bluuebunny/crossref_metadata_2025 huggingface dataset:
CTNI-67. DUAL INHIBITION OF POST-RADIOGENIC ANGIO-VASCULOGENESIS BY OLAPTESED PEGOL (NOX-A12) AND BEVACIZUMAB IN GLIOBLASTOMA – INTERIM DATA FROM THE FIRST EXPANSION ARM OF THE GERMAN PHASE 1/2 GLORIA TRIAL
Authors: Giordano Frank , Layer Julian , Leonardelli Sonia , Friker Lea , Schaub Christina , Turiello Roberta , Sperk Elena , Mildenberger Iris , Grau Franziska , Paech Daniel , Pietsch Torsten , Mueller Wolf , Grauer Oliver , Renovanz Mirjam , Tabatabai Ghazaleh , Kebir Sied , Glas Martin , Bisdas Sotirios , Hambsch Peter , Seidel Clemens , Hölzel Michael , Herrlinger Ulrich
Published: November 2022

BACKGROUND

We recently reported favorable safety, promising clinical efficacy and immunohistochemical indicators of response after radiotherapy (RT) plus escalating doses of the CXCL12-neutralizing RNA-Spiegelmer olaptesed pegol (NOX-A12) for glioblastoma in the German multicenter phase 1/2 GLORIA trial (NCT04121455). Here, we report outcomes after RT plus dual inhibition of vasculogenesis (NOX-A12) and angiogenesis (bevacizumab).

METHODS

After establishing safety in the monotherapy arm, we enrolled six patients with incompletely resected GBM, ECOG ≤ 2, age ≥ 18 and without MGMT promoter hypermethylation into a pre-planned expansion arm. Patients received standard RT (60 Gy in 30 fractions), continuous i.v. infusions of NOX-A12 (600 mg/week) and i.v. infusions of bevacizumab (10 mg/kg q2w). The primary endpoint was safety. Secondary endpoints included radiographic response, perfusion/diffusion imaging and neurologic performance.

RESULTS

Dual treatment was well-tolerated and safe. Of all G ≥ 2 AEs (n = 37), two G2 events (5.4%) were deemed related to NOX-A12. There were no dose-limiting toxicities and no treatment-related deaths. Longitudinal NANO assessment revealed stable neurologic functioning in all patients. Five out of six patients achieved partial responses (PRs) as per mRANO in week 9. All PRs remained durable at a median follow up of 5.6 months (range 3.6 to 9.3 months). No progression occurred. The mean best response was -65.9% (-13.3% to -99.9%) for target lesion sums and -92.1% (-76.2% to -100%) for non-target lesion (NTL) sums. In all three patients with NTL at least one lesion disappeared. The mean best change from baseline of the highly perfused-tumor fraction was -84.5% (-51.9% to -100%) and the mean best change of the apparent diffusion coefficient was 20.1% (-24.5% to 59.1%).

CONCLUSION

Interim data of the ongoing trial confirm the previously established safety profile of NOX-A12 and suggest improved efficacy of dual inhibition of post-radiogenic angio- and vasculogenesis by the addition of bevacizumab.
DNAR-08. THE ROLE OF SMARCAL1 AS A SYNTHETIC LETHAL VULNERABILITY IN ATRX-DEFICIENT GLIOMAS
Authors: Waitkus Matthew , Liu Heng , Brown Alexandrea , Strickland Laura , Khambati Taher , Fraley Casey , Diplas Bill , Keir Stephen , Ashley David
Published: November 2022

Mechanisms to maintain telomere length over successive cell divisions are a requirement for cancer cell immortalization. Although many cancers maintain telomere length through the activation of telomerase, ~10-15% of human cancers use telomerase-independent mechanisms of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). In gliomas, the ALT phenotype is associated with loss-of-function mutations in the ATRX gene in IDH-mutant astrocytomas, adult and pediatric glioblastomas (GBM), high-grade astrocytomas with piloid features, and pleomorphic xanthoastrocytomas. In adult GBM, we previously identified a relatively rare subset of ALT-positive ATRX wildtype tumors that harbored loss-of-function mutations in the SMARCAL1 gene, and we found that loss of SMARCAL1 activity plays a causative role in the onset of ALT in these tumors. SMARCAL1 is an annealing helicase that localizes to sites of DNA damage and replication stress and catalyzes the reversal of stalled replication forks. To better understand the relationship between ATRX mutations, SMARCAL1 activity, and the ALT phenotype, we investigated the localization and function of SMARCAL1 in ALT-positive glioma cells and xenografts with native ATRX deficiency. We found that SMARCAL1 localizes to ALT-associated PML bodies in astrocytoma cell lines with concurrent ATRX and IDH1 mutations (derived from grade 3 and 4 tumors). Our data show that inducible suppression of SMARCAL1 via doxycycline-induced RNAi leads to increased DNA double-strand breaks, increased abundance of extrachromosomal telomeric repeats (c-circles), and increased sensitivity to irinotecan, a topoisomerase I inhibitor. In an orthotopic mouse model, SMARCAL1 depletion exhibited a chemosensitizing effect in combination with irinotecan against a patient-derived xenograft (astrocytoma, IDH-mutant Grade 4). Based on these data, we hypothesize that SMARCAL1 activity is critical for resolving ALT-associated replication stress in ATRX-deficient malignant gliomas. We therefore propose that SMARCAL1 functions as a synthetic lethal vulnerability in ATRX-deficient ALT-positive gliomas and that SMARCAL1 inhibition is a viable therapeutic strategy in these tumors.
DNAR-13. SONIDEGIB AND GANT61 INDUCE DNA DAMAGE IN PEDIATRIC SONIC HEDGEHOG MEDULLOBLASTOMA
Authors: Price Laura , Lau Bonnie
Published: November 2022

Medulloblastoma is the most common malignant brain tumor in children, accounting for 20-25% of all pediatric brain tumors. The current standard of care includes both craniospinal and focal radiation therapy which leads to long term consequences in the pediatric population such as bone and tissue hypoplasia, decreased neurocognitive functioning, as well as an increased risk for secondary radiation induced cancers. Thus, there is a need for novel approaches to radiation therapy that can effectively target and kill tumor cells while minimizing radiation toxicity in healthy tissue in the pediatric patient population. The dysregulation and constitutive activation of the Sonic Hedgehog (SHH) signaling pathway accounts for ~30% of all medulloblastomas. In addition to playing a role in tumorigenesis, the SHH pathway is known to play a role in the radiation sensitivity and risk of tumor recurrence. We hypothesize that inhibiting the SHH pathway at Smoothened (Smo) or Gli1 will increase DNA damage, resulting in increased cell killing following radiation therapy. We found that treatment of medulloblastoma cells with the drug Sonidegib or GANT61 decreased the expression of the respective targets Smo and Gli1. Both drugs also significantly decreased expression of FANCD2, a DNA damage repair gene essential for homologous recombination. Pretreatment with Sonidegib or GANT61 resulted functionally with decreased tumor cell viability and self-renewal following radiation. These data suggest that Smo and Gli1 inhibition are promising targets for increasing the radiation sensitivity of pediatric SHH medulloblastoma.
DNAR-14. Y-BOX BINDING PROTEIN 1 REGULATES MEDULLOBLASTOMA TUMOR BIOLOGY AND THERAPEUTIC RESPONSE
Authors: McSwain Leon , Chen Victor , Huang Tiffany , Zhou Grace , Kenney Anna
Published: November 2022

BACKGROUND

Medulloblastoma (MB) is the most common pediatric brain tumor. MB can be dividing into 4 genetically distinct subgroups (Sonic Hedgehog (SHH), Wingless (WNT), Group 3, and Group 4). Most patients receive radiation and cisplatin backbone therapy. SHH and Group3 patients demonstrate poor prognosis, especially in cases of MYC amplification or p53 mutation. Given the continued reliance on DNA damage therapies, and our groups previous findings that YB1 regulates IGF2 transcription in SHH MB, we chose to investigate the role of YB1 in the DNA damage response and we performed RIPseq to understand mRNAs regulated by YB1 that may be targeted as novel therapeutics.

METHODS

Cell lines include primary MBCs derived from NeuroD2-SmoA1 SHH mice, ONS-76 human SHH, Daoy human SHH, D341 and D425 Human Group 3. YB1 overexpression or knockdown followed by radiation and assessment of yH2AX (a marker of DNA de-condensation following damage), Comet assay (a marker of physical damage), and proliferation time courses were used to assess effects of YB1 modulation on radiation response. RIPseq and YB1 knockdown were used to assess the effects of YB1 on PLXND1 levels. Scratch assay and western blotting of EMT markers were used to assess effects of PLXND1 silencing on migration.

RESULTS

(Radiation Studies) Overexpression of YB1 in primary MBCs followed by allograft into BL6 mice results in decreased overall survival. YB1 silencing followed by radiation results in faster resolution of yH2AX, faster resolution of damage, and lack of pRPA32 accumulation. YB1 silencing sensitizes cells to radiation resulting in substantial decreases in proliferation. (RNA Binding Protein Studies) YB1 binds and positively regulates PLXND1 translation. Silencing PLXND1 results in decreased migration and EMT mark expression.

CONCLUSIONS

YB1 drives a more error prone non-homologous End-Joining based mechanism of repair and binds and regulates PLXND1 post-transcriptionally. PLXND1 drives a migratory phenotype in SHH MB.
DDEL-04. BIOMATERIAL SCAFFOLDS FOR THE DELIVERY OF NEURAL STEM CELL THERAPIES INTO THE GLIOBLASTOMA RESECTION CAVITY
Authors: Bomba Hunter , Kass Lauren , Carey-Ewend Abigail , Sheets Kevin , Valdivia Alain , Goetz Morgan , Findlay Ingrid , Mercer-Smith Alison , Khagi Simon , Hingtgen Shawn
Published: November 2022

Therapeutic neural stem cells (tNSCs) are a promising new platform for the treatment of glioblastoma (GBM). tNSCs exhibit a characteristic known as tumor tropism, in which they can migrate towards distant GBM foci via cytokine signaling. Complementarily, genetic engineering of NSCs may be performed to turn the cells into drug-producing therapeutics. Together, this results in NSCs that act as targeted drug delivery vehicles that can seek out and kill invasive GBM lesions post-resection. However, one limitation of this cell therapy platform is that tNSCs delivered directly into the GBM resection cavity are rapidly cleared. We hypothesized that the commercially available, FDA-approved hemostat FLOSEAL® may be used as a drug delivery system for improving cell persistence in the brain, thus resulting in improved therapeutic efficacy. It was found that tNSCs encapsulated in FLOSEAL® were detectable in the brain for over 95 days in mice, a drastic improvement compared to directly injected cells and cells encapsulated in other existing hemostat systems, which persisted 2 weeks or less. However, two in vivo efficacy studies of tNSCs encapsulated in FLOSEAL® yielded contrasting results. While the FLOSEAL®-tNSC system was significantly more efficacious against a GBM8 tumor model in mice compared to directly injected tNSCs, it was not significantly more effective against a U87 tumor model. This could be due to a variety of factors, including the tumor type (diffuse vs. solid for GBM8 and U87, respectively) and negative impacts of FLOSEAL® on tNSC markers of proliferation, migration, drug production, and anti-apoptosis. While FLOSEAL® is a promising material for the delivery of tNSCs in the treatment of post-operative GBM, alternative systems that allow for improved persistence while maintaining the therapeutic activity of the cells would be optimal for long-term treatment with tNSCs.
DDEL-05. PHARMACOKINETICS AND CNS DISTRIBUTION OF A NOVEL ATM INHIBITOR, WSD0628, FOR THE TREATMENT OF BRAIN TUMORS
Authors: Rathi Sneha , Oh Ju-Hee , Sarkaria Jann , Elmquist William F
Published: November 2022

The treatment of brain tumors using chemo- and radio-sensitizers may be limited by a lack of effective drug delivery. Radiation and some cytotoxic therapies induce DNA damage, and DNA damage response (DDR) pathways modulate resistance to effective chemo-radiation therapy. Inhibiting DDR pathways by targeting ATM leads to sensitization and enhances cytotoxicity. The purpose of this study is to determine the key pre-clinical pharmacokinetic parameters and mechanisms that influence the CNS delivery of WSD0628, a novel ATM inhibitor. Pharmacokinetics and CNS distribution studies were conducted in FVB wild-type mice following intravenous (IV) and oral (PO) dosing (5 mg/kg). Plasma, brain, and spinal cord samples were harvested after dosing and drug concentrations were measured using LC-MS/MS. The free fraction of WSD0628 was measured in plasma, brain, and spinal cord using rapid equilibrium dialysis. Our results indicate that WSD0628 has a half-life of 4.11 hrs and a high oral bioavailability (0.95). The brain partition coefficient (AUCbrain/AUCplasma, i.e., Kp, brain) is 0.05 and 0.11 after IV and PO dosing, respectively. The free fraction in plasma is 2.01 ± 0.02 % and in brain it is 1.22 ± 0.40 %. The unbound brain partition coefficient (Kpuu brain) is 0.03 and 0.06 after IV and PO dosing, respectively. The differential binding to plasma and brain tissues results in a relatively low Kpuu, a key parameter describing brain penetrability. These data show that WSD0628, a highly potent inhibitor of ATM, reached sufficient concentrations in brain after a single dose of 5 mg/kg to engage ATM over the desired time frame and elicit a response after DNA damage by chemo-radiotherapy. WSD0628 shows promising potential and further evaluations are underway to determine if efflux transporters at the BBB limit its brain distribution.
DDEL-11. DETERMINING THE DOSE OF REGADENOSON MOST LIKELY TO TRANSIENTLY ALTER THE INTEGRITY OF THE BLOOD-BRAIN BARRIER IN PATIENTS WITH GLIOMAS
Authors: Romo Carlos , Ellingson Benjamin , Strowd Roy , Lesser Glenn , Raymond Catalina , Kral Brian , Ye Xiaobu , Desideri Serena , Fisher Joy , Grossman Stuart
Published: November 2022

BACKGROUND

The blood brain barrier (BBB) is a major obstacle to the delivery of chemotherapy to the CNS. Regadenoson is an FDA approved adenosine A2 agonist used for cardiac stress tests. In murine models, it transiently increases BBB permeability to a 70KD dextran. This multi-institutional, NIH funded, Adult Brain Tumor Consortium trial was designed to discover a dose of regadenoson that substantially increases vascular permeability in normal appearing white matter (NAWM) where drug delivery is particularly challenging.

METHODS

Adults ages 18-45 with supratentorial gliomas at low-risk for regadenoson complications were recruited (n = 7). One patient was treated at each of seven dose levels (from 0.05 to 1.4 mg) that are known to be safe in humans. The primary outcome measure is change in vascular permeability via dynamic contrast enhanced (DCE) perfusion MRI estimates of Ktrans. The primary outcome measure was a 10-fold higher Ktrans in NAWM than reported in literature (Ktrans > 0.04 min-1). Contrast-enhanced T1 subtraction map estimates of change in contrast enhancement and other measurements in normal brain and non-enhancing tumor were quantified.

RESULTS

Ktrans measures in NAWM averaged 1.13x10-3 ± 0.44x10-3 (SEM) min-1, lower than the target of 0.04 min-1. Normalized, contrast enhanced T1-weighted MR signal intensity in NAWM increased an average of 74.0 ± 22.4% min-1 (SEM) min-1, which was significantly higher than zero (P = 0.0163). Data available from this limited sample failed to meet the target goal in Ktrans increase or change in contrast enhancing signal intensity.

CONCLUSION

Administration of regadenoson at seven different doses did not significantly elevate Ktrans for gadolinium in NAWM. This data suggests that single doses of regadenoson are unlikely to substantially increase the delivery of therapeutic agents in non-enhancing brain tissue. This trial design is appropriate for further human testing of other regadenoson schedules and other novel approaches aimed at transiently modifying BBB permeability.
DDEL-13. ULTRASOUND-ENHANCED DELIVERY OF LIPOSOMAL DOXORUBICIN ACROSS THE BLOOD BRAIN BARRIER INDUCES AN IFN-GPHENOTYPE IN MICROGLIA, MACROPHAGES, AND T CELLS AND IMPROVES RESPONSE TO PD-1 BLOCKADE IN GLIOMAS
Authors: Arrieta Victor , Gould Andrew , Kim Kwang Soo , Dmello Crismita , Zhang Daniel , Castro Brandyn , Chen Li , Pandey Surya , Kai Li , Duffy Joseph , McCord Matthew , Ward Rachel , Muzzio Miguel , Canney Michael , Balyasnikova Irina , Zhang Bin , Horbinski Craig , Miska Jason , Stupp Roger , Lee-Chang Catalina , Sonabend Adam M
Published: November 2022

INTRODUCTION

Given the limited drug penetration across the blood-brain barrier (BBB), the therapeutic potential of new and existing therapies has not been fully exploited for the benefit of glioblastoma (GBM) patients.

METHODS

Here we employed a novel drug delivery technology based on low-intensity pulsed ultrasound combined with intravenous microbubbles (LIPU/MB) that temporarily opens the BBB to deliver liposomal doxorubicin (DOX) and anti-PD-1 therapy (aPD-1) in mouse glioma models and 3 recurrent GBM patients. Immunological variables were evaluated in tumor and immune cells as well as efficacy in glioma-bearing mice treated with DOX delivered by LIPU. These included measurement of HLA ABC and HLA DR protein expression by tumor cells, microglia, and macrophages and IFN-g production by glioma-associated microglia and macrophages in mouse and human tumors. We also assessed efficacy of LIPU/MB enhanced combination therapy in glioma-bearing mice.

RESULTS

Upregulation of HLA ABC and HLA DR was observed in GBM cell lines at low concentrations of DOX. Tumor cells from GBM patients treated with DOX, aPD-1 and LIPU/MB showed increased expression of HLA ABC and HLA DR compared to paired pretreatment samples. In both mice and humans, LIPU/MB liposomal DOX increased absolute brain drug concentrations and elicited a specific IFN-g phenotype and MHC I expression in glioma-associated microglia and macrophages in mice and humans. Furthermore, LIPU/MB-mediated BBB opening increased brain concentrations of aPD-1 in mice and in peritumoral regions of GBM patients. Combined treatment with liposomal DOX and aPD-1 delivered with LIPU/MB resulted in long-term survival of glioma-bearing mice that relied on the activity of CD8+ T cells for its efficacy.

CONCLUSIONS

Overall, this translational study demonstrates the utility of LIPU/MB to stimulate intracranial immune responses in the context of treatment with DOX and aPD-1 for gliomas.
DDEL-16. DELIVERY OF OT-101 - TGF-Β ANTISENSE- FOR THE TREATMENT OF GLIOBLASTOMA
Authors: Trieu Vuong
Published: November 2022

OT-101 - a TGF-beta antisense- is active against recurrent glioblastoma in G004- a phase 2 clinical trial [Uckun FM, Qazi S, Hwang L, Trieu VN. Recurrent or refractory high grade gliomas treated by convection enhanced delivery of a TGF-beta2 targeting RNA therapeutic: a post-hoc analysis with long-term follow up. Cancers. 2019, 11:1892]. OT-101 was delivered intratumorally by Convection Enhanced Delivery (CED). To further expand the application of OT-101, we explored the intrathecal delivery of tritiated OT-101 into Sprague-Dawley CD (albino) rats. Throughout the studies, there were no sex differences. After 1 hr intracerebral infusion in rats, OT-101 was limited to the infusion site. Whereas for the 1 hr itravesicular infusion, OT-101 was more widespread with 35X, 19X, 12X higher concentration found in cerebellum, remaining cerebrum, cerebrospinal fluid (CSF), respectively. OT-101 concentration was stable for the first 4 hours post infusion and decayed biexponentially with a slow terminal half life for tissue but not for CSF, suggestive of rapid penetration of the underlying tissue away from the CSF compartment. Minimum amount of OT-101 was detected in the plasma compartment. Intrathecal bolus administration of 0.1mL of OT-101 at 14, 30, 200, 300, and 500 uM into cynomolgus monkies did not result in any single dose toxicity. Histopathology examination revealed no substance-related histomorphological lesions in the cavum subarachnoideale of the lumbar region. No changes were noted in the grey and white matter of the spinal cord, the nerve trunk, and nerve cells did not show any abnormalities. These data suggest that intrathecal administration of OT-101 is a potentially effective delivery route for antisense therapeutics such as OT-101 to the midline ie. for Diffuse Midline Glioma (DMG). A phase 1b/2 clinical trial evaluating OT-101 against DMG is proposed and the trial design will be presented
DDEL-17. POOLED CANCER CELL SCREENING IDENTIFIES GENOMIC DETERMINANTS OF NANOPARTICLE DELIVERY TO CANCER CELLS: SUB ANALYSIS OF CENTRAL NERVOUS SYSTEM TUMOR CELL LINES
Authors: Straehla Joelle , Boehnke Natalie , Safford Hannah , Kocak Mustafa , Rees Matthew , Ronan Melissa , Rosenberg Danny , Cheah Jaime , Roth Jennifer , Koehler Angela , Hammond Paula
Published: November 2022

Background

There is great interest in utilizing nanoparticles to improve drug delivery to central nervous system (CNS) tumors, but the biologic features underlying successful delivery at the tumor site are not known.

METHODS

We developed a pooled screening assay to investigate cellular features predictive of nanoparticle delivery and screened 35 fluorescent nanoparticle formulations against 488 pooled cancer cell lines with DNA barcodes. The nanoparticle library was comprised of nonlethal liposomal or polymeric cores with varied surface functionality, including natural and synthetic polymers. Cells were profiled using fluorescence-activated cell sorting and barcodes sequenced and deconvolved to generate an association score for each nanoparticle-cell line pair.

RESULTS

Of 488 cancer cell lines, 22 cancer cell lineages (tissues of origin) were included. There were 35 cell lines derived from CNS tumors; of these 22 were classified as astrocytoma or glioblastoma, one as oligodendroglioma, and 2 as medulloblastoma. Of CNS tumor cell lines, 40% were from female patients and average age at cell line derivation was 52 years; TP53 hotspot mutations were detected in 25/35 lines, PTEN hotspot mutations in 15/25 lines. Using unsupervised hierarchical clustering by nanoparticle association profiles, CNS tumor lines did not cluster together by lineage, subtype, or mutation status but rather by baseline expression of genes involved in nanocarrier trafficking. Nanoparticle association profiles were heterogeneous among the CNS tumor lines for most formulations, though CNS tumor lines had high association with polystyrene formulations with respect to other lineages. Select CNS tumor lines had high uptake of liposomes coated with the natural polymers fucoidan, alginate, hyaluronic acid and chondroitin sulfate; these trends were further probed in a non-pooled screen of 5 additional cell lines.

Conclusions

Uptake of liposomal and polymeric nanoparticles is heterogeneous across CNS tumor lines, but can be predicted using baseline gene expression.
DDEL-18. PEG-AU-BP NANOCARRIERS FOR DBTRG HUMAN GLIOMA CELLS
Authors: Liu Szu-Yuan , Liu Szu-Yuan
Published: November 2022

n-butylidenephthalide (BP) has been verified to have the superior ability of cancer cell toxicity. Furthermore, gold nanoparticles (Au) are biocompatible materials as well as the effective carrier to deliver bio-active molecular for cancer therapeutics. In present research, Au nanoparticles were firstly conjugated with polyethylene glycol (PEG), and cross-linked with BP to obtain PEG-Au-BP nanocarriers. The physicochemical properties were characterized through Ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR) and Dynamic light scattering (DLS) to confirm the combination of PEG,Au,and BP. Through iv vitro, normal BAEC cells and DBTRG human glioma cells were treated with PEG-Au-BP nanocarriers to investigate the cytotoxicity, cell uptake efficiency, and mechanism of endocytic routes. According to the results of MTT assay, PEG-Au-BP could significantly inhibit DBTRG cells proliferation. Cell uptake efficiency and potential cellular transportation in both BAEC and DBTRG cell lines were observed to be significantly higher at the time point of 2hr and 24 hr. Moreover, the mechanisms of endocytosis, clathrin-mediated endocytosis and cell autophagy, were explored to routes for BAEC and DBTRG cells to absorb PEG-Au-BP nanocarriers. Next, the cell progression and apoptosis of DBTRG cells after PEG-Au-BP treatment was investigated by flow cytometry. The results figured out that PEG-Au-BP could remarkably regulate DBTRG cell cycle at Sub-G1 phase as well as induced more apoptotic cells. The expression of apoptotic-related proteins in DBTRG cells was determined. After the treatment of PEG-Au-BP, the apoptotic cascade proteins, p21, BAX, Act-caspase-3 were significantly expressed in DBTRG cells. Through in vivo, the tissue morphology and particle distribution in mice were examined by retro-orbital sinus injection with PEG-Au-BP nanocarriers. The results demonstrated the tissue integrity in brain (forebrain, midbrain, cerebellum), and other organs did not show significant destruction. The above finding elucidated PEG-Au-BP to be a potential nanodrug for brain tumor therapies.
DDDR-01. AZD9574 IS A NOVEL, BRAIN PENETRANT PARP-1 SELECTIVE INHIBITOR WITH ACTIVITY IN AN INTRACRANIAL XENOGRAFT MODEL OF TRIPLE NEGATIVE BREAST CARCINOMA WITH HOMOLOGOUS RECOMBINATION REPAIR DEFICIENCY
Authors: Jamal Kunzah , Staniszewska Anna , Gordon Jacob , Pilger Domenic , Illuzzi Giuditta , Wilson Joanne , Smith Aaron , Gosselin Eric , McWilliams Lisa , Wen Shenghua , McGrath Frank , Dowdell Gregory , Kabbabe Dominic , Griffin Matthew , Davies Barry , Hamerlik Petra , Schou Magnus , Pike Andy , Johannes Jeffrey
Published: November 2022

The Poly (ADP-ribose) polymerase (PARP) family has numerous essential functions in cellular processes such as transcription, chromatin remodelling, DNA damage response and repair as well as apoptosis. PARP inhibition blocks base excision repair and results in conversion of SSBs to DNA double-strand break (DSBs), the most deleterious form of DNA damage. DSBs can be repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). The physiological importance of HRR is underscored by the observation of genomic instability in HRR-deficient (HRD+) cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HRR genes. PARP1 and PARP2 are required for SSB repair, while PARP1 is also involved in the repair of DNA double-strand breaks (DSBs) and replication fork damage. AZD9574 is a novel brain penetrant PARP1 inhibitor that acts by selectively inhibiting and trapping PARP1 at the sites of SSBs. While AZD9574 inhibited PARP1 enzymatic activity in all tested cell lines irrespective of the HRR status (IC50 range between 0.3 – 2 nM), colony formation assay in isogenic cell lines pairs confirmed higher potency and selectivity towards HRD+ models. In vivo, AZD9574 demonstrated dose-dependent efficacy in a BRCA1 mutant MDA-MB-436 subcutaneous xenograft model. Anti-tumour effects of AZD9574 were manifested by significant growth regressions that were durable after treatment withdrawal. An intracranial xenograft model of breast cancer brain metastases was developed to assess the efficacy of AZD9574 in the context of blood-brain barrier penetrance. Treatment of animals with established intracranial lesions showed sustained tumour growth suppression resulting in a significantly extended survival of tumour-bearing mice. Collectively, we believe that our data support the development of AZD9574 as a potential therapy for patients with HRD+ breast cancer whose disease has spread to the brain.This abstract was previously presented at AACR 2022 (Hamerlik et al, AACR 2022, Abs #3880)
DDDR-05. ERK INHIBITION INCREASES SENSITIVITY TO ANTI-PD-1 THERAPY IN A MOUSE MODEL OF BRAF MUTANT MELANOMA BRAIN METASTASIS
Authors: de Sauvage Magali , Nayyar Naema , Torrini Consuelo , Nieblas-Bedolla Edwin , Brastianos Priscilla
Published: November 2022

BACKGROUND

Immune checkpoint inhibitors (ICI) have revolutionized the landscape of cancer treatment, including brain metastases. However, only a subset of patients with brain metastases respond to ICI. Activating mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, a key regulator of cell processes, are frequently found in brain metastases. LY3214996 (LY321) is a selective ERK 1/2 inhibitor that inhibits MAPK signaling and leads to regression of extracranial disease in preclinical models. Our objective was to evaluate whether with ERK inhibition (ERKi) can improve the efficacy of ICI in preclinical models of brain metastases.

METHODS

We tested sensitivity to LY321 in a BRAFV600E mutant, ICI resistant murine melanoma cell line (D4M) both in vitro and in vivo. To model brain metastases in vivo, we implanted D4M cells subcutaneously with subsequent intracranial implantation three days later. The treatment arms included LY321 and anti-PD-1 combination therapy, single agent therapies, and respective controls. We tested the efficacies of 2-week versus 4-week LY321 treatments, as well as 3-week treatment in an independent experiment.

RESULTS

D4M demonstrates in vitro sensitivity to LY321 (IC50 value of 4.75 µM). Our model demonstrated that LY321 in combination with anti-PD-1 improves both intracranial and extracranial disease control. Increased survival with combination therapy was observed with 3-week and 4-week LY321 administration. While individually, LY321 and anti-PD-1 produced better survival outcomes than control groups, combination therapy significantly outperformed both monotherapies.

CONCLUSION

Previous studies demonstrated that LY321 has minimal intracranial activity. However, we found that ERKi reduces intracranial tumor burden, with improved efficacy in combination with ICI in immunocompetent mice. Together, these data suggest that ERKi increases sensitivity to ICI therapy in pre-clinical brain metastasis models. Further investigation is needed to elucidate the mechanisms of this therapeutic approach, and to ultimately determine whether this combination will improve patient outcomes.
DDDR-07. EVALUATING THE ROLE OF THE PIK3CB PATHWAY IN TEMOZOLOMIDE RESISTANCE IN GLIOBLASTOMA USING THE GENOMICS OF DRUG SENSITIVITY IN CANCER (GDSC) AND CANCER CELL LINE ENCYCLOPEDIA (CCLE) DATABASES
Authors: Huang Leah , Sheng Zhi , Tegge Allison
Published: November 2022

Glioblastoma multiforme (GBM) is a lethal primary brain cancer with a median survival of 14.6 months despite standard treatment of maximal surgical resection, radiotherapy, and temozolomide (TMZ). Recent research has identified PIK3CB/p110β as the most important regulator of AKT activation and GBM cell survival. Utilizing the pharmacogenomic databases Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE), we sought to elucidate the relationship between PIK3CB mRNA expression or AKT phosphorylation and GBM treatment resistance. We first identified cell lines with matched PI3K isoform mRNA expression, protein expression (AKT phosphorylation at S473 and T308; reverse phase protein array) and drug sensitivity (defined by the area under the curve; AUC) from the GDSC1 and CCLE databases. Associations between PI3K isoforms, AKT phosphorylation, and drug sensitivities were reported by fitting univariable and multivariable linear regression models with and without interaction terms to communicate the effects of AKT phosphorylation conditional on PIK3CB levels. We found that AKT phosphorylation and PIK3CB mRNA data together performed better as a biomarker in predicting therapeutic response to p110β-selective inhibitors than each variable alone, specifically we found that AKT phosphorylation at T308 was negatively related to p110β-selective inhibitor AZD6482 sensitivity at low PIK3CB expression levels (B = -0.069, p = 0.0513) and this relationship disappeared at higher expression levels (B = 0.04, p = 0.176) with an interaction at near statistical significance (p = 0.0674). We found that PIK3CB mRNA levels were significantly positively associated with TMZ resistance in GBM (B=0.019, p=0.0211) while other PI3K isoforms were not. While AKT phosphorylation is currently used as a biomarker for PI3K pathway inhibitors, in this study, we demonstrate that both PIK3CB mRNA and AKT phosphorylation together was a significant biomarker in sensitivity to p110β inhibitors as compared to assessing each independently. Furthermore, PIK3CB was implicated in TMZ resistance in GBM as compared to other catalytic isoforms.
DDDR-08. ARF4-MEDIATED RETROGRADE TRAFFICKING AS A MECHANISM OF CHEMORESISTANCE IN GLIOBLASTOMA
Authors: Budhiraja Shreya , Baisiwala Shivani , Perrault Ella , Cho Sia , Chen Li , Dmello Crismita , Park Cheol , Sonabend Adam M , Ahmed Atique
Published: November 2022

Glioblastoma (GBM) remains a deadly disease with an extremely poor prognosis, owing largely to its high rate of resistance to conventional temozolomide (TMZ) chemotherapy. To identify the unknown drivers of this chemoresistance, we performed a genome-wide CRISPR knockout sensitivity screen. Results showed significant enrichment of ~200 novel genes, including ARF4, a previously unstudied gene involved in retrograde trafficking—a well-regulated process by which cargo is transported internally from endosomes to ultimately the nucleus. Initial investigation showed that ARF4-knockdowns resulted in significantly heightened susceptibility to TMZ in multiple GBM patient-derived xenograft lines and extended survival compared to the controls (p< 0.01) in vivo in both primary and recurrent lines. Live-cell imaging further revealed that ARF4-knockdowns significantly inhibited retrograde trafficking, while ARF4-overexpressions resulted in an untenable increase in retrograde trafficking (p < 0.001) in vitro. Enhanced trafficking was also observed in TMZ-treated cells (p < 0.001), suggesting that ARF4 may drive dysregulation of retrograde trafficking to promote chemoresistance.We then performed an unbiased proteomics screen in control and ARF4-knockdown GBM cells with and without therapy to identify which proteins were being uniquely transported to the nucleus during therapy as a result of ARF4-mediated retrograde trafficking. Results showed the greatest enrichment of EGFR signaling, as validated by an increase in EGFR trafficking and nuclear EGFR expression in ARF4-overexpression and TMZ-treated conditions and a decrease of this phenomenon in ARF4-knockdown conditions.We also show that DNA-PK, a DNA repair protein that is transcriptionally activated by nuclear EGFR, is similarly downregulated in ARF4-knockdown conditions and elevated in ARF4-overexpression and TMZ-treated conditions. Treatment with DNA-PK-inhibitor, KU57788, resulted in extended survival compared to the controls (p < 0.01) in vivo in a recurrent line, indicating potential clinical benefit from targeting this pathway. Overall, we present a novel understanding of how ARF4 acts during therapy to increase nuclear localization of chemoresistance-promoting proteins.
DDDR-15. SPIDER VENOM MOLECULES IMPAIR HUMAN GLIOMA CELLS MIGRATION: POTENTIAL APPLICATION IN CANCER THERAPY
Authors: Barreto Natalia , da Silva Monize Valeria Ramos , da Rocha-E-Silva Thomaz Augusto Alves , Sutti Rafael , Sciani Juliana Mozer , Veiga Jose Carlos Esteves , Verinaud Liana , Araujo Joao Luiz Vitorino , Raposo Catarina
Published: November 2022

Gliomas correspond to approximately 80% of malignant neoplasms of the central nervous system. Due to its aggressiveness, there is a growing need to seek new strategies to control the disease. The components of spider venoms are stable peptides with high specificity against biological molecular targets of therapeutic relevance. It has already been demonstrated by our group that two subfractions (SFs) isolated from the Phoneutria nigriventer spider venom (PnV) (called SF1 and SF11) can delay the migration of glioblastoma (GB) well-established cell lineage in invasions assays. Therefore, the present study aimed to evaluate the responsiveness of samples collected from Brazilian patients with a presumptive diagnosis of glioma (high and low grades), to PnV SFs, in order to contribute to the development of new drugs. After patient consent, 13 samples were collected for cultivation and attempt to reach the tenth passage, to ensure the establishment of the lineage. Four lineages were established: J01 and N07 – glioblastoma (high grade); C03 and L09 – astrocytoma and oligodendroglioma (low grade) respectively. Cells were incubated for 48 hours with SF1 and SF11, during the scratch wound-healing assay. The results demonstrated that the SFs, mainly SF11 (which proved to be a pure peptide with 1603,88 Da, sequenced as PyrKKDKKDKFY–GLM–NH2), harmed the migration of all these samples when compared to the control group. In addition, initial data with J01 lineage demonstrated that migration was also affected during the transwell invasion assay; as well as ROCK protein, involved in control of cell migration, was decreased (demonstrated by western blot) by both SFs, suggesting that ROCK is involved in the mechanism of the components. Ongoing tests continue to establish other samples collected so that the tests described above will also be carried out. These results will contribute to the development of new therapies against these tumors. Support: FAPESP #2019/10003-3.
DDDR-17. CHEMOTHERAPEUTIC STRESS INDUCES NONCANONICAL HIF1Α SIGNALING UNDER NORMOXIA IN GLIOBLASTOMA DRIVING THERAPEUTIC RESISTANCE
Authors: Ventarapragada Divya , Ahmed Atique , Perrault Ella , Park Cheol
Published: November 2022

Glioblastoma (GBM) is an aggressive brain tumor with no cure. Standard treatment includes adjuvant, concomitant temozolomide (TMZ) and radiation, but most patients do not survive more than 15 months. Tumor adaptations to temozolomide (TMZ) contribute to glioblastoma (GBM) chemoresistance and recurrence, and hypoxia inducible factor subunit alpha (HIF1α) and downstream effectors are implicated in attenuated therapeutic responses. We have previously shown that TMZ-dependent hijacking of HIF1α signaling under normoxia is independent from physiological hypoxia in the tumor core. Our results further showed that TMZ promotes HIF1α accumulation enhancing stem-like characteristics of GBM cells driving therapy-resistant populations involved in recurrence. However, noncanonical HIF1α signaling mechanisms are not well understood preventing the success of HIF1α inhibitors in GBM thus far. Here, we investigate how TMZ induces HIF1α in an oxygen-independent manner by characterizing the HIF1a binding profile post-therapy. We cultured patient-derived xenograft (PDX) models of GBM in vitro and found HIF1α expression and hypoxia response element (HRE) activity peak 96h post-therapy using western blot and luciferase assays. Our lab also performed a whole-genome CRISPR-Cas9 knockout screen in GBM cells over 28 days to determine genetic drivers of GBM aggression. HIF1α was found to be particularly enriched in this screen, further supporting the role of chronic hypoxia signaling for GBM proliferation (p-value < 0.05). We show TMZ therapy prevents proteasome-dependent degradation through reduced binding of PHD2 and VHL allowing nuclear translocation of HIF1a through western blot and immunocytochemistry. Thus, we performed immunoprecipitation of HIF1α 96h post-therapy for proteomic characterization to establish how TMZ uniquely regulates HIF1α stabilization in the absence of oxygen tension, and these results will be presented. We will subsequently characterize the regulatory role of TMZ-dependent post-translational modifications to HIF1α. Elucidating the molecular mechanisms of therapy-mediated HIF1α regulation can produce better therapeutic targets and increase TMZ efficacy.
DDDR-24. INTEGRATED ANALYSIS OF SINGLE CELL CHROMATIN ACCESSIBILITY AND RNA EXPRESSION IDENTIFIED COMMON VULNERABILITY DESPITE GLIOBLASTOMA HETEROGENEITY
Authors: Raviram Ramya , Raman Anugraha , Preissl Sebastian , Wu Shaoping , Koga Tomoyuki , Zhu Chenxu , Luebeck Jens , Van Deynze Kinsey , Han Jee Yun , Hou Xioameng , Ye Zhen , Mischel Anna , Li Yang Eric , Fang Rongxin , Baback Tomas , Mugford Joshua , Han Claudia , Glass Christopher , Barr Cathy , Mischel Paul , Bafna Vineet , Escoubet Laura , Ren Bing , Chen Clark
Published: November 2022

INTRODUCTION

In 2021, the World Health Organization (WHO) reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH) wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intra-tumoral heterogeneity is a key contributor to therapeutic failure.

METHODS

we applied integrated genome-wide chromatin accessibility (snATACseq) and transcription (snRNAseq) profiles to clinical specimens derived IDHwt glioblastomas and G4 IDHm) astrocytomas, with goal of therapeutic target discovery.

RESULTS

The integrated analysis achieved resolution of intra-tumoral heterogeneity not previously possible, providing a molecular landscape of extensive regional and cellular variability. snATACseq delineated focal amplification down to an ~40 KB resolution. The snRNA analysis elucidated distinct cell types and cell states (neural progenitor/oligodendrocyte cell-like or astrocyte/mesenchymal cell-like) that were superimposable onto the snATACseq landscape. Paired-seq (parallel snATACseq and snRNAseq using the same clinical sample) provided high resolution delineation of extrachromosomal circular DNA (ecDNA), harboring oncogenes including CCND1 and EGFR. Importantly, the copy number of ecDNA genes correlated closely with the level of RNA expression. Integrated analysis across all specimens profiled suggests that IDHm grade 4 astrocytoma and IDHwt glioblastoma cells shared a common chromatin structure defined by open regions enriched for Nuclear Factor 1 transcription factors (NFIA and NFIB). Silencing of NF1A or NF1B suppressed in vitro and in vivo growth of patient-derived IDHwt glioblastomas and G4 IDHm astrocytoma models that mimic distinct glioblastoma cell states.

CONCLUSION

Our findings suggest despite distinct genotypes and cell states, glablastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intra-tumoral heterogeneity.
DDDR-26. IDENTIFICATION OF OLIG2 AS A PREDICTOR OF OSIMERTINIB’S EFFICACY AGAINST EGFRVIII+ GLIOBLASTOMA
Authors: Adams Jackson , Kwatra Gita , Bordeaux Zachary , Kwatra Madan
Published: November 2022

BACKGROUND

Molecular characterization of glioblastomas by The Cancer Genome Atlas revealed molecular abnormalities in epidermal growth factor receptor (EGFR) in over 50% of glioblastomas. These included gene amplification, a large deletion in ligand binding domain (EGFRvIII), kinase duplications and gene fusions. Our laboratory has been focusing on targeting EGFRvIII in glioblastomas and we noted EGFRvIII heterogeneity. We found that growth inhibition of EGFRvIII+ glioblastoma stem cell lines D317 and D10-0171 by osimertinib was different, with D317 being more sensitive. Molecular analyses revealed that D10-0171 has 35-fold higher levels of the transcription factor OLIG2. Here we hypothesize that increased OLIG2 expression confers osimertinib resistance in EGFRvIII+ GBM.

METHODS

An OLIG2 knock out GSC, D10-0171 (OLIG2-KO) was generated using CRISPR. Efficacy of osimertinib in growth inhibition of D10-0171 (WT) and OLIG2-KO was examined in a subcutaneous xenograft model. Once tumors became visible, mice were treated daily with either 200 μl vehicle or 25 mg/kg osimertinib by oral gavage. RNA-seq and reverse phase protein array (RPPA) analyses were then performed on both GSC’s.

RESULTS

Assessment of tumor growth of both GSC’s demonstrated that OLIG2-KO cells formed tumors at a significantly lower rate than WT with a rate-based (T/C) ratio of 0.0077. Administration of osimertinib led to tumor growth inhibition in both GSCs but had a greater effect in OLIG2KO than WT, with rate-based T/C ratios of 0.099 and 0.1669, respectively. Transcriptomic analysis of OLIG2KO revealed downregulation of several pathways when compared to WT, including MYC targets V1 and V2, oxidative phosphorylation, and fatty acid metabolism, while proteomic analysis revealed downregulation of several growth-promoting pathways including ErbB and MAPK signaling.

CONCLUSION

Lower levels of OLIG2 increase osimertinib sensitivity of EGFRvIII+ glioblastoma. We propose a schema for selecting EGFRvIII+ patients who will respond to Osimertinib.
DDDR-30. UNRAVELING THE NOVEL ANTI-TUMOR MECHANISM OF ST-401, A BRAIN PENETRANT MICROTUBULE TARGETING AGENT
Authors: Khan Kainat , Vicente Juan , McFaline-Figueroa José , Wordeman Linda , Stella Nephi
Published: November 2022

Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of cancers. While evidence suggests that glioblastoma (GBM) is sensitive to disruptions in microtubule (MT) functions; most MTAs do not reach the brain. To address this limitation, we developed a brain-penetrant MTA, ST-401, that kills patient-derived GBM in culture at nanomolar concentrations, binds to colchicine sites through a unique interaction mode, gently and reversibly inhibits MT assembly and kills cancer cells through a novel anti-tumor mechanism as determined by analysis of its activity in the NCI 60 cancer cell panel compared to the Approved Oncology Agents library (Horne et al. (2020) Neuro-Onc Adv. 3(1): vdaa165).We used flow cytometry to confirm that nocodazole (10-100 nM) treatment of HCT116 cells triggered apoptosis, necrosis, and accumulation of autophagosomes, as well as increase in p-P53. By sharp contrast, ST-401 did not trigger these responses. Live cell imaging showed that NOC preferentially kills HCT116 cells in mitosis whereas ST-401 preferentially kills these cells in interphase. ScRNAseq analysis of NOC and ST-401 treatment of HCT-116 cells showed that ST-401 down-regulates MYC mRNA expression, suggesting that ST-401 might affect MYC function. MYC protein levels are regulated by multiple mechanisms, including phosphorylation at residue Thr58 that promotes its ubiquitination and proteasome degradation. We discovered that ST-401 increases MYC phosphorylation on Thr58 and down-regulates MYC protein expression in both HCT116 and the GBM cell line, U251.Our result suggests that the novel anti-tumor mechanism of ST-401 might involve cell death in interphase and reduced MYC expression. Funded by NIH (CA244213 to NS and NS106924 to NS).
DDDR-31. THE THERAPEUTIC POTENTIAL OF THE LONG NON-CODING RNA LNC-HLX-2-7 IN GROUP 3 MEDULLOBLASTOMAS IN CHILDREN
Authors: Katsushima Keisuke , Yuan Menglang , Lee Bongyong , Stapleton Stacie , Jallo George , Seal Sudipta , Eberhart Charles , Perera Ranjan
Published: November 2022

Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB formation and tumor progression. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 MBs. In this study, we report that lnc-HLX-2-7 RNA specifically accumulates in the HLX (host gene of lnc-HLX-2-7) promoter region and activates HLX expression by recruiting multiple factors including enhancer elements. RNA sequencing and chromatin immunoprecipitation revealed that HLX directly binds to the promoters of several tumor-promoting genes, including MYC, and activates their expression. Furthermore, intravenous treatment with antisense oligonucleotides targeting lnc-HLX-2-7 coated with cerium-oxide nanoparticle (CNP-lnc-HLX-2-7) reduced tumor growth (40-50%) in intracranial MB xenograft mouse model (n = 10, p < 0.01, t-test). We found that the combinatorial therapy of CNP-lnc-HLX-2-7 and cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared to CNP-lnc-HLX-2-7 monotherapy (n = 10, p < 0.01, t-test) only. We report here the importance of the lnc-HLX-2-7-HLX-MYC axis in regulating group 3 MB progression and provide a strong rationale for using lnc-HLX-2-7 as a specific and potent therapeutic target for the group 3 MBs in children.