bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3023: B7 H3 and autophagy in medulloblastoma
Authors: Kesaf Ayse E. , Shishido Katherine , Velpula Kiran K. , Asuthkar Swapna
Published: July 2021

B7-H3 and Autophagy in Medulloblastoma (MB) is the most common malignant pediatric brain tumor and we reported high expression of the immune checkpoint protein, B7-H3. We also showed that B7-H3 promotes exosome secretion, with a plausible cross-talk between exosomes and autophagy in tumor development. Autophagy promotes or suppresses tumorigenesis but its role in pediatric brain tumors has not be elucidated. As our recent research has shown, the elevated levels of B7-H3 in MB enhances exosome secretion that may regulate autophagy processes favoring tumorigenesis over tumor suppression. Therefore, the goal of this study is to investigate the relationship between B7-H3 and autophagy pathways in MB in efforts to improve treatment options and patient survival. Datamining studies conducted using MB patient datasets showed a negative correlation between B7-H3 and autophagy markers; Beclin1, LC3, and ATGs. Kaplan-Meier analysis further demonstrated an inverse relationship between autophagy markers and B7-H3 in the survival curves. Next, using human autophagy array, we observed significant reduction in the expression of Beclin1, LC3, and ATGs upon B7-H3 overexpression in D425 cells. In a separate experiment, we show that silencing B7-H3 using specific shRNA resulted in increased levels of LC3-II. Functional studies performed using FACs analyses and clonogenic assay on shB7-H3 treated- D425, UW228 cells revealed that inhibition of B7-H3 was associated with augmented apoptosis and reduction in colony formation, respectively. These results demonstrate the negative role of B7-H3 in MB autophagy regulation. Therefore, inhibition of B7-H3 may prove effective at promoting autophagy-mediated cell death in MB cells.

Citation Format: Ayse E. Kesaf, Katherine Shishido, Kiran K. Velpula, Swapna Asuthkar. B7 H3 and autophagy in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3023.
Abstract 3036: ALK protein expression and its prognostic significance in MYCN non amplified neuroblastoma
Authors: Aravindan Natarajan , Somasundaram Dinesh Babu , Shanmugarama Santny , Aravindan Sheeja
Published: July 2021

Anaplastic lymphoma kinase (ALK) point mutations (within the kinase domain), gene amplification and protein overexpression are identified (8-14%) both in familial and sporadic high-risk neuroblastoma (HR-NB). ALK mutations are much more frequent at time of relapse and were associated with poor clinical outcomes for children with HR-NB. Evidently, ALK is near exclusively co-amplified with MYCN, and the cooperative activity of ALK and MYCN are known to drive HR-NB pathogenesis. However, the ALK protein expression and its role in the prognosis of MYCN non-amplified NB remained unexplored. In this study, NB tumors representing varied stages, ages, sites, diagnosis/progressive disease, primary/metastatic site from a cohort MYCN non-amplified NB patients (n=92) were custom archived in TMAs and stained with ALK (immunohistochemistry) antibody. The correlations between ALK protein expression, and clinico-pathological and biological variables of MYCN non-amplified NB tumors were analyzed. Assessing the strong positivity, high expression of ALK protein could be detected in 47 (51%) of the 92 NB tumors. The majority of NBs with evident of ALK protein high expression exhibited undifferentiated or poorly differentiated histology (39/47, 82.9%). ALK protein high expression significantly predicted worse survival outcomes for NB (and HR-NB subset) patients with MYCN non-amplified status. Transcriptional data mining (R2: Genomics Analysis and Visualization Platform) across multiple cohorts of MYCN non-amplified NB patients revealed unique and defined associations of ALK to the adverse clinical prognostic factors (e.g. advanced disease stage, risk) and poor survival outcomes. Our study recognized the prognostic role of ALK protein expression in MYCN non-amplified NB. Since, ALK is indicated to transactivate MYCN, increase MYCN protein expression, and coordinate disease progression; further studies are warranted to define the mechanism(s) of ALK high-expression associated disease evolution in MYCN non-amplified HR-NB.Funding: This work was partially or in full, funded by Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-04; National Institutes of Health, NIH-P20GM103639 and; NIH-NCI- Cancer Center Support Grant P30CA225520

Citation Format: Natarajan Aravindan, Dinesh Babu Somasundaram, Santny Shanmugarama, Sheeja Aravindan. ALK protein expression and its prognostic significance in MYCN non amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3036.
Abstract 3045: Investigating the influence of glycosylation using cellular models of neuroblastoma
Authors: Cook Meghan , Garcia Alma , Konieczny Katie , Ferguson Meghan , Campbell Robert
Published: July 2021

Background: Neuroblastoma is a common childhood malignancy with approximately 800 new cases diagnosed each year in the United States. Treatment of neuroblastoma directly depends on patient risk categories and includes combinations of surgery, radiation therapy, chemotherapy, stem cell therapy, immunotherapy, and retinoid therapy. Various characteristics of cancer in general and some specific to neuroblastoma cell lines contribute to a difficulty in delivering clinically relevant concentrations of therapeutic agents to neuroblastoma masses. Amplification of the MYCN gene in certain neuroblastoma cell lines correlates with increased cellular surface glycosylation levels as well as a worse prognosis for patients. Overexpression of the MUC1 gene is also associated with increased glycosylation, specifically type-O, in various cancers, but the role of MUC1 in neuroblastoma has not been thoroughly defined. The objective of this study was to evaluate the influence of type-O glycosylation on therapeutic uptake in different neuroblastoma cell lines.

Methods: The neuroblastoma cell line SK-N-DZ, a MYCN-amplified line, was compared to SK-N-SH, a non-MYCN-amplified line (negative control) and BT-20, a highly glycosylated breast cancer cell line (positive control). Benzyl-α-GalNAc, an O-glycosylation inhibitor, was introduced to each cell line. To determine glycosylation levels in the respective cell lines, fluorescence assays were performed using CD227 (anti-MUC1) antibody. In vitro cytotoxicity assays were performed to determine maximum non-toxic concentrations of the glycosylation inhibitor (benzyl-α-GalNAc) and antibody (CD227) and to subsequently evaluate the functional role of glycosylation using cellular models of neuroblastoma.

Results: The maximum non-toxic concentrations per 10,000 cells for each neuroblastoma cell line was 0.05 mg/ml of benzyl-α-GalNAc, and 6 µl of CD227 stock preparation. Preliminary results from fluorescence detection studies showed an increase in CD227-FITC antibody uptake by the MYCN-amplified neuroblastoma cells (SK-N-DZ) following exposure to the O-glycosylation inhibitor (benzyl-α-GalNAc). Cellular uptake of CD227-FITC by non-MYCN-amplified cell line (SK-N-SH) was less by comparison.

Conclusion: Preliminary findings suggest that O-glycosylation is a barrier capable of limiting cellular uptake of agents in MYCN-amplified neuroblastoma cell lines. Ongoing studies are focused on investigating the influence of glycosylation inhibition on cytotoxicity of FDA-approved therapeutic agents.

Citation Format: Meghan Cook, Alma Garcia, Katie Konieczny, Meghan Ferguson, Robert Campbell. Investigating the influence of glycosylation using cellular models of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3045.
Abstract 3047: Spatial heterogeneity in diffuse intrinsic pontine gliomas treated with a PDGFR inhibitor
Authors: Arunachalam Sasi , Brady Samuel W. , Ma Xiaotu , Szlachta Karol , Mulder Heather , Easton John , Tinkle Christopher L. , Wetmore Cynthia , Baker Suzanne J. , Zhang Jinghui
Published: July 2021

Diffuse intrinsic pontine gliomas (DIPG) are lethal pediatric brain tumors that arise in the pons. To understand the spatial heterogeneity in DIPG, we performed ultra-deep sequencing and copy number profiling of 44 multi-region autopsy samples from 10 DIPG patients after phase I treatment with the PDGFR inhibitor crenolanib. Our study recapitulated the previously reported temporal order of driver mutation acquisition starting with histone H3 K27M followed by cell cycle (TP53 and PPM1D) or growth factor pathways (AVCR1, or PIK3R1), with mutations affecting PI3K and MAPK pathways being late, subclonal events. Further, we observed that aneuploidy was likely an early event as copy number heterogeneity was minimal in most patients. Compared to previous DIPG studies, we observed an enrichment in subclonal mutations and convergent evolution of PI3K alterations. This enrichment may have been due to selection for crenolanib resistance, or to the prior treatment history of the patients within the cohort. We also detailed intra-patient clonal composition among tumor samples and identified founder clones that were dominant in only a single location, which we considered to be non-migratory “occupier” clones, in four patients. By contrast, in six other cases, we observed clones that frequently invaded other sites. Interestingly, in one case with TP53 convergent evolution, aneuploidy clone carrying TP53 R273H mutation invades all other sequenced tumor sites whereas another clone with TP53 R248W does not. These results indicate the importance of understanding spatial heterogeneity within DIPG patients, which is crucial to the identification of effective treatments.

Citation Format: Sasi Arunachalam, Samuel W. Brady, Xiaotu Ma, Karol Szlachta, Heather Mulder, John Easton, Christopher L. Tinkle, Cynthia Wetmore, Suzanne J. Baker, Jinghui Zhang. Spatial heterogeneity in diffuse intrinsic pontine gliomas treated with a PDGFR inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3047.
Abstract 3057: Development of radiation-triggered phosphor platform for localized activation in combinatory cancer treatment
Authors: Papineni Rao V. , Krishnan Sunil , Pulickel Ajayan , Sahin Onur
Published: July 2021

A defined spatial and temporal control of induction of toxicity in response to external ionizing radiation such as X-rays has unique clinical benefits over conventional systems such as passive drug delivery or its activation. Here, we have combined X-rays with well characterized nanoscintilators emitting light that activates paired photosensitizers in photodynamic therapy (PDT) that improves the prowess of PDT to deep seated tumor treatment. Yttrium oxide nanoparticle construct doped with Europium (YOE) and coated with a silica layer entrapping methylene blue (MB) as the photosensitizer was fabricated. The nanoconstruct (YSM, for YOE, silica, MB) was decorated with polyethylene glycol (PEG) to make it biocompatible and characterized extensively for physicochemically parameters. Transmission electron microscopy (TEM) revealed a resultant average nanoparticle diameter of 24.1 ± 5.7 nm, and dynamic light scattering (DLS) a hydrodynamic diameter of 135.3 ± 17.9 nm. This nanoconstruct was evaluated for its ability to generate singlet oxygen reactive oxygen species upon irradiation using singlet oxygen sensor green (SOSG) fluorescence assay. Further, demonstrated that this nanoconstruct was taken up by cancer cells in vitro and by tumors in vivo. The uptake of YSM particles by Panc02 murine pancreatic cancer cells was tested by inductively coupled plasma – mass spectrometry. This demonstrated robust uptake in cells after 24hr incubation. No noticeable cell death occurred with doses of 0 Gy and 4 Gy, cell viability decreased significantly after a dose of 8 Gy irradiation in cells cultured with YSM when compared to controls. Since 8 Gy is within the range of a clinically applicable dose used routinely in stereotactic radiotherapy regimens, these results verified the potential of YSM to be used as scintillator-photosensitizer complexes for x-ray activated photodynamic therapy. Finally the biodistribution of YSM nanoconstructs in vivo in C57BL6 mice harboring subcutaneous Panc02 tumors show YSM accumulation in tumors. A versatile, biocompatible Yttrium oxide scintillator-photosensitizer complex (YSM) that can generate x-ray induced cytotoxic reactive oxygen species is fabricated. This is capable of facilitating x-ray activated photodynamic therapy (XPDT (external beam) and/or iXPDT (internal x-ray source)). It shows promise in maximizing the efficacy of the combinatory treatment and enhanced improvement of the quality of life and overall survival of cancer patients.

Citation Format: Rao V. Papineni, Sunil Krishnan, Ajayan Pulickel, Onur Sahin. Development of radiation-triggered phosphor platform for localized activation in combinatory cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3057.
Abstract 3059: Heavy ion radiation exposure perturbs intestinal stem cell homeostasis and induces accelerated tumorigenesis in apc mutant mouse intestine
Authors: Kumar Santosh , Suman Shubhankar , Datta Kamal , Fornace Albert J.
Published: July 2021

In a previous study, we demonstrated long-term decreased intestinal epithelial cell migration after low-dose heavy ion iron radiation, and this was associated with increased senescence-associated secretory phenotype (SASP) signaling. However, we know very little about the long-term effects of low dose space radiation on intestinal stem cell (ISC) senescence and SASP that have implications for intestinal homeostasis. Male Lgr5+ (Lgr5-EGFP-IRES-creERT) mice or Lgr5+/APC1638N/+ were exposed to 0.5 Gy of sham, γ-rays, or 28Si (69 keV/μm) radiation and were euthanized 60 or 150 days after irradiation. ISCs were FACS sorted for oxidative stress analysis and assessed for senescence and SASP signaling in tissue sections by immunostaining. Greater cellROX or mitoSOX staining was observed after 28Si indicating higher oxidative stress compared to γ-rays or control Lgr5 positive cells. β-gal co-staining with Lgr5, lysozyme, IL1R, or ILβ showed highest staining in 28Si irradiated compared to controls ISCs samples. γ-H2AX staining showed greater foci numbers in 28Si relative to γ-rays or control group. Higher expression of secretory cytokines IL8 and IL15 was observed in 28Si irradiated ISCs relative to γ-rays or control group at both time points. Elevated expression of DKK2 and activation of NF-κB and p38 MAPK were observed in senescent stem cells of 28Si irradiated relative to γ-rays or control group at these time points. A higher GI tumor count was observed in 28Si compared to γ-irradiated or control Lgr5+/APC1638N/+ mice. In summary, our results demonstrate low dose heavy ion exposure induces ongoing DNA damage, persistent oxidative stress, senescence and acquisition of the SASP in ISCs. Higher levels of oxidative stress, senescence and SASP were observed at 150 compared to the 60-day timepoint. Overall, space radiation-induced oxidative stress, senescence, SASP, and activated p38MAPK/NF-κB pathway may very well have implications in GI carcinogenesis, accelerated aging, and pathological conditions, such as intestinal dysfunction, in astronauts during deep space flights. Since similar effects have been seen in our studies with other particle beams including carbon ions, this has potential relevance to particle radiotherapy as well. This work was supported by NASA NNX15AI21G

Citation Format: Santosh Kumar, Shubhankar Suman, Kamal Datta, Albert J. Fornace. Heavy ion radiation exposure perturbs intestinal stem cell homeostasis and induces accelerated tumorigenesis in apc mutant mouse intestine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3059.
Abstract 3064: Investigation of ionizing radiation response in new resistance models of glioblastoma
Authors: Pinarbasi Nareg , Bagci-Onder Tugba , Erdem Ilknur Sur , Selek Ugur
Published: July 2021

Glioblastoma is one of the most aggressive central nervous system tumors, which constitutes 50% of all primary brain tumors. Glioblastomas have a very poor prognosis, approximately 12-15 months even with standard therapy. Current treatment protocols include surgery, chemotherapy, and radiotherapy. High rates of recurrence after treatment, genetic heterogeneity, and developing resistance to chemotherapy and radiotherapy are challenges to overcome in glioblastoma treatment. Therefore, developing models of chemo- or radio-resistance is of priority to understand the molecular mechanisms that operate during the transition of cells to therapy-refractory state, and to develop new therapeutic approaches. In this study, we generated ionizing radiation (IR)-exposed glioblastoma cell populations to investigate molecular alterations associated with acquired radiotherapy resistance. To generate a clinically relevant radioresistance model, we have exposed glioblastoma cells to a total dose of 60 Gy IR over a 6-week period and cultured age-matched parental cells. We successfully generated sustainable radioresistant cells, as demonstrated by cell viability and colony formation assays. RNA sequencing, performed on these matched cell lines revealed major alterations in the expression of genes of the cell cycle, DNA damage recognition, and repair machinery in the radioresistant cells. Specifically, elements of the DNA double strand break (DSB) recognition and repair, such as ATM, ATR, and CHK1 were markedly upregulated in the radioresistant cell populations. In line with these findings, DNA repair capacity of the radioresistant cell populations was increased, as revealed by the faster clearance of H2AX and 53BP1 in the radioresistant populations. Complementing this approach, pharmacological inhibition of major DNA damage response elements, such as ATM, ATR, as well as CHK1 markedly sensitized the radioresistant cells. Our results prepare a groundwork for studying the molecular mechanisms of radiotherapy resistance and developing novel combination therapy strategies for glioblastomas.

Citation Format: Nareg Pinarbasi, Tugba Bagci-Onder, Ilknur Sur Erdem, Ugur Selek. Investigation of ionizing radiation response in new resistance models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3064.
Abstract 3069: Radiation exposure of the base of the heart accelerates coronary atherosclerosis
Authors: Nepali Prerna R. , Mathieu Mickael , Kitz Sarah , Nakauchi Chiharuko , Russell James , Monette Sebastien , Rimner Andreas , Stewart Fiona A. , Jaimes Edgar A. , Haimovitz-Friedman Adriana
Published: July 2021

Purpose: To determine the mechanisms associated with the development of atherosclerotic lesions in different areas of the heart exposed to radiation therapy (RT). We hypothesize that accelerated development of cardiovascular disease (CVD) post-RT is dependent upon the differential sensitivity of regions of the heart to radiation.

Experimental procedures: Male Apolipoprotein E knockout (ApoE-/-) mice on a high fat diet received 16Gy cardiac irradiation targeted to the whole or partial (apical or basal) region of the heart at 9 weeks or 16 weeks of age. Atherosclerotic lesions and inflammatory changes in the hearts as compared to control unirradiated mice were assessed eight weeks following radiation.

Results: Our studies demonstrate that atherosclerotic lesion formation is accelerated by cardiac RT. Our results show that (1) Subendocardial atherosclerotic lesions at the base of heart of 9 weeks old mice after basal irradiation are comparable to those formed post whole heart irradiation. (2) A greater number of atherosclerotic lesions were present in the basal coronary arteries and basal subendocardial vasculature after irradiating the cardiac base as compared to unirradiated controls in older mice at 8 weeks post-RT. (3) Apical or whole heart irradiation had no impact on the development or acceleration of lesions in the basal region of the hearts of 16 week old mice, thus demonstrating the adverse impact of basal irradiation. (4) Infiltration of inflammatory cells (CD45 and CD3) and enhanced expression of endothelial adhesion molecules (CD31) were differentially and locally regulated based upon the site of irradiation. This also demonstrates a long-term inflammatory response to RT which may be crucial in the development of RT-related CVD.

Conclusion: Our results indicate that the base of the heart is more prone to development of RT-induced atherosclerotic lesions that could lead to CVD and thus omitting this area from RT direct exposure may improve the quality of life for cancer patients receiving thoracic RT.

Citation Format: Prerna R. Nepali, Mickael Mathieu, Sarah Kitz, Chiharuko Nakauchi, James Russell, Sebastien Monette, Andreas Rimner, Fiona A. Stewart, Edgar A. Jaimes, Adriana Haimovitz-Friedman. Radiation exposure of the base of the heart accelerates coronary atherosclerosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3069.
Abstract 3075: Effect of reducing AXIN2 levels in CRC cells with APC mutation and activated WNT signaling
Authors: Zhang Chi , Facey Caroline O. , Funk Katherine , Opdenaker Lynn M. , Boman Bruce M.
Published: July 2021

Regulation of the WNT/beta-catenin signaling pathway occurs by a negative feedback loop that keeps beta-catenin levels in check in normal cells. Dysregulation of this feedback mechanism due to APC mutation is well known. However, the role of AXIN2, another component of this negative feedback loop is less known, particularly in APC mutant tissues. Our goal is to study how AXIN2 functions as part of the negative WNT/beta-catenin signaling feedback loop and understand of how it contributes to CRC development. Our bioinformatics analysis suggests that CRC cells carrying APC-truncating mutations still retain residual function of the destruction complex, but there are significant changes between AXIN2 and APC expression levels. Therefore, we hypothesize that the interaction between truncated-APC and AXIN2 is responsible for the dysregulation of the feedback loop. We found, using a TCF/LEF reporter assay, that siRNA-mediated knockdown of AXIN2 leads to increased WNT/beta-catenin signaling. This result confirmed the functional integrity of the downstream part of the WNT pathway from destruction complex to transcription activation by TCF/beta-catenin complex. It also narrowed down the dysregulated component(s) to within the destruction complex. Protein analysis further demonstrated that AXIN2 siRNA-mediated knockdown changed expression of several WNT/beta-catenin target genes. Surprisingly, siRNA-mediated knockdown led to decreased cell proliferation of HT29 cells. We surmise that AXIN2 knockdown in the setting of mutant APC leads to a high level of WNT signaling that reduces the self-renewal capacity of colonic cancer stem cells and lowers the rate of cell proliferation. Overall, our results indicate that it is important to understand how WNT signaling levels play a role in SC self-renewal and stemness. We conjecture that if WNT signaling is not strictly controlled in CSCs, high WNT levels can be detrimental to CSC proliferation and/or survival, which could have important clinical implications.

Citation Format: Chi Zhang, Caroline O. Facey, Katherine Funk, Lynn M. Opdenaker, Bruce M. Boman. Effect of reducing AXIN2 levels in CRC cells with APC mutation and activated WNT signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3075.
Abstract 3077: ATRXLoss and IDH1R132H impair self-renewal of glioma stem cells after proton radiotherapy
Authors: Garcés Ángel Adrian , Bronk Lawrence , Bhat Krishna P. , Grosshans David R.
Published: July 2021

Background: Glioblastoma Multiforme (GBM-Grade IV) is the most aggressive form of glioma in the USA carrying only a 15 month median survival time. IDH1R132H is an early onset mutation present in 70-80% of low grade gliomas (LGG-Grade I and II) and secondary GBM patients that causes intracellular oxidative stress and impairment of post-radiation DNA damage repair (DDR) via Homologous Recombination (HR). Although IDH1R132H is associated with improved patient survival and clinical response to chemotherapy, astrocytoma (LGG) patients specifically also harbor ATRXLoss, a genetic alteration that inactivates the ATRX chromatin remodeling protein and impairs DDR via non-homologous end joining (NHEJ). Importantly, the recent discovery of glioma stem cells (GSCs), a subpopulation of chemoradioresistant, self-renewable, tumor initiating cells known to promote tumor recurrence, presents a new target for therapeutic intervention. However, the mechanisms by which IDH1R132H and ATRXLoss contribute to GSC sensitivity to proton radiotherapy (PRT), a more tumor specific and cytotoxic form of radiation therapy than conventional X-rays (XRT), is not well understood. We hypothesize that both IDH1R132H and ATRXLoss contribute to the impairment of GSC self-renewal after PRT, but not after XRT.

Methods: Isogenic MGG18-IDH1WT, MGG18-IDH1R132H, TS543-ATRXWT, and TS543-ATRXLoss patient derived glioma cells were cultured as neurospheres and treated with 3-6 Gy of XRT or PRT. GSC self-renewal, based on neurosphere formation frequency, was quantified using extreme limiting dilution analysis (ELDA).

Results: TS543-ATRXLoss significantly inhibited GSC self-renewal over TS543-ATRXWT when subject to PRT (Pairwise Testing Pr(>χ2)<0.01). However, TS543-ATRXWT displayed lower GSC self-renewal compared to TS543-ATRXLoss when subject to XRT (Pr(>χ2)<0.0001). Interestingly, similar results were found in the isogenic MGG18 cell lines. MGG18-IDH1R132H displayed higher radiosensitivity to PRT (Pr(>χ2)<0.05) while MGG18-IDH1WT displayed higher radiosensitivity to XRT (Pr(>χ2)<0.01), as defined by GSC self-renewal.

Conclusions: We conclude that ATRXLoss and IDH1R132H both substantially contribute to PRT sensitivity in GSCs, thereby supporting the benefits of PRT for astrocytoma patients. Our future experiments will further elucidate the biological mechanisms by which ATRXLoss and IDH1R132H confer PRT sensitivity, especially via dsDNA damage and apoptosis.

Citation Format: Ángel Adrian Garcés, Lawrence Bronk, Krishna P. Bhat, David R. Grosshans. ATRXLoss and IDH1R132H impair self-renewal of glioma stem cells after proton radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3077.
Abstract 3079: The antitumoral activity of metformin in glioblastoma stem cells is mediated by tmCLIC1 protein
Authors: Verduci Ivan , Cianci Francesca , Cannavale Gaetano , Castiglione Stefania , Ranucci Matteo , Mazzanti Michele
Published: July 2021

Glioblastoma (GB) is the deadliest brain tumor, representing a major clinical challenge in the field. Despite standard of care together with cutting-edge strategies, life expectancy stands invariantly poor. Tumor heterogeneity and its high-grade of invasiveness make it difficult to be surgically resected and recurrence is inevitable. There is scientific consensus that tumor relapse originates from progenitor/stem-like cells known as glioblastoma stem cells (GSCs) displaying properties of self-renewal and multi-lineage differentiation that contribute to tumor mass heterogeneity. Thus, GSCs represent the target of newly designed therapies. In recent years, it has been highlighted the anti-cancer properties of metformin, the first-line drug in Type-2 diabetes (T2D) treatment. Metformin assumption in T2D patients has been positively linked with a decrease in the risk of several cancer types and cancer-related mortality, including glioblastoma. Additionally, numerous studies demonstrated metformin's anticancer properties against GSCs. Despite metformin's mechanism of action on cancer cells has not been clarified yet, its effect has been proposed to target the oxidative phosphorylation (OXPHOS) pathway. Considering that GSCs have been reported to shuttle between glycolysis and OXPHOS energetic pathways, their metabolic plasticity should prevent the antiproliferative effect of metformin. However, metformin-treated GSCs show a phenotype superimposable to that caused by the blockage of the transmembrane form of the chloride intracellular channel 1 (tmCLIC1). tmCLIC1 involvement in the progression of GB has been assessed deeply in vitro as well as in vivo. In addition, its peculiar localization and enrichment on GSCs' membrane renders it a valuable pharmacological target for GB. The goal of the present work is to attest tmCLIC1 as the solely interactor with metformin in glioblastoma stem cells.

Ivan Verduci was supported by a AIRC fellowship for Italy.

Citation Format: Ivan Verduci, Francesca Cianci, Gaetano Cannavale, Stefania Castiglione, Matteo Ranucci, Michele Mazzanti. The antitumoral activity of metformin in glioblastoma stem cells is mediated by tmCLIC1 protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3079.
Abstract 3080: ILEI regulates LIFR expression and CSC self-renewal
Authors: Streitfeld William S. , Dalton Annamarie C. , Howe Philip H.
Published: July 2021

Our lab has previously identified a mechanism of transforming growth factor beta (TGFβ)-mediated epithelial-to-mesenchymal transition (EMT) in normal murine mammary gland (NMuMG) cells. Reduced expression of heterogenous nuclear ribonucleoprotein E1 (hnRNP E1, hereafter “E1”) in NMuMG cells induces EMT, tumorigenesis, and metastasis. Our experimental data has revealed that increased expression of interleukin-like EMT inducer (ILEI) and of leukemia inhibitory factor receptor (LIFR) proteins occurs following E1 knockdown (E1KD). ILEI and LIFR have been shown to interact by coimmunoprecipitation/radiography experiments, and there is a correlation between ILEI expression and phosphorylation of the LIFR effector STAT3. ILEI is necessary to induce EMT in NMuMG cells, and spheroid formation capacity in non-adherent serum-depleted cell culture conditions is attenuated following either ILEI or LIFR knockdown. Loss of spheroid formation following ILEI knockdown is rescued by treatment with exogenous recombinant ILEI protein (rILEI); however, spheroid formation is not rescued by rILEI following LIFR knockdown. In immunocompromised mice, orthotopic grafts of NMuMG cells with E1KD (E1KD cells) with additional knockdown of either ILEI or LIFR display a decrease in tumor initiation, growth, and metastasis relative to control cells. These data suggest that ILEI-induced spheroid formation capacity involves LIFR, and that an ILEI/LIFR signaling axis promotes breast cancer stem cell (BCSC) phenotype. Characterization of the mechanisms that allow BCSCs to persist during chemotherapeutic interventions will advance our understanding of drug resistant and dormant breast cancer. The precise mechanisms involved in ILEI-mediated EMT and ILEI/LIFR-mediated BCSC induction are unknown. Semi-quantitative PCR data demonstrates that the increase in LIFR expression in E1KD cells is regulated at the transcriptional level. Additionally, E1KD cells with CRISPR-Cas9-mediated ILEI knockout (E1KD-ILEI-KO cells) show decreased luciferase signal relative to control cells following transfection of LIFR promoter DNA sequences placed upstream of a luciferase gene. Further, E1KD-ILEI-KO cells show attenuation of spheroid formation when compared to unaltered E1KD cells. Our data allow us to conclude that ILEI participates in the regulation of LIFR gene transcription, which plays a role in ILEI-induced maintenance of spheroid formation and self-renewal. Continued investigation will allow development of studies aimed at revealing the mechanisms involved in ILEI/LIFR axis-mediated disease progression in vivo.

Citation Format: William S. Streitfeld, Annamarie C. Dalton, Philip H. Howe. ILEI regulates LIFR expression and CSC self-renewal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3080.
Abstract 3081: Secretory human ribonuclease 1 functions as Eph receptor A4 ligand to promote breast tumor initiation
Authors: Wang Ying-Nai , Lee Heng-Huan , Yang Wen-Hao , Hortobagyi Gabriel N. , Yu Dihua , Hung Mien-Chie
Published: July 2021

The pancreatic human ribonuclease (hRNase) A superfamily is comprised of eight canonical hRNases, including hRNase 1, all of which can be detected in various body fluids, e.g., serum and plasma. Secretory hRNase 1 exerts its ribonucleolytic activity to function in extracellular RNA clearance. Moreover, hRNase 1 regulates hemostasis, inflammation, and innate immunity, indicating that hRNase 1 possesses multiple functions in addition to RNA clearance. Studies on hRNase 1 have been extensively investigated the biochemical properties and post-translational modifications, such as glycosylation. Nonetheless, the biological function of hRNase1 and whether it plays a role in cancer have not yet been completely defined. Recent studies indicated that hRNase 5 serves as a ligand for the receptor tyrosine kinase (RTK) epidermal growth factor receptor and plexin-B2 receptor in solid and hematopoietic cancers. Those findings reveal a role of the hRNase A superfamily in tumor progression and an unconventional ligand-receptor relationship between RNase and RTK families. Here, we demonstrate that hRNase 1, independently its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the RTK Eph receptor A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical ligand-receptor ephrin-EphA4 juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from the paired breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 to enhance breast cancer stemness suggests a potential treatment strategy for breast cancer by inactivating the hRNase 1-EphA4 axis.

Citation Format: Ying-Nai Wang, Heng-Huan Lee, Wen-Hao Yang, Gabriel N. Hortobagyi, Dihua Yu, Mien-Chie Hung. Secretory human ribonuclease 1 functions as Eph receptor A4 ligand to promote breast tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3081.
Abstract 3105: 4-Methylumbelliferone (4Mu) turns CD133+ lung cancer stem cells (CSCs) more susceptible to conventional chemotherapy
Authors: Bezazian Ana L. , Escribano Maria Amanda , Gutierrez Marco A. Diaz , Fusco Mariel , Roselló Paula , Menendez Juan Braga , Fiore Esteban J. , Fina Juan M. Bayo , Rizzo Manglio M. , Malvicini Mariana
Published: July 2021

Non-small cell lung cancer (NSCLC) is the most common cause of cancer death worldwide. Taxane-platin chemotherapy is widely used for NSCLC treatment; however, the majority of patients will ultimately progress or relapse. In the tumor microenvironment (TME) cancer cells co-exist with cellular and non-cellular components that drive tumor processes such as chemotherapy resistance. A small group of tumor cells called cancer stem cells (CSCs) that express some markers such as CD133, CD44 and ALHD1 have particularly different proliferative, differentiation and self-renewal potential. In fact, CD133+ NSCLC cells showed significantly higher abilities of self-renewal and drug resistance characteristics when compared to CD133- cells. It has been partly described how the TME component hyaluronan (HA) regulates CSCs function. We have previously demonstrated that 4-Methylumbelliferone (4Mu), a modulator of HA synthesis, reduces CSCs properties in hepatocarcinoma. Here, we observed that HA was present Lewis Lung Carcinoma (LLC) tumors from mice. We found HA+ LLC cells; thus cancer cells produced, at least in part, the HA observed in tumors. We also observed about 6.58 ± 0.83 % of CD133+ CSCs on in vitro cultured LLC cells. Isolated CD133+ cells showed higher expression of HA and CD44 in comparison with non-CSCs population (p<0.05). Analysis of HA synthases (HAS), hyaluronidases (HYAL) and the CSCs transcription factors KLF4 and SOX2 expression from NSCLC patients using The Cancer Genome Atlas data showed that while HAS3 positively correlates with levels of KLF4 and SOX2, HYAL2 inversely correlates with SOX2 expression. Also, 4Mu treatment of whole, CD133+ or CD133- LLC cells showed an inhibitory concentration 50 (IC50) of 0.90mM, 0.60mM and 1.06mM respectively, while non tumor cells showed an IC50 of 4.22mM (p<0.01). When CD133+ cells were treated with 4Mu plus pemetrexed or paclitaxel we observed a significant decrease in viability with an IC50 of 0.4nM pemetrexed (Pe) vs. 0.05nM Pe+4M, and 9.3nM paclitaxel (Pa) vs. 0.80nM Pa + 4Mu. (p<0.05). Our results suggest that HA a principal ligand of CD44 is involved in CD133+ cells phenotype and their modulation by 4Mu could increases chemo sensibility of lung cancer stem cells.

Citation Format: Ana L. Bezazian, Maria Amanda Escribano, Marco A. Diaz Gutierrez, Mariel Fusco, Paula Roselló, Juan Braga Menendez, Esteban J. Fiore, Juan M. Bayo Fina, Manglio M. Rizzo, Mariana Malvicini. 4-Methylumbelliferone (4Mu) turns CD133+ lung cancer stem cells (CSCs) more susceptible to conventional chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3105.
Abstract 3115: Adora2a inhibition via caffeine as a novel therapeutic target for cisplatin-induced chemobrain
Authors: Oliveros Alfredo , Corujo-Ramirez Ana M. , Yoo Ki-Hyun , Tang Jason , Brogren Danielle , Rashid Mohammad A. , Liu Yuanhang , Jang Mi-Hyeon
Published: July 2021

Cisplatin chemotherapy, although efficacious in resolution of cancer, potentiates cognitive impairments (known as chemobrain) affecting approximately 14 million cancer survivors in the United States. Cisplatin is a frontline chemotherapy used to treat ovarian, testicular, breast and brain cancers. Despite the high prevalence of clinical reports of cognitive impairments during and following chemotherapy, there is little information on how learning/memory are detrimentally affected and no known cure. To elucidate the mechanisms by which chemobrain impairs cognition, we implemented a mouse model resembling intermittent cisplatin chemotherapy in humans. Our preliminary studies revealed that repeated intermittent cisplatin treatment detrimentally affects the structure of neuronal brain cells in the hippocampus, a brain region known for its ability to control learning/memory, emotional behaviors and motor function. Although the molecular pathways vulnerable to cisplatin in the brain remain elusive, gene sequencing from the hippocampus of mice administered cisplatin identified the adenosine A2A receptor (Adora2a) protein was concentrated in elevated quantities in this brain region. Importantly, the Adora2a receptor protein is known for its critical role in learning/memory and has been found to play a role in neurodegenerative conditions. To investigate Adora2a functionality in cisplatin-induced chemobrain, we assessed the efficacy of the non-selective Adora2a antagonist caffeine, in preventing cisplatin-induced cognitive dysfunction. Caffeine was selected as a therapeutic due to its safe pharmacological profile, its universal availability, and low cost. Our translational approach employing the non-selective Adora2a antagonist caffeine, protected against cisplatin-induced body weight deficits and motor dysfunction, both of which are common clinical symptoms of chemotherapy. Interestingly, relative to cisplatin alone, mice treated with caffeine in combination with cisplatin exhibited improved dendritic spine densities in the hippocampus, suggesting improved neuronal morphology by caffeine. Given that Adora2a antagonists are known to enhance anti-tumor activity while also showing neuroprotective promise in neurodegenerative conditions, inhibiting Adora2a during cancer treatment may have far-reaching synergistic effects on quality of life improvement for cancer survivors.

Citation Format: Alfredo Oliveros, Ana M. Corujo-Ramirez, Ki-Hyun Yoo, Jason Tang, Danielle Brogren, Mohammad A. Rashid, Yuanhang Liu, Mi-Hyeon Jang. Adora2a inhibition via caffeine as a novel therapeutic target for cisplatin-induced chemobrain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3115.
Abstract 3120: MMP-7 increases migration by decreasing intercellular cell junction complexes in prostate cancer microtumors formed by perlecan/HSPG2
Authors: Cruz Lissette A. , Tellman Tristen V. , Igoshin Oleg , Carson Daniel , Farach-Carson Mary (Cindy)
Published: July 2021

Background: Polarized epithelium is stabilized by lateral cell-cell interactions via cell adhesion molecules (CAMs) and by cell-matrix interactions with basement membrane including with perlecan/HSPG2 (pln). Prostate cancer (PCa) patients with bone metastases have circulating pln fragments, with inverse correlation between MMP-7 staining and loss of pln in cancer tissue. Cleavage of pln by MMP-7 increases cell-matrix interactions and dysregulates cell signaling, permitting migration. We earlier showed pln domain IV-3 (DmIV-3) drives cell-cell cohesion and, when digested with MMP-7, drives cell dyscohesion.

Methods: To evaluate the impact of MMP-7 and pln fragments on microtumor dyscohesion and cell migration, uniformly sized PCa microtumors were pre-formed in a microwell system. Pre-formed microtumors were transferred to Dm IV-3 or full length perlecan (FL pln) coated wells for 16-24 hrs. Microtumors were treated with MMP-7 alone or MMP-7 plus predigested Dm IV-3 fragments or MMP-7 plus FL pln fragments. For live cell imaging, tracking of migratory cells leaving microtumors was performed with Imaris software. Co-location of cell adhesion complex components was assessed with Imaris Spots.

Results: Pre-formed microtumors cultured with DmIV-3 cleaved by MMP-7 showed lower Pearson's correlation values at cell boundaries compared to microtumors treated with intact Dm IV-3. Line scan analysis revealed E-cadherin and F-actin fluorescent signals were enriched and co-aligned in microtumors treated with Dm IV-3; enrichment and co-alignment were reduced when DmIV-3 fragments and MMP-7 were present. Track number detected per cell cluster was highest in the presence of FL pln fragments plus MMP-7 along with a measurable change in distribution of track displacement lengths of cells toward high values.

Conclusion: Boundary reorganization promotes a migratory cell phenotype in microtumors treated with MMP-7 and DmIV-3 fragments. DmIV-3 fragments generated by MMP-7 cleavage can enhance cell dyscohesion by disrupting interactions between CAMs. Ongoing work will identify DmIV-3 fragment(s) positively associated with tumor dyscohesion that play key roles in secondary metastasis formation. Following patterns of dyscohesion of pre-formed microtumors provides a good model to study dynamic changes in intercellular junction components and quantitate cell migration patterns that foster circulating tumor cell formation and metastasis.

Citation Format: Lissette A. Cruz, Tristen V. Tellman, Oleg Igoshin, Daniel Carson, Mary (Cindy) Farach-Carson. MMP-7 increases migration by decreasing intercellular cell junction complexes in prostate cancer microtumors formed by perlecan/HSPG2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3120.
Abstract 3140: Tumor evolution selectively inactivates microRNA machinery for immune evasion
Authors: Zhao Haixin , Song Tian-yu , Long Min , Fan Hong-Jie , Liu Yang , Geng Chen-Lu , Xie Xiaoxiao , Huang Dawei , Peng Zhengang , Peng Bo , Cang Yong
Published: July 2021

Cancer arises after somatic mutations override cellular and immunological control of cell proliferation1. Mutations in oncogenes and tumor suppressor genes endow cancer cells intrinsic growth advantage over neighboring cells. Mutations can also enable cancer cells to escape from immune surveillance during cancer evolution, but a targeted approach to uncover such mutations is lacking. We hypothesized that genetic heterogeneity intrinsic to cancer cells affects the cell fitness to T cell-mediated cytotoxicity. By deep sequencing of murine tumors grown under different conditions of immune editing, we identified 59 genetic mutations in clonal populations that were specifically enriched under host immune pressure. To differentiate driver from passenger mutations against immune selection, we performed in vivo and T cell co-culture CRISPR screens with a guide RNA library targeting the corresponding genes. We identified ANKRD52 as required for PD-1-independent T cell-mediated cytotoxicity, which enhanced the JAK-STAT-interferon-γ signaling and antigen presentation in cancer cells. ANKRD52, in complex with PPP6C, dephosphorylates AGO26 and promoted microRNA (miRNA)-targeted silencing of suppressor of cytokine signaling 1 (SOCS1), and deletion of SOCS1 reinstalled the sensitivity of the mutant cells to T cell killing. Introduction of patient hotspot ANKRD52 mutations, or deletion of AGO2, DICER1 or XPO5 in cancer cells compromised the interferon-γ and T cell response. Remarkably, expression of the core miRNA biogenesis and targeting machinery strongly correlates with intratumoral T cell abundance across nearly all human cancer types. Our data indicate that the evolutionarily conserved miRNA pathway is exploited by cancer cells for immune escape.

Citation Format: Haixin Zhao, Tian-yu Song, Min Long, Hong-Jie Fan, Yang Liu, Chen-Lu Geng, Xiaoxiao Xie, Dawei Huang, Zhengang Peng, Bo Peng, Yong Cang. Tumor evolution selectively inactivates microRNA machinery for immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3140.