in patients with melanoma, lung, and breast cancer. Currently, there are limited options for GBM and BM patients who have failed the first-line standard treatment, underscoring the importance of developing new therapeutic strategies. Last year, we and other groups evaluated the neoadjuvant timing of anti-PD-1 checkpoint blockade therapy in recurrent GBM (rGBM) patients, which resulted in a modest survival benefit. In light of the known effectiveness of anti-PD-1 as a systemic therapy to control melanoma and non-small cell lung cancer BM, we set out to study the anti-tumor immune response of BM patients to anti-PD-1 in the neoadjuvant setting. We posited that neoadjuvant anti-PD-1 in patients with BM would result in a stronger antitumoral immune response, which could be quantified at the single cell level. To test this, we made use of contemporary single cell techniques, including multiplex immunofluorescence, time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq), to characterize the intratumoral immune cell populations and their transcriptomic profiles. We found that neoadjuvant anti-PD-1 significantly increased the number of tumor infiltrating T lymphocytes in BM compared to rGBM (2.5 fold in BM, p= 0.02 vs. 1.4 fold in rGBM, p= 0.19). Multiplex immunofluorescence analysis of T cells in BM samples revealed a change from T cell exclusion to a diffusely infiltrating phenotype after anti-PD-1 treatment. Importantly, BM showed a higher fraction of effector/cytotoxic T cells compared to rGBM (7.3% vs. 0.9% of lymphoid cells, p= 0.005) and anti-PD-1 further enhanced this population. In the myeloid compartment of BM, neoadjuvant anti-PD-1 increased the frequency of HLA-DR+CD206- M1-like macrophages, implicating a pro-inflammatory microenvironment. In summary, our study delineated the immune cell subtypes altered by neoadjuvant anti-PD-1 and offers insights into new combination therapies that can help understand the clinical efficacy of immunotherapy for BM and GBM patients.

IMMU-16. TWO DISTINCT SUBSETS OF NATURAL KILLER CELLS ARE ENRICHED IN THE TUMOR MICROENVIRONMENT AND CORRELATE WITH SURVIVAL OUTCOME IN HUMAN GLIOBLASTOMA.

Abstract Glioblastomas (GBM) account for up to 60% of all adult primary brain tumors. With few advances in therapeutics, median overall survival (mOS) remains at 15-months post diagnosis. Success of immunotherapy in peripheral solid tumors may offer an alternative therapeutic approach for patients with GBM tumors; however, no predictive immune features currently inform therapeutic stratification for GBM. Recently, we have identified radiographic tumor contact with the lateral ventricle (LV) as a prognostic indicator of OS, as patients with LV+ GBM survive 7 months less than patients with LV- gliomas. This disparity was independent of known prognostic factors (e.g. KPS, extent of resection). Further, we have identified a correlation between greater immune infiltration and the frequency of tumor subtypes with more favorable prognosis. We therefore hypothesized that differences in overall survival between patients with LV+ and LV- are due, in part, to a uniquely immunosuppressive microenvironment within the LV. Using 35-parameter single-cell mass cytometry (CyTOF) we profiled the immune infiltrate of human GBM tissue acquired in accordance with the Declaration of Helsinki and with the approval of the institutional review board (IRB #131870). Computational approaches (tSNE, Citrus, RAPID) correlated natural killer (NK) cell populations correlating with prognosis. NK cells made of 1–14% of the total immune infiltrate in GBM. Ninety percent of NK cells infiltrating LV- tumors were CD16+CD56dim cytotoxic NK cells (cNK). LV+ gliomas, however, were enriched in CD16+CD56bright immunoregulatory NK cells (irNK). The presence of cNK cells correlated with a 2.5-fold improvement of OS, whereas irNK cells correlated with a 2.2-fold reduction in OS. Further, 30–60% of NK cells infiltrating LV+ tumors expressed checkpoint receptors (TIGIT, TIM3, B7-H3) compared to only 10–20% in LV- tumors. These results suggest that NK cells contribute to immunosuppression in the LV and may serve as alternative targets to T cell-based therapies for GBM.

PATH-24. DETECTION OF POINT MUTATIONS AND GENE FUSIONS FROM CIRCULATING CELL-FREE DNA (CFDNA) OF GLIOBLASTOMA (GBM) PATIENTS

Abstract BACKGROUND GBM is characterized by intratumoral heterogeneity. Tumor heterogeneity, clonal diversity and mutation acquisition hamper the ability to tailor personalized therapy for GBM. Tumor sampling has limited ability to accurately capture the molecular landscape of the tumor and to disclose acquired molecular aberrations. Mutation analysis of cfDNA is a non-invasive procedure which may overcome these limitations as it may reflect the real composition of the tumor and track the molecular evolution. We sequenced cfDNA of GBM patients and assessed mutation patterns and fusion genes. METHODS We collected blood and respective tumor samples from 27 GBM patients and blood samples from 14 healthy controls. Tumor DNA, cfDNA and WBC DNA were sequenced using deep sequencing procedures. The data were analyzed for detection of single nucleotide polymorphism (SNPs) and gene-gene fusions. RESULTS GBM cfDNA concentrations were significantly elevated (median: 23.63 ng/mL; range 12.6–137) compared to healthy controls (median 2.06; range 1.68–7.62) (p < 0.0001). We identified unique SNPs in each glioma patient’s cfDNA and the corresponding tumor DNA including the top-10 most frequently mutated genes in GBM. For example, mutation of TP53 was detected in18.75%; EGFR in 37.5%; NF1-12.5%; LRP1B-25% and IRS4 in 25%. For gene-gene fusion we used the in-house fusion gene database, ChiTaRS 5.0, and identified fusions in cfDNA and tumor DNA. Thus, KMT2A-FLNA was the most frequent fusion found in 16.4% of samples. BCR-ABL1 in 8.82% and FGFR1-BCR in 2.94%. Other fusions included COL1A1-PDGFB (5.88%), NIN-PDGFRB (5.88%), KIF5B-RET (5.88%) and also TPM3-ROS1(2.94%), TFG-ALK(2.94%), MSN-ALK (2.94%) and NPM1-ALK (2.94%) which may be targeted by brain penetrating drugs that are ROS1 and ALK inhibitors. CONCLUSIONS Our study suggests that plasma cfDNA analysis may help to uncover real time mutational and gene fusion status of GBM by a non-invasive procedure. It may identify drug targets based on personalized gene-gene fusions.

DDRE-11. DEVELOPMENT OF THE FIRST-IN-CLASS INHIBITOR OF CHD4 - SENSITIZING RESISTANT GLIOMAS

Abstract Glioblastoma is a lethal brain tumor with high recurrence rate. CHD4 overexpression, which drives resistance to DNA damage, is one of the major sources of recurrence. Since standard GBM treatments like radiation and temozolomide chemotherapy create DNA damage, inhibition of CHD4 offers a new therapeutic option for resensitizing GBM. CHD4 is a ubiquitously expressed ATP-dependent chromatin remodeler, which plays a crucial role in epigenetic regulation of gene expression and in DNA damage repair. Structurally, CHD4 contains an HMG-like domain, PHD domains, two chromodomains, a catalytic ATPase module, two domains of unknown function (DUF) and a C-terminal domain CHDCT2. Currently, no specific inhibitors targeting this chromatin remodeler have been reported yet. We aim to develop the first-in-class inhibitor targeting chromo-domain of CHD4. We have performed in silico screens to identify small molecules binding to the chromo-domains of CHD4. We present our growing in vitro data demonstrating biophysical properties and mechanism-of-action of these novel inhibitors. We expect that the experiments proposed here will result in the development of the first-in-class CHD4 inhibitor which can be used in the future not only to better study the physiological role of CHD4 but also to determine its potential as a novel targeted therapy for GBM.

STEM-07. IDENTIFICATION OF A NOVEL CSC SIGNALING AXIS CONTAINING JAM-A/SERPINB3/TGF-BETA

Abstract Despite therapeutic interventions including surgery, radiation, and chemotherapy, multiple clones of treatment resistant cells repopulate the tumor, resulting in recurrence and a high rate of mortality in glioblastoma. Effective targeting of these cancer stem cells (CSCs) has been limited by our incomplete understanding of the intracellular signaling mechanisms maintaining the CSC state. Our laboratory previously identified junctional adhesion molecule-A (JAM-A) on CSCs and, through functional studies, demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. We determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the intermediate signaling network is yet to be fully elucidated. Therefore, our goal was to delineate the pathway downstream of JAM-A. To achieve this, we immunoprecipitated JAM-A from GBM CSCs and performed mass spectrometry leading to the identification of the serine/cysteine protease inhibitor SerpinB3. While SerpinB3 has a functional role in lung adenocarcinoma, breast, pancreas, as well as in hepatocellular carcinoma, there is limited information in GBM or CSCs, and its relationship to JAM-A is yet to be elucidated. Using in vitro CSC functional assays, we observed that SerpinB3 is necessary for the maintenance of CSCs. Additionally, in an intracranial implantation model of GBM the knockdown of SerpinB3 extended the survival of mice. Knockdown of SerpinB3 in multiple GBM CSCs models also led to decreased expression of TGF-β, a known regulator of the CSC state. We also observed that SerpinB3 stabilizes JAM-A expression and further studies are investigating the mechanism of this stabilization, identifying the specific interaction sites of JAM-A and Serpin B3, and determining how TGF-β alters the association through positive and negative feedback mechanisms. Taken together, these data highlight the role of SerpinB3 in GBM CSCs and provides a novel target to inhibit the JAM-A-mediated CSC maintenance as a next-generation GBM therapy.

CSIG-17. PRMT5 INHIBITION SENSITIZES GLIOBLASTOMA MODELS TO TRAMETINIB TREATMENT

Abstract INTRODUCTION Glioblastoma (GBM) is the most common malignant primary brain tumor. With limited effective therapeutic strategies, prognosis for GBM is very poor. Our previous study shows that the expression of Protein Arginine Methyltransferase 5 (PRMT5) is upregulated in GBM; its inhibition promotes anti-GBM effect through apoptosis and senescence of mature and immature tumor cells, respectively. In GBM, RAS-RAF- MEK-ERK signaling is aberrantly activated and promotes tumor growth. Therefore, MEK inhibitors, including trametinib are currently under investigation for GBM therapy. In this study, we tested whether inhibition of PRMT5 can enhance the anti-tumor efficacy of trametinib in GBM. METHODS Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, and western blot analysis were conducted. In vivo, PRMT5-intact and -depleted GBMNS were intracranially implanted in NSG mice and treated with trametinib by daily oral gavage, and tumor progression and mice survival rate were analyzed by MRI and Kaplan-Meier survival curve, respectively. RESULTS Trametinib treatment upregulated the expression of PRMT5 in GBMNS. Depletion of PRMT5 increased the cytotoxic effect of trametinib in GBMNS. In concurrence with the trametinib-induced PRMT5 upregulation, trametinib treatment increased the activity of AKT that was blocked with PRMT5 knockdown. In vivo, PRMT5-depletion extended the survival of the tumor bearing mice that further increased in combination with trametinib treatment. Interestingly, trametinib treatment alone had no survival benefit. CONCLUSION Trametinib treatment induces PRMT5 expression. Depletion of trametinib-induced PRMT5 expression sensitizes GBMNS for trametinib by inhibiting AKT activity.

Multiple Emulsions as a Biomaterial-based Delivery System for the Controlled Release of an Anti-cancer Drug

This paper focuses on developing multiple emulsions as a pH-responsive drug delivery system, for glioblastoma multiforme-GBM therapy, with reduced toxicity to healthy cells. The multiple emulsions with a stimuli-responsive biopolymer (CMC- sodium carboxymethylcellulose) were prepared in a Couette-Taylor flow contactor. As an external stimulus, the difference in pH of the cancer environment, and normal tissue, was investigated by adding salts as a triggering agent. The cancer cell lines of glioblastoma multiforme were investigated: U87MG, LN229, T98G, in order to verify emulsions’ components cytotoxicity to cells. Also normal (healthy) cells, K21-fribroblast, were analysed. Rhodamine B was used as a model drug instead of the clinically used chemotherapeutics (e.g. doxorubicin) in oncology. Results showed that multiple emulsions by themselves had no adverse effect on the viability of investigated cells, excluding one cell line: LN229. The control and modulated release rates of a model drug, by stimuli-responsive biopolymer, were established. Results confirmed the possibility of controlling the release rates of a drug in the acidic environment of the cancer cells. The proposed multiple emulsion could be explored for the potential delivery of chemotherapeutics in GBM therapy.

Targeting TRIBBLES1 (TRIB1) Pseudokinase in GBM: A New Therapeutic Strategy

GBM (WHO grade IV) is the most aggressive form of glioma with a 5-year survival rate of 5%. The current therapeutic regimen involves radiation therapy and concurrent and adjuvant TMZ (Stupp protocol). Due to dismal survival outcomes, there is an urgent need to identify druggable therapeutic targets in GBM for drug development. In this study we identified TRIB1, a Ser/Thr pseudokinase that acts as a scaffold protein to initiate Ubiquitin Proteasome System-mediated degradation of target proteins in the cell. TRIB1 mRNA and protein have been found upregulated in several cancers (e.g. NSCLC, prostate etc.). It has also been shown to contribute towards chemotherapy resistance in NSCLC and CRC. It has also been observed that COP1, a TRIB1 associated E3-ligase is also upregulated in glioma cells. We utilized a patient-centered reverse translational approach to identify novel therapeutic targets. TRIB1 was identified by statistical association (Cox regression analysis) and logic-based network analyses of the patient-derived methylation data generated using EPIC methylation array. TRIB1 was functionally validated in vitro by overexpression and knockdown approaches. For knockdown of TRIB1, a doxycycline inducible system was used. Stable cell lines were generated by puromycin selection and cell sorting. Protein levels were detected by western blotting. The global methylation analysis on a Utrecht GBM cohort revealed that TRIB1 promoter methylation was associated with better OS of GBM patients (HR = 0.81 (0.62-1.05); p = 0.11). We also observed that overexpression of TRIB1 caused a decrease in apoptosis of PDX GBM cell lines after radiation exposure and TMZ treatment. Overexpression of TRIB1 also increased the phosphorylation of ERK and Akt in PDX cell lines. However, in TRIB1 knockdown cell lines only phosphorylation of Akt was decreased. It was also observed that TRIB1 levels were upregulated in p53-mutant cell lines suggesting a correlation between TRIB1 and p53. TRIB1 is a potential therapeutic target for GBM therapy as targeting TRIB1 could promote radio-sensitivity of p53-mutant glioma cells by enhancing TMZ action. Targeting TRIB1 would also reduce oncogenic signaling in these cells; overall providing an additional treatment strategy for GBM.

Decorin expression is associated with predictive diffusion MR phenotypes of anti-VEGF efficacy in glioblastoma

Previous data suggest that apparent diffusion coefficient (ADC) imaging phenotypes predict survival response to anti-VEGF monotherapy in glioblastoma. However, the mechanism by which imaging may predict clinical response is unknown. We hypothesize that decorin (DCN), a proteoglycan implicated in the modulation of the extracellular microenvironment and sequestration of pro-angiogenic signaling, may connect ADC phenotypes to survival benefit to anti-VEGF therapy. Patients undergoing resection for glioblastoma as well as patients included in The Cancer Genome Atlas (TCGA) and IVY Glioblastoma Atlas Project (IVY GAP) databases had pre-operative imaging analyzed to calculate pre-operative ADCL values, the average ADC in the lower distribution using a double Gaussian mixed model. ADCL values were correlated to available RNA expression from these databases as well as from RNA sequencing from patient derived mouse orthotopic xenograft samples. Targeted biopsies were selected based on ADC values and prospectively collected during resection. Surgical specimens were used to evaluate for DCN RNA and protein expression by ADC value. The IVY Glioblastoma Atlas Project Database was used to evaluate DCN localization and relationship with VEGF pathway via in situ hybridization maps and RNA sequencing data. In a cohort of 35 patients with pre-operative ADC imaging and surgical specimens, DCN RNA expression levels were significantly larger in high ADCL tumors (41.6 vs. 1.5; P = 0.0081). In a cohort of 17 patients with prospectively targeted biopsies there was a positive linear correlation between ADCL levels and DCN protein expression between tumors (Pearson R2 = 0.3977; P = 0.0066) and when evaluating different targets within the same tumor (Pearson R2 = 0.3068; P = 0.0139). In situ hybridization data localized DCN expression to areas of microvascular proliferation and immunohistochemical studies localized DCN protein expression to the tunica adventitia of blood vessels within the tumor. DCN expression positively correlated with VEGFR1 & 2 expression and localized to similar areas of tumor. Increased ADCL on diffusion MR imaging is associated with high DCN expression as well as increased survival with anti-VEGF therapy in glioblastoma. DCN may play an important role linking the imaging features on diffusion MR and anti-VEGF treatment efficacy. DCN may serve as a target for further investigation and modulation of anti-angiogenic therapy in GBM.