bluuebunny/crossref_metadata_2025 huggingface dataset:
In utero choline exposure alters growth, metabolism, feed efficiency, and carcass characteristics of Holstein × Angus cattle from weaning to slaughter
Authors: Brown William E , Holdorf Henry T , Johnson Sara J , Kendall Sophia J , Green Sophia E , White Heather M
Published: June 2023

Feeding rumen-protected choline (RPC) to late gestation dairy cows has potential to affect growth in offspring. The objective of this study was to evaluate the effects of in utero choline exposure on the growth, feed efficiency (FE), metabolism, and carcass quality of Angus × Holstein cattle. Multiparous Holstein cows pregnant with male (N = 17) or female (N = 30) Angus-sired calves were enrolled 21 d prepartum and randomly assigned to one of four dietary treatments varying in quantity and formulation of RPC. The treatments included a control with 0 g/d supplemental RPC (CTL), supplemental RPC fed at the recommended dose (RD) of 15 g/d from either an established RPC product (RPC1RD; ReaShure; Balchem Corp.) or choline ion from a concentrated RPC prototype (RPC2RD; Balchem Corp.), or a high dose (HD) of RPC2 fed at 22 g/d (RPC2HD). From 2 to 6 mo of age, calves were group housed and offered 2.3 kg grain/hd/d (42% CP) with ad libitum grass hay, and stepped up to a complete finishing diet by 7 mo (12.0% CP; 1.34 Mcal/kg NEg). Weight and height were measured monthly. Animal FE was measured in individual pens for 35 d at 8 mo. Feed intake was measured daily, and blood was obtained on day 18 during the FE period. Afterwards, cattle were group housed and offered a free-choice finishing diet until slaughter, where carcass yield and quality characteristics were measured. Mixed models were used in PROC MIXED (SAS, 9.4) with the fixed effects of treatment, sex, time, their interactions, and the random effect of calf. Month was the repeated measure, and preplanned contrasts were used. Blood and FE data were analyzed with the fixed effect of dam choline treatment, calf sex, and the interaction. Increasing dose of RPC tended to increase weight over the entire study period. Feeding any RPC increased hip and wither height compared with CTL, and increasing RPC dose linearly increased hip and wither height. Treatment and sex interacted on DMI whereby increasing RPC intake linearly increased DMI for males but not females. Compared with control, feeding any RPC decreased plasma insulin, glucose, and an insulin sensitivity index (RQUICKI). In utero choline exposure increased kidney–pelvic–heart fat and marbling score. Mechanisms of action for intrauterine choline exposure on offspring growth, metabolism, and carcass characteristics should be explored as they have direct implications for profitability for cattle growers and feeders.
The effects of disagreement and unfriending on political polarization: a moderated-mediation model of cross-cutting discussion on affective polarization via unfriending contingent upon exposure to incivility
Authors: Lin Han , Wang Yi , Lee Janggeun , Kim Yonghwan
Published: June 2023

Cross-cutting discussion is the foundation of deliberative democracy. However, previous research has reported inconsistent results regarding the effects of exposure to dissimilar perspectives on political polarization. This study aims to extend the literature by exploring how cross-cutting discussion influences affective polarization through unfriending and how this indirect effect is contingent upon exposure to incivility. The study analyzes panel data from a two-wave online survey conducted in South Korea (N = 890). The results show a significantly positive indirect effect of unfriending, suggesting that cross-cutting discussion further reinforces affective polarization via unfriending. Furthermore, the study identifies the boundary conditions for this mediating mechanism, showing that the mediated relationship of cross-cutting discussion on affective polarization via unfriending is stronger for those who are more exposed to incivility on social media.
‘A slashing review is a thing that they like’: Vivisection and Victorian Literary Criticism
Authors: Hornsby Asha
Published: June 2023

In nineteenth-century Britain, the antivivisection movement attracted a striking number of authors, poets, and playwrights, who attended meetings, signed petitions, contributed funds, and lent their pens to the cause. However, the language of vivisection extended far beyond literature with a purpose, seeping into the heart of late-Victorian literary debates. This article explores analogies of writing as vivisection in literary-critical discourse. Surveying the newspapers and periodicals of the period demonstrates that such terminology was remarkably sprawling in terms of the genres and authors it was applied to and the meanings it conveyed. Essayists and reviewers also used metaphors relating to experimental physiology’s modus operandi to shape and articulate key methodological and ideological principles that were emerging in late-Victorian literary-critical theory and practice. These included discussions of how to analyse living authors and contemporary works, conceptualizations of whether critical operations should produce social benefits, and considerations of the aesthetic and technical opportunities that literary or critical vivisection offered or, indeed, prevented.
Breakthrough candidemia with hematological disease: Results from a single-center retrospective study in Japan, 2009–2020
Authors: Nishida Ruriko , Eriguchi Yoshihiro , Miyake Noriko , Nagasaki Yoji , Yonekawa Akiko , Mori Yasuo , Kato Koji , Akashi Koichi , Shimono Nobuyuki
Published: June 2023

Breakthrough candidemia (BrC) is a significant problem in immunocompromised patients, particularly those with hematological disorders. To assess the characteristics of BrC in patients with hematologic disease treated with novel antifungal agents, we collected clinical and microbiological information on said patients from 2009 to 2020 in our institution. Forty cases were identified, of which 29 (72.5%) received hematopoietic stem cell transplant (HSCT)-related therapy. At BrC onset, the most administered class of antifungal agents were echinocandins, administered to 70% of patients. Candida guilliermondii complex was the most frequently isolated species (32.5%), followed by C. parapsilosis (30%). These two isolates were echinocandin-susceptible in vitro but had naturally occurring FKS gene polymorphisms that reduced echinocandin susceptibility. Frequent isolation of these echinocandin-reduced-susceptible strains in BrC may be associated with the widespread use of echinocandins. In this study, the 30-day crude mortality rate in the group receiving HSCT-related therapy was significantly higher than in the group not receiving it (55.2% versus 18.2%, P = .0297). Most patients affected by C. guilliermondii complex BrC (92.3%) received HSCT-related therapy and had a 30-day mortality rate of 53.8%; despite treatment administration, 3 of 13 patients had persistent candidemia. Based on our results, C. guilliermondii complex BrC is a potentially fatal condition in patients receiving HSCT-related therapy with echinocandin administration.
ATRT-01. PARP INHIBITION AS A THERAPEUTIC TARGET IN ATRT
Authors: Alimova Irina , Mardok Gillian , Pierce Angela , Wang Dong , Madhavan Krishna , Brunt Breauna , Vibhakar Rajeev
Published: June 2023

Atypical teratoid rhabdoid tumors (ATRTs) are highly aggressive pediatric brain tumors with poor survival. The available treatments rely on toxic chemotherapy and radiotherapy, which alone can cause poor outcomes for young patients. Poly (ADP-ribose) polymerases (PARP), multifunctional enzymes which play an important role in DNA damage repair and genome stability have emerged as a new target in cancer therapy. An FDA approved drug screen revealed that Rucaparib, a PARP inhibitor, is important for ATRT cell growth.In this study we investigated the effect of Rucaparib treatment in ATRT. Using MTS and colony formation assays we found that Rucaparib treatment decreased ATRT cell growth and inhibited clonogenic potential of ATRT cells. Flow cytometry and Western blot analysis showed that Rucaparib treatment led to cell cycle arrest and apoptosis. Moreover, Rucaparib treatment led to DNA damage accumulation as seen by increased expression of yH2AX by immunofluorescence. In vivo, Rucaparib treatment significantly decreased tumor growth and prolonged survival in orthotopic mouse models. Furthermore, Immunohistochemistry revealed an increased number of P53 and Caspase3positive cells in the Rucaparib treatment group, confirming the induction of apoptosis in vivo. Furthermore, Rucaparib pretreatment sensitizes ATRT cells to radiation and significantly increased survival of xenograft-bearing mice. Thus, we demonstrated that Rucaparib has potential to be a new therapeutic strategy for ATRT as shown by its ability to decrease ATRT tumor growth both in vitro and in vivo.
BIOL-12. ENHANCING IMMUNE-MEDIATED TUMOR CONTROL IN MURINE MODELS OF GROUP 3 MEDULLOBLASTOMA
Authors: Abbas Zahra , Hii Hilary , Buck Jessica , Johnson Iley , Byrne Jacob , Elaskalani Omar , Kuchibhotla Mani , George Courtney , Arias Clara Andradas , Howlett Meegan , Lesterhuis Joost , Johns Terrance , Gottardo Nicholas , Endersby Raelene
Published: June 2023

Stagnation in clinical outcomes as well as severe side-effects of current treatment approaches for Group 3 medulloblastoma have prompted the search for novel therapies. To date, immunotherapies have not demonstrated success in medulloblastoma. Due to the highly aggressive nature of relapsed medulloblastoma, we suggest that it would be advantageous to introduce new immunotherapies first-line, alongside conventional therapies. To design immunotherapeutic strategies that are compatible in combination with standard therapies, we first determined the effect of standard-of-care craniospinal irradiation (CSI) and chemotherapy on the immunology of these tumours in immune competent and deficient murine models. We show that standard treatment approaches deplete adaptive immune cells, while macrophage and brain-resident microglia are not depleted. Furthermore, medulloblastomas are myeloid dominant tumors, and blocking the anti-phagocytic CD47-SIRPa interaction has shown preclinical efficacy. Therefore, we tested the combination of anti-CD47 immunotherapy with radiotherapy in a patient-derived xenograft model of Group 3 medulloblastoma. We found that anti-CD47 monotherapy had limited efficacy, while CSI results in only a temporary reduction in tumor size. In the cerebellum of relapsed tumors post-CSI, flow cytometry revealed increased peripheral myeloid populations. Remarkably, the combination of anti-CD47 and CSI was effective in clearing tumors and maintaining long-term tumor-free survival. Using RNA sequencing and immunohistochemistry, we found that the combination therapy results in enhanced cytokine signalling and phagocytic myeloid cell infiltration into the tumors compared to the monotherapies. We have also developed and characterized novel age-appropriate preclinical models for medulloblastoma and have observed altered myeloid cell phenotypes in these age-appropriate models. We are now testing this novel combination therapy in our new neonatal murine models to determine efficacy in age-appropriate hosts.
BIOL-18. NEWLY DEVELOPED REPLICATION REPAIR DEFICIENT (RRD) MOUSE MODELS PROVIDE INSIGHTS INTO MEDULLOBLASTOMA/GLIOMAGENESIS AND RESPONSE TO IMMUNOTHERAPY
Authors: Aamir Zoya , Galati Melissa , Gattoni Emma , Crump Owen , Nunes Nuno M , Das Anirban , Fernandez Nicholas R , Wong Angel K Q , Fortin Jerome , Stengs Lucie , Bianchi Vanessa , Edwards Melissa , Negm Logine , Chung Jiil , Malkin David , Egan Sean , Hawkins Cynthia , Tabori Uri
Published: June 2023

Replication Repair Deficiency (RRD), caused by germline monoallelic (Lynch Syndrome) or biallelic (Constitutional Mismatch Repair Deficiency, CMMRD) mutations in MMR genes, is present in 5-10% of glioblastomas in children, adolescents, and young adults. RRD glioblastomas are chemoradiation-resistant, but respond favorably to immune checkpoint inhibition (ICI). Representative immunocompetent animal models are urgently needed for 3 recently identified subgroups based on specific somatically-acquired mutations, survival, and immunotherapy response (RRD1: MMRD with POLE mutations, RRD2: MMRD associated with TP53 mutations, and RRD3: MMRD harboring IDH1 mutations). Using germline mutations and brain-specific Cre-drivers, we genetically engineered mouse models that recapitulate each human RRD-subgroup. RRD1 (Nestin- and Olig2-Cre+/ Msh2LoxP/LoxP/PoleS459F/+ and LSL-PoleP286R/+): CMMRD-like Nestin-Cre-driven mice develop posterior-fossa glioma-like or medulloblastoma (MB)-like tumors at ~2.7 months. Olig2-Cre-driven mice display hemispheric gliomas at ~10 months, suggesting distinct cell-of-origin. RRD2 (Nestin-Cre+/Trp53LoxP/LoxP and Msh2LoxP/LoxP or Mlh1-/-): CMMRD-like tumors develop in heterogenous locations at ~4.5 months (p<0.0001), classifying primarily as MB-like in hindbrain, and glioma-like in other brain regions. Strikingly, germline Mlh1 tumors occur earlier than Nestin-Cre-driven RRD2 tumors, indicating early developmental mutation accumulation in CMMRD-patients. Lynch-like RRD1/2 mice succumb exclusively to gliomas >13 months (p<0.0001). RRD3 (Olig2-Cre+/Msh2LoxP/LoxP/Trp53LoxP/LoxP/LSL-Idh1R132H/+): brain tumors occur later and are hemispheric. These observations recapitulate human data, where CMMRD-patients develop glioblastoma/MB earlier than Lynch-patients (8.6 vs. 14-years; p<0.0001), and posterior-fossa glioblastoma/MB presents earlier than hemispheric gliomas (p=0.04). Additionally, tumor onset and location vary (RRD1: 7.6-years, RRD2: 8.3-years, hemispheric/posterior-fossa; RRD3: 12-years, hemispheric; p=0.005). In both mice and humans, RRD1 exhibits ultra-hypermutation, high immune infiltration, and response to ICI, whereas RRD2 harbors lower mutational burden, are immune-cold, and ICI-monotherapy resistant. Temporal dynamics of RRD tumor development is currently being tracked by serial MRI to define biologically relevant time points. Our models accurately mimic the human condition and provide unique insights into RRD tumorigenesis, allowing optimization of subgroup-tailored therapeutic approaches.
DIPG-02. A NOVEL STRATEGY TO RADIOSENSITISE DIFFUSE MIDLINE GLIOMAS VIA DUAL-TARGETING OF GLUCOSE METABOLISM
Authors: Shen Han , Chang Cecilia , Ing Prunella , Nguyen Sandy , Mudassar Faiqa , Cook Kristina , Lai Joey , Gloss Brian , O’Neill Geraldine , Gee Harriet , Hau Eric
Published: June 2023

Diffuse Midline Gliomas (DMG) is the leading cause of brain tumor-related death in children. Currently, radiation is the only treatment that offers transient benefit but almost all DMG relapse due to radioresistance. Compared to normal brain tissue, DMG are hypo-perfused with tumor cells being exposed to hypoxia, a low oxygen microenvironment serves as a potent barrier to effective radiotherapy. Biguanides are hypoglycemic agents that can reduce the oxygen consumption rate (OCR) in mitochondria, thereby sparing more oxygen and alleviating hypoxia. Our previous study has shown that metformin significantly improves the radiosensitivity of DMG and extends survival in a patient-derived xenograft (PDX) model. In comparison with metformin, phenformin demonstrated greater efficacies in anti-proliferation, OCR and hypoxia inhibition, and radiosensitization in a panel of DMG cultures. These effects were possibly induced by phenformin through inhibition of mitochondrial complex I in DMG cells. The anti-tumor effect of phenformin was further enhanced by combining a second drug dichloroacetate that simultaneously attenuated phenformin-induced acidification. Specifically, the combination treatment induced higher levels of reactive oxygen species and DNA damage, which subsequently resulted in a much stronger effect in cell-cycle and proliferation arrest, apoptosis, and hypoxia inhibition. Metabolically, the co-treatment simultaneously targeted multiple pathways, which significantly reduced ATP production and further triggered metabolic catastrophe. RNA sequencing demonstrated significant alterations induced by the combination in cell-cycle, DNA repair, unfolded protein response and alternative energetic pathways. Furthermore, the combination treatment significantly improved the radiosensitivity of DMG cells, evidenced by higher levels of DNA damage, apoptosis, and clonogenic inhibition. Our preliminary in vivo data demonstrated a possible reduction in tumor burden following 4 weeks of combination treatment, with no elevation of serum L-lactate level in phenformin-treated groups. This therapeutic efficacy is currently being validated in multiple orthotopic DMG models with optimized treatment schedules.
DIPG-05. EVALUATION OF TELOMERE-MODIFYING COMPOUND THIO AS A RADIOSENSITIZER AND IMMUNOMODULATOR TO TREAT DIPG
Authors: Mishra Deepak , Umaru Banlanjo , Kumar Shiva Senthil , Gryaznov Sergei , Drissi Rachid
Published: June 2023

Diffuse intrinsic pontine glioma (DIPG) is one of the most aggressive tumors of the central nervous system in children. Radiotherapy remains the only standard treatment but is rarely curative. In recent years, several immunotherapy strategies have emerged as an option to treat DIPG. However, the low mutational burden and rare infiltration of T lymphocytes, render these tumors immunologically “cold” and therefore pose challenges for general immunotherapy. We have previously reported in pediatric brain tumors the preclinical validation of THIO (6-thio-2’-deoxyguanosine), a telomerase substrate precursor analog. Treatment of cancer cells with THIO caused both telomeric and genomic DNA damage and rapid cell death. In vivo treatment delayed tumor growth in mouse models of high-risk medulloblastoma and DIPG. Importantly, THIO crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. The cGAS-STING pathway is a cellular mechanism that senses cytosolic double stranded DNA, stimulating type I interferons (IFNs) and IFN-stimulated genes, thereby activating the innate and adaptive anti-tumor immune responses. Micronuclei resulting from cell division in the presence of unresolved DNA damage, has been shown to be recognized by the cGAS-STING pathway. THIO was shown to induce anti-tumor immunity through micronuclei formation in colon and lung tumor models. We hypothesized that THIO treatment will activate anti-tumor immunity in DIPG. Our results indicate that THIO treatment sensitized DIPG cells to ionizing radiation (IR) leading to a significant decrease in DIPG cell proliferation. We are currently testing this combination in vivo in an orthotopic mouse model of DIPG. We will present results on the potential of THIO to activate anti-tumor immunity through the activation of Sting pathway in a syngeneic mouse model of DIPG. These preclinical studies will support potential use of THIO in combination with IR to treat children with high-risk pediatric brain tumors.
DIPG-07. ROBUST CGAS-STING PATHWAY MEDIATED-INDUCTION OF ANTITUMOR ADAPTIVE IMMUNE RESPONSE: A POTENTIAL FOR DMG IMMUNOTHERAPY
Authors: Umaru Banlanjo , Mishra Deepak , Kumar Shiva Senthil , Devine Brendan , Drissi Rachid
Published: June 2023

Immunotherapy has emerged as groundbreaking in cancer treatment showing improved overall survival of patients with advanced cancers. However, its application for diffuse midline glioma (DMG) is still challenging due to the non-inflammatory immune environment with lack of T lymphocyte infiltration, low mutation burden associated with low or absence of neo-antigen expression and suppressive immune microenvironment in DMG. Therefore, determination of novel factors that not only trigger the immune response but also evading the tumor immunosuppression is imperative. The cGAS-STING pathway was shown to elicit the maturation and activation of dendritic cells (DCs) in response to the release of double stranded DNA (dsDNA) from tumor cells leading to effective cytotoxic T-cell infiltration and activity. Here, we aimed to determine the effects of a small molecule PTC596 (BMI-1 modulator and tubulin biding molecule) in cGAS-STING pathway activation and induction of antitumor immunity. We have previously reported the single agent efficacy for PTC596 in controlling tumor growth and improving survival compared to vehicle-treated mice. Our preliminary results using PTC596, indicate the presence of multinucleated cells and the formation of micronuclei, colocalizing with cGAS and induction of STAT1 activation (marker of inflammation through type 1 IFN). We are currently evaluating purified DCs post-PTC596 treatment for the activation of the cGAS-STING pathway and interferon-stimulated genes (ISGs) and determining their cross-priming capacity. In vivo, using our established syngeneic orthotopic DIPG models, we are evaluating the T cell infiltration (CD3, CD4 and CD8), and characterizing the tumor microenvironment in response to PTC596. Data collected from the above experiments will be presented and discussed.
DIPG-18. AI HUNTING FOR NEW DRUGS IN PEDIATRIC GLIOMA
Authors: Hashizume Rintaro , Newbury Patrick , Ishi Yukitomo , Zao Guisheng , Glicksberg Benjamin , Paithankar Shreya , Nazarian Javad , Pacold Michael , Watanabe Jun , Uchida Eita , Nicolaides Theodore , Chen Bin
Published: June 2023

Diffuse intrinsic pontine glioma (DIPG) is one of the most devastating pediatric gliomas, with nearly all patients succumbing to progressive tumor growth within two years of diagnosis. The identification of effective therapeutics based on the genetic features is of high importance for improving treatment outcomes for DIPG patients. We have developed machine learning algorithms that allow profiling thousands of genetic features, which can all contribute towards drug discovery. This study aimed to identify potential therapeutic agents using a computational pipeline to perform an in-silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines. Using a systematic computational approach with publicly available databases of gene signature in DIPG patients and cancer cell lines treated with a library of clinically available drugs, we identified drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. The biological and molecular effects of drug treatment was analyzed by cell viability assay and RNA sequence. In vivo DIPG mouse model survival studies were also conducted. As a result, two of three identified drugs showed potency against the DIPG cell lines. Triptolide and mycophenolate mofetil (MMF) demonstrated significant inhibition of cell viability in DIPG cell lines. Guanosine rescued reduced cell viability induced by MMF. In vivo, MMF treatment significantly inhibited tumor growth in orthotopic DIPG xenograft models. In conclusion, we identified clinically available drugs with the ability to reverse DIPG gene signatures and anti-DIPG activity in vitro and in vivo. This novel approach can repurpose drugs and significantly decrease the cost and time normally required in drug discovery.
DIPG-23. THE ROLE OF FOXO3 IN DMG RESISTANCE AND RESPONSE TO THERAPIES TARGETING DMG METABOLISM
Authors: Petrovic Antonela , Fernandez Solène , Uhr Nora Maria , Laternser Sandra , Müller Sabine , Nazarian Javad
Published: June 2023

Pediatric diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), are the most difficult childhood brain cancers to treat, with an overall survival rate of less than 12 months from diagnosis. Despite the H3K27M mutation being the main tumor driver, activation of the PI3K/Akt pathway also occurs, altering cancer cell metabolism, increasing glycolysis, and proliferation. Our research aims to understand the underlying mechanisms of resistance to therapies targeting DMG metabolism and to develop more effective treatment strategies. We have studied the effectiveness of ONC201 in targeting DMG cancer metabolism through mitochondrial degradation, reactive oxygen species increase, and oxidative phosphorylation impairment. Our findings suggest that despite promising results in patients, DMG tumor cells switch to glycolysis after treatment through PI3K/Akt pathway activation, which contributes to chemoresistance. We have found that the FOXO3 transcription factor plays a crucial role in determining DMG tumor sensitivity to therapy, specifically in the response to combined ONC201 and PI3K inhibitors. We have shown that FOXO3 activity is abolished via PI3K pathway activation in resistant DMG cell lines, leading to cytoplasmic translocation and tumor cell survival. Additionally, we found that the expression of FOXO3 is dysregulated in DMG patient tumors. Our data implies that a failure of FOXO3 to reach the nucleus may be a contributing factor to the inadequate response seen in certain ONC201 treated DMG patients. By utilizing CRISPR-modified patient-derived DIPG cells in vitro, we investigated the impact of FOXO3 knockout/overexpression on tumor sensitivity to various treatment regimens. We have validated new PI3K inhibitors and FOXO3 activators as potential therapeutic strategies for DMG patients. Our findings suggest that targeting FOXO family of transcription factors may be a promising approach to improve the efficacy of metabolic-targeting therapies for DMGs and ultimately improve the survival of patients diagnosed with this challenging pediatric brain cancer.
DIPG-29. A CONNECT CONSORTIUM PRECLINICAL STUDY IDENTIFIES MEK INHIBITION AND RADIATION AS POTENTIAL COMBINATION PARTNERS FOR THE ALK2 INHIBITOR TP-0184 IN ACVR1-MUTANT DMG
Authors: Rogers Rebecca , Tsoli Maria , Kumar Shiva Senthil , Potente Elisabet Fernandez , Biery Matt , Grabovska Yura , Mackay Alan , Carvalho Diana , Pereria Rita , Burford Anna , Sejdiu Drenusha , Ruddle Ruth , Gabel Florian , Raynaud Florence , Yadavilli Sridevi , Mizoguchi Sean , Subramaniam Bavani , Kilburn Lindsey , Hwang Eugene , Nazarian Javad , Vitanza Nicholas , Ziegler David , Drissi Rachid , Jones Chris
Published: June 2023

Somatic mutations in ACVR1, which encodes the serine/threonine kinase ALK2, are found in 20-25% of DMG-H3K27 patients. Treatment of ACVR1-mutant orthotopic xenografts with multiple chemotypes of ALK2 inhibitors (ALK2i) results in extended survival in vivo but, as single agents, these inhibitors are unable to achieve a complete anti-tumour response. To identify novel combination strategies alongside ALK2 inhibition, five international laboratories pooled resources to evaluate the ALK2i TP-0184 across 26 patient-derived DMG-H3K27 models in vitro, demonstrating GI50 values of ~0.5-10uM, although with no significant difference between ACVR1-mutant and wild-type. Genome-scale CRISPR screens of ALK2i-treated ACVR1-mutant models revealed specific genetic dependencies in critical nodes of several signalling pathways including MTOR, as well as PPP2R1A, known to play a role in MAPK pathway activation. Biomarker analysis showed inhibition of phospho-SMAD1/5/9 by western blot, with RNAseq and proteomic analysis indicating downstream signalling consequences in BMP/SMAD signalling following TP-0184 treatment. We observed synergistic pharmacological interactions between TP-0184 and both everolimus (mTORi) and trametinib (MEKi) in 7 to 8 ACVR1-mutant DMG models, respectively, with the latter validated across laboratories using orthogonal assays. Importantly, combining TP-0184 with radiation in vitro showed a profound radiosensitisation effect of the ALK2i in three ACVR1-mutant models using both clonogenic and cell viability assays in multiple laboratories, in part due to an observed activation of phospho-SMAD1/5/9 by radiation. Finally, in vivo tolerability and pharmacokinetic assays in multiple immuno-compromised and immune-competent mouse strains suggested a dose of 150mg/kg TP-0184 and 0.4mg/kg trametinib for use in in vivo efficacy studies, with good CNS penetrance of both drugs in excess of in vitro GI50 values. Such studies have been initiated in six DMG-H3K27 models across our laboratories to robustly assess the combination of TP-0184 and trametinib/radiation to support rapid potential translation to clinical trial.
DIPG-34. IMMUNE SYNAPSE OPTIMIZED GRP78-SPECIFIC CAR T CELLS ELICIT AN IMPROVED ANTITUMOR RESPONSE AGAINST DIPGS
Authors: Ibanez Jorge , Houke Haley , Meehl Michaela , Hebbar Nikhil , Thanekar Unmesha , Velasquez Paulina , Krenciute Giedre
Published: June 2023

Diffuse intrinsic pontine gliomas (DIPGs) are highly lethal pediatric brain tumors and a leading cause of cancer-related death in children. There is currently no cure for DIPG, highlighting an urgent need for novel therapeutics. Chimeric antigen receptor (CAR)-engineered T-cell therapy offers great promise for DIPG treatment; however, several limitations need to be addressed.

In this study, we targeted a novel tumor associated antigen GRP78 after showing it is reliably expressed on the cell surface of DIPGs. To test the anti-DIPG activity of GRP78-CAR T cells, we evaluated cytotoxicity, persistence, and cytokine secretion in vitro. We found that GRP78-CAR T cells kill DIPGs but fail to proliferate and secrete cytokines compared to positive control, U87. In vivo studies further confirmed that GRP78-CAR T cells eradicated U87 tumors but not DIPGs. Based on these findings, we concluded that DIPGs cannot trigger efficient CAR T cell signaling.

Because CAR T cell effector function is influenced by antigen density, we quantified the relative GRP78 expression on DIPGs and other brain tumors, including U87. We found that GRP78 is comparatively lowly expressed on DIPGs. Live-cell imaging of calcium flux in CAR T cells further revealed that T cell activation is lower upon interaction with DIPGs than U87s, suggesting that CAR-T cells form incomplete or dysfunctional immune synapses (IS) against DIPGs. To improve IS function, we knocked-out RASA2, a TCR downstream signaling inhibitor. Our results show that T cells lacking RASA2 have an improved IS reflected by bigger synaptic areas, increased lytic granules, and pZAP70 accumulation at the synapse. RASA2 KO further increased calcium influx and proliferation capacity of GRP78-CAR T cells against DIPGs. Thus, this study confirms that antigen density controls CAR T cell antitumor activity, and improving the IS formation of CAR T cells elicits enhanced antitumor response against low antigen-expressing tumor cells.
DIPG-40. HYPOXIC STRESS EXPOSES EPIGENETIC TARGETS TO ENHANCE RADIOTHERAPY RESPONSE IN H3K27M-POSITIVE PEDIATRIC DIFFUSE MIDLINE GLIOMAS
Authors: de Freitas Amanda Pereira , Dzwigonska Monika , Trubicka Joanna , Grajkowska Wieslawa , Mieczkowski Jakub , Kaminska Bozena , Leszczynska Katarzyna
Published: June 2023

Diffuse midline gliomas (DMGs) are the most challenging to treat pediatric high-grade gliomas, with a dismal prognosis of 9 to 15 months of median survival. The ones localized in the pons are not accessible to the surgery and rely on radiotherapy treatment. The majority of DMGs confer a mutation in histone 3 variants (H3.3 or H3.1), leading to lysine 27 to methionine substitution (H3K27M) and a consequent global decrease in H3K27 trimethylation (H3K27me3), increased H3K27 acetylation (H3K27ac) and oncogenic changes in gene expression. An intrinsic feature of solid tumors, including DMGs, is the occurrence of hypoxic (low oxygenated) regions, which interferes with the efficiency of radiotherapy. Identifying the vulnerabilities of hypoxia-exposed tumor cells might bring new approaches in radiosensitizing DMG tumors. Hypoxia increases histone methylation in multiple solid tumors but the consequences of hypoxia-induced histone hypermethylation in H3K27M bearing tumors are unknown. Using syngeneic patient-derived DMG cell lines, we show that the presence of H3K27M prevents the hypoxia-induced hypermethylation of H3K27me3, but also impairs the hypoacetylation of histones H3 and H4 normally caused by hypoxic stress. We show that enhancing histone acetylation in hypoxic conditions with histone deacetylase (HDAC) inhibitors has a stronger sensitization effect in cells expressing H3K27M than in H3 wild-type cells. While HDAC inhibitors have been shown problematic in pediatric DMGs due to high systemic toxicity, our data points out that HDAC inhibition in the context of hypoxia may improve the therapeutic efficacy. Recent studies showed the possibility of targeting hypoxic cells with hypoxia-activated pro-drugs, including hypoxia-activated panobinostat. Our data support that such approach might be an option to target DMGs and eliminate the radiotherapy-resistant hypoxic tumor fraction, while minimizing the systemic toxicity caused by all-time active HDAC inhibitors. Studies were supported by the grant UMO-2019/33/B/NZ1/01556 (KL) from National Science Center (Poland).
DIPG-48. DIPG THROUGH THE LOOKING GLASS
Authors: Senga Sasi S
Published: June 2023

Diffuse intrinsic pontine glioma (DIPG) has a peculiar pathogenesis with canonical Histone variants H3.1/H3.2 wherein Lysine 27 (H3K27) is substituted to methionine (H3K27M). It is restricted to pons and affects young children whereas non canonical H3.3 mutations result in tumours across the midline and affects older children. This discrepancy in the spatiotemporal distribution provides cues to target DIPG. Based on altered neuronal signalling which is a hallmark of cancer, a deeper understanding of neuron tumour interaction may unravel numerous new targets. One such target is light. The restriction of specific spectrum of light may hinder the progression and even the development of tumours in young children. It provides a lucrative therapeutic option which can be implemented merely by blocking a certain spectrum of light with glasses in an otherwise baffling disease. It warrants a deeper understanding of the light exposure on DIPG tumour progression.
DIPG-49. A SYSTEMS BIOLOGY APPROACH TO DEFINING AND TARGETING CELL STATE-SPECIFIC MASTER REGULATOR DEPENDENCIES IN DIFFUSE MIDLINE GLIOMA
Authors: Fernández Ester Calvo , Wang Junqiang , Zhang Xu , Wei Hong-Jian , Minns Hanna , Griffin Aaron , Obradovic Aleksandar , Vlahos Lukas , Martins Timothy , Becker Pamela , Crawford John , Gartrell Robyn , Szalontay Luca , Zacharoulis Stergios , Zhang Zhiguo , Wechsler-Reya Robert , Wu Cheng-Chia , Califano Andrea , Pavisic Jovana
Published: June 2023

Diffuse Midline Glioma (DMG) are fatal pediatric brain tumors. We leveraged network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct cell states. We produced a DMG regulatory network from 122 publicly available RNAseq profiles with ARACNe, and inferred sample-specific MR protein activity with VIPER. A CRISPR/Cas9 KO screen across 3 DMG patient cell lines identified a set of 73/77 essential genes that were enriched in the MR signature of 80% of patient samples (GSEA p=0.000034). FOXM1 emerged as an essential MR, significantly activated across virtually all patients. We then generated RNAseq profiles following perturbation with ~300 oncology drugs in 2 DMG cell lines most representative of patient MR signatures, and used this to identify drugs that invert patient MR activity profiles using the NYS/CA Dept.of Health approved OncoTreat algorithm. OncoTreat predicted sensitivity to HDAC, MEK, CDK, PI3K, and proteosome inhibitors in subsets of patients. 80%of OncoTreat-predicted drugs (p<10-5) from 3 DMG patient tumor biopsies showed in vitro sensitivity in cultured tumor cells from the respective patients, with overall 68% accuracy among 223 drugs evaluated by both OncoTreat and in vitro (Fisher’s Exact Test p=0.0449). Further analysis of DMG intra-tumor heterogeneity via protein activity inference from published scRNAseq profiles identified 6 tumor clusters with unique MR signatures representing distinct cellular states. Targetable MRs and OncoTreat-predicted drugs were distinct between these states. Bulk RNAseq analysis recapitulated predictions seen in the more prevalent Oligodendrocyte progenitor cell-like states, but failed to capture MR and drug predictions for the Astrocyte-like states. Ongoing validations of cell state-specific drug predictions in vivo in subcutaneous patient-derived xenograft and orthotopic syngeneic DMG models have already shown tumor volume and subpopulation differences (e.g. Trametinib-treated). This provides a platform to nominate much-needed novel drugs to treat DMG.
DIPG-50. INSIGHTS INTO TUMORIGENESIS OF DIFFUSE INTRINSIC PONTINE GLIOMA-ONCOHISTONE AND SIGNALING
Authors: Sun Ye , Yan Kun , Wang Dan , Zhou Wei , Wang Yi , Xu Cheng , Han Yujie , Tang Yujie , Zhang Liwei , Xi Qiaoran
Published: June 2023

Diffuse intrinsic pontine glioma (DIPG) is a rare and incurable pediatric brain cancer with survival of less than one year. Understanding the tumorigenesis mechanisms of DIPG and identifying potential therapeutic strategy are major research foci in the DIPG field. The most lethal subtype of diffuse intrinsic pontine glioma (DIPG) is the H3K27M subtype. Although ACVR1 mutations have been implicated in the pathogenesis of this presently incurable disease, the impacts of BMP signaling on more than 60% of H3K27M DIPG carrying ACVR1 wild-type remain unknown. Here, we show that BMP ligands exert potent tumor suppressive effects against H3.3 K27M and ACVR1 WT DIPG in a SMAD-dependent manner. Specifically, clinical data revealed that many DIPG tumors have exploited CHRDL1’s capacity to hijack BMP ligands. We discovered activation of BMP signaling promotes the exit of DIPG tumor cells from “prolonged-stem-cell-like” state to differentiation by epigenetically regulating CXXC5, which acts as a tumor suppressor and positive regulator of BMP signaling. Beyond showing how BMP signaling impacts DIPG, our study also identified potent anti-tumor efficacy of Dacinostat for DIPG. Thus, our study delineates context-dependent features of BMP signaling pathway in DIPG subtype.
DIPG-57. TESTING FOR SYNERGY AMONG RADIATION AND HIGHLY CLINICALLY RELEVANT CHEMOTHERAPEUTIC AGENTS IN PATIENT-DERIVED MODELS OF DIFFUSE MIDLINE GLIOMA
Authors: Slade Mathew , Link Gabrielle , DeSisto John , Feddersen Charlotte , Bargas Damien , Knox Aaron , Green Adam
Published: June 2023

Diffuse midline gliomas (DMG) are aggressive pediatric brain tumors with minimal long-term survival. Radiation is currently the only standard-of-care treatment and is non-curative. Several chemotherapy agents have shown preclinical and/or early clinical promise, and we hypothesize that finding effective chemoradiotherapeutic combinations is likely to be necessary to achieve long-term survival, based on experience from other previously incurable childhood cancers. We treated 8 DMG/DIPG cell lines with one, two, or three treatments among the HDAC inhibitor panobinostat, the CDK4/6 inhibitor ribociclib, the DRD2 inhibitor ONC201, the XPO1 inhibitor selinexor, the PI3K inhibitor paxalisib, and radiation. We then measured the cell survival with MTS assays. Cells were treated with drug concentrations ranging from 1 nM to 100 uM. To assess for synergy among our combinations, we used the Combination Index (CI) model, in which a score of <1 implies synergy. Of the 15 2-treatment combinations, ONC201 + radiation, ONC201 + paxalisib, ribociclib + ONC201, selinexor + paxalisib, and ribociclib + paxalisib met our criteria as successful, synergistic combinations: at least three cell lines with at least 90% cell death achieved, a CI value of 0.5 or lower at doses with at least 50% cell death, and at least 66% of dose levels with a CI value of 0.5 or lower. Early analysis suggests that ribociclib + ONC201 + paxalisib, ONC201 + paxalisib + radiation, and ONC201 + selinexor + paxalisib are the most effective three-treatment combinations. Patient-derived cell culture data in a heterogeneous pool of DMG/DIPG cell lines demonstrate synergy using several combinations of clinically relevant treatments, especially those including paxalisib. We are now optimizing treatment sequencing in synergistic combinations and testing in orthotopic xenograft models. Our goal is to find combinations that can become cycles of therapy in a multimodality clinical trial.
EPEN-02. OPTIMISING BIOMARKERS FOR ACCURATE EPENDYMOMA DIAGNOSIS, PROGNOSTICATION AND STRATIFICATION WITHIN INTERNATIONAL CLINICAL TRIALS: A BIOMECA STUDY
Authors: Chapman Rebecca J , Ghasemi David R , Andreiuolo Felipe , Zschernack Valentina , Espariat Arnault Tauziede , Buttarelli Francesca , Giangaspero Felice , Grill Jacques , Haberler Christine , Paine Simon M L , Scott Ian , Jacques Thomas S , Sill Martin , Stephan Pfister , Kilday John Paul , LeBlond Pierre , Massimino Maura , Modena Piergiorgio , Varlet Pascale , Pietsch Torsten , Grundy Richard G , Pajtler Kristian W , Ritzmann Timothy A
Published: June 2023

Accurate identification of brain tumour molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European “Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)” study. Across six European BIOMECA laboratories we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via FISH and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH. DNA Methylation profiling classified 65.3% (n=96/147) of cases as EPN-PFA and 15% (n=22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99-100% across three centres). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP1- fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-centre concordance and low sensitivity and specificity whilst MIP, MLPA and DNA methylation-based approaches demonstrated greater accuracy. We confirm, in a prospective trial cohort, that H3K27me3 IHC is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q and CDKN2A loss whilst FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. We propose CORE and CORE Plus test sets to guide future diagnostic approaches. CORE tests represent those that can currently be used to stratify and inform clinical trials and diagnosis and include IHC and DNA methylation profiling. CORE Plus tests have additional advantages for challenging cases and for use in the research setting and comprise of MIP and RNA-NGS sequencing.
EPEN-11. CHARACTERIZATION OF THE SPATIAL ARCHITECTURE OF INTRATUMORAL CELL STATES IN PEDIATRIC HIGH-RISK EPENDYMOMA
Authors: Bruckner Katharina , Madlener Sibylle , Biagi Junior Carlos A O , Gabler Lisa , Senfter Daniel , Jeong Daeun , Jiang Li , Englinger Bernhard , Jaunecker Carola , Bilban Martin , Haberler Christine , Dorfer Christian , Rössler Karl , Kool Marcel , Berger Walter , Filbin Mariella G , Lötsch-Gojo Daniela , Gojo Johannes
Published: June 2023

By single-cell analyses, we previously uncovered distinct intratumoral transcriptomic ependymoma (EPN) cell states and developmental trajectories correlating with molecular subtype and biological behavior. However, the relation of these cell states to characteristic EPN tissue architecture and tumor microenvironment remains widely unknown. We aimed to characterize the spatial architecture and elucidate regulatory circuits in EPN via spatial transcriptomics (10X Visium) of tumor specimens matching our previously generated single-cell transcriptome data spanning all EPN subtypes (ZFTA, YAP1, PFA, PFB). We investigated whether spatially restricted cell-cell interactions correlate with our previously identified cell states. Analyses revealed distinct spatially resolved cell populations in supratentorial and posterior fossa EPN. All subgroups showed several (5-9) spatially discrete cell clusters. Via non-negative matrix factorization (NMF), cell-specific spatial clusters emerged correlating with previous single-cell programs. ZFTA EPN highly correlates with ZFTA-specific metaprograms whereas all other subgroups did not show ZFTA-specific gene expression. Explicit genes such as DLK1 are exclusively upregulated in ZFTA EPN indicating spatially distinct aberrant Notch pathway signaling. Interestingly, this subgroup revealed spatial zones with upregulation of metabolic genes and endothelial markers anti-correlating with a ZFTA-specific program. Ependymal and neuronal-precursor-like signatures were enriched in our unique YAP1 EPN sample in a spatially restricted pattern, whereas GFAP was ubiquitously expressed. PFA was characterized by a neural stem cell (NSC)-like niche accompanied by metabolic and immune-reactive gene signatures. PFB showed a similar cell niche associated with NSC-like and glial-progenitor-like genes enriched simultaneously, further highlighting the NSC characteristic of posterior fossa tumors. Ependymal signatures were absent in PFA and present in PFB indicating more differentiated cell populations in PFB. Summarizing, we confirm presence of distinct intratumoral cell states within ependymoma tissue and reveal their spatial organization. In depth analysis and functional validation in matched ependymoma models will enable interrogation of underlying tumor promoting signaling events driving intratumoral developmental trajectories.
METB-06. HIGH FREQUENCY OF CLINICALLY RELEVANT ALTERATIONS DETECTED BY INTEGRATED TUMOR AND GERMLINE GENE PANEL TESTING IN CHILDREN WITH CNS TUMORS – RESULTS FROM THE TEXAS KIDSCANSEQ STUDY
Authors: Lin Frank Y , Desrosiers Lauren , Mangum Ross , Reuther Jacquelyn , Mohila Carrie , Blessing Melissa M , Aldave Guillermo , Muzny Donna M , Bernini Juan Carlos , Gill Jonathan , Griffin Timothy , Tomlinson Gail , Vallance Kelly , Chintagumpala Murali M , Scollon Sarah , López-Terrada Dolores H , Fisher Kevin E , Roy Angshumoy , Plon Sharon E , Parsons D Williams
Published: June 2023

BACKGROUND

Tumor and germline alterations detected by sequencing have the potential to inform management of children with CNS tumors. Comparative clinical utility data for available molecular sequencing modalities are needed to optimize testing strategies.

METHODS

Cancer patients less than 18 years of age were eligible to enroll in the Texas KidsCanSeq (KCS) clinical genomics study at six Texas institutions. Blood testing was performed on all patients using a pediatric solid tumor targeted DNA panel (124 genes), which included 35 genes associated with known cancer predisposition syndromes. Tumor testing was conducted on high-risk or recurrent tumors using the same DNA panel as well as an 81-gene RNA fusion panel. Tumor variants were classified as being of strong (Tier 1) or potential (Tier 2) clinical significance following AMP/ASCO/CAP guidelines. Germline pathogenic (P) or likely pathogenic (LP) variants per ACMG/AMP guidelines were considered clinically relevant.

RESULTS

A total of 224 children with CNS tumors were enrolled, including 146 patients with high-risk or recurrent tumors. Tissues were sufficient for analysis from 132 tumors and 211 germline samples. Clinically relevant alterations were identified in 90 of 132 (68%) tumors tested. Of 50 with therapeutic implications, 21 (42%) were not known from prior clinical testing. Therapeutically relevant tumor variants most frequently involved BRAF (n=20), CDKN2A/B (n=16), SMARCB1 (n=9), PIK3CA (n=8), and NF1 (n=7). One high grade glioma (in a patient with Lynch syndrome) was found to be hypermutated (tumor mutation burden >190 mut/Mb). Germline analysis detected P/LP variants in cancer predisposition genes in 25 of 211 (12%) patients tested, including 12 of 135 (9%) with high-risk/recurrent tumors.

CONCLUSION

Combined tumor and germline panel testing revealed alterations of clinical relevance according to consensus guidelines in approximately 70% of children with high-risk or recurrent CNS tumors.