Pediatric central nervous system (CNS) tumors are the leading cause of cancer-related mortality in children. Development of more effective therapies for pediatric CNS tumors has been slow, underscoring an urgent need for novel and innovative approaches. This review summarizes current pediatric clinical trials of oncolytic viruses for pediatric brain tumors including high-grade glioma (HGG), diffuse midline glioma (DMG), medulloblastoma (MDB), atypical teratoid rhabdoid tumors (ATRT), and other high-grade tumors, while highlighting limitations of early-phase data, exploratory biomarkers, imaging challenges, pseudoprogression, and future directions. Key platforms include HSV-based agents (HSV1716, G207, and M032); adenoviral vectors (DNX2401, Ad-TD-nsIL12, and ICOVIR-5); MV-NIS (measles virus); PVS-RIPO (poliovirus); and Reolysin (reovirus). We review trial status, innovations in viral engineering and delivery, combinatorial strategies and translational challenges to establish oncolytic virotherapy as part of the future standard care for pediatric brain tumors. Oncolytic virotherapy or immunovirotherapy offers a promising strategy to selectively kill tumor cells and generate antitumor immune response while minimizing toxicity to healthy cells compared to conventional treatments. Although still emerging in pediatric neuro-oncology, preclinical studies and early-phase clinical trials show encouraging safety and efficacy signals. Not applicable.

Rosai-Dorfman disease (RDD) is a rare, non-Langerhans cell histiocytosis that classically presents with cervical lymphadenopathy. Neurological involvement occurs in less than 5% of cases and is often associated with extra-axial lesions. Diagnosis of RDD in the setting of neurological deficits is challenging, as RDD often mimics meningioma and neurosarcoidosis on neuroimaging. Herein, we describe a case report of a 50-year-old African American man who was previously treated with radiation for presumed meningioma of the brainstem 12 years prior to the current presentation. The brainstem lesion progressed to include the pachymeninges despite radiation, and the patient developed papilledema and involvement of the bilateral cochlea. Lack of response to radiation, involvement of the pachymeninges, and development of papilledema and bilateral hearing loss led to an updated diagnosis of neurosarcoidosis. On current presentation, the patient was brought to the emergency department for altered mental status, seizures, and left-sided hemiparesis. Initial workup was negative for stroke, meningitis, and toxic or metabolic encephalopathies. Long-term electroencephalography confirmed seizures originating from the right frontal lobe, which was controlled with antiseizure medications. Magnetic resonance imaging revealed worsening diffuse brainstem and pachymeningeal enhancement of the bilateral cerebral convexities, suggesting progression of the disease. Workup for systemic malignancy revealed a hypodense soft tissue mass encasing the left kidney, raising concern for metastatic disease. Biopsy of the meninges and the renal mass confirmed RDD with multiorgan involvement. Diagnosis of RDD in this patient marks the longest duration from onset to diagnosis ever reported, indicating the need for tissue diagnostics in uncertain cases.

Toxicity from IVT oHSV in the left lateral ventricle and third and fourth ventricles at different time points.

Comparison of the protective effect of different pretreatment low doses of IVT oHSV from toxicity after a subsequent treatment dose.

IHC staining of the ependymal lining for CD8+ cells and confirmation of CD8 depletion.

Body weights of CBA/J, BALB/c and C57BL/6 mice (N = 5/group) after inoculation with 1x107 PFU of IVT oHSV.

Toxicity with repeated doses of IVT oHSV.

Effects of poly I:C alone and combined with oHSV in vitro and in vivo.

Time to recover baseline body weight in mice pretreated with low-dose oHSV prior to a standard treatment dose.

Interferon β (IFN β) levels over time in the CSF after a single 50 µg IVT dose of poly I:C or saline (N = 2 mice/group/time point).

Glioblastoma (GBM) is an immunologically cold tumor, but several immunotherapy-based strategies show promise, including the administration of ex vivo expanded and activated cytotoxic gamma delta T cells. Cytotoxicity is partially mediated through interactions with natural killer group 2D ligands (NKG2DL) on tumor cells. We sought to determine whether the addition of the blood–brain barrier penetrant PARP inhibitor niraparib to the standard of care DNA alkylator temozolomide (TMZ) could upregulate NKG2DL, thereby improving immune cell recognition. Changes in viability were consistent with prior publications as there was a growth inhibitory effect of the combination of TMZ and niraparib. However, decreases in viability did not always correlate with changes in NKG2DL mRNA. ULBP1/Mult-1 mRNA was increased with the combination therapy in comparison to either drug alone in two of the three cell types tested, even though viability was consistently decreased. mRNA expression correlated with protein levels and ULBP1/MULT-1 cell surface protein was significantly increased with TMZ and niraparib treatment in four of the five cell types tested. Gamma delta T cell-mediated cytotoxicity at a 10:1 effector-to-target ratio was significantly increased upon pretreatment of cells derived from a GBM PDX with TMZ and niraparib in comparison to the control or either drug alone. Together, these data demonstrate that the combination of PARP inhibition, DNA alkylation, and gamma delta T cell therapy has the potential for the treatment of GBM.

Safety and preliminary efficacy have been demonstrated with intratumoral oncolytic, engineered HSV immunotherapy; however, few responses have been durable, suggesting that augmented, prolonged anti-tumor T cell responses are likely required to maintain tumor remission. We hypothesized that combining the direct oncolytic effect and innate immune stimulus of oHSV with a vaccine that fosters T cell mediated immunity would lead to more prolonged tumor responses. We examined the preclinical efficacy of combining intratumoral oHSV with an intravenous (via tail vein) self-assembling nanoparticle vaccine co-delivering peptide antigen to E7 and Toll-like receptor-7 and -8 agonists (TLR-7/8a), termed ‘SNAPvax™’, compared to either therapy alone or control in a TC-1 E7-expressing tumor model in immunocompetent mice. We also assessed how SNAPvax™ timing (before or after oHSV) affected efficacy, viral replication by viral recovery assay, and T cell activation and responses via intracellular cytokine staining of CD8+ T cells in the blood and tetramer staining. The oHSV-vaccine combination prolonged survival compared to control or either agent alone. Critically, survival was significantly enhanced when SNAPvax was given before oHSV compared to after oHSV. Increased effectiveness was associated with reduced tumor volume, increased tumor antigen specific CD8+ T cells, and amplified viral recovery. These results demonstrate the criticalness of combination immunotherapy timing with improved efficacy seen when vaccine was given prior to oHSV, which was mediated by tumor specific CD8+ T cells and increased viral replication. Our data provide preclinical support for translation of this novel combination therapy to clinical trial.

Oncolytic virotherapy or immunovirotherapy is a strategy that utilizes viruses to selectively infect and kill tumor cells while also stimulating an immune response against the tumor. Early clinical trials in both pediatric and adult patients using oncolytic herpes simplex viruses (oHSV) have demonstrated safety and promising efficacy; however, combinatorial strategies designed to enhance oncolysis while also promoting durable T-cell responses for sustaining disease remission are likely required. We hypothesized that combining the direct tumor cell killing and innate immune stimulation by oHSV with a vaccine that promotes T cell-mediated immunity may lead to more durable tumor regression. To this end, we investigated the preclinical efficacy and potential synergy of combining oHSV with a self-assembling nanoparticle vaccine codelivering peptide antigens and Toll-like receptor 7 and 8 agonists (referred to as SNAPvax),which induces robust tumor-specific T-cell immunity. We then assessed how timing of the treatments (i.e., vaccine before or after oHSV) impacts T-cell responses, viral replication, and preclinical efficacy. The sequence of treatments was critical, as survival was significantly enhanced when the SNAPvax vaccine was given prior to oHSV. Increased clinical efficacy was associated with reduced tumor volume and increases in virus replication and tumor antigen-specific CD8+ T cells. These findings substantiate the criticality of combination immunotherapy timing and provide preclinical support for combining SNAPvax with oHSV as a promising treatment approach for both pediatric and adult tumors.

Abstract Pediatric high-grade gliomas (HGGs) represent a significant clinical challenge with poor survival rates. Engineered oncolytic herpes simplex virus (oHSV) has shown potential in several clinical trials at targeting HGGs through their direct lytic effect and induction of an immune response. We have learned from our clinical trial experience (NCT02457845) that a critical barrier to more durable response is augmenting and sustaining the anti-tumor immune response generated by oHSV. LB-100, a protein phosphatase 2A (PP2A) inhibitor, is known to enhance T-cell activity. We hypothesized that LB-100 with anti-PD-1 therapy would overcome the immunosuppressive barriers, enhancing the immunotherapeutic effects of oHSV and improve survival in an immunocompetent murine model. We investigated the therapeutic impact of clinically available, next-generation oHSV, M002, which expresses murine interleukin-12, combined with LB-100 and PD-1 inhibition compared to M002 with LB-100 or anti-PD-1 antibody, each therapy alone, or control by measuring median survival and changes in the tumor immune microenvironment. The triple combination of M002, LB-100, and anti-PD-1 antibody significantly extended median survival compared to control (45 days vs 22.5 days, P=0.001) or each treatment alone, and compared to M002 and LB-100 (44 days vs 28 days, P=0.017) or M002 and anti-PD-1 (45.5 days vs 41 days, P=0.023). This increase in survival was correlated with a robust increase in CD45+ immune cell infiltration and a reduction in T-regulatory cells. We will further investigate the changes in the tumor microenvironment to better understand the specific contributions of different immune cell populations. These data support the hypothesis that LB-100 enhances the efficacy of oHSV by amplifying T-cell-mediated immune responses against tumor cells. This combination therapy demonstrates a promising strategy that warrants further exploration and potential clinical application, emphasizing the critical role of targeting multiple aspects of immune regulation to enhance the efficacy of oncolytic virotherapy in pediatric tumors.

Jisu Kim, MD; Sang Mee Lee; Da Eun Kim; Sungjin Kim, MD; Myung Jin Chung, MD, PhD; Zero Kim, MD, PhD; Taeyoung Kim, PhD; Kyeong-Tae Lee, MD, PhD

Drug repurposing is promising because approving a drug for a new indication requires fewer resources than approving a new drug. Signature reversion detects drug perturbations most inversely related to the disease‐associated gene signature to identify drugs that may reverse that signature. We assessed the performance and biological relevance of three approaches for constructing disease‐associated gene signatures (i.e., limma, DESeq2, and MultiPLIER) and prioritized the resulting drug repurposing candidates for four low‐survival human cancers. Our results were enriched for candidates that had been used in clinical trials or performed well in the PRISM drug screen. Additionally, we found that pamidronate and nimodipine, drugs predicted to be efficacious against the brain tumor glioblastoma (GBM), inhibited the growth of a GBM cell line and cells isolated from a patient‐derived xenograft (PDX). Our results demonstrate that by applying multiple disease‐associated gene signature methods, we prioritized several drug repurposing candidates for low‐survival cancers.

Active learning and peer instruction contribute to positive learning outcomes. We developed a 25-week, question-based program for first-year medical students (MS1). Senior students developed weekly question and answer sets. Second-year peer educators helped MS1s learn to collaboratively problem solve by working through the questions and answer explanations. Controlling for average MCAT 2015 score, attendance significantly correlated with improved academic performance in basic science coursework (beta = 0.196, p < .001) and one organ systems module (beta = 0.104, p = .033). Academic outcomes and an 83

Abstract RATIONALE Outcomes for patients with high-grade glioma (HGG) are dismal despite standard of care surgical resection, radiation, and temozolomide (TMZ) chemotherapy. RESULTS from our Phase I trial of pediatric patients with recurrent HGG suggest oncolytic herpes simplex virus-1 (oHSV) is safe with promising efficacy. However, optimization of oHSV therapy is needed to achieve more durable responses. Based on our prior work with matched pair HGG cell lines and patient-derived xenografts (PDXs) with and without acquired TMZ resistance, oHSV efficacy is enhanced in TMZ-resistant cells with no difference in oHSV entry receptor (CD111) expression. Microarray transcriptomic data of four PDXs with and without TMZ resistance revealed downregulation of interferon (IFN) responses. Cytochrome c oxidase subunit 4 isoform 1 (COX4I1) is expressed in recurrent HGG samples and cell lines. While COX4I1 positively regulates influenza virus replication, no study has investigated the role of COX4I1 in mediating oncolytic virotherapy efficacy. HYPOTHESIS We hypothesized that COX4I1 expression mediates HGG sensitivity to oHSV through suppression of IFN signaling. METHODS Using previously published models, oHSV cytotoxicity was assessed in vitro using Alamar Blue and CellTiter-Glo assays. oHSV infectivity and CD111 protein expression was assessed by flow cytometry. COX4I1 protein expression was measured by immunoblot. mRNA expression of IFN-stimulated genes was assessed by qPCR. RESULTS COX4I1 overexpression increased oHSV cytotoxicity and infectivity compared to empty vector controls. Short hairpin RNA knockdown of COX4I1 decreased oHSV cytotoxicity and infectivity in TMZ-resistant cells compared to pLKO controls. IFN-stimulated genes IFNB1, TMEM173, DDX58, IFIH1, and OAS1 were all significantly decreased in COX4I1 overexpressing cells and TMZ-resistant cells compared to parental lines. CONCLUSIONS Overexpression of COX4I1 sensitizes HGG cells to oHSV, and COX4I1 knockdown de-sensitizes TMZ-resistant HGG cells to oHSV, suggesting a novel role of COX4I1 in mediating HGG sensitivity to oHSV.