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The unique cancer-associated immunosuppression in brain, combined with a paucity of infiltrating T cells, contributes to the low response rate and poor treatment outcomes of T cell-based immunotherapy for patients diagnosed with glioblastoma multiforme (GBM). Here, we report on a self-assembling paclitaxel (PTX) filament (PF) hydrogel that stimulates macrophage-mediated immune response for local treatment of recurrent glioblastoma. Our results suggest that aqueous PF solutions containing aCD47 can be directly deposited into the tumor resection cavity, enabling seamless hydrogel filling of the cavity and long-term release of both therapeutics. The PTX PFs elicit an immune-stimulating tumor microenvironment (TME) and thus sensitizes tumor to the aCD47-mediated blockade of the antiphagocytic “don’t eat me” signal, which subsequently promotes tumor cell phagocytosis by macrophages and also triggers an antitumor T cell response. As adjuvant therapy after surgery, this aCD47/PF supramolecular hydrogel effectively suppresses primary brain tumor recurrence and prolongs overall survivals with minimal off-target side effects.

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Purpose:High-grade glioma (HGG) treatment is limited by the inability of otherwise potentially efficacious drugs to penetrate the blood–brain barrier. We evaluate the unique intracavity delivery mode and translational potential of a blend of poly(DL-lactic acid-co-glycolic acid; PLGA) and poly(ethylene glycol; PEG) paste combining temozolomide and etoposide to treat surgically resected HGG.Experimental Design:To prolong stability of temozolomide prodrug, combined in vitro drug release was quantitatively assessed from low pH–based PLGA/PEG using advanced analytic methods. In vitro cytotoxicity was measured against a panel of HGG cell lines and patient-derived cultures using metabolic assays. In vivo safety and efficacy was evaluated using orthotopic 9L gliosarcoma allografts, previously utilized preclinically to develop Gliadel.Results:Combined etoposide and temozolomide in vitro release (22 and 7 days, respectively) was achieved from a lactic acid–based PLGA/PEG paste, used to enhance stability of temozolomide prodrug. HGG cells from central-enhanced regions were more sensitive to each compound relative to primary lines derived from the HGG-invasive margin. Both drugs retained cytotoxic capability upon release from PLGA/PEG. In vivo studies revealed a significant overall survival benefit in postsurgery 9L orthotopic gliosarcomas, treated with intracavity delivered PLGA/PEG/temozolomide/etoposide and enhanced with adjuvant radiotherapy. Long-term survivorship was observed in over half the animals with histologic confirmation of disease-free brain.Conclusions:The significant survival benefit of intracavity chemotherapy demonstrates clinical applicability of PLGA/PEG paste-mediated delivery of temozolomide and etoposide adjuvant to radiotherapy. PLGA/PEG paste offers a future platform for combination delivery of molecular targeted compounds.

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OBJECTIVE Atypical teratoid rhabdoid tumors (ATRTs) are aggressive pediatric brain tumors with no current standard of care and an estimated median patient survival of 12 to 18 months. Previous genetic analyses have implicated cyclin D1 and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase that is implicated in many cancers, as key drivers of tumorigenicity in ATRTs. Since the effects of EZH2 and cyclin D1 are facilitated by a host of cyclin-dependent kinases (CDKs), the authors sought to investigate the potential therapeutic effects of targeting CDKs in ATRTs with the multi–CDK inhibitor, TG02. METHODS Human ATRT cell lines BT12, BT37, CHLA05, and CHLA06 were selected for investigation. The effects of TG02 on cell viability, proliferation, clonogenicity, and apoptosis were assessed via Cell Counting Kit-8 assays, cell counting, clonogenic assays, and flow cytometry, respectively. Similar methods were used to determine the effects of TG02 combined with radiation therapy (RT) or cisplatin. Synergism indices for TG02-cisplatin combination therapy were calculated using CompuSyn software. RESULTS TG02 was observed to significantly impair ATRT cell growth in vitro by limiting cell proliferation and clonogenicity, and by inducing apoptosis. TG02 inhibited ATRT cell proliferation and decreased cell viability in a dose-dependent manner with nanomolar half maximal effective concentration (EC50) values (BT12, 207.0 nM; BT37, 127.8 nM; CHLA05, 29.7 nM; CHLA06, 18.7 nM). TG02 (150 nM) dramatically increased the proportion of apoptotic ATRT cells 72 hours posttreatment (TG02 8.50% vs control 1.52% apoptotic cells in BT12, p < 0.0001; TG02 70.07% vs control 15.36%, p < 0.0001). Combination therapy studies revealed that TG02 acted as a potent radiosensitizer in ATRT cells (BT12 surviving fraction, RT 51.2% vs RT + TG02 21.7%). Finally, CompuSyn analysis demonstrated that TG02 acted synergistically with cisplatin against ATRT cells at virtually all therapeutic doses. These findings were consistent in cell lines that cover all three molecular subgroups of ATRTs. CONCLUSIONS The results of this investigation have established that TG02 is an effective therapeutic against ATRTs in vitro. Given the lack of standard therapy for ATRTs, these findings help fill an unmet need and support further study of TG02 as a potential therapeutic option for patients with this deadly disease.

OBJECTIVE Medulloblastoma, the most common pediatric brain malignancy, has Sonic Hedgehog (SHH) and group 3 (Myc driven) subtypes that are associated with the activity of eukaryotic initiation factor 4E (eIF4E), a critical mediator of translation, and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and master regulator of transcription. Recent drug repurposing efforts in multiple solid and hematologic malignancies have demonstrated that eIF4E and EZH2 are both pharmacologically inhibited by the FDA-approved antiviral drug ribavirin. Given the molecular overlap between medulloblastoma biology and known ribavirin activity, the authors investigated the preclinical efficacy of repurposing ribavirin as a targeted therapeutic in cell and animal models of medulloblastoma. METHODS Multiple in vitro assays were performed using human ONS-76 (a primitive SHH model) and D425 (an aggressive group 3 model) cells. The impacts of ribavirin on cellular growth, death, migration, and invasion were quantified using proliferation and Cell Counting Kit-8 (CCK-8) assays, flow cytometry with annexin V (AnnV) staining, scratch wound assays, and Matrigel invasion chambers, respectively. Survival following daily ribavirin treatment (100 mg/kg) was assessed in vivo in immunodeficient mice intracranially implanted with D425 cells. RESULTS Compared to controls, ribavirin treatment led to a significant reduction in medulloblastoma cell growth (ONS-76 proliferation assay, p = 0.0001; D425 CCK-8 assay, p < 0.0001) and a significant increase in cell death (flow cytometry for AnnV, ONS-76, p = 0.0010; D425, p = 0.0284). In ONS-76 cells, compared to controls, ribavirin significantly decreased cell migration and invasion (Matrigel invasion chamber assay, p = 0.0012). In vivo, ribavirin significantly extended survival in an aggressive group 3 medulloblastoma mouse model compared to vehicle-treated controls (p = 0.0004). CONCLUSIONS The authors demonstrate that ribavirin, a clinically used drug known to inhibit eIF4E and EZH2, has significant antitumor effects in multiple preclinical models of medulloblastoma, including an aggressive group 3 animal model. Ribavirin may represent a promising targeted therapeutic in medulloblastoma.

Cotton balls are used in neurosurgical procedures to assist with hemostasis and improve vision within the operative field. Although the surgeon can reshape pieces of cotton for multiple intraoperative uses, this customizability and scale also places them at perpetual risk of being lost, as blood-soaked cotton balls are visually similar to raw brain tissue. Retained surgical cotton can induce potentially life-threatening immunologic responses, impair postoperative imaging, lead to a textiloma or misdiagnosis, and/or require reoperation. This study investigated three imaging modalities (optical, acoustic, and radiographic) to find the most effective method of identifying foreign bodies during neurosurgery. First, we examined the use of dyes to increase contrast between cotton and surrounding parenchyma (optical approach). Second, we explored the ability to distinguish surgical cotton on or below the tissue surface from brain parenchyma using ultrasound imaging (acoustic approach). Lastly, we analyzed the ability of radiography to differentiate between brain parenchyma and cotton. Our preliminary testing demonstrated that dark-colored cotton is significantly more identifiable than white cotton on the surface level. Additional testing revealed that cotton has noticeable different acoustic characteristics (eg, speed of sound, absorption) from neural tissue, allowing for enhanced contrast in applied ultrasound imaging. Radiography, however, did not present sufficient contrast, demanding further examination. These solutions have the potential to significantly reduce the possibility of intraoperative cotton retention both on and below the surface of the brain, while still providing surgeons with traditional cotton material properties without affecting the surgical workflow.

Background Hydrocephalus is managed by surgically implanting flow-diversion technologies such as differential pressure valves and antisiphoning devices; however, such hardware is prone to failure. Extensive research has tested them in flow-controlled settings using saline or de-aerated water, yet little has been done to validate their performance in a setting recreating physiologically relevant parameters, including intracranial pressures, cerebrospinal fluid (CSF) protein content, and body position. Objective To more accurately chart the episodic drainage characteristics of flow-diversion technology. A gravity-driven benchtop model of flow was designed and tested continuously during weeks-long trials. Methods Using a hydrostatic pressure gradient as the sole driving force, interval flow rates of 6 valves were examined in parallel with various fluids. Daily trials in the upright and supine positions were run with fluid output collected from distal catheters placed at alternating heights for extended intervals. Results Significant variability in flow rates was observed, both within specific individual valves across different trials and among multiple valves of the same type. These intervalve and intravalve variabilities were greatest during supine trials and with increased protein. None of the valves showed evidence of overt obstruction during 30 d of exposure to CSF containing 5 g/L protein. Conclusion Day-to-day variability of ball-in-cone differential pressure shunt valves may increase overdrainage risk. Narrow-lumen high-resistance flow control devices as tested here under similar conditions appear to achieve more consistent flow rates, suggesting their use may be advantageous, and did not demonstrate any blockage or trend of decreasing flow over the 3 wk of chronic use.

Interstitial chemotherapy plays a pivotal role in the treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain cancer, by enhancing drug biodistribution to the tumor and avoiding systemic toxicities. The use of new polymer structures that extend the release of cytotoxic agents may therefore increase survival and prevent recurrence. A novel core-sheath fiber loaded with the drug carmustine (BCNU) was evaluated in an in vivo brain tumor model. Three-dimensional discs were formed from coaxially electrospun fiber membranes and in vitro BCNU release kinetics were measured. In vivo survival was assessed following implantation of discs made of compressed core-sheath fibers (NanoMesh) either concurrently with or five days after intracranial implantation of 9L gliosarcoma. Co-implantation of NanoMesh and 9L gliosarcoma resulted in statistically significant long-term survival (>150 days). Empty control NanoMesh confirmed the safety of these novel implants. Similarly, Day 5 studies showed significant median, overall, and long-term survival rates, suggesting optimal control of tumor growth, confirmed with histological and immunohistochemical analyses. Local chemotherapy by means of biodegradable NanoMesh implants is a new treatment paradigm for the treatment for brain tumors. Drug delivery with coaxial core-sheath structures benefits from high drug loading, controlled long-term release kinetics, and slow polymer degradation. This represents a promising evolution for the current treatment of GBM.

Purpose: High-grade glioma (HGG) treatment is limited by the inability of otherwise potentially efficacious drugs to penetrate the blood–brain barrier. We evaluate the unique intracavity delivery mode and translational potential of a blend of poly(DL-lactic acid-co-glycolic acid; PLGA) and poly(ethylene glycol; PEG) paste combining temozolomide and etoposide to treat surgically resected HGG. Experimental Design: To prolong stability of temozolomide prodrug, combined in vitro drug release was quantitatively assessed from low pH–based PLGA/PEG using advanced analytic methods. In vitro cytotoxicity was measured against a panel of HGG cell lines and patient-derived cultures using metabolic assays. In vivo safety and efficacy was evaluated using orthotopic 9L gliosarcoma allografts, previously utilized preclinically to develop Gliadel. Results: Combined etoposide and temozolomide in vitro release (22 and 7 days, respectively) was achieved from a lactic acid–based PLGA/PEG paste, used to enhance stability of temozolomide prodrug. HGG cells from central-enhanced regions were more sensitive to each compound relative to primary lines derived from the HGG-invasive margin. Both drugs retained cytotoxic capability upon release from PLGA/PEG. In vivo studies revealed a significant overall survival benefit in postsurgery 9L orthotopic gliosarcomas, treated with intracavity delivered PLGA/PEG/temozolomide/etoposide and enhanced with adjuvant radiotherapy. Long-term survivorship was observed in over half the animals with histologic confirmation of disease-free brain. Conclusions: The significant survival benefit of intracavity chemotherapy demonstrates clinical applicability of PLGA/PEG paste-mediated delivery of temozolomide and etoposide adjuvant to radiotherapy. PLGA/PEG paste offers a future platform for combination delivery of molecular targeted compounds.

OBJECTIVEVentricular shunt infection remains an issue leading to high patient morbidity and cost, warranting further investigation. The authors sought to create an animal model of shunt infection that could be used to evaluate possible catheter modifications and innovations.METHODSThree dogs underwent bilateral ventricular catheter implantation and inoculation with methicillin-sensitive Staphylococcus aureus (S. aureus). In 2 experimental animals, the catheters were modified with a polymer containing chemical “pockets” loaded with vancomycin. In 1 control animal, the catheters were polymer coated but without antibiotics. Animals were monitored for 9 to 11 days, after which the shunts were explanted. MRI was performed after shunt implantation and prior to catheter harvest. The catheters were sonicated prior to microbiological culture and also evaluated by electron microscopy. The animals’ brains were evaluated for histopathology.RESULTSAll animals underwent successful catheter implantation. The animals developed superficial wound infections, but no neurological deficits. Imaging demonstrated ventriculitis and cerebral edema. Harvested catheters from the control animal demonstrated > 104 colony-forming units (CFUs) of S. aureus. In the first experimental animal, one shunt demonstrated > 104 CFUs of S. aureus, but the other demonstrated no growth. In the second experimental animal, one catheter demonstrated no growth, and the other grew trace S. aureus. Brain histopathology revealed acute inflammation and ventriculitis in all animals, which was more severe in the control.CONCLUSIONSThe authors evaluated an animal model of ventricular shunting and reliably induced features of shunt infection that could be microbiologically quantified. With this model, investigation of pathophysiological and imaging correlates of infection and potentially beneficial shunt catheter modifications is possible.

Glioblastoma multiforme (GBM) has few clinically approved therapeutic regimens. One of these therapeutic options includes placing biodegradable wafers releasing BCNU (Gliadel®) into the tumor bed at the time of surgical removal of the tumor. Due to the significant benefit this polymer technology has had clinically, we have prepared wafers releasing Temozolomide (TMZ), an anticancer drug used systemically for treating GBM. TMZ delivered via polymer wafer could be used as a complementary treatment with or as an alternative to Gliadel®. TMZ is an alkylating agent which is water soluble. To remain comparable with the preclinical studies that led to Gliadel® the same size of wafers were formulated with TMZ. Wafers were loaded with 50% w/w TMZ in poly(lactic acid-glycolic acid) (PLGA) and showed reliable release of high dose TMZ for a period of 4 weeks. To achieve this 30-day release of the highly water soluble drug, we developed an encapsulation method, where the drug powder was first coated with the polymer to form core-shell particles in which the coating shell served as a rate controlling membrane for the drug particles. Wafers were also made with a co-loading of TMZ and BCNU. All wafers were tested in vivo by treating an intracranial 9 L gliosarcoma model in F344 rats. Rats that were either untreated or treated with blank wafer died within 11 days while the median survival for rats treated with systemic TMZ was 18 days. The group that received the BCNU alone wafer had a median survival of 15 days, the group that received the TMZ wafer alone had a median survival of 19 days, and the group treated with the BCNU-TMZ wafer had a median survival of 28 days with 25% of the animals living long term (p .0038 vs. Control; p .001 vs. Blank Polymer). These findings demonstrate the potential of this newly designed wafer for treating GBM. Moreover, this concept, can pave the way for other drug combinations that may improve the clinical application of numerous agents to treat solid tumors.

Background Medulloblastoma (MB) is the most common brain malignancy in children, and is still responsible for significant mortality and morbidity. The aim of this study was to assess the safety and efficacy of Disulfiram (DSF), an FDA-approved inhibitor of Aldehyde-Dehydrogenase (ALDH), and Copper (Cu++) in human SSH-driven and Group 3 MB. The molecular mechanisms, effect on cancer-stem-cells (CSC) and DNA damage were investigated in xenograft models. Methods The cytotoxic and anti-CSC effects of DSF/Cu++ were evaluated with clonogenic assays, flow-cytometry, immunofluorescence, western-blotting. ONS76, UW228 (SHH-driven with Tp53m), D425med, D283 and D341 (Group 3) cell-lines were used. In vivo survival and nuclear protein localization protein-4 (NPL4), Ki67, Cleaved-Caspase-3, GFAP and NeuN expression were assessed in two Group 3 MB xenografts with immunohistochemistry and western-blotting. Results Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations. DSF/Cu++ induced cell-death through NPL4 accumulation in cell-nucleus and buildup of poly-ubiquitylated proteins. Flow-cytometry demonstrated a significant decrease in ALDH+, Nestin+ and CD133+ following treatment, anti-CSC effect was confirmed in vitro and in vivo. DSF/Cu++ prolonged survival, and increased nuclear NPL4 expression in vivo. Conclusions Our data suggest that this combination may serve as a novel treatment, as monotherapy or in combination with existing therapies, for aggressive subtypes of pediatric MB.

ObjectiveVentricular collapse is a prevalent yet poorly understood complication of ventriculo-peritoneal shunting (VPS) in patients with idiopathic intracranial hypertension (IIH). By identifying and describing the risk factors of ventricular collapse (VC), this study aims to characterize the clinical progression, etiopathogenesis and treatment of IIH and its complications. The relationships between ventricular size, symptoms and treatments were also assessed longitudinally with ventricular segmentation on MRI/CT imaging, and correlated with other risk factors of IIH and VC. MethodsWe retrospectively reviewed records of 147 patients who underwent VPS for IIH at our Institution, and identified 73 shunt-naïve subjects included in the final analysis. Manual segmentation of CT/MRI scans was performed longitudinally at each clinical stage (baseline, post-shunting, post-collapse and after each intervention). Variables collected included valve type and opening-pressure, shunt revisions, use of anti-siphoning devices (ASD), comorbidities, venous sinus hypoplasia/stenosis and stenting, and general demographics. Linear univariate regression models were used to determine the association between individual risk factors and VC, and to quantitatively assess the effect of treatment. Two multivariate models were also tested, including Pre-Shunting and Post-Shunting variables, to quantify their association with VC. ResultsOf 73 IIH patients with new shunts, 32 experienced collapse (uni- or bilateral, 26.5% of the total). In shunt-naïve patients, collapse was associated with pre-shunting (rho=-0.36; p=0.001) and post-shunting ventricular size (rho=0.62; p=0.0002). Both collapse and ventricular area were correlated with shunt-related symptoms at 6 months (rho=-0.29; p=0.01). Shunt adjustment, addition of ASDs, and valve replacement proved to be the most effective strategies to re-expand the ventricles and reduce symptoms. Nonetheless, a significant fraction of patients remained symptomatic after multiple treatments, suggesting a complex and multifactorial etiology for VC. On univariate analysis, catheter revisions were more common in the VC group, while the multivariate model with Post-Shunting risk factors proved to be significantly associated with VC. ConclusionsIn newly VP-shunted IIH patients, small ventricular size predisposed to collapse and headaches, while higher valve OPs and ASDs may reduce the risk of collapse and promote symptomatic improvement. Within the restraints of a retrospective analysis, this study is the first to analyze the risk factors of VC in IIH patients, longitudinally integrating the clinical progression with ventricular imaging. Further prospective studies are warranted to better understand the etiopathogenesis and clinical progression of collapse.

Deregulation of mRNA translation is a widespread characteristic of glioblastoma (GBM), aggressive malignant brain tumors that are resistant to conventional therapies. RNA-binding proteins (RBPs) play a critical role in translational regulation, yet the mechanisms and impact of these regulations on cancer development, progression and response to therapy remain to be fully understood. Here, we showed that hnRNP H/F RBPs are potent regulators of translation through several mechanisms that converge to modulate the expression and/or the activity of translation initiation factors. Among these, hnRNP H/F regulate the phosphorylation of eIF4E and its translational targets by controlling RNA splicing of the A-Raf kinase mRNA, which in turn modulates the MEK-ERK/MAPK signaling pathway. The underlying mechanism involves RNA G-quadruplex (RG4s), RNA structures whose modulation phenocopies hnRNP H/F translation regulation in GBM cells. Our results highlighted that hnRNP H/F are essential for key functional pathways regulating proliferation and survival of GBM, highlighting its targeting as a promising strategy for improving therapeutic outcomes.

Neurosurgical operations are long and intensive medical procedures, during which the surgeon must constantly have an unobscured view of the brain in order to be able to properly operate, and thus must use a variety of tools to clear obstructions (like blood and fluid) from the operating area. Currently, cotton balls are the most versatile and effective option to accomplish this as they absorb fluids, are soft enough to safely manipulate the brain, act as a barrier between other tools and the brain, and function as a spacer to keep anatomies of the brain open and visible during the operation. While cotton balls allow neurosurgeons to effectively improve visibility of the operating area, they may also be accidentally left in the brain upon completion of the surgery. This can lead to a wide range of post-operative risks including dangerous immune responses, additional medical care or surgical operations, and even death. This project seeks to develop a unique medical device that utilizes ultrasound technology in order to minimize cotton retention after neurosurgical procedures in order to reduce undesired post-operative risks, and maximize visibility.

Spinal cord injury (SCI) affects approximately 2.5 million people worldwide. The primary phase of SCI is initiated by mechanical trauma to the spinal cord, while the secondary phase involves the ensuing tissue swelling and ischemia that worsen tissue damage and functional outcome. Optimizing blood flow to the spinal cord after SCI can mitigate injury progression and improve outcome. Accurate, sensitive, real-time monitoring is critical to assessing the spinal cord perfusion status and optimizing management, particularly in those with injuries severe enough to require surgery. However, the complex anatomy of the spinal cord vasculature and surrounding structures present significant challenges to such a monitoring strategy. In this study, Doppler ultrasound was hypothesized to be a potential solution to detect and monitor spinal cord tissue perfusion in SCI patients who required spinal decompression and/or stabilization surgeries. This approach could provide real-time visual blood flow information and pulsatility of the spinal cord as biomarkers of tissue perfusion. Importantly, Doppler ultrasound could be readily integrated into the surgical workflow, because the spinal cord was exposed during surgery, thereby allowing easy access for Doppler deployment, while keeping the dura intact. Doppler ultrasound successfully measured blood flow in single and bifurcated microfluidic channels at physiologically relevant flow rates and dimensions in both in-vitro and in-vivo porcine SCI models. Furthermore, perfusion was quantified from the obtained images. Our results provide a promising and viable solution to intraoperatively assess and monitor blood flow at the SCI site to optimize tissue perfusion and improve functional recovery in SCI patients.

Paroxysmal dysarthria and ataxia (PDA) is a rare syndrome characterized by brief, stereotyped episodes of slurred speech, clumsiness with extremities, or vertigo. It is usually observed in young patients suffering from multiple sclerosis with numerous lesions. PDA is challenging to identify in those presenting with atypical patterns. Here, a non-ataxic variant of PDA in an otherwise neurologically healthy elderly man is presented who had a single midbrain lesion. A broad diagnostic workup illustrates the challenges of identifying PDA. Teaching points emphasize the significance of the midbrain lesion and response to anti-epileptic medication., Highlights • Paroxysmal Dysarthria and Ataxia (PDA) is a very rare MS-related paroxysm • Case of a non-ataxic PDA variant in an otherwise neurologically healthy elderly man • Video clearly shows the symptoms of this Spontaneous Paroxysmal Dysarthria • Solitary midbrain lesions and responsiveness to anti-epileptics are key features

There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance. Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed PLGA/PEG thermo-sensitive biodegradable paste. Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study is ongoing where we already observe a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (50 days vs. 44 vs 30 respectively). A more pronounced survival benefit was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy. RNAseq data from 10 GBM patients show significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence–activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells. Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.

BACKGROUND There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance. Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed PLGA/PEG thermo-sensitive biodegradable paste. MATERIAL AND METHODS Metabolic and clonogenic assays were used to assess effects on proliferation and clonal growth upon in vitro glioma exposure to olaparib. Flow cytometry and Annexin V/Propidium iodide were used to determine apoptosis. The 9L high-grade glioma orthotopic allograft model was utilised to assess survival upon intra-cavity olaparib delivery. RESULTS Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study revealed a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (95 vs. 44 vs. 30 days respectively). A more pronounced survival benefit, as measured by number of animals surviving long-term, was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy, relative to standard-of-care (95 vs. 44 days). Clinical correlation was determined using RNAseq data from 10 GBM patients, showing significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence-activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells. CONCLUSION Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma with a proclivity for systemic dissemination, leading many patients to present with advanced stage disease and fail available treatments. There is a notable lack of targeted therapies for NPC, despite working knowledge of multiple proteins with integral roles in NPC cancer biology. These proteins include EZH2, Snail, eIF4E, and IMPDH, which are all overexpressed in NPC and correlated with poor prognosis. These proteins are known to be modulated by ribavirin, an FDA-approved hepatitis C antiviral that has recently been repurposed as a promising therapeutic in several solid and hematologic malignancies. Here, we investigated the potential of ribavirin as a targeted anticancer agent in five human NPC cell lines. Using cellular growth assays, flow cytometry, BrdU cell proliferation assays, scratch wound assays, and invasion assays, we show in vitro that ribavirin decreases NPC cellular proliferation, migration, and invasion and promotes cell-cycle arrest and cell death. Modulation of EZH2, Snail, eIF4E, IMPDH, mTOR, and cyclin D1 were observed in Western blots and enzymatic activity assays in response to ribavirin treatment. As monotherapy, ribavirin reduced flank tumor growth in multiple NPC xenograft models in vivo. Most importantly, we demonstrate that ribavirin enhanced the effects of radiotherapy, a central component of NPC treatment, both in vitro and in vivo. Our work suggests that NPC responds to ribavirin-mediated EZH2, Snail, eIF4E, IMPDH, and mTOR changes and positions ribavirin for clinical evaluation as a potential addition to our NPC treatment armamentarium.

BACKGROUND Medulloblastoma (MB), the most common brain malignancy among children, is classified into four molecular subtypes, WNT-driven, Shh-driven, Group 3 and 4. The aim of this study was to assess the effects of the combination of Disulfiram, an inhibitor of Aldehyde Dehydrogenase, and Copper in Shh-driven and Group 3 MB, in vitro and in vivo. METHODS The mechanisms of action and anti-cancer stem-cell effects of disulfiram were evaluated with clonogenic assays, flow-cytometry (FC), western blotting (WB), and immunofluorescence (IF) using established MB cell-lines - ONS76, UW228, D425med, D341 and D283- representing the main molecular subtypes. Survival, tumor volume, nuclear protein localization protein-4-expression (NPL4) and markers of proliferation/apoptosis were assessed in multiple models of Group 3 MB in vivo and ex vivo with WB and immunohistochemistry (IHC). RESULTS Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations of DSF in all lines. DSF/Cu++ induced cell death (increased AnnV/PI, cleaved-Poly(ADP-ribose)polymerase fraction, and Apoptosis Inducing Factor on WB/FC/IF, in vitro and ex vivo) through NPL4 accumulation in cell nucleus and intracellular buildup of poly-ubiquitylated proteins. DNA damage was also detected with WB and H2AX foci on immunofluorescence. Flow-cytometry analysis demonstrated a significant reduction in ALDH, Nestin- and CD133-positive cells in Group 3 lines, confirmed with WB in vitro and ex vivo. DSF/Cu++-toxicity was tested in vivo in multiple dose-escalating trials, and the combination significantly prolonged survival and reduced tumor volume in D425 and D341 xenografts. IHC showed lower Ki67 and increased Cleaved-Caspase-3 expression, higher NPL4-positive cells and no difference in NeuN and GFAP-positive cells after treatment. CONCLUSIONS DSF/Cu++ demonstrated a potent therapeutic effect on Shh-driven and Group 3 MB cell lines by determining apoptosis, targeting cancer stem cells and inducing DNA damage. Our data suggest that this combination may serve as a novel treatment, alone or with existing therapies, for pediatric MB.

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma with a propensity for metastasis, leading many patients to fail available treatments and/or present with advanced-stage disease. There is currently a scarcity of targeted therapies for NPC, despite working knowledge of several proteins with key roles in NPC cancer biology. These include EZH2, Snail, eIF4E, and IMPDH, which are all overexpressed in NPC and correlated with poor patient prognosis. These proteins are known to be targeted by ribavirin, a well-characterized anti-viral drug that has recently been repurposed as an anti-cancer agent in several solid and hematologic malignancies. In the present study, we investigated the potential of ribavirin as a targeted therapy and radiosensitizing agent in five human NPC cell lines. We show in vitro, using cellular growth assays, flow cytometry, BrdU cell proliferation assays, scratch wound assays, and invasion assays, that ribavirin decreases NPC cellular proliferation, migration, and invasion and promotes cell cycle arrest and cell death. Modulation of EZH2, Snail, eIF4E, IMPDH, and mTOR were observed in western blots and enzymatic activity assays in response to ribavirin treatment. In vivo, monotherapy with ribavirin reduced flank tumor growth in multiple NPC xenograft models. Given that radiation therapy is a mainstay of NPC treatment, we next investigated the effects of combining ribavirin with radiation. Using clonogenic assays and flow cytometry, we demonstrate that ribavirin enhanced the cytotoxic effects of radiation on NPC cells in vitro. Most importantly, using a flank tumor xenograft model, we show that pre-treatment with ribavirin potentiated the effects of radiation therapy in vivo. Our work suggests that NPC responds to ribavirin-mediated EZH2, Snail, eIF4E, IMPDH, and mTOR modulation and positions ribavirin for clinical evaluation as a targeted therapy and radiosensitizing agent in this cancer. Citation Format: Sakibul Huq, Joshua Casaos, Riccardo Serra, Michael Peters, Yuanxuan Xia, Andy Ding, Jeffrey Ehresman, Jayanidhi Kedda, Manuel Morales, Noah Gorelick, Tianna Zhao, Wataru Ishida, Alexander Perdomo-Pantoja, Arba Cecia, Chenchen Ji, Ian Suk, David Sidransky, Mariana Brait, Henry Brem, Nicolas Skuli, Betty Tyler. Use of the anti-viral drug ribavirin as a radiosensitizing agent in nasopharyngeal carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6268.

Purpose: Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that is often diagnosed at an advanced stage, leading to poor disease-free and overall survival. Accumulating literature suggests that elevated protein expression of enhancer of zeste homolog 2 (EZH2), eukaryotic initiation factor 4E (eIF4E), and inosine-5’-monophosphate dehydrogenase (IMPDH)—proteins implicated in myriad cancers—correlates with poor prognosis in NPC. These three proteins are modulated by the Food and Drug Administration-approved antiviral drug ribavirin, which has recently been repositioned by our laboratory and others as a promising anticancer agent. Based on this intersection of molecular signature and drug targets, we investigated the potential of ribavirin as a therapeutic agent for NPC. Experimental Design: We assessed antineoplastic efficacy of ribavirin on six human NPC cell lines in vitro using cellular growth assays, flow cytometry, and scratch wound assays. Mechanistic pathways involved were investigated using genomic expression datasets, Western blots, and enzymatic activity assays. The effects of combining ribavirin with radiation were assessed using clonogenic assays and flow cytometry. Finally, we evaluated the effects of ribavirin on tumor growth in vivo using two human cell line-derived xenograft models. Results: Ribavirin significantly decreased NPC cellular proliferation and migratory capacity in addition to promoting cell cycle arrest and cell death. Modulation of the EZH2, Snail, eIF4E, and IMPDH pathways was observed in response to ribavirin treatment. Ribavirin significantly enhanced the cytotoxic effects of radiation therapy in NPC. Most importantly, ribavirin significantly reduced flank tumor growth in two NPC xenograft models. Conclusions: Our work suggests that ribavirin has potent anticancer effects in NPC and could represent a safe and promising addition to current NPC treatment regimens. Citation Format: Sakibul Huq, Joshua Casaos, Michael Peters, Yuanxuan Xia, Andy Ding, Manuel Morales, Noah Gorelick, Riccardo Serra, Tianna Zhao, Wataru Ishida, Alexander Perdomo-Pantoja, Arba Cecia, Chenchen Ji, Ian Suk, David Sidransky, Mariana Brait, Henry Brem, Nicolas Skuli, Betty Tyler. Repositioning the FDA-approved antiviral drug ribavirin as targeted therapy for nasopharyngeal carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B06.

Gliomas are the most common solid tumors and the greatest cause of cancer-related deaths among children in the U.S. Treatment for this group of heterogeneous malignancies involves surgery and chemotherapy; however, tumor recurrence is inevitable. Immunotherapy is a treatment modality that can stimulate the intrinsic immune defenses of the body to eliminate tumor cells. This has been challenging for gliomas, though, due to the exclusionary anatomic barriers and the immunologically quiescent environment of the brain. This project aims to develop an injectable hydrogel patch for controlled local delivery of combination immunotherapy directly to the postsurgical formed cavity of the brain to treat residual disease and to prevent tumor recurrence. Using the hydrogel patch, we seek to overcome the delivery and immunosuppressive barriers of the brain by locally releasing, as a programmed regimen, the following immunomodulatory entities: i) C-X-C motif chemokine 10 (CXCL10; to promote T-lymphocyte recruitment), ii), a new generation of programmed death-ligand 1 (PD-L1) inhibitors (to prevent T-cell exhaustion), iii) FMS-like tyrosine kinase 3 ligand (FLT3L; to induce differentiation and expansion of dendritic cells), and iv) a stimulator of interferon genes (STING) agonist (to trigger cross-priming of CD8+ cytotoxic T cells). Our novel technology possesses the flexibility to personalize an array of antigens/adjuvants or other components of immunotherapy and enables the prevention of brain metastasis by creating an immunologically inhospitable setting for circulating tumor cells. As pediatric gliomas are notoriously resistant to treatment, our hydrogel formulation seeks to address an unmet clinical need for more effective therapeutic modalities. Note:This abstract was not presented at the conference. Citation Format: Shiran Ferber, Alexander M. Cryer, Noah Gorelick, Betty Tyler, Henry Brem, Robert S. Langer, Natalie Artzi. Training an immuno-army: Exploiting immunoengineering for the treatment of glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A27.

Together, medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT) represent two of the most prevalent pediatric brain malignancies. Current treatment involves radiation, which has high risks of developmental sequelae for patients under the age of three. New safer and more effective treatment modalities are needed. Cancer gene therapy is a promising alternative, but there are challenges with using viruses in pediatric patients. We developed a library of poly(beta-amino ester) (PBAE) nanoparticles and evaluated their efficacy for plasmid delivery of a suicide gene therapy to pediatric brain cancer models-specifically herpes simplex virus type I thymidine kinase (HSVtk), which results in controlled apoptosis of transfected cells. In vivo, PBAE-HSVtk treated groups had a greater median overall survival in mice implanted with AT/RT (P = 0.0083 vs. control) and MB (P 0.0001 vs. control). Our data provide proof of principle for using biodegradable PBAE nanoparticles as a safe and effective nanomedicine for treating pediatric CNS malignancies.

The growing cost of medical care worldwide, particularly in oncology, has incentivized researchers and physicians to repurpose clinically used drugs to alleviate the financial burden of drug development and offer potential new therapeutics. Recent works have demonstrated anticancer properties of the FDA-approved drug ribavirin, a synthetic guanosine analogue and antiviral molecule used over the past four decades for the treatment of hepatitis C. The efficacy of ribavirin in cancer has been explored through several preclinical models and ongoing clinical trials in multiple cancers, including acute myeloid leukemia, oropharyngeal squamous cell carcinoma, and metastatic breast cancer. In this review, we summarize the role of ribavirin as an antiviral medication and focus our attention on its recent use as an antitumoral agent. We highlight current knowledge of the potential use and mechanisms of action of ribavirin in cancer. Because current therapeutics for patients with cancer still fail to cure, introducing new forms of treatment is essential. Converging evidence suggests that ribavirin represents a promising addition to a generation of newly repurposed safe and effective anticancer agents.

There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance (Fulton et al 2018). Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) (PLGA/PEG) thermo-sensitive biodegradable paste. Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study is ongoing where we already observe a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (120 days vs. 44 vs 30 respectively). A more pronounced survival benefit, as determined by the total number of long-term surviving animals, was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy. RNAseq data from 10 GBM patients show significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence–activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells. Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.

Medulloblastoma (MB), an aggressive pediatric brain malignancy, is commonly classified into four subtypes. Although survival has improved remarkably, Group 3 and Shh-driven tumors still experience high rates of recurrent and metastatic disease. Disulfiram (DSF), a safe and inexpensive drug, has shown an antitumor effect when administered with copper (Cu++) against other brain malignancies, and its repurposing to MB could benefit current standards of treatment. ONS76 (Shh-driven Tp53 wild-type), UW228 (Shh-driven Tp53-mutated), D425med and D341med (Group 3) lines were treated with DSF-Cu++and tested with viability, proliferation and clonogenic assays. Flow cytometry (FACS) with AnnexinV-PI, Ki67, PI/RNAse for necrosis and cell cycle, and CD133 and Aldefluor for cell-stemness were carried out. MB lines were tested with western blotting (WB) and immunofluorescence (IF) for NPL4(Nuclear protein localization protein 4 homolog), AIF (apoptosis-inducing factor), cleaved-PARP(Poly-ADP-ribose-polymerase), H2AX and other molecular targets. In vivo safety (n=8) and efficacy studies (n=16) were carried out on Nu/Nu athymic mice implanted intracranially (IC) with D425med cells, along with Hematoxylin/Eosin and immunohistochemistry analyses on brain samples. DSF-Cu++showed remarkable cytotoxic and anti-proliferative activity against all MB lines. In particular, NPL4, a protein involved in the ubiquitin signaling, appears to be the primary DSF target, as previously shown in other malignancies. Apoptosis quickly follows the Heat-Shock Response, as confirmed by FACS, WB and IF, and cell-stemness is decreased significantly by nanomolar doses of DSF-Cu++(p=0.002). Oral administration of DSF-Cu++was proven safe at doses of 100 mg/kg and significantly prolonged survival in IC xenografts (median 28.5 days; p=0.02) compared to controls (median 24 days). DSF, a compound used in the treatment of chronic alcoholism, has recently shown a remarkable effect in a number of malignancies when co-administered with Cu++. We provide proof of its potential as a chemotherapeutic for pediatric MB, and describe its molecular mechanism of action.

Immune checkpoint blockade targeting programmed cell death protein 1 (PD-1) is emerging as an important treatment strategy in a growing list of cancers, yet its clinical benefits are limited to a subset of patients. Further investigation of tumor-intrinsic predictors of response and how extrinsic factors, such as iatrogenic immunosuppression caused by conventional therapies, impact the efficacy of anti-PD-1 therapy are paramount. Given the widespread use of corticosteroids in cancer management and their immunosuppressive nature, this study sought to determine how corticosteroids influence anti-PD-1 responses and whether their effects were dependent on tumor location within the periphery versus central nervous system (CNS), which may have a more limiting immune environment. In well-established anti-PD-1-responsive murine tumor models, corticosteroid therapy resulted in systemic immune effects, including severe and persistent reductions in peripheral CD4+ and CD8 + T cells. Corticosteroid treatment was found to diminish the efficacy of anti-PD-1 therapy in mice bearing peripheral tumors with responses correlating with peripheral CD8/Treg ratio changes. In contrast, in mice bearing intracranial tumors, corticosteroids did not abrogate the benefits conferred by anti-PD-1 therapy. Despite systemic immune changes, anti-PD-1-mediated antitumor immune responses remained intact during corticosteroid treatment in mice bearing intracranial tumors. These findings suggest that anti-PD-1 responses may be differentially impacted by concomitant corticosteroid use depending on tumor location within or outside the CNS. As an immune-specialized site, the CNS may potentially play a protective role against the immunosuppressive effects of corticosteroids, thus sustaining antitumor immune responses mediated by PD-1 blockade.

INTRODUCTION: The blood brain barrier is a critical limiting step to achieving therapeutic CNS drug concentrations. We have developed a poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG)-based Platform Technology to interstitially deliver multiple chemotherapy agents from a single thermo-setting paste, potentially filling the 3-week therapy gap that currently exists between surgery and commencement of radiotherapy in the treatment of high grade gliomas. Here we evaluate mouldable PLGA/PEG paste for combined temozolomide (TMZ) and etoposide (ETOP) delivery in an orthotopic high grade glioma model. METHODS: Drug release and preserved stability of the active TMZ molecule (AIC) was evaluated by fluoroscopic and LC-mass spectrometer based methods. In vitro cytotoxicity of released TMZ/ETOP was evaluated against high grade glioma cell lines and patient-derived primary cultures using metabolic assays. In vivo efficacy and overall survival were evaluated in the syngenic orthotopic 9L glioma rat model of intra cavity chemotherapy. RESULTS: TMZ and ETOP were released from PLGA/PEG alone or in combination over 2 weeks in vitro. Cytotoxicity of released drugs in vitro is comparable to directly applied agents, demonstrating the retained molecular integrity of TMZ/ETOP upon loading and releasing from PLGA/PEG. Both high (20% w/w TMZ / 50% w/w ETOP) and low (10% w/w TMZ / 25% w/w ETOP) doses were well tolerated in vivo, with no observable weight loss nor neurological deficits. Significant survival benefits from PLGA/PEG/TMZ/ETOP therapy were observed compared to surgery alone (49 vs. 14 days; p < 0.001), surgery with blank paste (49 vs. 14 days; p < 0.001) or surgery with daily oral TMZ (49 vs. 33 days; p < 0.004). CONCLUSIONS: This study demonstrates that interstitial delivery of TMZ/ETOP achieves significant glioma survival benefits. As such, PLGA/PEG paste applied to the post-surgical resection cavity offers a realistic opportunity for localised control of residual disease in pediatric and adult high grade gliomas.

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive, malignant tumors and are the most common malignant brain tumor in children under 6 months of age. Currently, there is no standard treatment for AT/RT. Recent studies have reported potential anti-tumoral properties of ribavirin, a guanosine analog and anti-viral molecule approved by the Food and Drug Administration for treatment of hepatitis C. We previously demonstrated that ribavirin inhibited glioma cell growth in vitro and in vivo. Based on these results and the fact that no pre-clinical model of ribavirin in AT/RT exists, we decided to investigate the effect of ribavirin on several human AT/RT cell lines (BT12, BT16, and BT37) both in vitro and in vivo. We provide evidence that ribavirin has a significant impact on AT/RT cell growth and increases cell cycle arrest and cell death, potentially through modulation of the eIF4E and/or EZH2 pathways. Interestingly, using scratch wound and transwell Boyden chamber assays, we observed that ribavirin also impairs AT/RT cell migration, invasion, and adhesion. Finally, we demonstrate that ribavirin significantly improves the survival of mice orthotopically implanted with BT12 cells. Our work establishes that ribavirin is effective against AT/RT by decreasing tumoral cell growth and dissemination and could represent a new therapeutic option for children with this deadly disease.

Tumor progression, limited efficacy of current standard treatments, and the rise in patient mortality are associated with gene expression caused by the synergistic action of intratumoral hypoxia and HIF-1α activation. For this reason, recent investigations have focused on HIF-targeting therapeutic agents, with encouraging preclinical and clinical results in solid tumors. Here we describe the efficacy of a HIF-1α inhibitor, Acriflavine, and demonstrate its potency against brain cancer. This safe antibacterial dye induces cell death and apoptosis in several glioma cell lines, targets HIF-1α–mediated pathways, and decreases the level of PGK1, VEGF and HIF-1α in vitro and in vivo. Administered locally via biodegradable polymers, Acriflavine provides significant benefits in survival resulting in nearly 100% long term survival, confirmed by MRI and histological analyses. This study reports preclinical evidence that this safe, small molecule can contribute to brain tumor therapy and highlights the significance of HIF-1α-targeting molecules.

INTRODUCTION: We previously developed a humanized mouse model of chordomas, which allowed us to investigate the interaction between human chordomas and human immune cells. This platform is particularly useful for studying immunotherapy against rare cancers such as chordomas, where murine equivalents are currently unavailable. We aim to utilize this model to study synergism between PD-1 blockade and LXR-623, a synthetic agonist of liverX receptors, a class of anti-neoplastic agents disrupting cancer cholesterol-metabolism. Our preliminary data suggest that LXR-623 downregulates PD-L1 on chordoma cell lines and reduces immunosuppressive myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. METHODS: To achieve immune system reconstitution/humanization, 20 NSG-SGM3 mice were engrafted with human fetal thymus and CD34+ stem cells, whose HLA-types were partially-matched with those of the U-CH1 chordoma cell lines. They were divided into the following groups (n=5 for each group): control group (isotype antibodies (Abs) + vehicle), LXR-623 monotherapy group (isotype Abs + LXR-623 100mg/kg, i.p., daily for 4 weeks), anti-PD-1 Abs monotherapy group (anti-human-PD-1-Abs (Clone: J116), 10 mg/kg, i.p., 3 times/week for 4 weeks), and combination group (anti-human-PD-1 Abs + LXR-623). Anti-tumor activities will be monitored via tumor size measurement, flow cytometric analyses of peripheral blood and spleen as well as chordomas, using various Abs including anti-mouse-CD45, anti-human-CD3, anti-human-CD4, anti-human-CD8, anti-human-CD11b, anti-human-CD14, anti-human-CD15, anti-human-CD19, anti-human-CD25, anti-human-CD45, anti-human-CD45RA, anti-human-CD45RO, anti-human-PD-1, and anti-human-FoxP3, multiplex cytokine analyses including IFN-gamma and TGF-beta, and multiplex-immunohistochemistry. We expect to observe the synergistic inhibitory effect of LXR-623 and anti-PD1-Abs against chordomas through PD-L1 downregulation and reduction in immunosuppressive MDSC levels mediated by LXR-623. We plan to sacrifice the last animal group on August 24th. Due to the lengthy nature of the experiment (humanization and treatment take 13–14 weeks), the data are not fully available at the time of submission but will be available by the late-breaking deadline.