Sarcomas are malignant cancers of soft tissue or bone predominantly affecting children and adolescents. The most common subtypes are osteosarcoma and Ewing family tumours (EFTs). The most unfavorable prognostic factor is the presence of metastases, which accounts for 9 out of 10 sarcoma cancer deaths. Identifying factors and/or drugs that have an impact on metastatic spread have tremendous potential to affect outcome by reducing disease burden to the primary site, which can be more effectively treated by surgery and radiation. Pertinent animal models are critical for translating in vitro findings to clinical trials. Xenotransplantation of human cancer cells into transparent zebrafish embryos provides a novel in vivo platform for visualizing tumor micro-environment interactions contributing to sarcoma proliferation and spread in real time, which is not easily provided by other animal models. We recently demonstrated the effectiveness of the zebrafish xenotransplantation model for the study of specific drug-tumor interactions for both chronic myelogenous leukemia and acute promyelocytic leukemia and used a rapid and novel ex-vivo proliferation assay to quantify therapeutic responses (Corkery et al, BJH 2011). We have now applied this technology to EFTs. Human EFT TC-32 cells were fluorescently labeled with CmDiI, and microinjected into the yolk sac of two day old casper embryos, a double pigment mutant that prevents any auto-fluorescence that might interfere with image quality. EFT cells successfully engrafted, survived and proliferated over 96 hours post-injection (hpi). Migration of cells from the yolk sac to the tail occurred between 48 and 144 hpi with evidence of vascular extravasation and tissue infiltration. Y-box binding protein 1 (YB-1) is implicated in the metastatic spread of epithelial cancers due to its key role in promoting an epithelial-to-mesenchymal transition (EMT). In contrast to parental TC-32 cells, xenografted YB-1 knockdown (KD) TC-32 cells showed absent or significantly delayed migration, suggesting that YB-1 also regulates this process in zebrafish xenografts. Moreover, using transgenic fli-EGFP casper embryos that display fluorescent vasculature, we saw evidence of vascular recruitment into the tumor mass in WT TC-32 cells but not in YB-1 KD, potentially implicating angiogenesis as a mechanism that contributes to tumor spread in YB-1 expressing sarcomas. Exposure of TC-32 xenografted embryos to 5-40 Gy of ionizing radiation effectively reduced cell proliferation in a dose-dependent manner. These studies highlight the utility of the zebrafish xenograft model to elucidate the mechanisms underlying the metastatic behavior of EFTs and position this system as an in vivo tool for drug discovery to identify novel anti-proliferative and anti-metastatic agents to improve outcome in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1398. doi:1538-7445.AM2012-1398
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no approved targeted therapies. Tumor endothelial marker 8 (TEM8), initially identified as a marker of tumor endothelial cells in colorectal cancer and other solid tumors has recently been shown to be upregulated in TNBC and breast cancer stem cells (BCSCs). We investigated whether TEM8 specific chimeric antigen receptor (CAR) T cells recognize and kill both tumor endothelial cells as well as TNBC tumor cells. TEM8 specific CAR molecules were generated using single chain variable fragment derived from the monoclonal antibody, L2. L2 CAR T cells selectively recognized TEM8, secreted immunostimulatory cytokines and effectively killed both TEM8 positive TNBC and tumor endothelial cell lines. Moreover, L2 CAR T cells targeted breast cancer stem cells significantly reducing the number of mammospheres relative to non-transduced T cells. In vivo, adoptive transfer of L2 CAR T cells induced regression of established vascularized TNBC xenografts. Hence, TEM8 may serve as an attractive target for immunotherapy of TNBC. Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Kevin Bielamowicz, Steven Seaman, Daniel Landi, Nino Rainusso, Poul Sorensen, Joachim Koch, Winfried Wels, Bradley Fletcher, Meenakshi Hegde, Brad St Croix, Nabil Ahmed. TEM8/ANTXR1 specific T cells co-target tumor stem cells and tumor vasculature in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2312.
Background: Rhabdomyosarcoma (RMS) has a high unmet need in terms of precision therapy development as there are currently no approved immunotherapies or targeted therapies, and few in the developmental pipeline. Here we sought to identify cell surface oncoproteins as a target for novel RMS-directed immunotherapies. Methods: We first performed plasma membrane enrichment followed by mass spectrometry to define the cell surface landscape of 7 fusion(+) and 14 fusion(-) RMS patient-derived xenograft (PDX) models. The surfaceome data was filtered to only “high confidence” surface proteins by querying protein localization databases, Compartments (https://compartments.jensenlab.org/) and CIRFESS (https://gundrylab.shinyapps.io/cirfess/). We then developed a prioritization algorithm that uses a rank-product approach to score surface proteins. The input to the algorithm is a matrix that integrates multiple datasets to score the surface proteins based on their suitability to be an optimal immunotherapeutic target. In addition to the surfaceome data generated here, we also integrated matched RNA-sequencing data from eleven of the RMS PDX models, RNA-sequencing data from GTEx (n=15,253) and a recently developed normal tissue proteomics dataset (n=201) [Jiang. Cell. 2020], a list from Gene Ontology that included genes involved in muscle development pathways, and the gene dependency list for RMS in DepMap (https://depmap.org/portal/). Results: A total of 913 and 937 high confidence surface proteins were annotated from the mass spectrometry data for fusion(+) and fusion(−) samples, respectively. A dendrogram separated the surface protein profiles into two clusters based on fusion(+) and fusion(−) RMS subtypes, thus the algorithm was run separately on each subtype. Within the top 50% of prioritized targets, 88% and 86% of the targets overlapped and 12% and 14% were identified exclusively in fusion(+) and fusion(−) subtypes, respectively. ALK, a previously putative protein marker in fusion(+) RMS, scored in the top 10% of the fusion(+) targets based on the algorithm, and surprisingly we saw abundant ALK expression in 6/14 fusion(−) PDX samples. MEGF10, a novel target, was ranked as the top target for both fusion(+) and fusion(−) RMS. MEGF10 plays a role in cell adhesion, motility, and proliferation. It scored as a significant dependency in DepMap for RMS (p-value=0.0002). Based on RNA-sequencing and proteomics, MEGF10 shows no expression in most healthy tissues surveyed, with several orders of magnitude lower expression detected in RNASeq in muscle and brain tissue, but not in the proteomic datasets. Conclusion: Here, we defined the surfaceome of RMS, and found substantial overlap in surface proteins between fusion(+) and fusion(−) RMS subtypes. We validated previous observations that ALK is expressed in RMS, here verifying that the protein is expressed on the plasma membrane. MEGF10 appears to be a strong novel candidate target for RMS immunotherapies, and ongoing work to validate our proteogenomic findings will be reported. Citation Format: Rawan Shraim, Amber K. Weiner, Karina L. Conkrite, Alexander B. Radaoui, John M. Maris, Yael P. Mosse, Sharon J. Diskin, Ahmet Sacan, Benjamin A. Garcia, Peter J. Houghton, Raushan T. Kurmasheva, Poul Sorensen, Gregg B. Morin, Brian Mooney. Proteogenomic prioritization of immunotherapeutic targets in rhabdomyosarcoma nominate MEGF10 for preclinical development [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 A009.
Background: Ewing sarcoma (ES), an osteogenic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. In the clinical setting, prognosis for patients with metastatic ES at diagnosis is clearly worse than for those without metastases (5-year survival > 30%). Hence, there is an urgent need to understand the fundamental molecular mechanisms of ES differentiation, invasion, and metastasis to possibly identify novel therapeutic strategies to prevent metastasis. The purpose of this study was to shed further light into the function of Chondromodulin 1 (CHM1) on ES pathogenesis, especially on metastasis, and at best to establish new therapeutic targets. Material and Methods: Expression of CHM1 was analyzed using microarrays and its function was examined by RNA interference (RNAi). To analyze resulting changes qRT-PCR, ELISA, FACS, IHC, proliferation and invasion assays, as well as a xeno-transplant model in immune deficient mice were applied. Results: In this study, we investigated the role of the BRICHOS chaperon domain containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES and ChIP data demonstrate CHM1 to be directly bound by EWS-FLI1. Using RNA interference we demonstrate that CHM1 enhanced contact-dependent as well as independent proliferation and the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated MMP9 expression. In a xenograft mouse model CHM1 was essential for the establishment of lung metastases, which is in line with the observed increased CHM1 expression in patient specimens with ES lung metastases. Mechanistically, CHM1 promoted chondrogenic differentiation capacity of ES cells but suppressed endothelial differentiation. Further, CHM1 suppressed the number of TRAP+ osteoclasts in an orthotopic model of tumor growth in line with suppression of osteolytic genes such as HIF1A, IL6, JAG1, and VEGF, indicating that CHM1-blocked osteomimicry might play a role in homing, colonization, and invasion into bone tissues. Conclusions: Our results suggest that CHM1 is an important player suppressing endothelial differentiation capacity and seems essential for the invasive and metastatic capacities of ES. Citation Format: Kristina von Heyking, Julia Calzada-Wack, Stefanie Göllner, Oxana Schmidt, Tim Hensel, David Schirmer, Annette Fasan, Carsten Müller-Tidow, Poul Sorensen, Stefan Burdach, Günther H.S. Richter. The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype promoting lung metastasis in Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B03.
Introduction: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), though clinical trials with olaparib failed to show significant clinical benefit. While single agent therapy proved inefficacious in the clinical treatment of ES, combination therapies may show anti-tumour activity. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs have been correlated to more malignant phenotypes in sarcomas and inhibition of HDAC in ES has been shown to be effective in inhibiting tumor growth. HDAC inhibition combined with PARP inhibition has been shown to sensitize cells to treatment in vitro, however clinically, combination therapies often require sequential administration due to different pharmacokinetic profiles and overlapping toxicities, severely limiting clinical utility. Here, we evaluate the activity and efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibiting activity. Methods: PARP1 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit and PARP2 activity was measured using the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit. HDAC activity was measured using HeLa nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. Accumulation of phospho-histone H2AX (pH2AX) was detected by western blot using anti-phospho histone H2AX (Ser139) antibody from Cell Signaling Technologies. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low µM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in the increased accumulation of pH2AX by western blot and increased S and G2/M cell cycle arrest compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may result in a novel treatment opportunity for Ewing sarcoma. Citation Format: Sarah Truong, Beibei Zhai, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Poul Sorensen, Mads Daugaard. In vitro efficacy of a novel dual PARP-HDAC inhibitor 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 A024.
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