Osteosarcoma (OS) is the most common human primary bone cancer, primarily affecting children and young adults. While the survival rate of patients diagnosed with localized OS is ∼65%, this decreases to ∼20% for patients with metastatic disease, and recurrent disease remains largely incurable. Therefore, identifying new therapeutic strategies is urgently needed for metastatic and refractory OS. This study analyzes the surfaceomes and global proteomes of 22 unique OS patient-derived xenografts (PDXs) using mass spectrometry to identify surface proteins for potential immunotherapeutic targeting. Both methods identify known OS-associated surface candidates including LRRC15, MMP14, MRC2, and CADM1, and several poorly characterized targets, including ROR2 and TMEM119. Both ROR2 and TMEM119 display robust expression in OS tissues but only limited or no expression in normal pediatric tissues, and loss of both targets reduces the migration of OS cells. These data provide a resource of surface proteins as potential immunotherapeutic targets in OS.

Abstract Background Relapsed or refractory (RR) pediatric CNS embryonal tumors, including medulloblastoma (MB), embryonal tumor with multilayered rosettes (ETMR), atypical teratoid/rhabdoid tumor (AT/RT), and pineoblastoma, have dismal outcomes with 5-year overall survival ranging from ∼10% in RR-MB to < 1% in ETMR and AT/RT. Glypican-2 (GPC2) is an oncofetal antigen and promising immunotherapeutic target expressed in pediatric cancers including CNS embryonal tumors. Preclinical studies demonstrating potent antitumor activity of GPC2-CAR T cells in MB models prompted initiation of a first-in-human Phase I clinical trial (NCT07087002) evaluating their feasibility and safety using intracerebroventricular administration in children and young adults with RR-CNS embryonal tumors. Methods Because antigen density is critical for GPC2-CAR T-cell potency, enrollment is restricted to GPC2-positive disease, predicted to exceed CAR detection thresholds using an H-score of > 100 as surrogate eligibility cut-off. Tumor specimens undergo CLIA-certified GPC2 immunohistochemistry (IHC) and are scored using a standard H-score (0–300) derived from staining intensity (0–3+) and percentage of GPC2-positive tumor cells. In parallel, an advanced scoring method segmenting intratumoral intensity differences (formula = (1 × % weak) + (2 × % moderate) + (3 × % strong)) is performed to assess potential clinical utility. Results To date, twelve patients were screened; 75% met eligibility by standard H-score, and two patients have enrolled. Screened tumors include MB (n = 2/median H-score 190), ETMR (n = 4/median 170), pineoblastoma (n = 2/median 200), and AT/RT (n = 3/median 2). Except for AT/RT, all tumors exceeded the enrollment threshold by either scoring method, with advanced H-scores trending higher than standard scores (p = 0.0977). Conclusion These findings demonstrate feasibility of GPC2 antigen prescreening using archival tumor tissue to support early-phase trial enrollment, enabling coherent safety and early efficacy assessment of GPC2-CAR T-cells in pediatric CNS Embryonal tumors. Larger cohorts are needed to determine whether advanced scoring provides added benefit in borderline cases.

Gene fusions generated by chromosomal rearrangements function as oncogenic drivers in human cancers. We previously showed that EWSR1-ETS oncofusions of Ewing sarcoma directly induce surface expression of IL1 receptor accessory protein (IL1RAP), which along with limited expression in healthy tissues except in the placenta nominate IL1RAP as a promising Ewing sarcoma immunotherapy target. We therefore engineered antibody-drug conjugates (ADC) with different cytotoxic payloads to target IL1RAP. ADCs potently blocked tumor growth and induced durable regression of Ewing sarcoma xenografts in mice and diminished metastatic dissemination in vivo. Moreover, we show that other oncofusions also induce IL1RAP expression in diverse cancers, including NPM-ALK in anaplastic large cell lymphoma and ETV6-NTRK3 in multiple tumor types. IL1RAP expression rendered these malignancies similarly vulnerable to IL1RAP-targeting ADCs, which effectively blocked the growth of ALCL xenografts and syngeneic ETV6-NTRK3+ sarcomas. Lack of detectable normal tissue toxicity, including in nonhuman primates, supports the further clinical translation of IL1RAP-targeting ADCs. SIGNIFICANCE:The IL1RAP surface protein is induced by multiple oncogenic fusions, including EWSR1-ETS, NPM-ALK, and EN in distinct malignancies, rendering these cancers vulnerable to anti-IL1RAP ADCs. We demonstrate that IL1RAP-targeting ADCs have potent efficacy in both primary and metastatic diseases and are well tolerated in nonhuman primates, warranting their further clinical translation.

A pathological role of α-synuclein (aSyn) aggregation in the etiology of Parkinson disease (PD) is well established. Here, we applied spatial transcriptomics (ST) on brain sections derived from a rodent mouse model of α-synucleinopathy (transgenic M83+/+ line). Our ST data revealed that induction of aSyn pathology in the brainstem of rodents triggered upregulation of pathways controlling energy metabolism. At a later stage, characterized by movement disability, the ST data indicated a drastic downregulation of mitochondrial metabolic pathways along with perturbed expression of mRNA translation machinery. Furthermore, analyses of microarrays datasets derived from 4 independent cohorts of PD patients led to the identification of aberrant osteopontin (SPP1) signaling and increased expression of CREB-binding protein as consistent markers associated with the progression of aSyn pathology in the rodent model and in PD brains. We anticipate that our findings hold promise for biomarker discovery and/or mechanism-based therapies in PD and related neurodegenerative disorders.

Tumors must adapt to high levels of endoplasmic reticulum (ER) stress to sustain tumor growth and metastases. The chaperone GRP78 (BiP/HSPA5) is a key component of the unfolded protein response (UPR) and essential for ER stress management and adaptive signaling supporting pro-survival UPR activities. Here, we report that oncofetal chondroitin sulfate (CS) glycosaminoglycans are required for ER stress adaptation in osteosarcoma. When osteosarcoma cells encounter ER stress, they upregulate 4-O-sulfated CS at the expense of other glycosaminoglycans leading to a reconfiguration of the glycocalyx in favor of an oncofetal CS subtype. Genetic ablation of the CS synthesis pathway impairs the UPR by preventing osteosarcoma cells from mounting GRP78 expression in response to ER stress. CS deficiency makes osteosarcoma cells hypersensitive to inhibition of GRP78 under both ambient and ER stress conditions, and acute ER stress drives CS-defective osteosarcoma cells into an apoptotic cell death that can be rescued by re-instating CS 4-O-sulfation capacity. This has direct implications for the metastatic progression of osteosarcoma, whereby oncofetal CS protects dissociated osteosarcoma cells from anoikis to allow pulmonary colonization in mice. Our data identify CS glycosaminoglycans as a critical component of the UPR that permits osteosarcoma cells to manage ER stress.

Posterior fossa type A (PFA) ependymoma is an unusual infantile brain tumour with few known somatic mutations, thought to be driven by epigenetic mechanisms1. PFA ependymoma has a markedly higher incidence and worse prognosis in male children than in female children2. The mechanisms that underlie these sex differences are at present unknown. Here we show that the cellular hierarchy of PFA ependymoma is less differentiated in male individuals than it is in female individuals. In the normal developing mouse hindbrain, male gliogenic progenitors are less differentiated than matched female sibling controls. To further parse the effects of chromosomal versus gonadal contributions in the male hindbrain, we used the four-core genotype mouse model3, which showed that androgen signalling, rather than sex chromosomes, prolongs hindbrain differentiation in male mice. Androgen supplementation promotes the growth of PFA ependymoma, but not that of other brain tumours. Conversely, androgen blockade diminishes both the stem-like potential and the proliferation of PFA ependymoma. We conclude that androgen signalling in both the normal developing hindbrain and PFA ependymoma is sufficient to promote growth and delay differentiation. Anti-androgen therapies represent a potential clinical avenue to target this currently untreatable childhood cancer.

Background Relapsed medulloblastoma remains a significant therapeutic challenge as it is near universally fatal. The tumor microenvironment of medulloblastoma plays a critical role in tumor progression, influencing tumor growth, immune evasion, and therapeutic resistance. We hypothesized that defining tumor-immune interactions in diagnostic and relapsed medulloblastoma may uncover mechanisms of immune evasion and identify novel therapeutic targets.Methods We analyzed paired primary and recurrent RNA-sequencing data from 140 medulloblastoma patients to profile immune cell composition and validate spatial relationships within the TME. To identify key tumor-immune interactions, we developed a novel algorithm to detect receptor-ligand pairs using single-cell RNA-sequencing data. These interactions were validated across RNA and proteomic datasets. Their functional significance was empirically demonstrated in newly developed immunocompetent models of recurrent medulloblastoma that closely recapitulate the human disease.Results We observed a shift toward a heightened immunosuppressive TME at relapse. Using our algorithm, we identified biologically significant receptor-ligand interactions, most notably MIF-CD74, constitutively expressed at RNA and protein levels across medulloblastoma subgroups, at diagnosis and relapse. Disrupting MIF-CD74 interactions led to significant alterations in the tumor microenvironment, highlighting its functional significance.Conclusions Our multifaceted approach identified key tumor-immune interactions in medulloblastoma. Among these, MIF-CD74 was validated as a targetable interaction, demonstrating the utility of our integrative approach for identifying novel therapeutic targets across multiple tumor types.

Distinct molecular variants of the brain cancer ependymoma are distributed along the rostral-caudal extent of the central nervous system (CNS). Historically proposed to arise from ventricular ependyma, recent studies have suggested conflicting cellular origins, including the neural radial glia and the roof plate lineages. Using single-cell transcriptomics, immunohistochemistry, and lineage tracing, we demonstrate that ependymomas across all CNS compartments transcriptionally mirror MSX1 +ve pre-neural crest/roof plate (Pre-NC/RP) lineage derivatives. Ependymoma subgroups recapitulate the spatial and molecular diversity of regional Pre-NC/RP populations, while retaining conserved MSX1 expression. Expression of the oncogenic fusion ZFTA-RELA within the murine Pre-NC/RP lineage generated tumors that faithfully resembled human ependymoma. These findings identify a common embryonic cellular origin for ependymomas and reconcile previously conflicting models of tumorigenesis.

MYCN amplification is associated with poor outcomes in neuroblastoma (NB). MYCN encodes a transcription factor that induces the expression of mitochondrial one-carbon enzymes and chaperones, promoting metabolic reprogramming and aggressiveness in NB tumor cells. While the contribution of MYCN to changes in the mitochondrial proteome is well established, how mitochondrial proteostasis contributes to MYCN activity remains poorly understood. Mitochondrial HSP60 (HSPD1) is a conserved mitochondrial chaperone promoting the mitochondrial one-carbon pathway and mitochondrial translation by folding the one-carbon enzyme MTHFD2 and preventing aggregation of mitochondrial ribosomal proteins. Here, we show that HSPD1 depletion in MYCN-amplified NB cells led to reduced mitochondrial translation, MYCN expression, and tumorigenesis in vitro and in vivo. In addition, HSPD1 mRNA and protein correlate with MYCN expression in NB tumors. These results establish HSPD1 as a promising target for the treatment of MYCN-amplified NB.

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor and is comprised of four molecular subgroups (WNT, SHH, Group 3, and Group 4). Regardless of subgroup, MB patients are treated with the same standard of care (SoC) (surgical resection, chemotherapy, craniospinal irradiation) that can lead to lifelong neurocognitive deficits in survivors. A subset of Group 3 MBs harbors focal amplifications of the MYC oncogene (MYC-G3MB) that invariably lead to disease recurrence which currently remains incurable. Therefore, there is an urgent need for development of therapies that are safe for the vulnerable developing brain while exerting potent anti-tumor efficacy against therapy-resistant subtypes. Here, we identify nuclear envelope (NE) proteins LBR and TMPO to be highly expressed in MYC-G3MBs in contrast to normal tissue and human neural stem cells (hNSCs), and a context-specific mislocalization of NE proteins to the plasma membrane (PM), only in MYC-G3MBs. NE protein expression correlated to worse MB patient prognosis and survival and was significantly enriched in recurrent tissue compared to the patient matched primary samples. High resolution microscopy and transcriptomic analysis implicated hyperproliferative cell states and endogenous chromosomal instability to drive NE deformation and the creation of DNA double strand break-capturing nuclear envelope tubules (dsbNETs), critical recently described factors in genome stability that mediate the repair of rapidly accumulating DNA damage in cancers such as MB. CRISPR Cas9-mediated endogenous tagging of LBR and TMPO revealed exposure of N-termini to the extracellular matrix and linked ER or ER-like vesicles to be directly trafficked to the cell surface in MYC-G3MBs. We developed single domain antibodies (sdAbs) specific to the N-terminus of LBR and TMPO and engineered second generation chimeric antigen receptor (CAR) T cell mono (LBR or TMPO) and bispecific (LBR and TMPO) therapies. We demonstrate the potent anti-tumor efficacy of NE protein-targeted CAR T cell therapies in patient derived xenograft models (PDX) of MYC-G3MBs. We present nuclear envelope proteins aberrantly mislocalized to the plasma membrane exclusively in G3MB as a novel class of cancer-selective therapeutic targets, for the development of a new class of CAR T cell therapies for treatment-refractory childhood MB. Citation Format: Sheila Kumari Singh, Yujin Suk, Martin A. Rossotti, Jorge Ibañez-Vega, Muhammad Vaseem Shaikh, Yiyun Chen, Hardikkumar Patel, Laura Escudero, Alberto Delaidelli, Aapti Khanna, Sarah Slassi, Carlos Barba Bazan, Erika Apel, Stefan Custers, Shan Grewal, William Maich, Lucas Asselstine, Dillon McKenna, Yuxi Xiao, William Gwynne, Jason Moffat, Hiromichi Suzuki, Poul H. Sorensen, Ray Truant, Kevin Henry, Elena Sotillo-Piñeiro, Chitra Venugopal, Crystal L. Mackall, Giedre Krenciute. Surface localized nuclear envelope proteins define a therapeuticvulnerability in MYC-driven Group 3 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4028.

Abstract Key determinants for efficacious CAR-T antigens remain near absence in vital tissues and that antigen levels in clinical specimens exceed CAR-T detection thresholds. CAR-T cells meeting these criteria have demonstrated clinical benefit in aggressive pediatric brain tumors, as seen in H3K27M-mutant diffuse midline glioma responding to GD2-directed CAR-T cell therapy. Medulloblastoma (MB) is a devastating childhood brain tumor, for which standard-of-care is fails in 30% of patients, and leaves many survivors with debilitating long-term effects. Survival rates of recurrent disease reside below <10%. Increasing cure rates and diminishing treatment-related morbidity necessitates targeted therapies, but success using CAR-T cells in MB has been limited by a dearth of suitable cell-surface targets. We profiled the CAR-T antigen landscape of MB through integrating unbiased surfaceome analysis from tumor and normal tissue transcriptomes and quantitative antigen density mapping of candidate targets on n= 24 MB tumor biopsies across all molecular subtypes. We found that MB closely resembles prenatal brain and credentialled oncofetal antigens as the predominant target pool in MB. A quantitative flow cytometry-based assay to precisely quantify antigen densities of candidate targets in clinical specimens prioritized GPC2 as the top candidate. GPC2 antigen densities ranged from ∼1700-24500 molecules/cell across MB subgroups (mean: 6374 +/-4968). Because antigen density greatly influences CAR-T potency, we previously engineered GPC2-CAR-Ts tuned towards clinical densities on neuroblastoma and demonstrated that cJUN-overexpression (OE) augments their potency by lowering antigen detection thresholds (Heitzeneder et. al, 2022). Here, we employ orthotopic xenograft models and locoregional CAR-T delivery to study the interplay between GPC2 antigen density and CAR potency and to inform a phase-1 trial design. We found that both GPC2-CAR-T constructs (+/-cJUN) achieved durable disease control against GPC2-intermediate GR4-MB (ICB1299: ∼9000 mol/cell and MBT375, a newly established PDX: ∼6500 mol/cell). In a highly aggressive, MYC-amplified, GPC2-high GR3-MB model (SU_MB002: ∼18800 mol/cell), recurrence occurred in 3/5 mice post GPC2-CAR, whereas repeated dosing or cJUN-OE significantly improved persistence and long-term anti-tumor control. In a GPC2-low, GR3-MB model (HDMB03: ∼3000 mol/cell) used to further dissect antigen detection limits, only cJUN.GPC2-CAR-T cells maintained anti-tumor responses. This work informed an ongoing phase-1 clinical trial at Stanford (NCT07087002), currently testing repeated, locoregional GPC2-CAR-T infusions and is planned to employ cJUN overexpression in patients with recurrent/refractory MB, aiming to provide a targeted treatment option for this devastating malignancy. Citation Format: Diren Usta, William Gwynne, Yujin Suk, Yiyun Chen, Molly T. Radosevich, Daria Chernova, Yushen Feng, Emon Nasajpour, Maria Trissal, Rebecca Poetschke, Alberto Delaidelli, Christopher Dunham, Louai Labanieh, Jasper van der Lugt, Stefan Nierkens, Mariella G. Filbin, Claudia Petrisch, Poul H. Sorensen, Katie Ryan, Chitra Venugopal, Elena Sotillo-Piñeiro, Crystal L. Mackall, Sheila Kumari Singh, Sabine Heitzeneder. Decoding the medulloblastoma surfaceome prioritizes the oncofetal antigen GPC2 for potent CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4009.

The c-SN6j/TRC105 antibody does not recognize murine or rhesus endoglin.

List of antibodies and reagents used in the study.

Introduction: Hemangioblastomas (HBs) are benign, highly vascular tumors that can be found intracranially or in the spinal region, representing around 2–15% of primary intramedullary tumors. They can occur sporadically or in association with Von Hipple–Lindau (VHL) disease. Despite recent of advancement of nonsurgical treatments, complete surgical resection remains the gold standard of care for the spinal HBs. Materials and Methods: We conducted an international multicenter retrospective analysis of adult patients surgically treated for spinal HBs in four European referral centers between January 2000 and September 2024, with a minimum post-operative follow-up duration of 6 months. Patients’ sex and age at surgical intervention, clinical presentation, and duration symptoms prior to clinical diagnosis were identified. The pre- and post-operative neurological status at 1 and 6 months and at the last visit was assessed using the modified McCormick score (MCS). The extent of surgical resection was divided into gross total resection (GTR) and subtotal resection (STR). Finally, post-operative complications were inspected as well, namely cerebrospinal fluid leaks, infections, hemorrhages and post-operative spinal stability. Results: A total of 35 patients were included in the cohort, with an age median of 52 years (34.5–60) and a slight male predominance (21/35, i.e., 60%). The median follow-up period was 37.5 months (12–75). More than half were located in the cervical region, making it the most common (54.3%). Syrinxes were observed in 23 cases (72%), and HBs were more commonly intramedullary (80%). GTR was achievable in around 88% of cases. Post-operative complications were observed in nine patients (25.7%). Nearly half of patients were discharged into rehabilitations centers (48.5%). Tumor recurrence was seen in 10.3% only. At the last follow-up, an excellent overall post-operative neurological status (positive ∆ McCormick) was observed in most of patients (88%) and was found to be associated with a relatively younger age group. Tumor location and presence of syrinxes did not show any statistical significance regarding clinical outcome. In patients having benefited from intra-operative monitoring, only D-wave changes showed statistical significance regarding post-operative outcome (p < 0.05). Conclusions: A large majority of patients operated for a spinal HB demonstrated favorable outcome after surgery, with unchanged or improved neurological status. Advanced age could have an impact on the post-operative neurological outcome. Other factors such as tumor size, location, and the presence of syrinx did not seem to significantly impact the neurological outcome. Finally, the surgery of these vascular lesions with no possibility of debulking or piece-meal removal and requiring “en bloc” resection is technically demanding and should be performed by experienced teams in spine and spinal cord surgery only.

There are limited therapeutic options for patients with advanced sarcomas, which leads to dismal outcomes for children and adults. Although chimeric antigen receptor (CAR) T cells hold promise for treating advanced sarcomas, this approach is constrained by a paucity of effective targets. Our previous clinical study identified endoglin (ENG/CD105), a TGFβ coreceptor, as a target of the endogenous immune response in a patient with sarcoma who exhibited an exceptional response to HER2-targeted CAR T-cell therapy. ENG is expressed on various sarcomas, cancer-associated fibroblasts, and neoangiogenic vessels and therefore offers comprehensive tumor targeting. Furthermore, ENG knockout in sarcoma cells reduces their invasiveness, highlighting its potential as a therapeutic target. Accordingly, we designed a second-generation human ENG-targeting CAR molecule signaling through the CD28 endodomain and retrovirally transduced primary human T cells with this CAR. ENG CAR T cells exhibited strong antigen-specific cytokine release, robust proliferation, memory formation, and cytotoxic function against various sarcoma cell lines. Their cytotoxicity remained unaffected by the presence of soluble ENG or its natural ligand, bone morphogenetic protein-9. Furthermore, ENG CAR T cells disrupted multicellular tumor spheroids in vitro, overcoming tumor compactness and the stromal barrier created by cancer-associated fibroblasts, which are critical challenges in sarcoma CAR T-cell therapy. In orthotopic xenograft models of sarcomas, ENG CAR T-cell treatment resulted in control of tumor growth and metastasis, leading to survival extension. In summary, our study describes the involvement of ENG in sarcoma metastasis and validates our human ENG CAR T cells as a potential therapeutic for advanced sarcomas.

ENG CAR design and molecular modeling.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and is stratified into four molecular groups ‒ Wingless and Int-1 (WNT), Sonic hedgehog (SHH), Group 3 and Group 4. Group 3 MB patients exhibit the poorest prognosis, with a 5-year overall survival of <60%, followed by Group 4 MB patients. Apart from MYC amplification in a subset of Group 3 MBs, the molecular pathomechanisms driving aggressiveness of these tumors remain incompletely characterized. The gene encoding the mTOR substrate and mRNA translation inhibitor eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) represents a possible MYC target gene whose corresponding protein, 4EBP1, was shown to be more active in Group 3 versus Group 4 MBs. However, the prognostic role of 4EBP1 in MB and the mechanisms supporting 4EBP1 overexpression in Group 3 MB are still elusive. We analyzed EIF4EBP1 mRNA expression in publicly available data sets and found an upregulation in MB as compared to non-neoblastic brain. EIF4EBP1 mRNA expression levels were higher in Group 3 compared to Group 4 MBs. EIF4EBP1 mRNA expression was correlated with MYC expression, most prominently in Group 3 MBs. Survival analyses highlighted that high EIF4EBP1 mRNA expression was associated with reduced overall and event-free survival across all MB patients and in Group 3/Group 4 MB patients. Immunohistochemical evaluation of 4EBP1 protein expression in MB tissues confirmed that high levels of 4EBP1 are associated with poor outcome. Functional analyses revealed that MYC directly regulates EIF4EBP1 promoter activity, providing a mechanism for increased EIF4EBP1 mRNA levels in Group 3 MBs. Finally, we observed that 4EBP1 may support colony formation of in vitro cultured MB cells. Our data highlight that transcriptional upregulation of EIF4EBP1 by MYC promotes in vitro tumorigenicity of MB cells and associates with shorter survival of MB patients.