bluuebunny/crossref_metadata_2025 huggingface dataset:
CBMT-29. PROMOTING FATTY ACID STORAGE BY DIACYLGLYCEROL-ACYLTRANSFERASE 1 PROTECTS GLIOBLASTOMA AGAINST OXIDATIVE STRESS AND MAINTAINS LIPID HOMEOSTASIS FOR RAPID TUMOR GROWTH
Authors: Cheng Xiang , Geng Feng , Zhong Yaogang , Tian Zhihua , Pan Meixia , Cheng Chunming , Horbinski Craig , Mo Xiaokui , Han Xianlin , Guo Deliang
Published: November 2019

Fatty acids are essential building blocks and energy substrates of lipids in cells. They constitute the major structural components of membrane lipids, i.e., glycerophospholipids and sphingolipids, and also serve as an important energy resource. Whereas excess fatty acids or dysregulation of fatty acid metabolism causes lipotoxicity, leading to severe cell damage. We previously identified that lipid metabolism is dramatically altered in glioblastoma (GBM), especially fatty acid synthesis, which is greatly elevated in various types of cancers. However, how tumor cells prevent excessive fatty acids accumulation from inducing lipotoxicity has rarely been studied. We recently identified that GBM greatly upregulates diacylglycerol-acyltransferase 1 (DGAT1) to direct excess fatty acids into triglycerides and lipid droplets to prevent oxidative stress. Inhibiting DGAT1 disrupts lipid homeostasis and shifts excessive fatty acids moving into mitochondria for oxidation, resulting in a high level of reactive oxygen species (ROS), mitochondrial damage, cytochrome c release and apoptosis. Inhibiting carnitine palmitoyltransferase to reduce fatty acids shuttling into mitochondria significantly decreases ROS and rescues cell death caused by DGAT1 inhibition. Xenograft models show that genetic or pharmacological inhibition of DGAT1 blocks lipid droplet formation, induces tumor cell apoptosis and markedly suppresses GBM tumor growth. Together, our study demonstrates that DGAT1 upregulation protects GBM from oxidative damage and maintains lipid homeostasis by facilitating excess fatty acids storage. Our data further show that targeting DGAT1 specifically induces lipotoxicity in tumor cells, while sparing normal brain, which is a very promising therapeutic strategy antagonizing GBM and has a high potential to shift current paradigm in treating GBM.
CBMT-32. EGFR SUPPRESSES p53 FUNCTION THROUGH DNA-PKcs BINDING TO p53: NOVEL CROSSTALK BETWEEN EGFR AND TP53 IN GLIOBLASTOMA
Authors: Ding Jie , Li Xiaolong , Zhang Chen , Gao Feng , Wu Shaofang , Wasylishen Amanda , Baggerly Keith , Lozano Guillermina , Koul Dimpy , Yung Alfred
Published: November 2019

Comprehensive analysis of the TCGA Glioblastoma database has identified 3 prognostically relevant subgroups (Proneural, Classical and Mesenchymal). Interestingly, it has also revealed a near mutual exclusivity of EGFR amplification and TP53 mutations. There are only a few tumors with both amplified EGFR and mutant TP53. We decided to investigate the functional relationship between EGFR and TP53 relative to tumorigenesis and tumor proliferation. We selected from our molecularly characterized GSC bank a series of EGFR amplified+TP53wt/TP53mut lines for this study. Here we report that EGFR amplification overrides the effect of p53wt but co-regulates tumorigenesis with TP53R175H mutation. We show that CRISPR EGFR knockout in TP53wt cells leads to complete loss of cell growth suggesting that EGFR is essential for cell survival, providing growth signal and suppression of p53wt function. We further show that EGFR induces physical binding between p53wt and DNA-PKcs, which suppresses p53wt by inhibiting p53 phosphorylation at S15. The knockdown of DNA-PKcs restores p53wt anti-tumor function. In contrast, GSC262 harboring TP53R175H mutation is the only GSC that survives after EGFR knockout, suggesting that this specific mutation has a gain of function (GOF) to sustain growth without EGFR. This is confirmed by dual knockout of EGFR and TP53R175H showing no surviving cells. Further investigation shows both EGFR and p53R175H induce tumor glycolysis, the major energy metabolism and heightened growth signal for this group. We further show that EGFR mediated suppression of p53R175H is through DNA-PKcs binding to p53R175H and reduced S15 phosphorylation. Taken together, this study reveals 1) EGFR amplification is essential for tumor growth in TP53wt GSCs; 2) novel crosstalk between EGFR and p53 through DNA-PKcs. 3) EGFR suppresses p53wt function by providing the energy required for tumor growth through inducing glycolysis, and 4) TP53R175H is a GOF mutation that sustains life without EGFR by inducing glycolysis.
CSIG-04. PAN-RTK INHIBITION OF sLRIG1 MEDIATES AXL DOWNREGULATION IN GLIOBLASTOMA
Authors: Neirinckx Virginie , Hau Ann-Christin , Schuster Anne , Fritah Sabrina , Chevigné Andy , Schmidt Mirko H , Niclou Simone
Published: November 2019

INTRODUCTION

Aberrant regulation of receptor tyrosine kinase (RTK) activity is characteristic of Glioblastoma (GBM). However, RTK-based targeted therapies have been largely unsuccessful in GBM patients, partially due to the complexity and redundance of RTK signaling. LRIG1 (Leucine-rich Repeats and ImmunoGlobulin-like domains 1) is an important endogenous inhibitor of epidermal growth factor receptor (EGFR) during health and disease, however its mechanism of action is poorly understood. We previously showed that the soluble form of LRIG1 potently inhibits GBM growth in vivo, irrespective of EGFR expression level and status, suggesting the involvement of other RTKs. Here, we aimed to shed light on the molecular mechanisms underlying its anti-cancer activity.

MATERIAL AND METHODS

We generated a recombinant human soluble LRIG1 protein (rh-sLRIG1) by expressing LRIG1 ectodomain in insect cells via baculovirus infection and subsequent His-tag purification. rh-sLRIG1 was used to treat patient-derived GBM stem-like cells, classical GBM cell lines and patient-derived 3D tumor organoids. Using different cell-based assays, cell proliferation, invasion, cell morphology, as well as protein expression and protein-protein interactions were investigated.

RESULTS

We find that recombinant sLRIG1 efficiently reduced proliferation, invasion and viability of GBM cells and patient-derived organoids, and modulated cytoskeleton proteins and cell shape. In line with previous data, the effect of recombinant sLRIG1 was independent of EGFR expression. Interestingly sLRIG1 impacted multiple RTKs including AXL, by direct protein downregulation, while EGFR expression was not affected. At the molecular level, we find that sLRIG1 interfereed with AXL dimerization, while no protein interaction with EGFR was detected.

CONCLUSION

We identify AXL as a novel LRIG1 target and provide evidence that sLRIG1-mediated RTK downregulation requires direct protein-protein interaction. These data pave the way for a potential therapeutic application of recombinant sLRIG1 in the inhibition of growth factor signaling in GBM.
CSIG-10. ROLE OF c-Met/β1 INTEGRIN COMPLEX IN THE ESTABLISHMENT OF BRAIN METASTASES
Authors: Wadhwa Harsh , Lau Darryl , Chandra Ankush , Nguyen Alan , Shah Sumedh , Spatz Jordan , Safaee Michael , Sudhir Sweta , Patel Akshar , Cheng Justin , Aghi Manish
Published: November 2019

INTRODUCTION

Metastases cause 90% of human cancer deaths. The metastatic cascade involves five steps: invasion, intravasation, extravasation, colonization, and proliferation. While individual mediators of these processes have been investigated, their interactions remain undefined. We previously demonstrated increased formation of a structural complex between receptor tyrosine kinase c-Met and β1 integrin in metastases compared to primary tumors. We used novel cell culture models and in vivo assays to define the role of this complex in individual steps of the metastatic cascade.

METHODS

The iDimerize heterodimer system was inserted into MDA-MB-231 breast adenocarcinoma cells, allowing c-Met/β1 heterodimerization induction via A/C heterodimerizer treatment. Scratch assays and novel transwell assay modifications were used to measure migration, invasion, intravasation, and extravasation. Proximity ligation assay was performed to measure c-Met/β1 complex. Nanostring panel was used to transcriptionally profile cells.

RESULTS

c-Met/β1 complex induction promotes breast cancer invasion (p< 0.001), migration (p< 0.05), intravasation (p< 0.01), and adhesion to the vessel wall (p< 0.01). However, it does not increase extravasation in culture or in vivo. These effects may be driven by the ability of c-Met/β1 to increase mesenchymal character (p< 0.05) and stem cell population (p< 0.001). Nanostring analysis revealed upregulated Wnt and hedgehog pathways after c-Met/β1 complex induction, particularly WNT7B (p< 0.05). OS2966, an antibody preventing c-Met/β1 binding, decreased invasion (p< 0.05), intravasation (p< 0.05), and mesenchymal morphology (p< 0.001) and gene expression (p< 0.001). Brain- and bone-seeking breast cancer cells have higher c-Met/β1 complex than controls (p< 0.05) and preferentially adhere to tissue-specific matrix (p< 0.01).

CONCLUSIONS

The c-Met/β1 complex drives breast cancer cell intravasation. While extravasation is not affected by the complex, preferential affinity for tissue-specific matrix enables the c-Met/β1 complex to drive breast cancer metastases to brain and bone. Pharmacological targeting of the complex may prevent metastases, particularly to the brain and bone.
CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO
Authors: Koch Marilin , Czemmel Stefan , Lennartz Felix , Beyeler Sarah , Przystal Justyna , Govindarajan Parameswari , Canjuga Denis , Neumann Manfred , Rizzu Patrizia , Tatagiba Marcos , Nahnsen Sven , Heutink Peter , Raineteau Olivier , Tabatabai Ghazaleh
Published: November 2019

OBJECTIVE

The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms.

METHODS

Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile.

RESULTS

The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways.

CONCLUSIONS

A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.
CSIG-19. CYTOMEGALOVIRUS INFECTION LEADS TO NF-kB DEPENDENT UPREGULATION OF c-MET AND MGMT IN GLIOBLASTOMA AND RESISTANCE TO TEMOZOLOMIDE IN VIVO
Authors: Zdioruk Mykola , Krenzlin Harald , Oskar Nowicki M , Cook Charles H , Chiocca Ennio , Lawler Sean
Published: November 2019

INTRODUCTION

Cytomegalovirus (CMV), a member of the betaherpesvirinae subfamily widespread in human populations has been reported to be detectable in glioblastoma (GBM). We have recently shown that CMV latent infection promotes GBM growth in a mouse model. The mechanisms involved are not well-defined and treatment responses have not been evaluated.

METHODS

To investigate the effects of CMV on GBM cells we performed an RNAseq study. Differentially expressed genes were identified and validated in vitro and in vivo.

RESULTS

RNAseq analysis showed multiple alterations induced by CMV infection of GBM cells including significant upregulation of c-MET (8.2 log2 fold change) and MGMT (3.9 log2 fold change) transcripts after CMV infection. c-MET is a tyrosine kinase receptor known to promote GBM growth, and MGMT expression leads to resistance to the alkylating agent temozolomide (TMZ). These findings were validated in vitro in both mouse and human GBM cells, and shown to be dependent on CMV-induced upregulation of NF-kB by siRNA knockdown of the essential RelA NF-kB subunit. The c-MET co-receptor CD44 was also upregulated suggesting a role for the c-MET signalling axis in CMV induced tumor progression. CMV-infected tumor cells were resistant to TMZ treatment as expected due to MGMT upregulation. Treatment of our murine GBM model with TMZ led to a significant increase in survival in control mock-infected mice (median survival 38 days control, 48 days TMZ), whereas the CMV infected mice did not respond (median survival 35 days control, 35 days TMZ; p value between TMZ treated groups = 10–4).

CONCLUSIONS

Our results define novel and relevant mechanisms by which CMV may influence both GBM growth and resistance to treatment. Further therapeutic and mechanistic studies are underway to investigate these pathways in the context of CMV in GBM. These data support further study of CMV targeting in GBM.
CSIG-23. NOTCH ACTIVATION INDUCED BY HSV-1 ENCODED miRNA-H16 SENSITIZES oHSV-TREATED TUMORS TO NOTCH INHIBITOR
Authors: Otani Yoshihiro , Young Yoo Ji , Chao Samantha , Liu Joseph , Cristina Jaime-Ramirez Alena , Jin Lee Tae , Hurwitz Brian , Yan Yuanqing , Dai Hongsheng , Glorioso Joseph C , Caligiuri Michael A , Yu Jianhua , Kaur Balveen
Published: November 2019

INTRODUCTION

Glioblastomas (GBMs) are resistant to traditional therapies, and new treatment is urgently needed. NOTCH signaling is highly activated in glioma and activated NOTCH signaling is associated with increased tumor growth and therapeutic resistance. Effect of oncolytic virus therapy on NOTCH signaling activation has not been reported to our knowledge.

METHODS

Western blotting, real time Q-PCR, reporter assay, and bioluminescence mice imaging were used to evaluate oHSV-mediated activation of NOTCH signaling. High throughput analysis of HSV-1-encoded microRNAs and genes were performed to identify the activator of NOTCH signaling, and in silico analysis, 3’ untranslated region (3’UTR) assay, and immunoprecipitation were used to uncover the down-stream signaling. Anti-tumor efficacy of NOTCH inhibitor, gamma-secretase inhibitor (GSI) combined with oHSV was evaluated using intracranial glioma-bearing mouse xenografts.

RESULTS

oHSV infection induced NOTCH activity in glioma PDX cells. High throughput analysis uncovered this to be due to HSV-1-encoded miR-H16 (miR-H16). 3’UTR analysis of Factor Inhibiting HIF-1 (FIH-1) indicated miR-H16 directly target FIH-1 and repress its protein expression. Immunoprecipitation analysis also uncovered that FIH-1 reduction liberated Mild bomb 1 (Mib1) from FIH-1 mediated sequestration, permitting it to activate NOTCH signaling by ubiquitination of NOTCH ligands. Forced expression of either antago-miR-H16 or FIH-1 attenuated the oHSV-induced NOTCH activation. Furthermore, oHSV therapy-induced activation of NOTCH signaling sensitized tumors to GSI, resulting in significantly improved therapeutic response to oHSV therapy and prolonged the survival in several intracranial GBM xenografts.

CONCLUSION

In this study, we identified the mechanism of oHSV therapy-mediated activation of NOTCH signaling, and showed the therapeutic benefit of combining oHSV with GSI.
CMET-01. IMMUNE CHECKPOINT INHIBITORS FOR NON-SMALL CELL LUNG CANCER WITH BRAIN METASTASIS: THE ROLE OF GAMMA KNIFE RADIOSURGERY
Authors: Ho Lee Min , Cho Kyung-Rae , Lee Jung-Il
Published: November 2019

PURPOSE

Immune checkpoint inhibitors (ICIs) are approved for the treatment of non-small cell lung cancer (NSCLC), but the safety and efficacy of the combined treatment with ICIs and gamma knife radiosurgery (GKS) remain undefined.

METHODS

We retrospectively reviewed the medical records of patients with brain metastases from NSCLC who were treated with ICIs between January 2015 and December 2017 at our institute. Of the 134 patients identified, 77 who were assessable for brain responses were categorized into three groups based on the medication administered and GKS: Group A, ICI alone; Group B, ICI with concurrent GKS within 14 days; and Group C, ICI with non-concurrent GKS.

RESULTS

The median follow-up duration after the diagnosis of brain metastases was 19.1 (range, 1–68.7) months. At the last follow-up, 48 patients (62.3%) died. The overall survival was shorter in Group A than in Groups B and C (P=0.001). There were no statistically significant differences in the intracranial disease progression-free survival (P=0.514), local progression-free survival (P=0.358), and complication rates among the three groups. Twelve patients presented with leptomeningeal seeding (LMS) during follow-up. There was a significant difference in the LMS-free duration from the time of administering ICIs (P=0.011) and the diagnosis of metastasis (P< 0.001).

CONCLUSION

GKS in combination with ICI showed no favorable overall survival outcome in the treatment of brain metastasis from NSCLC. However, GKS with ICI did not increase the risk of complications. Furthermore, compared with ICI alone, GKS with ICI may be associated with a reduced incidence of LMS.
CMET-02. ESTABLISHING THE SAFETY AND EFFICACY OF A NEW MULTI-AGENT INTRATHECAL TREATMENT PROTOCOL FOR PATIENTS WITH NEOPLASTIC MENINGITIS
Authors: Zammar Samer , Eby Richard , Zacharia Brad , Strowd Roy , Grossman Stuart , Aregawi Dawit , Michael Glantz
Published: November 2019

BACKGROUND

The prognosis for neoplastic meningitis (NM) remains dismal and single-agent (rather than multi-agent) chemotherapy remains the standard of care. Objective: To analyze survival of patients with NM receiving multi-agent vs. single-agent intrathecal chemotherapy and evaluate the safety of antitumor intrathecal biological agents.

METHODS

We compared a standardized multi-agent intrathecal treatment cohort to the corresponding patient level data acquired from 6 of the 7 randomized controlled trials (RCTs) conducted in patients with NM, all of which used single-agent intraventricular therapies. The toxicity of intrathecal biological agents was studied by analyzing the outcome data form patients with NM treated with at least 1 intrathecal biological drug.

RESULTS

283 patients receiving multi-agent therapy were compared to 290 patients from the RCTs. Patients and tumor characteristics did not differ between groups. All patients from the multi-agent patient group were included in an intent-to-treat model. For all solid tumors, median survival (multi-agent vs. single-agent treatment) was 211 vs. 97 days, hazard ratio (HR) 3.39, p< 0.001. For lymphoma, survival was 304 vs. 81 days, HR 2.10, p< 0.001. Stratified by tumor histology, median survival for breast cancer NM was 315 days [95% CI 248–449], for lung cancer 193 days [127- 200]; for melanoma 307 days [65–1492], and for primary brain tumors 253 days [187- 348]. No additional toxicity was seen in the multi-agent compared to the single-agent treatment groups. In 110 patients, grade III toxicity occurred in 5.3%, 3.1%, 0% and 0% of patients receiving rituximab, trastuzumab, panitumumab, and alpha interferon. No difference in survival was seen when comparing patients with and without treatment related toxicity in any histological groups.

CONCLUSION

Multi-agent intraventricular chemotherapy was associated with dramatically increased survival in patients with solid tumor and lymphomatous NM. Incorporating biologic agents as part of a multi-agent intrathecal treatment regimen is very safe.
CMET-12. EFFICACY OF UPFRONT IMMUNE CHECKPOINT INHIBITORS IN LUNG CANCER BRAIN METASTASIS
Authors: Barnett Addison , Lauko Adam , Li Hong , Ali Assad , Sagar Soumya , Chao Samuel , Pennell Nathan , Peereboom David , Stevens Glen , Angelov Lilyana , Yu Jennifer , Murphy Erin , Mohammadi Alireza , Suh John , Barnett Gene , Ahluwalia Manmeet
Published: November 2019

INTRO/OBJECTIVE

Immune checkpoint inhibitors (ICI) have improved outcomes in a subset of patients with lung cancer. However, data describing the efficacy of ICI in lung cancer brain metastasis (LCBM) is limited. We analyzed overall survival (OS) in patients with LCBM treated with upfront ICI, defined as having received ICI within 90-days of LCBM diagnosis, compared to non-ICI therapies.

METHODS

We reviewed 665 patients with LCBM diagnosed between 2000 and 2018 at a major tertiary care institution. Of those patients, 240 received ICI, 164 of which received ICI after 90-days and 76 received ICI within 90-days. Propensity score (PS) was calculated by logistic regression model including age, KPS, number of baseline brain lesions, and presence of extra-cranial metastasis (ECM) at time of LCBM diagnosis. OS from LCBM diagnosis between PS matched cohorts were compared using Kaplan-Meier, the Log-Rank test, and Cox proportional hazards models.

RESULTS

Prior to PS matching, median survival between ICI and non-ICI cohorts was not significantly different (10.9 months for both, p=0.81), although more ICI patients had ECM (57.1% vs 40.9%, p=0.006). Following PS matching, the ICI (n=76) and non-ICI (n=76) cohorts had median age (62.4 vs 62.3 years), KPS (80 for both), lesion number (2 for both), and ECM (56.6% for both). Of matched patients, 94% received SRS, 52% received WBRT, and 29% underwent surgical resection. Compared to non-ICI, the ICI cohort had a 2-year OS hazard ratio=0.87 (95% CI=0.58–1.31, p=0.51). Median and 1-year survival were not significantly different between ICI and non-ICI cohorts (median: 10.9 vs 9.1 months; 1-yr: 43.0% vs 42.4%).

CONCLUSION

Patients with LCBM who received ICI within 90-days of their diagnosis did not have improvement in OS compared to patients who received non-ICI therapies. Evaluation of clinical factors that may affect the efficacy and durability of immunotherapy is ongoing and will be presented.
CMET-16. A PHASE II TRIAL OF BEVACIZUMAB IN PATIENTS WITH RECURRENT SOLID TUMOR BRAIN METASTASES WHO HAVE PROGRESSED FOLLOWING WHOLE BRAIN RADIATION THERAPY (WBRT): FINAL RESULTS
Authors: Kumthekar Priya , Dixit Karan , Grimm Sean , Lukas Rimas , Schwartz Margaret , Helenowski Irene , Sharp Laura , Nelson Valerie , Raizer Jeffrey
Published: November 2019

BACKGROUND

Brain metastases (BM) are the most common intracranial tumor with limited treatment options following the progression after WBRT.

METHODS

This open label phase 2 study for patients have progressed following WBRT enrolled participants who received bevacizumab 10 mg/kg intravenously every two weeks until CNS disease progression (one cycle=4 weeks). The primary endpoint was objective radiographic tumor response as defined by modified Response Assessment in Neuro-oncology (RANO) criteria. Secondary endpoints included safety, progression free survival (PFS), time to response, duration of response, overall survival (OS), and quality of life (QOL) as measured by FACT-G and FACT-Br.

RESULTS

A total of 27 patients were registered of which 24 were evaluable for ORR (3 came off study prior to first follow up MRI brain). Median age was 53 (range 27–73), median number of cycles was 5.5 (range 1–20) with a median follow up of 8.7 months (range 2.4–47.9mo). Of the 24 evaluable patients, 6 showed radiographic response (Partial response=6, stable disease=16, progressive disease=2, 81% (22/24) experienced clinical benefit). The 6 month PFS: 46% (95% CI: 25% - 67%) and median PFS was 5.3 months. Median OS was 9.5 months (95% confidence interval 6.3m – 15.0m). For the patients who completed sequential QOL assessments, there no decline in QOL seen secondary to treatment and there was a non statistically significant improvement seen in the FACT-Br questionnaire. Overall, treatment was well tolerated with 3 grade 3 adverse events seen: hypertension (n=3), headache (n=1) and thrombotic event (n=1).

CONCLUSION

For this pretreated BM population with historically poor clinical outcome and survival, we showed disease response with bevacizumab therapy, drug tolerability and improved survival as compared to historical controls. While larger studies are needed to confirm, bevacizumab therapy could be a viable option for solid tumor BM patients who experience progression following WBRT.
CMET-27. EVALUATION OF DYNAMIN 2 (DNM2) AS A THERAPEUTIC TARGET IN LEPTOMENINGEAL METASTATIC DISEASE
Authors: Chernikova Sophia , Polyak Dina , Deng Jiaojiao , Tsau Sheila , Casey Kerriann , Johnson Eli , Bhambhvani Hriday , Khoeur Lina , Stanley Geoff , Tran Kevin , Connolly Ian , Joyce Annie , Li Yingmei , von Eyben Rie , Nagpal Seema , Hayden Gephart Melanie
Published: November 2019

Leptomeningeal metastasis (LM), a spread of cancer to the meninges and cerebrospinal fluid, carries extremely poor prognosis due to fast progression and no effective treatment. Given that LM mostly develops in specific types of cancer, notably melanoma, breast and lung cancer, it is likely that predisposition for LMD is shaped by specific genetic footprints and/or anti-cancer therapies. In this regard, it is interesting that among all breast cancers the triple-negative (i.e. estrogen and progesterone receptor-negative and HER2 overexpression-negative) breast cancer (TNBC) type develops a disproportionally high percentage of LM, accounting for the majority (~40%) of all breast cancer LM cases. TNBCs trace their origin to the genomic instability stemming from defects in DNA repair (notably homology-directed repair, HDR). We have recently shown that the efficiency of HDR depends on dynamin 2 (DNM2) best known for its role in endocytic protein trafficking. Higher DNM2 was associated with more efficient HDR and the resistance to DNA-crosslinking chemotherapy. Importantly, elevated DNM2 was associated with lower relapse-free survival and shorter times to relapse after chemotherapy only in TNBCs and not in other types of breast cancer. As DNM2 also fuels migration and invasion, the cells with high DNM2 are not only the most resistant to chemotherapy but also are the most mobile, and thus may represent the core population of LM. Here we test the inhibition of the DNM2-dependent endocytic trafficking as a potential therapeutic strategy to halt LM in TNBC. As a model of LM we use the human-in-mouse model of brain metastasis based on internal carotid injection of MDA-MB-231-BR3 cells, which we have shown to faithfully recapitulate human LM. We show that DNM2 knockdown delays metastatic spread to the brain and potentiates the effect of DNA-crosslinking chemotherapeutic cyclophosphamide, providing justification for further testing of DNM2 inhibitors for targeted therapy of LM.
CMET-29. DETECTING TUMOR MUTATIONS IN CEREBROSPINAL FLUID: LIQUID BIOPSY FOR THE DIAGNOSIS OF CENTRAL NERVOUS SYSTEM METASTASES
Authors: Shah Mauli , Zorofchian Soheil , Lan Chieh , Duose Dzifa , Hu Peter , Zhu Jay-Jiguang , Luthra Rajyalakshmi , Esquenazi Yoshua , Ballester Leomar Y
Published: November 2019

There is a critical need for better methodologies to diagnose and monitor patients with central nervous system (CNS) tumors. Recent advancements in the use of circulating tumor DNA (ctDNA) show that it is a promising tool for the evaluation of patients with primary or metastatic CNS tumors. Cerebrospinal fluid (CSF) is a more suitable sample type to evaluate metastatic CNS cancers than plasma. The current techniques for diagnosing metastatic CNS tumors include imaging (e.g., MRI), examination of tumor cells in CSF (CSF-cytology) and biopsies. However, MRI and CSF-cytology have poor specificity and sensitivity, respectively, and brain biopsies are invasive. The objective of this study is to optimize and validate a next generation sequencing (NGS) assay to detect ctDNA in the CSF of patients with metastatic CNS tumors. We have compared several methods to isolate ctDNA from CSF and identified the optimal method based on the quantity and quality of ctDNA obtained. CSF-ctDNA was extracted using QIAamp Circulating Nucleic Acid kit from CSF patient samples (CNS tumor= 10, Breast cancer=17, Lung cancer=8, Ovarian cancer=1, Melanoma=1, B-cell lymphoma=2, and Uterine cancer=1). We analyzed CSF-ctDNA mutations with the OncomineTM Pan-Cancer cell-free assay and found mutations in 19/40 (47.5%) CSF samples. The targeted panel detected 22 single nucleotide variations involving 11 genes and TP53 was the most commonly mutated gene. Copy number variations were detected in FGFR3, MYC, CCND1, CCND3, FGFR1, and CDK4 genes. We compared the sensitivity of the NGS assay to detect ctDNA with CSF-cytology and found that 7/22 (31.8%) CSF-cytology negative patients were positive for CSF-ctDNA. In conclusion, CSF-ctDNA targeted sequencing is more sensitive than CSF-cytology in the evaluation of patients with CNS metastasis. This approach will improve the diagnosis and management of primary and metastatic CNS tumors.
CMET-35. COMPETING RISKS ANALYSIS OF FACTORS INFLUENCING DEVELOPMENT OF LEPTOMENINGEAL METASTASIS IN BREAST CANCER PATIENTS RECEIVING STEREOTACTIC RADIOSURGERY FOR LIMITED BRAIN METASTASES
Authors: Zhang Michael , Lazar Ann , Chan Jason , Xu Chelsea , Anderson August , Villanueva-Meyer Javier , McDermott Mike , Melisko Michelle , Sneed Patricia , Morin Olivier , Braunstein Steve
Published: November 2019

Leptomeningeal metastasis (LM) is a late stage manifestation of advanced breast cancer frequently managed with whole brain radiotherapy (WBRT) and/or intrathecal chemotherapy. A subset of breast cancer patients who undergo stereotactic radiosurgery (SRS) for limited brain metastases (BM) ultimately develop LM. We hypothesized that this subset of high-risk patients may be identified by patient, disease, and/or treatment parameters. Clinical records from 133 breast cancer patients from a single institution who underwent SRS for BM between February 2010 and March 2018 were retrospectively analyzed. Variables including histopathology, BM features, systemic disease burden, and prior treatments were analyzed. Cumulative incidence rates were estimated with death as a competing risk. Dichotomous variable cutoffs were based on the 75th percentile value. In our cohort, 27 (20.3%) patients ultimately developed LM. With a median follow up of 21.2 months after diagnosis of BM, the actuarial rate of LM at 24 months was 15.2% (95% CI, 8.7%-21.7%). Median OS after diagnosis of LM was 7.0 (95% CI, 3.1–15.4) months. There was significantly increased risk of LM with ≥9 vs < 9 BM at BM diagnosis (28.1% vs 10.8% [24-month actuarial risk], subdistribution HR 2.4, p=0.027), and ≥11 vs < 11 cumulative number of BM treated (25.7% vs 11.7% [24-month actuarial risk], subdistribution HR 2.7, p=0.01). Variables not significantly associated with the risk of LM included tumor receptor status (ER, PR, HER2, triple negative), graded prognostic assessment, KPS, extracranial metastases, total BM volume, prior WBRT, or prior surgical resection. Time intervals between SRS treatments immediately preceding LM diagnosis was not significantly different from other time intervals. In conclusion, patients with a larger number of brain metastases at BM diagnosis (≥9) or cumulatively treated (≥11) appear to be at higher risk of developing LM and may benefit from stronger consideration of WBRT, intrathecal chemotherapy, and/or brain-penetrating systemic therapy.
CMET-37. CLINICAL UTILITY OF ENCYCLOPEDIC TUMOR ANALYSIS TO TREAT PATIENTS WITH BRAIN METASTASIS IN REFRACTORY CANCERS
Authors: Crook Timothy , Patil Darshana , Akolkar Dadasaheb , Ranade Anantbhushan , Bhatt Amit , Datta Vineet , Bhatia Jatinder , Schuster Stefan , Patil Revati , Srinivasan Ajay , Kathuria Harjeet , Datar Rajan
Published: November 2019

Brain metastasis in solid organ cancers is associated with adverse prognosis, which is further aggravated by limited systemic treatment options. Such patients are also often excluded from clinical trials since their poor prognosis is perceived to unfavorably impact trial outcomes and misrepresent efficacy data. We retrospectively evaluated the efficacy of treatment guided by Encyclopedic Tumor Analysis (ETA) in patients with advanced refractory malignancies and brain metastases to determine the impact on outcomes. Freshly biopsied tumor tissue (primary / lymph node / liver) and peripheral blood of patients were used for integrational multi-analyte investigations as part of ETA, which included gene mutations, gene expression, and in vitro chemosensitivity profiling of viable tumor cells. Based on ETA, patients received individualized therapy recommendations. All patients underwent a PET-CT scan as well as MRI scan prior to treatment start to determine extent of disease. All patients underwent follow-up PET-CT scans and brain MRI scans every 6–8 weeks. Of the ten patients with brain metastases, which were evaluated after receiving ETA-guided treatment, the median follow-up duration was 97 days (range 79 – 180 days) during which all ten patients remained progression-free. Median time to progression for these patients on the last (failed) line of treatment was 91 days (range 30 - 176 days). Five patients showed partial response and five patients showed stable disease while on ETA-guided treatment. During the follow-up period, all brain metastases were either stable (n=7) or had regressed (n=3), and none of the patients reported new brain lesions. Personalized ETA guided treatments imparted clinical benefit by halting disease progression in this cohort of high-risk patients who would have otherwise been considered for palliative regimens due to perceived unfavorable prognosis.
COMP-08. COMPREHENSIVE RNA ANALYSIS OF CEREBROSPINAL FLUID FROM LEPTOMENINGEAL METASTASES
Authors: Polyak Dina , Li Yingmei , Liu Boxiang , Connolly Ian , Khoeur Lina , Wasunga Kakusa Bina , Johnson Eli , Andersen Stephanie , Pan Wenying , Nagpal Seema , Montgomery Stephen B , Hayden Gephart Melanie
Published: November 2019

Leptomeningeal metastases (LM), a diffuse form of brain metastases is rare and fatal progression of non-small cell lung cancer (NSCLC). In LM, metastatic cancer cells spread and resign on the brain meninges, the cerebrospinal fluid (CSF), cranial and spinal nerves. Rapid disease progression and scarce tissue availability hinder the progress of scientific study of LM and its treatment. To overcome the critical lack of tissue and to determine the genetic profile of NSCLC LM, we have developed methods to extract tumor-associated cell-free RNA from CSF, and isolated and sequenced circulating single cells from CSF. Herein, we used high throughput qPCR to target lung and brain-associated genes and identified NSCLC LM metastases-related RNA. Brain-specific gene signature (GFAP, NRGN, SNCB, ZBTB18) was detected in all CSF sample (control and metastases), whereas lung-specific genes (MUC1, SFTPB, SFTPD, SLC34A2) were detected in CSF of brain metastases patients. Normal, healthy CSF lacks cellular component, but CSF of patients with LM metastases inhabited with very low amount of circulating tumor cells. Single cells from CSF of 4 patients with NSCLC LM metastases were captured with microfluidic chip. Cells (n = 197) were clustered by significantly differential expressed genes demonstrating two distinct populations of white blood and tumor cells. These data identified specific cfRNA and single cell transcriptome profiles compared to normal cells or patients without NSCLC LM metastases, and highlighted metastases-associated carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) as highly expressed in patients with NSCLC LM metastases. CEACAM6 mRNA was detected in CSF of 86% of patients with NSCLC LM but not in the CSF of control patients. In vitro inhibition of CEACAM6 protein lead to decreased invasion in NSCLC cells which was rescued by overexpression of the protein. We have developed sensitive and robust techniques to leverage human CSF to study NSCLC LM.
DDIS-03. DEVELOPMENT OF A NOVEL CLASS OF cGAS AGONISTS TO TRIGGER STING PATHWAY-DEPENDENT INNATE IMMUNE RESPONSES AGAINST GLIOBLASTOMA
Authors: Stegh Alexander
Published: November 2019

The Stimulator of Interferon Genes (STING) pathway represents a major innate immune sensing mechanism for tumor-derived DNA. Modified cyclic dinucleotides (CDNs) that mimic the endogenous STING ligand cGAMP are currently being explored in patients with solid tumors that amenable to intratumoral delivery. Inadequate bioavailability and insufficient lipophilicity are limiting factors for clinical CDN development, in particular when consideration is given to systemic administration approaches. We have shown that the formulation of oligonucleotides into Spherical Nucleic Acid (SNA) nanostructures, i.e., the presentation of oligonucleotides at high density on the surface of nanoparticle cores, lead to biochemical and biological properties that are radically different from those of linear oligonucleotides. First-generation SNAs conjugated with TLR9-agonsitic DNA oligonculeotides (NCT03086278; solid cancers) and intravenously administered, brain-penetrant siRNA-based SNAs (NCT03020017, recurrent GBM) have recently entered clinical trials. Here, we report the development of a STING-agonistic immunotherapy by targeting cGAS, the sensor of cytosolic dsDNA upstream of STING, with SNAs presenting dsDNA at high surface density. The strategy of using SNAs exploits the ability of cGAS to raise STING responses by delivering dsDNA and inducing the catalytic production of endogenous CDNs. SNA nanostructures carrying a 45bp IFN-simulating dsDNA oligonucleotide, the most commonly used and widely characterized cGAS activator, potently activated the cGAS-STING pathway, as evidenced by increased IRF responses, elevated protein marker expression indicative of the activated M1 macrophage state, and enhanced expression of pro-inflammatory cytokines in macrophage cultures in vitro, and in intracranial isogenic GBM explants in vivo. Our use of SNAs addresses the challenges of nucleic acid delivery to intracranial tumor sites, exploits the binding of closely-spaced, neighboring dsDNA molecules on the SNA surface to enhance the formation of a dimeric cGAS:DNA complex, and establishes cGAS-agonistic SNAs as a novel class of immune-stimulatory modalities for triggering innate immune responses against tumor.
DDIS-17. DEVELOPMENT OF BRAIN-PENETRANT EGFR INHIBITORS FOR CNS MALIGNANCIES
Authors: Tsang Jonathan , Urner Lorenz , Tse Christopher , Baufeld Lynn , Faull Kym , Clark Peter , Cloughesy Timothy , Jung Michael , Nathanson David
Published: November 2019

The epidermal growth factor receptor (EGFR) is altered in nearly 60% of glioblastoma (GBM) tumors, however, EGFR tyrosine kinase inhibitors (TKIs) have failed to improve outcomes for patients with GBM. This can be attributed to the inability of clinically available EGFR TKIs (e.g., erlotinib, gefitinib, lapatinib, afatinib, cetuximab) to effectively cross the blood-brain-barrier (BBB) and reach adequate pharmacological levels for a tumor response. Herein, we performed a structure-activity relationship (SAR) to obtain EGFR TKIs with both high brain penetrance and potency against EGFR-activated GBM cells. From over 80 novel compounds synthesized, our lead EGFR TKI—JCN068—exhibited exceptional BBB penetration (350% brain to plasma) while also having optimal ADME properties (60% oral bioavailability, ~5 hr half-life, >100 µg/mL solubility, etc). Moreover, JCN068 demonstrated picomolar potency against purified EGFR kinase, 1000-fold selectivity for EGFR relative to other kinases, and nanomolar activity against EGFR-altered, GBM patient-derived cells in culture. Importantly, JCN068 demonstrated superior efficacy—with negligible toxicity—compared to clinically available small molecule EGFR TKIs (erlotinib and lapatinib) against multiple EGFR-altered patient-derived orthotopic GBM xenografts. Due to these excellent drug-like properties, JCN068 is currently progressing towards clinical development for EGFR-activated GBM patients.
DDIS-19. USING MATCHED PRIMARY AND RECURRENT PEDIATRIC BRAIN TUMOR ORTHOTOPIC MOUSE MODELS(PDOX) TO ILLUSTRATE THE GENETIC REVOLUTION AND TO INFORM NEW ANTI-RECURRENT PERSONALIZED THERAPIES
Authors: Du Yuchen , Kogiso Mari , Braun Frank , Qi Lin , zhang Huiyuan , Lindsay Holly B , zhao Sibo , Kool Marcel , Stephan Clifford , Li Xiaonan
Published: November 2019

BACKGROUND AND PURPOSE

Despite standard treatment largely benefits patients’ survival, tumors may recur as aggressive, therapy resistant relapses. It is imperative to obtain a better understanding of molecular changes that occur after primary tumor resection and treatment. We established a serial of PDOX models using patient tumor tissues procured at different time of patient clinical history.

METHODS

Dissociated tumor cells were cultured in vitro and transplanted into mouse brains. DNA and RNA were then extracted from tumor tissues or cells from patient and established xenografts. Whole genome methylation and transcriptomic profiling as well as histological phenotypes were examined. Standard therapies were conducted in xenografts.

RESULTS

Currently, three sets of matched primary and recurrent tumor xenografts (19850PNET, 100815GGG/GBM and 5958MB) were established. Tumor-take rate increased from 50–70% of primary tumors to 100% of recurrent tumors with decreased tumor onset time (6 months to 3 months), which demonstrated progressive phenotypes. RNA sequencing reveals preserved and newly changed genetic changes in recurrent tumors. High-throughput drug screening discovered a shared and a diverse panel of therapeutic targets that could be selected further for in vivo drug treatment especially for recurrent tumors. Tumor xenografts responded to standard therapy either same or different from clinic outcome, which might indicate the intrinsic or inducible evolutional molecular changes during cancer progression and treatment at different stages.

CONCLUSION

Three sets of matched primary and recurrent pediatric brain tumors, PNET/PNET, GGG/GBM and MB/MB, were successfully established in pediatric orthotopic mice tumor models (PDOXs) providing clinically-relevant and biologically-accurate animal model systems for development of novel therapeutic targets especially for recurrent tumors. Responses of primary and recurrent tumor xenografts to standard chemo-therapy were different possibly due to evolutionarily molecular changes in recurrent tumors during progression or induced by therapies.
DDIS-22. DRUG SCREEN IN PATIENT-DERIVED IDHMUT GLIOMA STEM CELLS IDENTIFIES SEVERAL FDA-APPROVED ANTINEOPLASTIC AGENTS
Authors: Dao Trong Philip , Pusch Stefan , Unterberg Andreas , Herold-Mende Christel , Warta Rolf
Published: November 2019

OBJECTIVE

The discovery of the Isocitrate Dehydrogenase (IDH) mutation in glioma has led to a paradigm shift on how we see glioma biology. While it is clear, that IDH mutated (IDHmut) and wildtype (IDHwt) tumors have to be viewed as separate entities, the underlying biological differences are still matter of extensive research. Difficulties in cultivating IDHmut glioma stem cells (GSC) have led to a paucity of preclinical models in IDHmut glioma making the discovery of new effective chemotherapeutic agents problematic. We therefore sought to perform a repurposing drug screen in five patient-derived IDHmut GSC lines to discover potential effective antineoplastic agents, already approved by the FDA.

METHODS

Patient tumor tissue was obtained in our neurosurgical department to isolate and establish IDHmut GSC lines. (D)-2-hydroxyglutarate (2HG) levels were measured in the cell culture supernatant of IDHmut GSCs using an enzymatic diaphorase/resazurin system. The drug library provided by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI) consisting of 146 FDA-approved drugs was used for the screen. Cell viability was assessed with the CellTiterGlo assay (Promega).

RESULTS

Despite several passages, the IDHmut GSCs showed stable production of 2HG and were therefore suitable for the drug screen. Cells were cultured as neurospheres and subjected to the test compounds for 72h in concentrations ranging from 0.1nM – 1µM. We identified several compounds in two IDHmut GSC lines (NCH551b, NCH1681) that had a half maximal inhibitory concentration (IC50) below 1µM and could confirm its cytotoxic potential in additional three IDHmut GSC lines (NCH612, NCH620, NCH3763).

CONCLUSION

In this study, we present a feasible preclinical model for a high-throughput drug screen in patient-derived IDHmut GSCs and identified several FDA-approved antineoplastic agents which warrant further investigations.
DDIS-23. CMV-BASED PLASMID DNA VACCINE FOR GBM USING THE UNITE PLATFORM
Authors: Adhikari Amit , Toll Barb , Johnson Yoshimi , Marketon Anthony , Wilson Greg , Macauley Juliete , Heiland Teri
Published: November 2019

Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months, remaining unchanged in spite of technological advances in the standard of care. The presence of the partial CMV genome, specifically in GBM cells, provides a great opportunity for a targeted therapy. We have utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to build a polyvalent DNA vaccine which includes HCMV proteins, pp65, gB and IE-1. The UNITE platform is based, in part, on a lysosomal targeting technology which can result in increased antigen presentation, a balanced T cell response, and subsequent immunologic benefit. Using an orthotopic GBM mouse model expressing CMV proteins in the CT2A cell line, we have shown up to 45% survival when treated therapeutically with the pp65-IE-1 vaccine. In order to further improve the vaccine’s efficacy, and also to understand the mechanism of action, we are evaluating the post-vaccination tumor microenvironment and testing combination therapies with immune checkpoint inhibitors. Preliminary data on treated tumors suggest an increase in the PD-1 immune checkpoint regulator and a higher number of regulatory T cells. Our immune response evaluation of the polyvalent vaccine in naïve mice showed generation of robust antigen specific T cell activation. The use of multiple antigens in this vaccine makes it better suited to prevent antigen escape by tumor cells. Encouraged by our non-clinical data and the promising outcomes from our collaborators’ clinical trials using pp65 mRNA transfected autologous dendritic cells (ATTAC and ATTAC-II), we are moving forward with a phase I trial. We believe that the immune boost from our UNITE platform combined with deactivation of the immunosuppressive microenvironment by checkpoint inhibition holds a promising treatment against GBM.