Abstract Osteosarcoma (OS) is the most common type of bone cancer affecting children and adolescents. Pulmonary metastasis is the primary cause of OS mortality, yet treatments have not improved patient outcomes in four decades. This gap emphasizes the urgent need to identify and vet novel anti-metastatic therapies. When OS tumor cells spread to the lung, they are subjected to oxidative stress caused by high levels of reactive oxygen species (ROS) secreted from microvascular endothelial cells and alveolar macrophages. This stress is further amplified by limited antioxidant capacity and other redox-imbalancing features of the lung microenvironment. We previously demonstrated that metastatic OS cells upregulate NFE2-like BZIP Transcription Factor 2 (NRF2), promoting cell survival in this stressful environment. In addition to being a master regulator of redox balance, NRF2 also influences mitochondrial function by controlling the expression of thioredoxin system members, including the antioxidant enzyme peroxiredoxin 3 (PRDX3) located in the mitochondrial matrix. Therefore, we hypothesize that PRDX3 upregulation promotes metastatic cell survival during the early stages of lung colonization, and disrupting this response will impair adaptation to the lung microenvironment. We evaluated thiostrepton (TS), a Streptomyces-derived thiopeptide antibiotic with demonstrated PRDX3-inhibitory activity in preclinical and clinical cancer studies, to determine its effect on metastatic OS tumor cells. To mimic the oxidative stress of the lung microenvironment in vitro, we treated cells with tert-butyl hydroperoxide (tBHP), which chemically induces oxidative stress. Through cell viability assays we showed that subtoxic levels of TS sensitize metastatic OS cells to tBHP-induced oxidative stress. In addition, we determined that PRDX3 is upregulated when metastatic OS cells are subjected to oxidative stress via confocal microscopy. Through protein immunoblots, we demonstrated that TS acts by dimerizing PRDX3, inactivating the catalytic site of the enzyme. Furthermore, we found that combining TS with the chemotherapeutic agents doxorubicin or etoposide, which are commonly used to treat pediatric pulmonary OS metastasis, produced a synergistic increase in cell death compared to either drug alone. Lastly, we studied TS as a single agent therapy using the pulmonary metastasis assay, an ex vivo lung explant system, and found that treatment significantly decreased OS lung tumor burden. Our findings provide promising preclinical data supporting the use of TS as a possible anti-metastatic therapeutic for pediatric OS. Citation Format: Elli Maria Tiliakou, Michael Lizardo, Yue-Zhou Huang, Taras Shyp, Poul Sorensen. Inhibition of the mitochondrial antioxidant response blocks ex vivo lung colonization in pediatric osteosarcoma [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 2248.

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.

Replicate graphs of HDAC, PARP1, PARP2 and PARylation assays

Replicates of cell cycle assays and statistics

Replicate data of DNA damage assays

Replicate graphs of spheroid assays and cell fluorescence data

Replicate graphs of cell viability assays and comparison of cell viability assay methods

Blood cell panel counts and weight loss percentage of in vivo toxicity study

T cell therapies, such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) transgenic (tg) T cells, are a promising approach in the treatment of solid malignancies but are limited by T cell exhaustion caused by chronic antigen stimulation. Clusterin (CLU) is a chaperone protein known to protect both normal and malignant cells, from metabolic stress and reactive oxygen species (ROS). In this study, we investigated whether overexpression (OE) of CLU in TCRtg T cells and CAR-T cells respectively can reduce exhaustion induced by chronic antigen stimulation and enhance T cell functionality against Ewing sarcoma (EwS). Among other cytoprotective genes, we found that CLU was significantly downregulated in dysfunctional tumor infiltrating lymphocytes. Therefore, we engineered EwS-directed TCRtg T cells targeting a Chondromodulin-1 (Chm1)-derived peptide and GD2 CAR-T cells to overexpress CLU. We show here that CLU is downregulated in T cells following activation by tumor cells. CLU-overexpressing T cells exhibit decreased expression of exhaustion markers (PD1, LAG3) and reduced apoptosis after repetitive stimulation. These cells demonstrated improved infiltration into tumor spheroids and maintained functionality under hypoxic conditions. In vivo, CLU-overexpressing T cells showed enhanced persistence and a trend towards reduced tumor growth. Mechanistically, proteomic analysis suggested that reduced ribosomal activity might delay T cell exhaustion, implicating metabolic reprogramming. In conclusion, CLU OE in tg T cells enhances their persistence and functionality by mitigating exhaustion, possibly through modulation of ribosomal activity and metabolic pathways. This strategy holds potential for improving adoptive T cell therapies against solid tumors. ### Competing Interest Statement S.E.G.B has ownership interest in PDL BioPharma and had US and EU intellectual properties in gene expression analysis. He served as consultant to EOS Biotechnology Inc. and serves as advisor to Bayer AG and Swedish Orphan Biovitrum AB. Other authors declare no conflict of interest. Cura Placida Children’s Cancer Research Foundation, CP101/22 German Research Foundation, INST 95/1650-1 FUGG

Clear cell ovarian cancer (CCOC) is a histological subtype of ovarian cancer that is resistant to standard chemotherapies. Although it accounts for only around 6% of all ovarian cancer cases, it is the second leading cause of mortality among ovarian cancers. ARID1A loss-of-function mutations have been identified as a hallmark (∼65%) of CCOC. ARID1A, a key component of the SWI/SNF chromatin remodeling complex, plays a critical role in regulating gene expression. Loss of ARID1A disrupts the expression of SLC7A11, leading to decreased glutathione (GSH) levels and elevated reactive oxygen species (ROS) levels, while making cancer cells highly dependent on oxidative phosphorylation (OXPHOS). EO3001, a synthetic small molecule, can selectively transport extracellular Cu(II) to mitochondria, increasing mitochondrial ROS and inducing cuproptosis. Recent studies suggest that ARID1A-deficient CCOC cells are more vulnerable to EO3001. In this study, an ex vivo model, the pulmonary metastasis assay (PuMA), along with other in vitro models, was employed to assess the effectiveness of EO3001 in CCOC. PuMA enables the study of cancer cell growth in a more physiologically relevant microenvironment. In PuMA, fluorescently tagged cancer cells are inoculated into mouse lungs, which are then inflated with agarose gel, sectioned, and treated under different conditions. The growth of cells is quantified by fluorescence intensity from lung sections. Isogenic ARID1A wild-type and mutant CCOC cell lines (RMG-1, OVCA429, and JHOC-5) were generated using CRISPR-Cas9. The effects of EO3001 were evaluated using in vitro assays and PuMA. Cell viability, proliferation, ROS levels, glycolysis and OXPHOS activity, migration, and invasion were measured with and without EO3001 treatment under various stress conditions. Drug efficacy was validated in both short- and late-harvest PuMA models. EO3001 demonstrated significant differential effects against ARID1A-deficient CCOC cells in both in vitro and ex vivo models. However, hypoxic conditions significantly diminished its efficacy, likely due to a metabolic shift between OXPHOS and glycolysis. Furthermore, Cu(II) concentration in the microenvironment remarkably impacts the efficiency of EO3001. Overall, EO3001 presents a promising therapeutic strategy for ARID1A-mutant CCOC as a single agent. Targeting the OXPHOS dependency in ARID1A-mutant CCOC cells and enriching Cu(II) concentration could further enhance the efficacy of EO3001. Yuchen Ding, Tsz Yin (Jacky) Lam, Yuting (Shary) Chen, Longyijie (Grace) Wei, Yuqin (Lucy) Li, Yuhan (Joyce) Zhang, Farhia Kabeer, Forouh Kalantari, Jeffrey Bacha, Dennis Brown, Sarath Kanekal, Neil Sankar, Michael Lizardo, Michelle Woo, Amal M. EL-Naggar, David Huntsman. Investigating the potential of EO3001 as a therapeutic agent for clear cell ovarian cancers harboring ARID1A mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4298.

Materials and methods for in vivo toxicity and cellular fluorescence assays

Fig. S4. CR-1-31B down-regulates known targets of rocaglate-based inhibitors

Abstract Glypican 2 (GPC2) chimeric antigen receptor (CAR) T cells are safe and efficacious in neuroblastoma and are currently being tested in a first-in-human Phase 1 clinical trial at the Children’s Hospital of Philadelphia. Tumor-derived extracellular vesicles (TEVs) are nanosized vesicles secreted by cancer cells, often enriched with glypicans. However, the presence of GPC2 on neuroblastoma TEVs and any interaction with GPC2 CAR T cells has not been explored. High-levels of GPC2 were found on TEVs isolated from neuroblastoma preclinical models [7 cell lines, circulating EVs from 6 patient-derived xenograft (PDX)-bearing mice] and from the peripheral blood of 16 neuroblastoma patients, but not on circulating EVs from 7 non-tumor bearing mice or 12 healthy human donors. The level of circulating GPC2+ EVs isolated from the peripheral blood of PDX-bearing mice positively correlated with PDX size (R=0.96; P<0.0001) and parent PDX GPC2 expression. To evaluate how GPC2 on EVs modulates CAR T cell functionality, we generated EVs with varied levels of GPC2 (Hi/Lo/Neg) and co-incubated each EV subset with GPC2 CAR T cells. Here using immunofluorescence and flow assays we found that EVs bind GPC2 CAR T cells proportional to the amount of GPC2 on their surface. Further, GPC2Hi EV-CAR T cell synapses induced potent T cell activation shown by CD69/Granzyme B expression and release of IL-2/IFN-γ. GPC2Hi EV pre-incubated CAR T cells also displayed enhanced target neuroblastoma cell cytotoxicity, inducing 4-fold more specific cytotoxicity than CAR T cells incubated with control GPC2Neg EVs. In vivo we observed that injection of GPC2Hi EVs into GPC2Lo neuroblastoma SK-N-AS xenografts genetically altered to have significantly decreased endogenous EV production (via RAB27A knock-out) greatly enhanced the efficacy of co-infused GPC2 CAR T cells (p<0.05). Similarly, in an isogenic SK-N-AS-GPC2 xenograft murine model treated with a limiting number of GPC2 CAR T cells concurrently with the EV-inhibiting drug GW4869, GPC2 CAR efficacy was dependent on the presence of GPC2Hi EVs. Here, pharmacologic EV inhibition in mice decreased GPC2 CAR efficacy which could be rescued by intratumoral injection of GPC2Hi EVs. In both in vivo models, the presence of intratumoral GPC2Hi EVs resulted in a significantly enhanced tumor infiltration of activated (CD69+/CD25+) GPC2 CAR T cells (p<0.05). Taken together, GPC2Hi EVs are selectively secreted from neuroblastomas and bind and activate GPC2 CAR T cells enhancing their anti-tumor cytotoxicity. To further capitalize on these findings, we engineered GPC2Hi EVs to have an extended circulation half-life through albumin binding or tumor targeting via GD2 binding that are currently being tested in vivo and results will be reported. EVs offer a versatile platform for CAR antigen presentation and should be further validated as a strategy to enhance CAR T cell efficacy for solid tumors. Citation Format: Anna M. Giudice, Stephanie Matlaga, Guillem Pascual-Pasto, Patrick M. Schuerch, Geoffrey Rouin, Brendan McIntyre, Vincent P. Zecchino, Kristopher R. Bosse. Neuroblastoma extracellular vesicles present GPC2 and activate GPC2 CAR T cells in an antigen-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5118.

Abstract The success of CD19-directed chimeric antigen receptor (CAR)-T cell therapy for ALL has yet to be matched in solid pediatric malignancies. Moreover, immune checkpoint blockade has shown limited benefit in pediatric cancer. Despite this unmet need, there is a lack of reliable preclinical models to facilitate novel target discovery, validate therapeutic benefit, anticipate on- or off-target toxicity, and understand mechanisms of resistance. Syngeneic murine model systems are a critical intermediate for preclinical translation, preserving the complex and heterogenous immune landscape and host-immunotherapy interactions. However, model interpretation and validity are limited as expression and function of murine targets and immune effector cells are not always conserved. We have developed a syngeneic immunocompetent C57BL/6 neuroblastoma system to study CAR efficacy, toxicity, and mechanisms of resistance, as well a variety of vaccination strategies. Specifically, we have built two murine CAR T constructs containing a 100% conserved antigen-directed scFv joined to orthologous murine TCR signaling machinery. In parallel, we engineered the murine neuroblastoma cell line 9464D to stably express surface antigen Gpc2 (Bosse, Cancer Cell 2017), or a novel chimeric single chain trimer of a 9mer peptide from intracellular oncoprotein PHOX2B discovered on HLA-A*24:02 (Yarmarkovich, Nature 2023) as Phox2b/A24*02/H2-kb to generate tumor-bearing B6 or B6-A24-Tg mice. We are currently using this system to demonstrate how vaccination approaches enhance the persistence and potency of CART directed to both targets and are extending our syngeneic murine constructs to additional TH-MYCN allograft models. To further study the innate and adaptive immune response to various therapeutics, we have developed peripheral blood mononuclear cell (PBMC) humanized models. Our autologous humanized PDX mouse models of neuroblastoma use immunocompromised NSG mice engrafted with a PDX and humanized with patient-matched PBMCs to evaluate personalized vaccines in vivo. We have shown the ability to stably engraft both lymphoid and myeloid populations in various human cytokine transgenic NSG variants (i.e. SGM3, Flt3L), allowing for T/B cell and APC crosstalk, thus providing a robust means to study targeted interventions. We are currently using this autologously humanized PDX model to test the ability of multivalent, personalized neoantigen vaccines developed specific to the neoantigen landscape of individual PDXs, with a focus on endogenous T cell responses of patient PBMC against their own PDX in vivo. Citation Format: Timothy T. Spear, Elisabeth Posthill, David Groff, Anna M. Giudice, Kristopher R. Bosse, Leyuan Ma, Stephen P. Schoenberger, John M. Maris. Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5121.

Fig. S9. Distribution of NFE2L2 and MYC transcripts in light and heavy polysomal fractions and ectopic expression of NRF2.

Fig. S1. Single agent activity of CR-1-31B in osteosarcoma (OS) cells

Supplementary Figure 3. Mouse retina expression of GPC2 and GD2, retinal CAR T-cell infiltration, systemic toxicities, and antigen loss in the intraocular model (see related main Fig. 4).

Domain structure of ENPP1 and CDH11.

Supplementary Data file containing Supplementary Methods and Materials and associated references.

Fig. S12. eFT226 inhibits proliferation of a metastatic PDX OS cell line model under oxidative stress.