{"paper_id":"ef6b14ff-a6a2-4fb2-b071-c381d536be3e","body_text":"3131\nONCOSTREAMS: NOVEL DYNAMICS PATHOLOGICAL\nMULTICELLULAR STRUCTURES INVOLVED IN\nGLIOBLATOMA GROWTH AND INVASION\nAndrea Comba1, Patrick Dunn, Anna E Argento, Padma Kadiyala,\nSebastien Motsch, Phillip Kish, Alon Kahana, Daniel Zamler,\nKarin Muraszko, Maria G Castro and Pedro R Lowenstein\n1University of Michigan\nOBJECTIVES/SPECIFIC AIMS: Oncostreams represent a novel\ngrowth pattern of GBM. In this study we uncovered the cellular\nand molecular mechanism that regulates the oncostreams function\nin GBM growth and invasion. METHODS/STUDY POPULATION:\nWe studied oncostreams organization and function using genetically\nengineered mouse gliomas models (GEMM), mouse primary patient\nderived GBM model and human glioma biopsies. We evaluated\nthe molecular landscape of oncostreams by laser capture microdis-\nsection (LCM) followed by RNA-Sequencing and bioinformatics\nanalysis. RESULTS/ANTICIPATED RESULTS: Oncostreams are\nmulticellular structures of 10-20 cells wide and 2-400 μm long.\nThey are distributed throughout the tumors in mouse and human\nGBM. Oncostreams are heterogeneous structures positive for GFAP,\nNestin, Olig2 and Iba1 cells and negative for Neurofilament. Using\nGEMM we found a negative correlation between oncostream density\nand animal survival. Moreover, examination of patient ’s glioma\nbiopsies evidenced that oncostreams are present in high grade but\nno in low grade gliomas. This suggests that oncostreams may play\na role in tumor malignancy. Our data also indicated that onco-\nstreams aid local invasion of normal brain. Transcriptome analysis\nof oncostreams revealed 43 differentially expressed (DE) genes.\nFunctional enrichment analysis of DE genes showed that “collagen\ncatabolic processes ”, “positive regulation of cell migration ”, and\n“extracellular matrix organization ” were the most over-represented\nGO biological process. Network analysis indicated that Col1a1,\nACTA2, MMP9 and MMP10 are primary target genes. These genes\nwere also overexpressed in more malignant tumors (WT-IDH) com-\npared to the less malignant (IDH1- R132H) tumors. Confocal time\nlapse imagining of 3D tumor slices demonstrated that oncostreams\ndisplay a collective motion pattern within gliomas that has not been\nseen before. DISCUSSION/SIGNIFICANCE OF IMPACT: In sum-\nmary, oncostreams are anatomically and molecularly distinctive,\nregulate glioma growth and invasion, display collective motion\nand are regulated by the extracellular matrix. We propose onco-\nstreams as novel pathological markers valuable for diagnosis, prog-\nnosis and designing therapeutics for GBM patients.\n3024\nOsteocyte-derived CXCL12 is Essential for Load-Induced\nBone Formation in Adult Mice\nPamela Cabahug Zuckerman 1, Chao Liu, Emily Fang and\nAlesha B Castillo\n1New York University - H þH Clinical and Translational Science\nInstitute\nOBJECTIVES/SPECIFIC AIMS: Our aim is to test whether osteo-\ncyte-specific CXCL12 expression is critical to exercise-driven bone\nformation. METHODS/STUDY POPULATION: All procedures\nwere approved by the NEW YORK UNIVERSITY Institutional\nAnimal Care and Use Committee. We generated male and female\nmice in which CXCL12 was deleted from OCYs (CXCL12 Δ OCY)\nby crossing CXCL12 floxed mice and 10kb DMP1-Cre transgenic\nmice (gifts from Drs. Geoffrey Gurtner and Lynda Bonewald,\nrespectively). The 10kb DMP1-Cre has been shown to be robustly\nexpressed in odontoblasts and OCYs, with little to no activity in cells\nfrom non-mineralized tissues (Luþ J Dent Res 2007). Growing male\nand female mice (n=3-8/group) were given fluorochrome labels\nevery two weeks between 4-16 weeks of age, to monitor the role of\nCXCL12 during development. A second group, of adult 16-week-old\nmice (n=5/group), were subjected to tibial axial cyclic loading\n(1200με, 2Hz, 120cycles, 3days/wk for 2 wks) (Liu þ Bone 2018).\nBasal and load-induced periosteal (Ps) and endosteal (Es) mineral-\nizing surface (MS/BS, %), mineral apposition (MAR, μm/day) and\nbone formation rates (BFR/BS, μm3/μm2/year) were calculated\n(Dempsterþ JBMR.2013) at mid-length. RESULTS/ANTICIPATED\nRESULTS: No significant differences were detected in basal bone\nformation during development. However, relative load-induced\nPs MAR (rMAR) was reduced by 50% in female (p=0.02) and 75%\nin male (p=0.002) CXCL12 Δ OCY mice; and similarly, Ps rBFR/BS\nwas reduced by 50% in female (p=0.01) and 70% in male (p=0.001)\nCXCL12Δ OCY mice (Figure 1). Es bone formation was not affected\nby CXCL12 deletion. DISCUSSION/SIGNIFICANCE OF IMPACT:\nIn summary, osteocyte-specific CXCL12 expression plays a critical\nrole in exercise-driven periosteal new bone formation, suggesting that\nCXCL12 signaling may positively regulate osteogenic differentiation\nand/or mature osteoblast function. Further underlying mechanisms\nare currently being explored. Thus, osteocyte-specific CXCL12 signal-\ning may be a promising target to enhance load-induced bone forma-\ntion in patients with compromised ability to form new bone.\n3332\nOverexpression of CD44 is involved in the development of\nthe early endometriotic lesion in a xenograft model\nJennifer Knudtson1, Jessica McLaughlin, Marlen Tellez Santos,\nRajeshwar R Tekmal and Robert Schenken\n1University of Texas Health Science Center San Antonio\nOBJECTIVES/SPECIFIC AIMS: Previously, we showed decreased\ndevelopment of endometriotic lesions in CD44 knockout mice com-\npared to control.(1) CD44 has 10 different variants and a standard\nform. Menstrual endometrial cells (MECs) from women with endo-\nmetriosis have increased adhesion and also express higher levels of\nCD44 variant 6 (v6) than v3, compared to MECs from women with-\nout endometriosis. (2) Here, we assessed the effects of CD44 standard\n(CD44s), CD44v3 and CD44v6 overexpression (OE) on immortal-\nized human endometrial epithelial (iEECs) and stroma cells (hESCs)\nin vivo attachment in a nude mouse xenograft model. 1. Knudtson JF,\nTekmal RR, Santos MT, et al. Impaired Development of Early\nEndometriotic Lesions in CD44 Knockout Mice. Reproductive scien-\nces (Thousand Oaks, Calif.). 2016;23(1):87-91. 2. Griffith JS, Liu YG,\nTekmal RR, Binkley PA, Holden AE, Schenken RS. Menstrual endo-\nmetrial cells from women with endometriosis demonstrate increased\nadherence to peritoneal cells and increased expression of CD44 splice\nvariants. Fertility and sterility. 2010;93(6):1745-1749. METHODS/\nSTUDY POPULATION: Overexpression of CD44s, CD44v3 and\nCD44v6 was carried out using lipofectamine and their expression\nverified with qRT-PCR in iEEC and hESCs. Nude mice, 8-10 week\nold, were injected with estrogen 1 week prior to injection of iEECs\nand hESCs (n=7 per group). The cells were counted after transfection\nand at least 300,000 iEECs and 300,000 hESCs were injected per\nmouse. The transfected cells were tagged with cell tracker red (iEECs)\nand green (hESCs). Forty-eight hours after injection into the\nJCTS 2019 Abstract Supplement 111\nhttps://doi.org/10.1017/cts.2019.255 Published online by Cambridge University Press\n\nxenograft, the mice were sacrificed. The cells were counted using\nfluorescent stereo microscopy (FSM). Percent attachment was calcu-\nlated based on the number of cells visualized by FSM divided by the\nnumber of transfected cells injected. Unpaired student t-test was per-\nformed to analyze differences in the percent attachment of the cells.\nRESULTS/ANTICIPATED RESULTS: The majority of cells were\nattached to the peritoneum. There was increased attachment of\nhESCs with OE of CD44v6 compared to control (p=0.03). CD44v6\nOE did not change attachment of iEECs. There was no difference in\nattachment in iEECs or hESCs with OE of CD44s or CD44v3.\nDISCUSSION/SIGNIFICANCE OF IMPACT: Overexpression of\nCD44v6 increases attachment of ESCs to PMCs in an in vivo xeno-\ngraft model. Menstrual endometrial cell type and CD44 variants play\na complex role in the development of the early endometriotic lesion.\n3468\nPredictive biomarkers of platinum-based chemotherapy\nresponse in Puerto Rican Hispanics with high-grade\nserous ovarian cancer.\nJeyshka M. Reyes-Gonzalez 1, Sharee Umpierre and\nPablo E. Vivas-Mejia\n1University of Puerto Rico-Medical Sciences Campus\nOBJECTIVES/SPECIFIC AIMS: High-grade serous ovarian carci-\nnoma (HGSOC) is the most common and malignant histological sub-\ntype of epithelial ovarian cancer. While the majority of HGSOC\npatients initially respond to platinum-based chemotherapy, they often\npresent with recurrent chemoresistant disease, which is extremely\nfatal. Therefore, there is an urgent need to identify predictive bio-\nmarkers of platinum response and to develop rational, targeted thera-\npies to improve the outcome of patients with HGSOC. The objectives\nof the present study are to profile and assess the clinical significance of\nMYC network dysregulation in HGSOC. METHODS/STUDY\nPOPULATION: We will conduct a retrospective cohort study of\nPuerto Rican Hispanics with HGSOC who underwent surgery fol-\nlowed by platinum-based chemotherapy at clinical institutions in\nPuerto Rico. Medical records, pathology reports, and cancer registries\nwill be reviewed to extract data on clinicopathological features, disease\nrecurrence, and death. For eligible patients, formalin-fixed, paraffin-\nembedded (FFPE) tissue samples will be processed and analyzed by\nquantitative Real Time PCR (qRT-PCR) and immunohistochemistry\n(IHC). RESULTS/ANTICIPATED RESULTS: Expression levels of\nMYC and MYC-related molecules are expected to correlate with clin-\nicopathological features and prognosis of HGSOC. DISCUSSION/\nSIGNIFICANCE OF IMPACT: The identification and validation of\nclinically-relevant alterations in HGSOC, such as dysregulation of\nthe MYC network, will be crucial to guide therapy regimen, maximize\nclinical benefit, and improve patient outcome.\n3506\nPRMT5 is a novel therapeutic target to enhance radiation\ntherapy for cancer treatment\nJake L Owens1, Elena Beketova, Samantha Tinsley, Andrew Asberry,\nXuehong Deng and Chang-Deng Hu\n1Indiana University School of Medicine\nOBJECTIVES/SPECIFIC AIMS: Prostate cancer is the second lead-\ning cause of cancer-related death among men in the U.S. and over\nhalf of all prostate cancer patients receive radiation therapy (RT).\nRT induces double-strand breaks (DSBs) in DNA which are lethal to\ncells if not repaired. While potentially curative, 10% of low-risk patients\nand 50% of high-risk patients treated with RT still experience tumor\nrecurrence. Thus, identification of novel therapeutic targets to enhance\nRT will likely reduce prostate cancer mortality. The only clinical\napproach to enhance RT is androgen deprivation therapy, which targets\nandrogen receptor (AR) signaling; however, its use is limited due to sys-\ntemic side effects. We recently reported that PRMT5 epigenetically acti-\nvates AR which led us to investigate if targeting PRMT5 sensitizes\nprostate cancer to RT. The goal of this project is to determine if\nPRMT5 is a therapeutic target for prostate cancer radiosensitization\nand analyze its mechanistic role in response to radiation. METHODS/\nSTUDY POPULATION: To evaluate if targeting PRMT5 may sensitize\nprostate cancer cells to radiation, we performed a clonogenic assay of\nirradiated cells. To determine if PRMT5 is required for repair of radi-\nation-induced DSBs, we performed foci analysis via immunocyto-\nchemistry. We then used RNA-seq, qPCR, western blot, and ChIP to\nevaluate a potential epigenetic role of PRMT5 in activating the expres-\nsion of genes critical to DSB repair. To extend our findings, we analyzed\nclinical data from around 18,000 of cancer patients encompassing 43\ncancer types to assess if PRMT5 expression correlates with the expres-\nsion of its putative target genes. RESULTS/ANTICIPATED RESULTS:\nTargeting PRMT5 sensitizes prostate cancer cells to radiation inde-\npendently of AR status. RNA-seq analysis revealed putative PRMT5\ntarget genes including several involved in DSB repair and G2 arrest.\nMechanistically, PRMT5 functions as a master epigenetic activator\nof DNA damage response (DDR) genes: PRMT5 maintains the basal\nexpression of several DDR genes including BRCA1, BRCA2, and\nRAD51 and is recruited upon radiation to DDR gene promoters to acti-\nvate their expression via histone methylation. Targeting PRMT5\ndecreases expression of these genes at the protein level and hinders\nrepair of radiation-induced DSBs in multiple cancer and non-cancer\ncell types. Clinically, PRMT5 expression positively correlates with\nthe expression of these DDR genes across all 43 cancer types analyzed.\nDISCUSSION/SIGNIFICANCE OF IMPACT: PRMT5 acts as a\nmaster epigenetic activator of genes involved in DDR and is critical\nfor cells to survive radiation treatment. Importantly, PRMT5 epigeneti-\ncally activates multiple genes that encode for well-characterized core\nrepair proteins involved in HR (RAD51, RAD51AP1, RAD51D,\nBRCA1 and BRCA2) and NHEJ (NHEJ1, Ku80, XRCC4, and\nDNAPKcs), which may explain why PRMT5 is essential to repair\nIR-induced DSBs in several cell lines. As PRMT5 is overexpressed in\nmany human cancers and its overexpression correlates with poor prog-\nnosis, our findings suggest that more efficient DSB repair via PRMT5\noverexpression in these cancers may confer survival advantages par-\nticularly following DNA damaging treatments. Lastly, because target-\ning DSB repair is a clinically validated therapeutic approach for cancer\ntreatment, our findings also suggest that PRMT5 targeting may be\nexplored as a monotherapy or in combination therapy with radiation\ntherapy or chemotherapy for cancer treatment.\n3204\nRenin-Angiotensin System Inhibitors Do Not Improve\nSurvival in Fibrillin-1 Hypomorphic Mice with Established\nAortic Aneurysm\nMary Burchett Sheppard 1, Jeff Zheying Chen, Debra L. Rateri,\nJessica J. Moorleghen, Mackenzie Weiland and Alan Daugherty\n1University of Kentucky Center for Clinical and Translational\nScience\nOBJECTIVES/SPECIFIC AIMS: Drugs to attenuate aortic growth\nare usually not initiated in patients with Marfan syndrome until\n112 JCTS 2019 Abstract Supplement\nhttps://doi.org/10.1017/cts.2019.255 Published online by Cambridge University Press","source_license":"CC0","license_restricted":false}