Intracranial mesenchymal tumors, FET::CREB fusion-positive (ICMT), show fusions involving FET RNA-binding protein family genes (EWSR1 or FUS) and CREB family of transcription factors (ATF1, CREB1 or CREM). The methylation signature(s), gene expression characteristics and clinical behavior of this important tumor type require further characterization. We study the methylation profiles of 81 ICMT cases (61 newly profiled cases and 20 cases from publicly available sources). Clinicopathologic and genomic data were recorded for each case when available. ICMT showed a relatively distinct methylation signature compared to related tumors. Among the 65 cases where fusion types were documented, the identified fusions included EWSR1::ATF1 (25 cases), EWSR1::CREB1 (12 cases), EWSR1::CREM (21 cases), FUS::CREM (3 cases) and SMARCA2::CREM (4 cases). We confirmed the prior description of two distinct subgroups of ICMT (subclasses A and B). The majority of the cases belonged to subclass A (n = 69; 85%), which showed higher median age compared to subclass B patients (26 years vs. 15 years). Subclass B cases (n = 12; 15%) showed shorter progression-free survival (p < 0.01). Gene expression analysis of ICMT showed key overexpressed markers in ICMT, with significant CREM overexpression regardless of fusion type, when compared to either meningioma alone, or a larger group of CNS tumors. This work provides further characterization of ICMT as an important CNS mesenchymal neoplasm that is prone to tumor recurrence, showing 2 prognostically relevant methylation subclasses, and warranting diagnostic distinction from other epigenetically and histologically related tumors. ICMTs show substantial overexpression of the CREM gene, independent of fusion type.

High-grade meningiomas (HGMs) recurring after X-ray treatment show poor prognosis. We assessed the effectiveness and safety of boron neutron capture therapy (BNCT) in patients with refractory recurrent HGMs. This phase II investigator-led randomized controlled trial utilized an accelerator-based BNCT system to treat refractory recurrent HGMs. Patients were randomly assigned in a 2:1 ratio to the BNCT (12 patients) and control (6 patients) arms. Progression-free survival (PFS) judged by an independent third-party committee was the primary endpoint and PFS judged by the investigators and overall survival of the BNCT arm were the secondary endpoints. The control arm received rescue BNCT if they show disease progression. Three and two patients with World Health Organization (WHO) grade 3 disease were assigned to the BNCT and control arms, respectively; the remaining patients had WHO grade 2 disease. Median PFS (primary endpoint) was 14.4 months (95% confidence interval (CI): 7.9-26.4) in the BNCT arm and 1.4 months (95% CI: 1.0-9.0) in the control arm. Median PFS (secondary endpoint) was 14.7 months (95% CI: 7.6-22.8) in the BNCT arm and 1.5 months (95% CI: 1.0-9.0) in the control arm. The differences were statistically significant (log-rank test, P = 0.0157 and P = 0.0002, respectively). Five patients in the control arm received rescue BNCT. The objective response rate in the BNCT arm was 27.3%. BNCT is an effective treatment for refractory recurrent HMGs. Compared with conventional therapy, PFS in both primary and secondary endpoints were considerably improved.

Abstract Background High-dose methotrexate (MTX)-based chemotherapy is the mainstay of treatment of primary central nervous system lymphoma (PCNSL). Only ∼60% of patients achieve a complete response to first line therapy with frequent relapses. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has shown promising antitumor activity in recurrent/refractory PCNSL. Methods: The goal of the current single-center phase 2 trial was to explore whether the addition of ibrutinib to the combination of rituximab, methotrexate, procarbazine, and vincristine ((R-MVP/i) increases complete response rate (CCR). Results Thirty newly diagnosed PCNSLs were enrolled; median age 69 (range 41-79), median ECOG = 1. 29 patients completed R-MVP/i, 1 withdrew consent after 2 cycles. A CR/CRu was achieved in 29 patients and a partial response in 1 for a CRR of 29/30 (97%, 95% CI : 83.3%, 99.8%)). Treatment was well tolerated with no grade 5 toxicity was observed. Eight patients experienced 13 grade 4 toxicities (lymphopenia (n = 3), neutropenia (n = 4), thrombocytopenia (n = 3) white cell count decrease (n = 3)). The most common toxicities were thrombocytopenia, anemia, lymphopenia and liver enzyme elevations. No Aspergillus or Pneumocystis infections occurred. No refractory disease was observed. For the 29 patients completing the trial, 19 received consolidation with cytarabine (Ara-C), 8 autologous stem cell transplant, 1 rituximab maintenance and 1 was observed without maintenance or consolidation. At a median follow up of 25.1 months (range 3.3-49.2), the median progression-free (PFS) and overall survival (OS) was not reached with a 2-year PFS of 84.2% (95% CI: 62.7%-93.9%). Conclusions R-MVP/i was well tolerated and associated with excellent disease control and survival.

The prognosis of patients with recurrent WHO grade 4 glioma is poor, particularly in glioblastoma (GBM), which has a median survival of approximately 6 months and no effective treatment options. We evaluated the short-term (28-day) safety and efficacy of ON-01, an engineered recombinant oncolytic herpes simplex virus type-1, in patients with recurrent WHO grade 4 glioma. In this single-arm, phase 1/2 clinical trial, eligible patients received intratumoral injections of ON-01 under stereotactic guidance. The primary endpoint was to assess the short-term safety profile of ON-01 treatment. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and the 2-year OS rate. An exploratory objective was to identify tumor-related biomarkers predictive of treatment efficacy. Of the 30 patients treated with ON-01, 13 (43.3%) were male, and the median age was 50.0 years (range, 22-75). A total of 36 grade 1, 12 grade 2, and 2 grade 3 adverse events were reported. Among all treated patients, the median OS was 12.0 months (95% CI, 10.1-13.9), median PFS was 3.0 months (95% CI, 1.7-4.3), and 2-year OS rate was 27.7% (95% CI, 12.6%-45.0%). Seven patients with recurrent multifocal gliomas demonstrated regression of non-injection site lesions following ON-01 therapy. Furthermore, patients with elevated expression of herpesvirus entry mediator exhibited significantly prolonged survival (p=0.015). Intratumoral infusion of ON-01 appeared safe and demonstrated efficacy in patients with recurrent malignant glioma, with no evidence of neurotoxicity. The therapeutic response to ON-01 may be associated with HVEM expression levels.

Approximately 10% of cancers achieve replicative immortality through a telomerase-independent mechanism of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). ALT is particularly prevalent in certain subtypes of malignant gliomas, such as IDH-mutant astrocytoma and pediatric glioblastoma, and frequently co-occurs with ATRX inactivating mutations. Although ALT is an adaptive mechanism through which cancer cells achieve proliferative immortality, the elevated levels of replication stress observed in ALT tumors constitute a potential therapeutic vulnerability. Leveraging CRISPR/Cas9 screening data from the Cancer Dependency Mapping Project, coupled with patient-derived cell lines and xenografts, we identified SMARCAL1 as a novel synthetic lethal vulnerability in ATRX-deficient glioma models that engage ALT. Using complementary molecular assays for DNA damage, telomere maintenance, and telomeric replication stress, we define the mechanisms underlying cytotoxicity induced by SMARCAL1 depletion in ALT-positive glioma cells. Our data demonstrate the annealing helicase SMARCAL1 is a highly specific synthetical lethal vulnerability in cancers that use ALT. SMARCAL1 localizes to ALT-associated PML bodies in ALT-positive glioma cell lines, including IDH-mutant astrocytomas. SMARCAL1 depletion, via doxycycline-induced RNAi, led to a hyperactivation of the ALT phenotype, high levels of DNA double-strand breaks in G2 phase, and cell death via mitotic catastrophe. In mice bearing intracranial xenografts derived from high-grade IDH-mutant astrocytoma, inducible SMARCAL1 depletion prolonged animal survival. Our findings demonstrate that the molecular processes orchestrating ALT-mediated telomere maintenance constitute a targetable synthetic lethal vulnerability that can be exploited by SMARCAL1 inhibition, thus supporting the future development of small molecule inhibitors of SMARCAL1 as anti-cancer therapeutics.

Glioblastoma is characterized by heterogeneous and plastic cellular populations that adopt transcriptional programs shaped by genetic alterations and microenvironmental cues. Recent studies have identified at least four partially inconvertible cell states-astrocytic-like, neural progenitor-like, oligodendrocyte progenitor-like, and mesenchymal-like-that represent aberrant developmental programs. Expanded analysis further reveals hybrid and intermediate states that form continuous transcriptional and metabolic gradients. These states exhibit spatial organization, assembling into three distinct microanatomical niches: a perivascular niche enriched with mesenchymal-like and oligodendrocyte progenitor-like cells, a hypoxic niche harboring quiescent and stressed cells of all states, and an invasive niche containing astrocyte-like or proneural populations. Niches continuously remodel as cell states transition, migrate, and re-establish new programming in response to angiogenesis, hypoxia, immune infiltration, and neuronal activity. This interplay between states and the microenvironment generates a self-renewing spatial architecture, maintaining expansion at the edge and protection within the core. This review integrates single-cell, single-nucleus, and spatial studies to describe a microenvironmental-driven model of cell state organization. Understanding how these multiscale drives converge to generate a continuum of cell state identities may reveal strategies to disrupt the spatial architecture underlying glioblastoma plasticity and recurrence.

Cellular heterogeneity is a defining feature of glioblastoma (GBM), shaping tumor progression and therapeutic response. While single-cell profiling resolves this heterogeneity, it remains impractical for large-cohort studies and clinical implementation. Conversely, DNA methylation-based classification is widely used for GBM diagnostics but does not provide cellular resolution. We introduce a hierarchical non-negative matrix factorization approach (ITHresolveGBM) to deconvolute bulk DNA methylation profiles, inferring the abundance of glial, immune, and neuronal cells of the microenvironment, and further distinguishing differentiation states of malignant cells. Using ITHresolveGBM, we find that low tumor cell content impairs methylation-based classification, most notably linking the mesenchymal subtype with high immune cell infiltration. By integrating multi-omic single-cell data, we show that epigenetic deconvolution captures a malignant differentiation continuum ranging from stem-like to more differentiated tumors. This continuum aligns prior GBM classification systems and is associated with distinct molecular drivers (e.g., PDGFRA, TP53, EGFR) and survival outcomes. Our framework reconciles DNA methylation- and RNA-based classification systems and provides a blueprint for unifying bulk tumor profiles with single-cell biology, thereby refining molecular stratification and enhancing GBM diagnostics.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and accounts for 15% of childhood cancer death. The nucleosome remodeling and deacetylase (NuRD) complex is a major chromatin remodeling complex that regulates chromatin accessibility and gene transcription. However, its role in the pathogenesis of neuroblastoma remains poorly understood. The genetic dependency and clinical significance of MBD3 in neuroblastoma was evaluated by analysis of public datasets. The function of MBD3 in neuroblastoma cell growth was evaluated by shRNA knockdown experiment. Cleavage under targets and tagmentation sequencing (CUT&Tag-seq), coupled with RNA-sequencing, was employed to explore the mechanisms involved in the epigenetic regulation executed by NuRD decommissioning following MBD3 deficiency. Here we find that MBD3 is the most lineage-selective dependency among the non-enzymatic subunits of the NuRD complex in neuroblastoma. Knockdown of MBD3 induces cell cycle arrest and apoptosis, and inhibits neuroblastoma growth in vivo. Mechanistically, MBD3 deficiency leads to decommissioning of the NuRD complex and dissociation of the EZH2-PRC2 complex from chromatin, thereby orchestrating the epigenetic regulation of gene expression by modulating the balance between histone acetylation and methylation. NuRD decommissioning upon MBD3 deficiency selectively downregulates the expression of core regulatory transcription factors and upregulates a tumor suppressor SRCIN1, collectively suppressing neuroblastoma progression. Our data identify MBD3 and the NuRD complex as potential therapeutic targets in neuroblastoma, highlighting the critical role of epigenetic regulation in tumor maintenance. Targeting this pathway may offer a novel strategy to selectively impair neuroblastoma cell survival and improve outcomes.

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) approved for metastatic HER2+ and HER2-low/ultralow breast cancer. It has shown impressive clinical activity for HER2+ brain metastases. We conducted preclinical brain metastasis experiments to understand T-DXd efficacy. Nude mice were intracardiacly injected with either JIMT1-BR (HER2-2+) or SUM190-BR (HER2-3+) brain-tropic breast cancer cells and dosed with 3 or 10 mg/kg T-DXd or 10 mg/kg control-ADC, with endpoints of metastasis number and size, in both the metastasis prevention and treatment of established disease settings. In the JIMT1-BR model, T-DXd at both doses reduced metastasis number by 48-88% and size by 32-88%; a reduction of HER2 expression by lesions remaining at the experimental endpoint and heterogeneous T-DXd distribution were observed. A distinct dose effect was observed in SUM190-BR with the 3 mg/kg dose inhibiting size and number by 24-39% and 10 mg/kg by 72-79%; HER2 expression was maintained together with heterogeneous T-DXd distribution. In both models widespread reduced tumor Ki-67 was observed, while increased cleaved caspase-3 primarily costained with T-DXd. We used an in vitro model of the blood-brain- and blood-tumor barriers (BBB/BTB) to ask how T-DXd crossed. Data demonstrated T-DXd endocytosis and transcytosis of brain endothelial cells partially reliant on the neonatal Fc receptor (FcRn). BTB transcytosis was accompanied by increased endothelial RAB11FIP5 expression in vitro and in vivo. The data confirm T-DXd activity in HER2+ brain metastases and identify important correlates including heterogeneous uptake, variable HER2 expression at endpoint, tumor cell cytotoxicity, decreased proliferation, and BTB transcytosis.

Glioblastoma (GBM) is a highly aggressive brain tumor with profound metabolic heterogeneity. However, a clinically actionable classification based on metabolic gene expression remains undefined. We conducted a comprehensive multi-omics analysis of IDH-wildtype GBMs from three publicly available datasets. Prognostic metabolism-related genes were used to define transcriptional subtypes, which were validated in independent datasets and patient-derived cell (PDC) models. Functional assays and drug sensitivity studies were performed to explore therapeutic relevance. We identified three distinct metabolic subtypes: M1, enriched for synaptic signaling and amino acid metabolism, exhibited leading-edge anatomical features; M2, characterized by mitochondrial metabolism and cell cycle activity, was associated with favorable survival; and M3, marked by hypoxia, immune activation and suppression, and broad metabolic pathway engagement, correlated with poor prognosis. These subtypes were reproducible across cohorts and faithfully recapitulated in PDC models. Metabolomic profiling confirmed distinct subtype-specific metabolic signatures. Notably, M3 cells showed high sensitivity to inhibitors targeting glycosaminoglycan degradation, nicotinamide metabolism, and retinoic acid pathways in both in vitro and in vivo models. Our study defines three biologically and clinically relevant metabolic subtypes of IDH-wildtype GBM. This classification reveals distinct metabolic programs and therapeutic vulnerabilities, providing a framework for precision metabolism-targeted strategies in glioblastoma.