bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract A012: Targeting the dysregulation of transcription and splicing in Ewing sarcoma
Authors: Lawrence Liesl A. , Miller Henry , Bishop Alexander 
Published: September 2022

Abstract
The goals of this study are to investigate how EWS-FLI1 disrupts regulation of transcription and splicing in Ewing sarcoma, and to evaluate this dysregulation as a potential therapeutic target. Ewing Sarcoma is an aggressive pediatric bone and soft tissue cancer driven primarily by the EWS-FLI1 fusion oncogene. EWS-FLI1 acts as an oncogenic transcription factor and also interferes with normal regulation of transcription and transcription-associated RNA processing. Chemotherapy has substantially improved overall survival, but the prognosis is still very poor for those with metastatic or recurrent disease, and toxicity is a major concern in pediatric patients. Consequently, effective, less toxic, targeted treatment strategies are much needed. We performed a genome-wide RNAi screen in the Ewing sarcoma cell line TC32 and found that one of the top pathways required for cell survival was RNA processing/splicing. Previous studies have reported increased amounts of alternative splicing events in Ewing sarcoma. RNA processing, including splicing, is functionally coupled with transcription. We recently reported that Ewing sarcoma cells have high levels of phosphorylated RNA polymerase II and increased transcription activity, accumulate R-loops, and fail to regulate transcription appropriately in response to DNA damage. We proposed that EWS-FLI1 driven hyper-activation of transcription causes a targetable dependence on RNA splicing in Ewing sarcoma. Ewing sarcoma cells showed increased sensitivity to depletion of key splicing factors compared to mesenchymal stem cell controls; this was partially rescued by depleting EWS-FLI1 or by overexpressing RNASEH1, which degrades R-loops. Ewing sarcoma cells were extremely sensitive to splicing inhibition, which showed synergy with multiple chemotherapeutic agents. Splicing inhibition in Ewing sarcoma cells caused cell cycle arrest, induced apoptosis, and altered global gene expression, alternative splicing, and R-loop profiles as seen by RNA-seq and DRIP-seq. This work provides novel insight into transcription regulation and its dysregulation by EWS-FLI1. In addition, our results point to RNA splicing as a potential new therapeutic target in Ewing sarcoma, which has the potential to benefit all Ewing sarcoma patients, especially those with chemo-resistant disease.
Citation Format: Liesl A. Lawrence, Henry Miller, Alexander Bishop. Targeting the dysregulation of transcription and splicing in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A012.
Abstract A026: PROX1 transcription factor regulates rhabdomyosarcoma growth, myogenic properties and therapeutic targets
Authors: Gizaw Nebeyu Y. , Kallio Pauliina , Gucciardo Erika , Haglund Caj , Bohling Tom , Lehti Kaisa , Sampo Mika , Alitalo Kari , Kivela Riikka 
Published: September 2022

Abstract
Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, novel RMS therapies are urgently needed. Here we show that the PROX1 transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and altered the RD (ERMS) transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, inhibition of either alone did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and decreased tumor xenograft growth. Our findings uncover the critical role of PROX1 in RMS and offer new insights to the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in unrelated clinical trials, our findings provide a new promising option for RMS treatment.
Citation Format: Nebeyu Y. Gizaw, Pauliina Kallio, Erika Gucciardo, Caj Haglund, Tom Bohling, Kaisa Lehti, Mika Sampo, Kari Alitalo, Riikka Kivela. PROX1 transcription factor regulates rhabdomyosarcoma growth, myogenic properties and therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A026.
Abstract A037: TT-00420, a novel kinase inhibitor potentially benefiting c-Myc amplified/overexpressed osteosarcoma
Authors: Hua Yingqi , Qiang Xiaoyan , Tian Kai , Jiang Yafei , Wu Frank , Peng Peng 
Published: September 2022

Abstract
Osteosarcoma (OS) is the most common bone tumor in pediatric patients, whose effective therapies are of great unmet medical needs, particularly for those of high-risk features. c-Myc, a well-known oncogene, is commonly amplified/overexpressed in OS patients, contributes to dismal prognosis and associates with lower 5-year overall survival rate, which is also confirmed by our retrospective analysis for the correlation between c-Myc gene amplification/overexpression and patient prognosis in our patient cohort. TT-00420, a clinical-stage small molecule targeting Aurora A/B, VEGFRs, FGFRs, JAKs and CSF1R, has demonstrated down-regulation on c-Myc expression in TNBC studies. Moreover, as reported, Aurora kinases and c-Myc regulate each other back and forth, triggering maintenance of the malignant state. In this study, we examined the efficacy of TT-00420 in c-Myc-amplified/overexpressed OS. In pre-clinical clonogenic assay, TT-00420 significantly inhibited the colony formation in c-Myc-overexpressed OS cell lines in a dose-dependent manner. In MNNG/HOS cell line, we demonstrated that TT-00420 dramatically suppressed Aurora kinase pathway and blunted c-Myc protein expression level, indicating TT-00420 might down-regulate c-Myc by Aurora kinases inhibition. In pre-clinical in-vivo efficacy studies, 143B cells were injected into the medullary cavity of the tibia to make the orthotopic OS xenograft model. With 15 mg/kg/day treatment of TT-00420, tumor growth was obviously inhibited as compared to vehicle group. Immunohistochemistry (IHC) analyses in tumors revealed that TT-00420-treated tumors exhibited increased apoptosis with upregulated expression of cleaved caspase 3 (CC3) and H2AX, and significantly decreased proliferations with raised expression of Ki67. Moreover, in a c-Myc-overexpressed OS patient-derived xenograft (PDX) model from a male pediatric patient, 15 mg/kg/day TT-00420 dramatically blunted the tumor growth with a TGI of 87.6%. In China, an investigator-initiated trial is currently ongoing to evaluate the safety and tolerability of TT-00420 in OS patients with c-Myc overexpression (ChiCTR2000036618). Taken together, TT-00420 depicted robust anti-tumor activity in c-Myc overexpressed OS both in vitro and in vivo with downregulation of c-Myc expression, indicating its potential as a novel monotherapy for OS patients with c-Myc overexpression. Future clinical trial result is of great expectation.
Citation Format: Yingqi Hua, Xiaoyan Qiang, Kai Tian, Yafei Jiang, Frank Wu, Peng Peng. TT-00420, a novel kinase inhibitor potentially benefiting c-Myc amplified/overexpressed osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A037.
Abstract A039: IGFBP5 is induced by tumor-suppressive naïve stromal cells and regulates GAS2-associated apoptosis in rhabdomyosarcoma
Authors: Ehnman Monika , Katkhada Karim , Meng Liu Zhen , Zhang Yue , Zhao Binbin , Tong Shanlin , Kallai Aditi , Östman Arne , Tobin Nick 
Published: September 2022

Abstract
Stromal cells can have either suppressive or stimulatory effects on tumor progression. However, this diverse collection of cells is still poorly characterized in soft-tissue sarcomas. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood with molecular features of skeletal muscle differentiation. In this study, we explored tumor growth-regulatory signaling between differentially educated non-malignant mesenchymal stromal cells and malignant tumor cells in RMS. Xenograft experiments in mice were designed to provide functional evidence for alterations in tumor behavior linked to tumor-associated stromal cells. Molecular characterization was performed on the Clariom D platform, by gene set enrichment analysis, cell culture validation of deregulated genes, and finally, by clinicopathological validation in a discovery cohort (N=101) and a validation cohort (N=147). The results revealed transient tumor growth-suppressive paracrine effects of naïve orthotopic stromal cells derived from skeletal muscle. These effects were easily abolished if the stromal cells were pre-exposed to RMS cells, either short-term in vitro, or long-term in hindlimb muscle in vivo. Explorative analyses of RMS cells exposed to naïve stromal cells identified insulin-like growth factor binding protein 5 (IGFBP5) as the top upregulated gene, with an ability to induce Caspase 3/7 activation and growth arrest. The gene coding for connective tissue growth factor (CTGF) was identified as the major prosurvival factor being downregulated. Analysis of patient cohorts consistently demonstrated an association between IGFBP5, growth arrest specific protein 2 (GAS2), lower disease stage and favorable survival. Altogether, our study identifies a novel anti-tumor growth mechanism with conditionally deregulated genes linked to apoptosis and differentiation in RMS. The results provide support for continued studies on tumor growth-regulatory mechanisms induced by tumor-surrounding stromal cell subsets at primary and metastatic sites in sarcoma.
Citation Format: Monika Ehnman, Karim Katkhada, Liu Zhen Meng, Yue Zhang, Binbin Zhao, Shanlin Tong, Aditi Kallai, Arne Östman, Nick Tobin. IGFBP5 is induced by tumor-suppressive naïve stromal cells and regulates GAS2-associated apoptosis in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A039.
Abstract B009: Establishing new cell lines from undifferentiated pleomorphic sarcoma for sarcoma research
Authors: You Hye Jin , Lee Eun-Young , Rayhan Abu , Kang Hyun Guy , Kim June Hyuk , Park Jongwoong 
Published: September 2022

Abstract
Undifferentiated pleomorphic sarcoma (UPS), also known as malignant fibrous histiocytoma (MFH), is a high-grade soft-tissue sarcoma (STS). Despite of importance, there are very few models for study. Here, we have established and characterized UPS-derived cell lines. Cells were obtained UPS tissues by mincing followed by extracting or dissociating using enzymes and cultured by regular culture environment. Cells were maintained and immortal for months without artificial treatment and tumorigenic in vivo study. The tissues from in vivo study and tissues from patients were compared if it is representative for UPS by immunohistochemistry. Transcriptome from tissues and cell lines were compared. Several genes were identified as specific in tumor tissues and cell lines. Fusion genes were also identified. This study showed that new UPS cell lines might be a good resource for UPS study to get new insights.
Citation Format: Hye Jin You, Eun-Young Lee, Abu Rayhan, Hyun Guy Kang, June Hyuk Kim, Jongwoong Park. Establishing new cell lines from undifferentiated pleomorphic sarcoma for sarcoma research [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B009.
Abstract B011: Two epigenetically distinct cellular states in osteosarcoma are regulated by a cluster-specific set of pioneer transcription factors
Authors: Fuentes Eunice Lopez , Clugston Andrew , Sayles Leanne C. , Sweet-Cordero E. Alejandro 
Published: September 2022

Abstract
The Osteosarcoma (OS) genome is characterized by aneuploidy and dramatic structural rearrangements often leading to copy number gain and loss. There is significant heterogeneity between patients. Additionally, there is less known regarding the epigenetic heterogeneity within osteosarcoma and how this may impact critical phenotypes such as therapy response and metastasis. To characterize the osteosarcoma epigenome, we integrated ATAC-seq and RNA-seq across a unique set of patient samples, PDXs and PDX-derived cell lines obtained across the disease continuum and including biopsies, resections, and metastases. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) analysis revealed the presence of two distinct epigenetic cell states. Differential chromatin accessibility between the clusters showed ~3000 unique open regions per cluster. Using GREAT to associate the differential genomic regions to genes, the enriched pathways of Cluster 1 (C1) are related to mesenchymal cell proliferation, skeletal system morphogenesis, osteoblast differentiation, and Wnt signaling pathway, whereas Cluster 2 (C2) enriched pathways are related to cell motility and migration, extracellular matrix organization and regulation of protein serine/threonine kinase activity. Accessible regions in the chromatin were enriched for a unique set of transcription factor binding sites. C1 accessible regions are enriched with transcription factor binding motifs (TFBM) related to development including RUNX2/3, MEOX2, HOXA2/5, VAX1/2, MEF, VSX and DLX; whereas members of AP1 complex including FOSL1/2, c-FOS, JUN are TFBM enriched in open regions in C2. This is correlated with high protein expression of these transcription factors in a cluster-specific manner and differential binding to chromatin. As expected, gene expression showed a positive correlation with accessible regions in promoters. RUNX2 plays a major role in chromatin remodeling in C1, modifying the chromatin accessibility of 5300 regions, most located in &lt;1kb from the promoters. In conclusion, we uncovered two epigenetically cell states regulated by a cluster-specific set of transcription factors which are able to remodel the chromatin.
Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Leanne C. Sayles, E. Alejandro Sweet-Cordero. Two epigenetically distinct cellular states in osteosarcoma are regulated by a cluster-specific set of pioneer transcription factors [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B011.
Abstract B017: Tumor-secreted collagen VI weakens endothelium and promotes metastasis
Authors: Liu Ying , Fuller Ashley M. , Murazzi Ileana , Devine Ann , Skuli Nicolas 
Published: September 2022

Abstract
Metastasis is the leading cause of cancer-related deaths. However, the mechanisms behind the metastatic cascade remain poorly understood. Tumor’s metastatic potential is strongly influenced by microenvironmental cues such as low oxygen (hypoxia). Our published work reported that lung metastases in sarcoma are associated with increased primary tumor expression of the hypoxia-inducible collagen-modifying lysyl hydroxylase, Plod2; in multiple subtypes of sarcoma, excessive collagen lysyl hydroxylation results in secretion of immature collagen aggregates able to physically associate with tumor cells and promote both intravasation and extravasation. In the current study, we observed that Plod2 is colocalized with collagen VI and is important for its extracellular network structure, suggesting that collagen VI is a putative substrate of Plod2. Using an impedance sensing assay and a zebrafish intravital microinjection model, we demonstrated that tumoral Plod2 and collagen VI weaken the endothelial barrier and promote extravasation. With a tail vein injection mouse model, we determined that collagen VI is essential for lung metastasis. Clinically, we detected high levels of collagen VI in metastatic sarcoma in surgically resected patient lungs. Furthermore, by analyzing patient data in The Cancer Genome Atlas (TCGA), we found a strong correlation between the expression of both PLOD2 and collagen VI with disease outcomes. Together, our study identifies a novel mechanism of sarcoma lung metastasis, opening up opportunities for therapeutic intervention.
Citation Format: Ying Liu, Ashley M. Fuller, Ileana Murazzi, Ann Devine, Nicolas Skuli. Tumor-secreted collagen VI weakens endothelium and promotes metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B017.
Abstract B018: A robust system to study human soft-tissue sarcoma lung metastasis
Authors: Muñoz Maria , Carr-Ascher Janai R. 
Published: September 2022

Abstract
Soft-tissue sarcoma (STS) is a rare connective tissue cancer that encompasses over 50 distinct subtypes including liposarcoma, undifferentiated pleomorphic sarcoma (UPS) and pleomorphic rhabdomyosarcoma. While these subtypes are histologically and genetically different, all are capable of metastasizing into the lungs. However, the mechanisms of metastasis are poorly understood, and patients with metastatic disease have low survival rates. Current statistics show that these patients have a 15% 5-year relative survival rate. It is crucial to understand the mechanisms of metastasis to improve patient therapeutics and survival. Thus, we developed a robust system to enrich and study spontaneous metastatic cells in STS. To do this, we started by screening multiple cell lines for metastatic activity in-vivo. First, immunocompromised mice were injected in the thigh (intramuscularly) with tumor cells. The liposarcoma cell line SW872 gave rise to metastatic tumors in the lung although with limited penetrance and a long latency. Therefore, we identified a mouse with a high metastatic burden and generated a cell line from the primary tumor that was highly metastatic. These serially passaged cells were injected intramuscularly into the thigh of immunocompromised mice and allowed to spontaneously metastasize. This resulted in earlier detection of tumors, and a decreased latency to metastatic disease. For downstream analysis, we enriched the metastatic population resulting from the serially passaged tumor. This was accomplished by first digesting lungs, then depleting mouse cells with magnetic beads. We further enriched the population by sorting HLA-APC positive (human) cells using fluorescence-activated cell sorting (FACS). This process was necessary to reduce mouse cell contamination, and ensure we solely analyzed human cells. Afterwards, metastatic cells can be compared to the primary tumor using RNA, ATAC or Whole-Exome sequencing. We hypothesize that a comparison between the primary and metastatic tumors will identify pathways that allow cells to metastasize. These pathways can be targeted in future therapeutics to improve patient outcomes. This system can also be applied to other STS subtypes. Overall, our robust system allows us to study spontaneous lung metastases, is reflective of the human disease and will provide insight into the mechanisms of metastasis in STS.
Citation Format: Maria Muñoz, Janai R. Carr-Ascher. A robust system to study human soft-tissue sarcoma lung metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B018.
Abstract B021: Long non-coding RNAs that are required for robust cell growth in ewing Sarcoma
Authors: Briones Martin del Campo Marcela C. , Lee Alex , Horlbeck Max , Dinh Truc , Roman-Moreno Marta , Sayles Leanne , Seong Bokyung , Stegmaier Kimberly , Gilbert Luke , Sweet-Cordero E. Alejandro 
Published: September 2022

Abstract
While long non-coding RNAs (lncRNAs) are upregulated in many cancers, their role in pediatric cancers is unclear. Among pediatric cancers, Ewing sarcoma is unique in that these tumors show upregulation of a large, specific number of lncRNAs. To perform an unbiased screen of lncRNA function in Ewing sarcoma, we used a pooled CRISPRi screen to identify lncRNAs that modify cell growth. We included in the pooled library lncRNAs either highly expressed in Ewing Sarcoma or directly regulated by the driving oncogene in Ewing Sarcoma, EWS-FLI. We identified 49 lncRNAs that when inhibited by CRISPRi led to decreased growth of Ewing cell lines. Among those that scored most significantly were the small nucleolar host genes SNHG1, SNHG12, and SNHG30. To validate the screening results, we individually cloned the two most depleted sgRNAs targeting for each of these three lncRNAs into a lentiviral vector. We expressed these sgRNAs in three different dCas9-KRAB+ Ewing cell lines and used internally controlled growth assays to test whether the observed phenotypes from the screens were reproducible. We confirmed that for all three lncRNAs hits significantly affected Ewing’s growth, and their depletion leads to an increase in apoptosis. To determine how lncRNAs influence the ability of Ewing sarcoma cells to growth and survive in vivo, we performed a subcutaneous xenograft CRISPRi screen using the same pooled library used in vitro. The lncRNAs with the strongest pro-growth phenotype after knock-down were PVT1 and the uncharacterized LINC02688. Ongoing studies are directed at determining the Ewing-specific role of these lncRNAs. Overall, our studies demonstrate the value of studying lncRNA functions in vitro and in vivo, provides a valuable resource of new lncRNAs targets to further research and establishes a framework for systematic discovery of functional lncRNAs in Ewing cells.
Citation Format: Marcela C. Briones Martin del Campo, Alex Lee, Max Horlbeck, Truc Dinh, Marta Roman-Moreno, Leanne Sayles, Bokyung Seong, Kimberly Stegmaier, Luke Gilbert, E. Alejandro Sweet-Cordero. Long non-coding RNAs that are required for robust cell growth in ewing Sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B021.