Daniels, David J.

Abstract BACKGROUND CNS ependymal tumors rarely demonstrate mixed subependymal and ependymal pathology. The clinical course of these tumors is difficult to predict, but prior studies have identified TERT mutations and loss of chromosome 6 as poor prognostic factors in these tumors when located in the posterior fossa. METHODS In this retrospective single-institution study, patients with a diagnosis of mixed subependymoma/ependymoma were identified and chart reviewed to describe demographics, pathological findings, treatment courses and clinical outcomes. RESULTS Eleven patients (median age 57, 2 female, 9 male) were identified. Ten tumors were in the posterior fossa and 1 in the cervical spine. At diagnosis, 2 underwent gross total resection, 8 underwent subtotal resection, and 1 was only biopsied. Radiotherapy was administered to 5 patients at initial diagnosis (54Gy in 30 fractions). Systemic therapy (temozolomide and bevacizumab) was administered at recurrence to 2 patients. Loss of chromosome 6 was present in 5 /11 patients and TERT mutations were present in 4/11 patients, with both present in 3/11 patients. Median progression-free survival (mPFS) was 90.1 months (95% CI: 90.1–NA), with 3 progression events observed at 22, 90, and 107 months. Median follow-up duration was 27.9 months. Receiving radiotherapy had a longer mPFS (98 vs 22 months, p=0.15), although non-significant. At the time of data cut-off none of the patients were deceased. CONCLUSIONS Mixed subependymoma/ependymoma is a rare diagnosis with varying clinical outcomes. Our case series demonstrated that the majority of patients did not exhibit the previously identified poor prognostic factors. Although non-significant due to limited case numbers, radiotherapy was suggestive of longer mPFS.

Abstract Pontine H3 K27-altered diffuse midline gliomas (DMGs) are rare, fatal pediatric brain tumors resistant to systemic therapies due to the blood-brain barrier (BBB) and efflux transporters (ETs), which actively clear drugs from the brain. Convection-enhanced delivery (CED) bypasses the BBB by directly infusing therapeutics into the tumor, but maintaining adequate drug concentrations remains a challenge. This study explores strategies to enhance drug-tumor residence time and improve treatment efficacy. Using chemical modeling, FDA-approved drugs identified in prior high-throughput screening efforts were classified based on physiochemical properties impacting CED pharmacokinetics, including BBB penetration and ET substrate status. Drug behavior was validated in patient-derived in vitro and in vivo DMG models. The impact of systemic drug co-administration on drug retention and therapeutic efficacy was evaluated using multiple patient-derived orthotopic xenograft (PDOX) models. ET substrate drugs exhibited rapid elimination from the brain post-CED, limiting their therapeutic potential. However, systemic everolimus administration prolonged drug retention and potentiated the effects of efflux substrate drugs delivered via CED (e.g., topotecan, ponatinib). This combinatorial approach significantly enhanced anticancer effects in multiple PDOX models, demonstrating improved tumor control compared to monotherapy. Overall, these findings suggest that pharmacological inhibition of ETs via systemic everolimus administration can enhance drug retention in DMG when combined with CED. The observed synergistic effects highlight a promising strategy to overcome drug clearance limitations and improve treatment outcomes. Further clinical investigation is warranted to validate this approach in patients.

Abstract Diffuse midline and diffuse hemispheric glioma contain the driver mutations H3.3 K27M and G34R/V, respectively. These mutations alter histone methylations, suggesting that dysregulation of gene expression may contribute to tumor formation. We recently reported that these mutations also inhibit the phosphorylation of H3.3 at Serine 31. Importantly, H3.3 S31A mice developed high-grade gliomas – similar to those from G34R and K27M mice – indicating that loss of phosphoS31 promotes gliomagenesis. Chk1, a key kinase in DNA damage response and mitotic fidelity, phosphorylates H3.3 S31. H3.3 S31 is phosphorylated at the pericentromere in metaphase and is typically absent during interphase. Reduction of S31 phosphorylation by H3.3 K27M, G34R, G34V, or S31A was sufficient to induce mitotic errors leading to aneuploidy. Using CRISPR/Cas9, we generated novel H3.3 WT/WT and WT/S31A lines from glioma patient-derived SF-8628 (H3.3 WT/K27M) and KNS-42 (H3.3 WT/G34V) cells. Immunofluorescence of metaphase spreads revealed H3.3 phosphoS31 at the pericentromeres as expected. Interestingly, total Chk1 and active Chk1 (pS296) were significantly enriched at the pericentromeres of H3.3 K27M and G34V cells, as compared to H3.3 WT cells. While total ATR and active ATR (pT1989) were present at the pericentromere, their levels were unaltered by the H3.3 mutations. Surface plasmon resonance of purified proteins revealed that Chk1 has two binding affinities for H3.3 - likely corresponding to the previously identified Chk1 phosphorylation sites (T11 and S31) on H3.3. Chk1 binding at one site was unaffected by H3.3 K27M, while the second site showed an altered dissociation from H3.3 K27M. In summary, our data suggest that the low pericentromeric pS31 levels associated with H3.3 glioma mutations result from Chk1 recognizing the mutant histone, but without completing a phosphorylation cycle, becoming trapped at the pericentromere. This result suggests that H3.3 mutations, by trapping Chk1, may impede Chk1 activities beyond just reducing H3.3 phosphorylation.

OBJECTIVE Neurosurgeon scientists play a unique role in advancing neuroscience research. While previous publications have explored trends in federal and foundation funding among neurosurgeons, funding is often dominated by neurosurgical oncologists and functional neurosurgeons. Less is known about the research efforts of pediatric neurosurgeons. The aim of this study was to survey the members of the American Society of Pediatric Neurosurgeons (ASPN) to provide an overview of past research experience, current involvement, funding, and research priorities among pediatric neurosurgeons, and to gather insights that could shape future efforts to advance pediatric neurosurgical research. METHODS A survey was developed using the REDCap platform and distributed to all ASPN members via email. Survey questions used branching logic and were organized into 5 sections: 1) demographics, 2) research experience during training, 3) research experience as an attending physician, 4) research priorities, and 5) multicenter consortiums. RESULTS One hundred thirty-nine respondents completed more than half of the survey, for an overall response rate of 52.1%. Most respondents (96.4%) participated in research during their training, but only 38.1% had received a grant during training. In contrast, 83.9% of respondents were actively engaged in research as an attending physician, and 48.7% reported active funding (60.7% federal, 41.8% from foundations, and 42.9% internal). Furthermore, 74.8% of respondents reported being a member of a multicenter research consortium, and 82.4% agreed that multicenter research is important. Seventy percent of respondents agreed that the ASPN should facilitate multicenter consortium-based pediatric neurosurgical research, offering free-text responses with the following suggestions: 1) set aside time at the annual meeting to discuss multicenter research (22.9%); 2) encourage collaboration and facilitate networking (42.9%); 3) provide centralized core services such as a data coordinator and biostatistician (12.9%); and 4) provide training, education, and mentoring (7.1%). CONCLUSIONS The survey provided a cross-sectional analysis of the pediatric neurosurgical research landscape, highlighting the current state of research experience, funding, and the perspectives of pediatric neurosurgeons regarding research priorities. Despite the challenges, there is clear recognition of the importance of multicenter research collaboration. These findings reinforce the ongoing necessity of organized initiatives to support pediatric neurosurgical research and offer actionable insights into how organized pediatric neurosurgery can contribute to this critical endeavor.

Abstract Background Atypical teratoid rhabdoid tumor (ATRT) is a deadly central nervous system embryonal tumor caused by loss of SMARCB1, a core subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes. SMARCB1-deficient cancers are defined by loss of cell differentiation-associated enhancers, but how SWI/SNF interacts with other arbiters of cell differentiation (specifically lineage-specific transcription factors [TFs]) remains poorly understood. Methods We leveraged a multi-omics approach, patient-derived ATRT cells, and patient-derived orthotopic xenografts to investigate the interplay of SWI/SNF with lineage-specific TFs in a clinically relevant setting. Results We observe that an activating protein 1 (AP-1)-dependent transcriptional regulatory network is lost in ATRT, and AP-1 and lineage-specific TFs TEAD1 and ZIC2 require SMARCB1 for enhancer binding. SMARCB1-dependent SWI/SNF integrates transcriptional functions of lineage-specific TFs into a core regulatory circuit that depends on the AP-1 subunit c-JUN, whose expression is determined by a SMARCB1-dependent super-enhancer that is lost in ATRT-MYC. In the absence of SMARCB1, lineage-specific TFs are sequestered to promoters, where they maintain core transcriptional programs necessary for cell survival. Targeting residual, promoter-proximal TF activity by a protein degrader of the SWI/SNF ATPase SMARCA4 or small-molecule inhibitors that indirectly inhibit AP-1 and TEAD activity abrogates expression of these networks, reducing cell viability in vitro and prolonging survival in an orthotopic patient-derived xenograft model. Conclusions These results demonstrate SWI/SNF complexes are critical for lineage-specific TF binding and activity at both promoters and enhancers. In the context of ATRT, these findings reveal a previously underappreciated therapeutic vulnerability in targeting residual promoter-proximal TF function in ATRT.

Abstract The prognosis for pediatric high-grade gliomas associated with mutations in the H3-A3 histone gene is very poor. To investigate whether peptide-pulsed dendritic cells (DC) together with checkpoint blockade might be a potential treatment modality for diffuse hemispheric glioma, H3 G34-mutant (DHG), we have developed a novel mouse model, and utilized a novel bioinformatics pipeline to identify tumor-associated peptide antigens (TAs). We used the RCAS/tv-A system to target the expression of H3/G34R and PDGFb, and to knock out p53 in neural progenitors in transgenic C57BL/6 neonatal mice. Three independent cell lines were obtained that expressed transcripts associated with oligodendrocyte and interneuron lineages, and formed lethal tumors after intracranial implantation. To identify TAs, we implemented a new computational workflow to identify nonapeptides generated from alternative splicing of mRNAs that were highly enriched in the tumor transcriptomes of two DHG patients, and predicted to bind the HLA-A*2:01 class I allele carried by these patients. Three of the candidate peptides were conserved in the mouse and also predicted to bind the H2-Kb MHC I allele carried by C57/BL6 mice. Using our syngeneic mouse model, we showed that DCs pulsed with these peptides together with anti-PD1 mAb provided a significant survival benefit compared with checkpoint inhibitor and DCs pulsed with peptides that were not predicted to bind the H2-Kb allele. Splenocytes isolated from successfully treated mice were analyzed through bulk TCR sequencing, and in comparison with control mice and publicly-accessible TCR libraries, 6 highly-represented TCR clonotypes were identified only in treated mice. In summary, we demonstrate that effective TAs can be extracted from RNAseq data, and can be targeted with DC vaccination to generate a systemic anti-tumor cytotoxic T cell response to eliminate DHG in a novel immunocompetent mouse model.

Abstract Advancements in understanding H3 K27-altered diffuse midline glioma (DMG) biology have not yet translated into effective therapies for these tumors likely due to delivery challenges across the blood-brain barrier (BBB) and variable tumor genetics. This study systematically probes convective-enhanced delivery (CED) as a means to administer small molecule drugs directly to DMG tumors, circumventing constraints imposed by the BBB. Our findings emphasize the necessity of strategic drug selection and pharmacokinetic analysis in refining CED approaches, advocating for precision medicine techniques specialized for DMG. We demonstrate that while most small molecule drugs are rapidly cleared from the brain following CED, their retention and effectiveness are substantially improved by co-administration of systemic efflux transporter (ET) inhibitors such as everolimus. The synergistic potential of combining everolimus with topotecan or ponatinib offers a novel therapeutic pathway, advocating for further investigation into this DMG management strategy.