bluuebunny/crossref_metadata_2025 huggingface dataset:
CTNI-72. PHASE 2 STUDY OF DIANHYDROGALACTITOL (VAL-083) IN PATIENTS WITH MGMT-UNMETHYLATED, BEVACIZUMAB-NAÏVE GLIOBLASTOMA IN THE RECURRENT AND ADJUVANT SETTING
Authors: O’Brien Barbara , Penas-Prado Marta , Kamiya-Matsuoka Carlos , Weathers Shiao-Pei , Yung W K Alfred , Loghin Monica , Harrison Rebecca , Majd Nazanin , Bacha Jeffrey , Brown Dennis , Johnson Greg , Langlands John , Schwartz Richard , Kanekal Sarath , Steino Anne , Lopez Lorena , de Groot John
Published: December 2020

Current standard-of-care for glioblastoma (GBM) includes surgery followed by concurrent therapy with radiation and temozolomide (TMZ) followed by adjuvant TMZ. Almost all GBM patients experience recurrent/progressive disease despite upfront standard of care treatment, with a median survival after recurrence of 3–9 months. Unmethylated promoter for O6-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for TMZ-resistance and is correlated with poor patient prognosis. VAL-083 is a bi-functional DNA-targeting agent which rapidly induces inter-strand cross-links at N7-guanine inducing double-strand breaks causing cell death and acts independent of MGMT DNA repair. This trial is an open-label two-arm biomarker-driven phase 2 clinical trial in MGMT-unmethylated bevacizumab-naïve GBM patients with either recurrent (Group 1) or newly diagnosed GBM requiring adjuvant therapy after chemo-radiation with temozolomide (Group 2). Patients receive VAL-083 IV at 30 or 40 mg/m2/d on days 1, 2, and 3 of a 21-day cycle. The primary objective of this study is to determine the effect of VAL-083 on median overall survival (mOS) in MGMT-unmethylated recurrent GBM patients (Group 1); and progression-free survival (PFS) in newly diagnosed GBM patients requiring adjuvant therapy after chemo-irradiation with temozolomide (Group 2), compared to historical controls in both groups. Tumor response will be assessed by MRI every 42 days, using RANO criteria. The initial starting dose in this study was 40 mg/m2/d on days 1, 2, and 3 of a 21-day cycle, which was subsequently reduced to 30 mg/m2/d to improve tolerance due to myelosuppression. As of June 2-2020, 35 patients with recurrent GBM (Group 1) have received 40 mg/m2/d and 39 patients have received 30 mg/m2/d VAL-083. In the adjuvant setting (Group 2), 25 patients have been enrolled (30 mg/m2/day). Enrollment, safety data and study updates will be presented at the meeting. Clinicaltrials.gov identifier: NCT02717962.
DDRE-03. INTERNATIONAL PRECLINICAL DRUG DISCOVERY AND BIOMARKER PROGRAM INFORMING AN ADOPTIVE COMBINATORIAL TRIAL FOR DIFFUSE MIDLINE GLIOMAS
Authors: Przystal Justyna , Yadavilli Sridevi , Abadi Christina Coleman , Yadav Viveka Nand , Laternser Sandra , Cosentino Chiara Cianciolo , Waszak Sebastian , Cartaxo Rodrigo , Biery Matt , Myers Carrie , Jayasekara Samantha , Olson James , Filbin Mariella , Vitanza Nicholas , Cain Jason , Koschmann Carl , Mueller Sabine , Nazarian Javad
Published: December 2020

INTRODUCTION

DMG-ACT (DMG- multi-arm Adaptive and Combinatorial Trial) aims to implement a highly innovative clinical trial design of combinatorial arms for patients with diffuse midline gliomas (DMGs) at all disease stages that is adaptive to pre-clinical data generated in ten collaborating institutions. Novel drug and drug combination were tested, predictive biomarkers were identified and incorporated in clinical trial design.

METHODS

In vitro (n=15) and in vivo (n=8) models of DMGs across ten institutions were used to assess single and combination treatments with ONC201, ONC206, marizomib, panobinostat, 5-Azacytidine, Val-083, GDC0084 and TAK228. In vivo drug toxicity screenings were conducted using larval zebrafish model and murine PDX models. Predictive biomarkers for ONC201 and ONC206 were identified using meta-analysis, and extensive molecular assays including CRISPR, RNAseq, FACS, and IHC.

RESULTS

Inhibitory concentrations (IC50) were established and validated multiple preclinical models. ONC201 and ONC206, ONC201 and TAK228, ONC201 and GDC0084 showed synergism. In vivo survival assays showed increased survival for: ONC201 (p=0.01), ONC206 (p=0.01), ONC201+ONC206 (p=0.02), and ONC201+panobinostat (p=0.01). Marizomib showed toxicity in murine/zebrafish PDXs models. Murine pharmacokinetic analysis showed peak brain levels of ONC201 and ONC206 above pre-clinical IC50. Molecular testing and analyses of existing drug screen across 537 cancer cell lines validated mitochondrial protease ClpP and ATF4 as ONC201/6 targets. Predictive biomarkers of response to drug were identified.

CONCLUSION

Thorough preclinical testing in a multi-site laboratory setting is feasible and identified ONC201 in combination with ONC206, TAK228 and GDC0084 as promising therapeutics for DMGs.
DDRE-05. DECREASED EXPRESSION OF DNA DAMAGE RESPONSE PATHWAYS CORRELATES WITH SYNERGISTIC ACTIVITY OF TEMOZOLOMIDE WITH ATR INHIBITORS IN GLIOBLASTOMA
Authors: Wu Shaofang , Li Xiaolong , Singh Sanjay , Koul Dimpy , Yung W K Alfred
Published: December 2020

The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most important gaps in enabling the development of effective targeted cancer therapies. Increasing evidence supports the notion that DNA damage response (DDR) signaling plays an important role in inducing radiation and temozolomide (TMZ) resistance in glioblastoma (GBM) and hence, it has emerged as a molecular target for therapeutic development. The cellular response to TMZ therapy is critically mediated by MGMT and DDR signaling networks that are regulated by Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3-related protein (ATR). ATR is a primary sensor of DNA damage initiating subsequent repair leading to cell survival. Here, we show that three clinical grade ATR inhibitors (Bay1895344, AZD6738 and VE-822) had similar selective sensitivity pattern across eight glioma-like stem cells (GSC) lines tested with three (GSC28, GSC11, GSC274) being the most sensitive, and three (GSC300, GSC272, and GSC20) being the most resistant cell lines. Interestingly, all three ATR inhibitors showed a significant synergism with TMZ (Combination index and Bliss model) in GSC300, GSC272, and GSC20, and antagonism in GSC28, GSC11, and GSC274. Further analysis showed that the synergistic group was resistant to single agent ATR inhibitor, whereas antagonistic group was inherently sensitive to ATR inhibitor. The synergistic response was not associated with any major genetic alterations such as MGMT status, EGFR amplification, PTEN deletion/mutation, or p53 deletion/mutation. However, GSEA analysis of baseline RNA-seq data of the 6 cell lines showed an enhanced expression of six DNA damage/repair/response pathways in the antagonistic group. It suggests that a low level of DNA damage/repair/response pathways could be a marker of synergy for TMZ with ATR inhibitors in GBM. Further studies are underway to validate this observation in more GSC lines in vitro and in vivo.
DDRE-06. CELLULAR STRESS RESPONSE IN DIPG THERAPY
Authors: Sridharan Sreepradha , Harmanci Arif , Siddaway Robert , Dobson Tara , Swaminathan Jyothishmathi , Sharma Ajay , Tarapore Rohinton S , Allen Joshua , Hawkins Cynthia , Khatua Soumen , Zaky Wafik , Gopalakrishnan Vidya
Published: December 2020

Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor of the pons and brainstem. Therefore, there is a desperate need for new therapeutics. Genomic profiling of tumors identified a highly prevalent dominant negative somatic mutation at lysine (K)-27 in histone genes HIST1H3B and H3F3A. Clonal evolution modeling suggests these mutations are truncal, and studies have demonstrated their contribution to tumorigenesis. ONC201, a first-in-class DRD2 antagonist and ClpP agonist is an anticancer drug developed by Oncoceutics, which targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling and is actively being investigated in patients with recurrent H3 K27M-mutant gliomas. In adults with recurrent glioma, single agent studies showed benign-safety, no dose-limiting toxicities and a durable objective response when administered orally. In addition, intra-tumoral drug levels exceeded therapeutic thresholds, and induced tumor cell apoptosis. Based on this and response seen in a pediatric patient with DIPG for whom compassionate use of ONC201 was approved, a multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) is actively accruing patients. However, the strength of UPR and ISR in DIPGs and their effect on DIPG response to ONC201 is not known. Our group employed bulk/single cell transcriptomic and single cell proteomic approaches to demonstrate substantial heterogeneity in UPR and ISR signaling in human DIPG samples. Consistent with this, DIPG cell lines exhibited considerable variability in sensitivity to ONC201. Single cell profiling identified tumor sub-populations with significant proliferative capacity even after ONC201 exposure. Incomplete response promotes recurrence. To target these cells, we performed a synthetic lethality screen with a library of 360 FDA-approved CNS penetrant compounds, which identified HDAC inhibitors and DNA damage-inducing chemotherapy as having synergy with ONC201. Thus, we suggest that tumor heterogeneity impacts sensitivity to ONC201 and that this can be reduced by combination treatments.
DDRE-08. POTENTIAL THERAPEUTIC EFFECTS OF ETOMOXIR IN COMBINATION WITH TEMOZOLOMIDE AGAINST HUMAN GLIOBLASTOMA TUMORSPHERES
Authors: Hyun Kim Eui , Shim Jin-Kyoung , Park Jungseong , Seon Jieun , Kim Hyun Sil , Moon Ju Hyung , Chang Jong Hee , Yook Jong In , Kang Seok-Gu
Published: December 2020

PURPOSE

Glioblastoma (GBM) is the most common and lethal brain tumor with the median survival between 10 and 14 months. Since current treatments including surgical resection, radiation and chemotherapy fail to cure GBM, a new strategy to maximize the efficacy is needed. Here, etomoxir, an inhibitor of fatty acid oxidation, has been combined with temozolomide, a standard treatment for GBM, to examine its anti-tumor effects. Etomoxir has shown anti-cancer effects against human bladder, prostate cancers as well as GBM. We hypothesize that etomoxir inhibits fatty acid metabolism in cancer cell to suppress tumor progression while temozolomide causes cancer cell death.

METHOD

Four kinds of human GBM tumorspheres (TSs) were treated with temozolomide or etomoxir alone, or in combination with each other. Therapeutic effects of two drugs were evaluated by measuring cell viability, apoptosis, neurosphere formation, 3D-invasion using collagen/matrigel matrix, and LC/MS analysis of energy metabolism. Protein and mRNA expression profiles after drug treatment were evaluated by western blotting and RNA-sequencing.

RESULTS

Combination treatment of temozolomide and etomoxir significantly inhibited the cell viability, stemness, and invasiveness in GBM TSs. Expression levels of stemness-, invasiveness-associated markers were also decreased by the combined treatment. The combined treatment also significantly inhibited ATP production in TCA cycle.

CONCLUSIONS

Our findings suggest that combined treatment of etomoxir and TMZ may be therapeutically effective in the treatment GBM
DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA
Authors: Bollu Lakshmi , Wainwright Derek , Zhai Lijie , Ladomersky Erik , Lauing Kristen , Bell April , Schlitz Gary
Published: December 2020

INTRODUCTION

Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein.

METHODS

A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway.

RESULTS

Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation.

CONCLUSIONS

This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.
DDRE-13. DECONVOLUTING MECHANISMS OF RESISTANCE TO BRAF INHIBITORS IN BRAF V600E HUMAN GLIOMA
Authors: Schreck Karisa , Morin Andrew , Zhao Guisheng , Sanford Bridget , Green Adam , Jones Kenneth , Banerjee Anurhada , Nazemi Kellie , Samuel David , Kilburn Lindsay , Solomon David , Pratilas Christine , Nicolaides Theodore , Levy Jean Mulcahy
Published: December 2020

BACKGROUND

While BRAF-targeted therapy can be effective in a subset of patients with glioma, resistance to treatment can emerge over time. The description and validation of mechanisms of resistance in BRAF-mutant glioma are not previously described.

METHODS

Pre- and post- BRAF inhibitor (BRAFi) or BRAFi/MEK inhibitor (MEKi) treated patient samples were obtained under IRB-approved protocols at University of Colorado Denver, UCSF, and Johns Hopkins. Targeted DNA sequencing or whole exome sequencing (WES), and RNA-seq were conducted on paired samples. Functional validation of putative resistance mechanisms was performed using established glioma cell lines with BRAF V600E mutation (DBTRG-5MG, AM38, B76).

RESULTS

Analysis of 15 tissue sample pairs identified point mutations in 15 genes (including CBL, NF1, PTEN, and MAP2K1) and expression changes in RAF1 leading to putative mechanisms of resistance. We performed functional validation of loss of NF1 and CBL as resistance mechanisms and demonstrated growth inhibition and cell death in response to BRAFi with siRNA/sgRNA-mediated knockdown of each gene. Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN-/-) restored sensitivity to BRAFi. Ingenuity pathway analysis suggested that a switch from BRAF to CRAF dependence mediated resistance in some tumors. Indeed, inhibition of CRAF expression using siRNA in a patient-derived glioma cell line re-sensitized cells to BRAFi.

CONCLUSIONS

Analysis of pre-/post-treatment BRAF mutant glioma sample pairs, primarily from pediatric patients, identified a variety of putative resistance mechanisms, some of which have been validated in vitro. Resistance mechanisms to BRAFi in glioma are varied and may be susceptible to different combinations of targeted therapy, highlighting the importance of a personalized approach.
DDRE-20. GENERATION OF A HIGH-AFFINITY EGFR/EGFRvIII-TARGETED D2C7 CAR-T CELL
Authors: Ryan Katherine , Wilkinson Daniel , Landi Daniel , Fecci Peter
Published: December 2020

BACKGROUND

The intent of this investigation is to validate a novel, high-affinity epidermal growth factor receptor (EGFR) chimeric antigen receptor (CAR) T-cell product for adoptive immunotherapy of EGFR-expressing malignancies of the central nervous system (CNS). Wild type EGFR is ubiquitously expressed on glial tumors in both children and adults, and the majority of solid tumors that metastasize to the CNS but is not expressed on healthy CNS tissues. EGFR and its isoform mutant, EGFRvIII, are hyperactivated or overexpressed receptor tyrosine kinases described in many human cancers. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) that binds to both wild type EGFR and EGFRvIII. We hypothesize that D2C7 adapted as a CAR on primary human T cells will generate potent, cytotoxic activity against EGFR-expressing tumors in vivo.

METHODS

In vitro interferon gamma release assays comparing the D2C7 CAR to the EGFRvIII CAR have proven that D2C7 CAR-T cells have high specificity and potent cytotoxicity against established murine glioblastoma (GBM) tumor cell lines, expressing both EGFR and EGFRvIII. Next steps include pre-clinical in vivo testing of the D2C7 CAR-T cells in murine models of CNS tumors expressing EGFR/EGFRvIII such as GBM, non-small cell lung carcinoma (NSCLC), breast carcinoma, and melanoma.

CONCLUSIONS

To improve and validate the effectiveness of CAR T-cell therapy for EGFR-expressing CNS tumors, our team has designed a high-affinity CAR-T cell that targets both EGFRvIII and wild-type EGFR, known as a D2C7 CAR-T cell. This investigation has established pre-clinical anti-tumor activity of D2C7-CAR T cells in vitro and we plan to present more mature data regarding efficacy in orthotopic murine models of GBM, NSCLC, breast carcinoma, and melanoma at the meeting this Fall.
DDRE-22. NOVEL LonP1 INHIBITORS FOR TARGETING GLIOMA STEM CELLS
Authors: Douglas Christopher , Di Kaijun , Lomeli Naomi , Bota Daniela
Published: December 2020

Glioblastoma (GBM) has an exceptional high rate of reoccurrence that largely explains its < 15 months median survival. LonP1 is a serine protease that degrades misfolded proteins and regulates mitochondrial DNA replication. It drives tumor progression towards a malignant cancer phenotype in colorectal cancer, melanoma, oral cancer and cervical cancer. Dr. Daniela Bota has previously shown that LonP1 is overexpressed in human malignant gliomas and is associated with higher tumor grade and poor survival prognosis. In collaboration with Professor Bhaskar Das, we have used structure activity Relationship (SAR) analysis to generate compounds with on-target inhibition of LonP1 protease activity. Preliminary work on these novel compounds shows that these proprietary inhibitors can drastically decrease cell viability in the established D54 and U251 GBM lines. The lead compound BT317, shows on-target LonP1 and exceptional chymotrypsin-like proteasome inhibition. This has led to further testing, which has shown that BT317 has enhanced activity against glioma stem cell lines (GSC) and can cause global downregulation of hypoxia inducible factor 1 alpha (Hif1α) in a heterogenous GSC-derived organoid model. Finally, we have demonstrated that BT317 has less activity against differentiated GSC lines (e.g. through successive passages) and appears to have enhanced activity against TNFa-induced, differentiated GSC. This preliminary data highlights combinatorial, pharmacological LonP1 and proteasome inhibition as a novel strategy for targeting GSC in GBM.
DDRE-23. THE COMBINATION PARP INHIBITOR OLAPARIB WITH TEMOZOLOMIDE IN AN EXPERIMENTAL GLIOBLASTOMA MODEL
Authors: Hwang Kihwan , Go Kyeong-O , Kim Sang Ho , Lee Hyunwoo , Han Jung Ho , Kim Chae-Yong
Published: December 2020

Poly (ADP-ribose) polymerase (PARP) inhibition could enhance the efficacy of temozolomide and prolong survival in patients with glioblastoma. The aim of this study was to evaluate the combination of the PARP inhibitor olaparib with temozolomide in the treatment of glioblastoma by evaluating in vitro and in vivo antitumor effects in an experimental glioblastoma model. The authors investigated antitumor effects of olaparib on temozolomide-induced cytotoxicity on O6-methylguanine methyltransferase (MGMT) promotor methylated (U87MG, U251MG) and MGMT promotor unmethylated (T98G) glioblastoma cell lines using in vitro cell viability and apoptosis assay. We found that the combination of olaparib with temozolomide enhanced temozolomide-induced cytotoxicity in all glioblastoma cell lines regardless of the status of MGMT promotor methylation. For in vivo studies, nude mice bearing orthotopically xenografted glioblastoma cell lines (U87MG) were randomized to four experimental groups: (i) untreated, (ii) temozolomide alone, (iii) olaprib alone and, (iv) olaparib+temozolomide. Mice were treated daily for 4 weeks and monitored for tumor growth, and survival. However, the addition of olaparib had no impact on the efficacy of temozolomide. The combination of PARP inhibitor olaparib with temozolomide could be an effective therapeutic approach for treatment of glioblastoma regardless of MGMT promotor methylation status, although the efficacy still should be evaluated by in vivo and clinical studies.
DDRE-24. ACQUIRED RESISTANCE TO TARGETED INHIBITORS IN EGFR-DRIVEN GLIOBLASTOMA: IDENTIFICATION OF DUAL KINASE TARGETS
Authors: Shelton Abigail , Smithberger Erin , Butler Madison , Stamper Allie , Bash Ryan , Angus Steven , East Mike , Johnson Gary , Berens Michael , Furnari Frank , Miller C Ryan
Published: December 2020

Glioblastoma (GBM) is a devastating primary brain tumor with 5-year survival < 5%. CDKN2A deletion (~60%) and EGFR amplification (55–60%) mutations frequently co-occur in these tumors. EGFR is an attractive therapeutic target due to its mutational frequency and availability of multiple brain-penetrant tyrosine kinase inhibitors (TKI). Several EGFR TKI have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used genetically engineered mouse astrocytes harboring Cdkn2a deletion and EGFRvIII, a common (35%) activating mutation. Resistant cells were generated via chronic exposure to gefitinib or erlotinib, either in vitro or in vivo. Resistance to these first-generation EGFR TKI conferred cross resistance (up to 36-fold ΔIC50) to a panel of second- and third-generation TKI relative to sensitive parental lines. Moreover, integrated RNA sequencing (RNA-seq) and chemical proteomics (multiplexed inhibitor beads and mass spectrometry (MIB-MS)) showed that the kinase transcriptome and proteome were rewired in resistant cells: 113 of ~300 detected kinases were differentially expressed (p< 0.05). We then used these techniques to examine acute (≤ 48 h) kinome changes in both sensitive and resistant cells upon treatment with a CNS-penetrant, second-generation EGFR TKI, afatinib. Whereas exposure of treatment-naïve, sensitive cells to afatinib significantly rewired the kinome (120 differentially expressed kinases), the response of resistant cells to drug re-challenge was significantly blunted (13 differentially expressed kinases). A subset of expressed kinases (35 of 263) dynamically responded to afatinib in both sensitive and resistant cells. Overall, upregulated kinases include those implicated in the biology of gliomas (Bmx, Fgfr2) and of other cancers (Pdgfrb, Mapk3/4, Ddr1/2, Pdk2). These kinases thus represent putative druggable targets for dual inhibition therapy. Integrated kinome profiling using MIB-MS and RNA-seq in GBM models with defined mutational profiles provides a powerful framework to identify novel therapeutic targets that could significantly alter current treatment paradigms.
DDRE-26. TARGETING THE UNFOLDED PROTEIN RESPONSE POTENTIATES MARIZOMIB ANTITUMOR ACTIVITY IN GLIOBLASTOMA
Authors: Severin Christophe , Benitez-Hernandez Jorge , Dumitru C Dan , Beltran-Raygoza Nishdallyh , Flandin-Blety Pierre , Escoubet Laure , Rolfe Mark
Published: December 2020

Glioblastoma (GBM) is the most prevalent and aggressive malignant tumor of the central nervous system in adults. Patients with GBM have a poor prognosis, with a median survival of 15 months with current standard-of-care treatment, consisting of surgery followed by radiotherapy with concomitant and adjuvant temozolomide. Marizomib is an irreversible, pan-proteasome inhibitor that crosses the blood–brain barrier, currently under investigation in a phase 3 trial for newly diagnosed GBM. This preclinical study investigated the mechanism of action of marizomib in patient-derived GBM spheres and explored a series of combination treatments to further enhance its antitumor potency. Marizomib inhibited the 3 proteolytic subunits of the 20S proteasome in a dose-dependent manner, and this correlated with reduced cell proliferation compared with vehicle control (dimethyl sulfoxide). Marizomib induced transient expression of unfolded protein response (UPR) pathway genes, with a sustained upregulation of the SQSTMI gene. Combining marizomib with the histone deacetylase (HDAC) inhibitors panobinostat and vorinostat demonstrated cooperative upregulation of UPR genes and enhanced inhibition of cell proliferation. Furthermore, marizomib treatment upregulated survivin, an inhibitor of apoptosis protein (IAP). Compared with marizomib alone, addition of the IAP inhibitors AZD5582 and LCL161 increased marizomib antiproliferative activity in GBM spheres. In conclusion, these findings indicate that combining marizomib with HDAC or IAP inhibitors can enhance the overall antitumor activity of marizomib in patient-derived GBM spheres. Additional studies in preclinical models of GBM are needed to corroborate these findings.
EPCO-01. LUNG ADENOCARCINOMA BRAIN METASTASIS PREDICTION, PREVENTION, AND NON-INVASIVE DIAGNOSIS USING METHYLATION SIGNATURES WITHIN TISSUE AND CIRCULATING TUMOUR DNA
Authors: Zuccato Jeffrey , Mamatjan Yasin , Patil Vikas , Nassiri Farshad , Voisin Mathew , Aldape Kenneth , Zadeh Gelareh
Published: December 2020

BACKGROUND

One quarter of lung adenocarcinoma (LUAD) patients develop brain metastases (BM) and experience a poorer median survival of 12 months despite treatment. Clinical variables do not robustly predict who will develop BM and targeted preventative treatments are limited. Tumour DNA methylation signatures predict outcomes in other cancers and can be detected in circulating tumour DNA (ctDNA). This work predicts BM development from LUAD using methylation data, identifies novel potential treatment targets to prevent metastases, and detects LUAD-BM ctDNA non-invasively.

METHODS

DNA methylation profiling was undertaken on N=124 LUAD tumours. A gradient boosted regression model built on differentially methylated CpGs (DMCs) between tumours with and without BM in 70% of samples was validated in an independent 30% testing cohort. Nine paired BM samples were profiled and DMCs between their corresponding LUAD tissue were identified along with copy number (CN) alterations. A total of 47 LUAD-BM plasma samples underwent sequencing of immunoprecipitated methylated ctDNA and differentially methylated regions (DMRs) between LUAD-BM and intrinsic brain lesions were identified.

RESULTS

The methylation-based model significantly predicted time to brain metastasis development within the testing cohort independently from cancer stage in a multivariate analysis (HR=4.3, 95%CI 1.1–17, p=0.038). Genes/pathways involved in the process of brain metastasis were identified through assessment of 83K DMCs (FDR< 0.2, mean difference >|0.1|) between paired samples as well as the CN losses found in chromosome 12q/19 of BM samples. A total of 5.5K DMRs were identified that distinguish BM samples from gliomas or primary CNS lymphomas (FDR< 0.05, logFC >1).

CONCLUSIONS

DNA methylation signatures in lung adenocarcinomas predict brain metastasis development independently from prognostic clinical factors. Genes and pathways involved in metastasis were identified as novel potential therapeutic targets. Methylated circulating tumor DNA signatures differentiate lung brain metastases from other ring-enhancing brain lesions and may have potential for non-invasive diagnosis.
EPCO-06. MOLECULAR DETERMINANTS OF EVOLUTION AND PROGNOSIS AMONG DIFFUSE LOWER GRADE ASTROCYTOMAS
Authors: Kumar Mehul , Cairncross Gregory , Blough Michael , Bose Pinaki
Published: December 2020

Gliomas with wild type (WT) isocitrate dehydrogenase (IDH) are considerably more aggressive than those with mutant IDH. To identify putative drivers of the distinct progression trajectories of IDH WT and mutant disease, we analyzed transcriptomes of lower grade astrocytomas (LGAs; grade 2–3) with retention of 1p and 19q from The Cancer Genome Atlas (TCGA; n = 347). Compared to IDH mutant LGAs, we found that PDGF signaling was significantly enriched in IDH WT LGAs and that PDGFA was the top overexpressed gene in this pathway. We identified copy number gains of chromosome 7 in WT LGAs, and methylation of the PDGFA promoter in mutant LGAs, as candidate mechanisms for the differential expression of PDGFA. High PDGFA expression and low PDGFA promoter methylation were significantly associated with poor survival in all LGAs. We also found that PDGFA expression was positively associated with aneuploidy and extracellular matrix-related immunosuppressive features in WT LGAs, underscoring the role of PDGFA as a secreted mitogen. Finally, we show that the proportion of p53 pathway mutations increase significantly with grade in IDH WT gliomas. Taken together, our findings suggest that IDH WT LGAs evolve to higher grades, and ultimately to GBM, by progressive inactivation of the p53 pathway - functioning in concert with a background of increased PDGFA expression. These data emphasize the scope of genomic reprogramming that occurs in gliomas in relation to IDH mutations and further highlight the role of PDGFA in glioma formation, progression, and prognosis. Going forward, this work provides critical biological insight that may inspire new therapeutic strategies to suppress the transformation of IDH WT LGAs to higher-grade cancers.
EPCO-09. STEREOTACTIC IMAGE-GUIDED EPIGENOME PROFILING REVEALS DIVERSE EVOLUTIONARY GROWTH ROUTES IN DIFFUSE GLIOMAS
Authors: Barthel Floris , Verburg Niels , Anderson Kevin , Koopman Thomas , Johnson Kevin , Wesseling Pieter , de Witt Hamer Philip , Verhaak Roel
Published: December 2020

Intratumoral heterogeneity is a hallmark of diffuse gliomas and inhibits effective prediction of clinical outcome. To devise a three-dimensional reconstruction of tumor lineage, we used neuronavigation to acquire eight to twelve image-guided and spatially separated stereotactic biopsy samples from 16 adult patients with a diffuse glioma, which we characterized using DNA methylation arrays. A total of 133 samples were obtained from regions with and without imaging abnormalities. Methylation profiles were analyzed to construct phylogenetic trees and subsequently projected on image-derived tumor maps. Lineage analysis of these evolutionary trees indicated that the sampled gliomas largely evolved stochastically, suggesting that critical tumor drivers were acquired early in time. These results were further validated using 102 multi-region samples from 24 independent patients. Patristic (evolutionary) and cartesian (spatial) distances between pairs of tumor samples from the same patient demonstrated strong correlations, suggesting that this information could be used to determine trajectories of tumor evolution. Evolutionary and spatial distance metrics were combined with histologically obtained and computationally quantified cellularity and proliferation rates to infer vectors representing the direction and magnitude of tumor growth. Using the resulting vector field we determined the minimum and maximum rates of change in order infer the tumor’s evolutionary trajectory. Using this metric we identified three distinct growth patterns: (1) tumors with a trajectory oriented towards the tumor core, (2) outward growing tumors with a linear trajectory pointing outside tumor boundaries, and (3) outward growing tumors with a branching trajectory directed outside tumor boundaries. Association of evolutionary growth patterns with survival demonstrated distinct impacts on outcome, suggesting that growth patterns are an important determinant of tumor aggressiveness. Taken together, although our analyses indicated that the observed glioma heterogeneity is small and largely stochastic, when spatially mapped these changes can be used to track tumor lineage and identify clinically relevant growth patterns.
EPCO-12. SINGLE CELL PROFILING OF LONG NON-CODING RNAS IN GLIOMA
Authors: Mehani Bharati , Chung Hye-Jung , Bandle Russell , Young Sarah , Kelly Michael , Aldape Kenneth
Published: December 2020

Non-coding RNAs have critical functions across biological processes that regulate glioma initiation and progression, and deregulated expression of long non-coding RNAs (lncRNAs) have been implicated in the onset and progression of malignancies. The majority of these transcripts exhibit tissue- and cancer-specific expression but little has been investigated at the single-cell level. We performed single cell RNA Sequencing (10x Genomics) for 9 IDH-wild-type glioblastomas from 7 patients. In total 66,825 cells dissociated from tumor tissues and not sorted were included in this analysis which encompassed 41,989 mean sequencing reads and 2,619 median coding genes per cell. Single cell analysis of lncRNAs in captured 190 median lncRNAs per cell and demonstrated a distinct lncRNA expression profile for glioma cells compared to the non-tumor cells with SOX2-OT significantly upregulated (2X) in glioma cells. Consistent with this finding, SOX2-OT is known to be overexpressed in a variety of cancers and has been previously implicated in glioma proliferation and migration. We then examined patterns of lncRNA expression in GBM expression subtypes. Subtype correlation indicated overexpression of RMST (classical subtype), PCED1B-AS1 (mesenchymal) and LINC00689 (proneural) lncRNAs in these expression subtypes. Consistent with these findings, upregulation of each of these 3 lncRNAs have previously been implicated on pro-tumorigenic effects, including in glioma. Examination of an independent published single cell GBM dataset also validated PCED1B-AS1 in the mesenchymal subtype. Comparison with bulk tumor GBM profiles (IDHwt TCGA GBM dataset) also showed correlations with the expression of RMST, PCED1B-AS1 and LINC00689 lncRNAs in the classical, mesenchymal and proneural subtypes respectively. Overall, these results indicate lncRNA expression can be determined in 10x-generated glioma single cell data and may reveal additional insights about cellular state and glioma biology.
EPCO-17. A SINGLE-CELL ATLAS OF GLIOBLASTOMA EVOLUTION UNDER THERAPY
Authors: Wang Lin , Babikir Husam , Shamardani Karin , Catalan Francisca , Sudhir Sweta , Kohanbash Gary , Aghi Manish , Chang Susan , Philips Joanna , Diaz Aaron
Published: December 2020

Most clinical trials for glioblastoma (GBM) enroll patients at recurrence. However, most pre-clinical animal models are of primary disease. Most studies have used tissues from primary tumors. Knowledge of GBM cellular composition at recurrence is limited. We profiled 80 human IDH-wild-type GBM specimens via single-nucleus RNA sequencing, 40 primary tumors and 40 patient-matched recurrent tumors. Select cohorts were also profiled via single-cell assay for transposase-accessible chromatin, single-cell digital-spatial, and single-cell spatial-transcriptomic assays. All patients were treated only with standard-of-care therapy: temozolomide, radiation and surgical resection. The cohort had a 1.2 male/female ratio, ages 35–76. We found a significant increase at recurrence in cells with the Verhaak mesenchymal phenotype and fewer proneural cells. Although there was an increase in the percentage of cycling cells overall, the fraction of cycling proneural cells decreased while the fraction of cycling mesenchymal cells increased. These changes were concomitant with a significant increase in the percentage of tumor-infiltrating monocytic-lineage cells derived from the periphery. Taken together, these findings support a proneural-to-mesenchymal shift at recurrence due to an increase in the birth rate of mesenchymal cells, supported by an expansion of myeloid-derived cells from peripheral blood. Although the percentages of glioblastoma-infiltrating T-cells are generally low (~1% on average in our data), we found a statistically significant increase in T-cell abundance at recurrence. We identified a cohort of T-cell outliers that demonstrate 2-to-8-fold increases in T-cells over average levels. We validated that T-cells in these tumors have extravasated beyond the perivascular space and into the cellular tumor via immunohistochemistry and single-cell spatial profiling. We present our ongoing analysis of the paracrine signals, upstream transcription-factor expression, and cis-regulatory grammars utilized in immune-outlier GBMs. We describe how these programs change under therapy. These studies shed light on the effect of standard therapy in shaping GBM composition at recurrence.
EPCO-18. MULTI-MODAL AND MULTI-TISSUE PROFILING OF GBM PATIENTS TREATED WITH CAR T CELL THERAPY TO ELUCIDATE MOLECULAR MECHANISMS OF VARIATION IN TREATMENT RESPONSE
Authors: Yahn Stephanie , Zhang Sophia , Peter Lance , Chung Mei-I , Gutierrez Austin , Starr Renate , Alizadeh Darya , Brown Christine , Banovich Nicholas
Published: December 2020

Recent advances in immunotherapy, particularly chimeric antigen receptor (CAR)-engineered T cell therapy, have shown promise for the treatment of many tumor types including progressive recurrent glioblastoma (GBM). While early phase clinical trials have illuminated the potential for CAR T cell therapy to effectively treat GBM, they have also highlighted the unique challenges regarding the efficacy and safety of immunotherapy for brain tumors, and many patients continue to progress during therapy. We seek to overcome these challenges and ultimately extend the time of survival for patients diagnosed with GBM by investigating the immune- and tumor-mediated mechanisms driving variation in response to CAR T cell therapy. We generated the first multi-omics time-series dataset of CAR T cells, endogenous immune cells, and tumor cells from 59 GBM patients treated with CAR T cell therapy. Using single cell RNA-sequencing and simultaneous quantification of nearly 200 cell surface proteins, we comprehensively profiled the cellular phenotypes and signaling pathways within tumor and circulating immune cells that are associated with treatment response. The combination of mRNA and protein expression allowed us to resolve cell states beyond what either modality was capable of alone. Additionally, we found differentially expressed genes and proteins between tumor biopsies collected before and after CAR T cell therapy as well as differential expression between pre-infusion CAR T cells and those identified within the tumor following infusion. By evaluating the CAR T cell phenotypes prior to and during treatment we sought to address the outstanding question of how intrinsic variability impacts the activity and persistence of CAR T cells and to determine the phenotypes that confer the greatest therapeutic benefit for patients with GBM. Our results have direct implications for precision medicine and future clinical trials investigating the use of CAR T cell therapy for GBM as well as other solid tumors.
EPCO-20. RELATING GLIOBLASTOMA HETEROGENEITY TO HUMAN FETAL GLIAL DEVELOPMENT THROUGH HIGH RESOLUTION SINGLE NUCLEI TRANSCRIPTOMICS
Authors: Mussa Zarmeen , Ramos Susana , Nabel Elisa , Allette Kimaada , Hamid Ammar , Cai Maggie , Zhao Will , Wang Ying-chih , Beaumont Kristin , Sebra Robert , Tsankov Alexander , Tsankova Nadejda
Published: December 2020

Glioblastoma (GBM) is thought to be driven by a therapy-resistant cancer stem cell population that recapitulates developmental phenotypes. Direct comparisons of GBM to glial states during human fetal development are limited due to paucity of data from late prenatal gestation, when gliogenesis is thought to occur. Here, we generated a comprehensive single nuclei RNA sequencing (snRNAseq) dataset of approximately 200,000 nuclei taken from the germinal matrix and the cortical plate of 16 fetal postmortem samples, ranging from 17 to 41 gestational weeks, enabling high spatiotemporal resolution of late neurogenesis and early-to-peak gliogenesis. We performed unbiased clustering to identify broad cell types within each sample and integrated all fetal samples to analyze evolving glial states and relationships across two regions and four developmental stages. Subclustering analysis of developing glia from the germinal matrix and cortical plate resolved developmental cell type signatures that are absent in the adult brain. Trajectory inference and pseudo-time analyses reconstructed relationships within these glial lineages and states, identifying a robust common glial progenitor population (GPC) with distinct signature, preceding both oligodendrocyte progenitor cell (OPC) and astrocyte lineage commitment during late prenatal development. We then performed snRNAseq on approximately 30,000 nuclei taken from the core and infiltrating edge of two surgically resected GBM samples with IDH-mutant and IDH-wildtype status and EGFR amplification. Uniform manifold approximation and projection (UMAP) dimensionality reduction revealed distinct neoplastic and non-neoplastic population clusters within each GBM sample. Projecting our previously defined neural stem cell / progenitor signatures onto each GBM UMAP identified notable predominance of the GPC-like developmental signature throughout both GBM tumors with focal minor contributions from the OPC-, transit amplifying-, and astrocyte-like signatures. The high spatial and temporal resolution of the generated roadmap dissolves GBM intratumoral heterogeneity into distinct developmental molecular states driven by potentially targetable regulatory networks.
EPCO-22. IDENTIFYING NEOANTIGENS FOR A PERSONALIZED MUTATION-DERIVED GENOMIC VACCINE IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA
Authors: Kodysh Julia , Rubinsteyn Alex , Blazquez Ana , Mandeli John , Bhardwaj Nina , Hormigo Adilia
Published: December 2020

BACKGROUND

Glioblastomas (GBM) are known for having a lower mutational burden than many other tumor types. Nevertheless, somatic variants that occur in GBM mutational processes may give rise to neoantigens, which can be targeted in a personalized genomic vaccine to elicit a patient-specific anti-tumor response.

METHODS

Normal and tumor DNA and tumor RNA are extracted from tumor specimens and PBMCs. Exome and RNA sequencing and HLA typing is performed for each patient. Neoantigens are identified using the OpenVax computational pipeline, which calls somatic variants in the DNA and prioritizes corresponding candidate neoantigens for vaccination based on tumor RNA expression and predicted MHC class I binding affinity for each of the patient’s HLA alleles. This work is the base for a phase I trial of personalized neoantigen vaccines in combination with Tumor Treating Fields for GBM (NCT03223103).

RESULTS

For each of the 9 patients enrolled in the trial, an average of 1005 somatic mutations were identified (range 299–2441), of which 118 were coding variants (range 52–198), 20 were coding and expressed in the tumor RNA (range 9–45), and 16 were coding, expressed and resulted in predicted MHC class I ligands (range 7–33). An average of 2.3% of all somatic variants identified in each tumor gave rise to predicted neoantigens. Sufficient numbers of neoantigens were identified in all tumor samples of the patients enrolled in the study. The overall somatic mutation landscape for the tumors revealed 4/9 PIK3R1 deletions, 2/9 IDH1 substitutions, as well as disruptions in PTEN, TP53, and ATRX, among others.

CONCLUSIONS

Identifying sufficient neoantigens for inclusion in the personalized genomic vaccine is computationally feasible despite a typically low GBM mutational burden. Driver and passenger mutations can be identified through the same computational pipeline utilized for other tumor types.
EPCO-23. COMPARATIVE TRANSCRIPTOMICS TO IDENTIFY TARGETED THERAPY CANDIDATES IN HIGH GRADE GLIOMA
Authors: Hundley Kelsey , Vaske Olena , Lyle Geoff , Learned Katrina , Beale Holly , Kephart Ellen , De Loose Annick , Lee Madison , Day J D , Rodriguez Analiz
Published: December 2020

Genomic characterization is often used for the identification of therapeutic targets in tumors. Recently, comparative transcriptomics has begun to be utilized for this purpose. In this pilot, we compare the transcriptome of a patient with recurrent high grade glioma (HGG) to our cohort to identify potential therapies. We reviewed transcriptomic profiles from patients who had resection of HGG at our institution over the past year as well as the UCSC cancer compendium. Briefly, tumor RNA was extracted from embedded tumor tissue sections with tumor cellularity higher than 20%. RNA libraries were sequenced to obtain approximately 65 million reads on an Illumina HiSeq 4000 System utilizing patterned flow cell technology. The RNA profile of a 24 male with Li-Fraumeni syndrome and recurrent HGG with leptomeningeal spread underwent comparative transcriptomics to identify targets. A Bayesian statistical framework for gene expression outlier detection was used. These comparisons allowed for the identification of genes and pathways that are significantly overexpressed. Our internal HGG cohort consisted of 44 adult patients and was evenly distributed among the 4 HGG Verhaak subtypes. Our patient of interest had druggable outlier expression in HDAC1, STAT1 and STAT2 in comparison to our internal cohort indicating vorinostat and ruxolitinib as potential therapies, respectively. We then compared our patient of interest to 12,747 patients in the cancer compendium and STAT2 expression was high but not an outlier. In comparison to 738 glioma samples, STAT1 and STAT2 were outliers but not HDAC1 again indicating ruxolitinib as a potential targeted therapy. The patient did not have outlier expression in notch transcriptional targets or immune checkpoint biomarkers when compared to all cohorts. In conclusion, comparative Transcriptomics can identify therapeutic targets in a patient with recurrent HGG even in small cohorts. In our pilot, we identified ruxolitinib as a potential candidate to treat leptomeningeal recurrence.