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.
Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. Recently, we found that high podocalyxin (gene name PODXL, protein name Podxl) expression is associated with high metastatic profile in OS cell lines. Consistent with this data, we found a significant association between high Podxl expression and poor outcome in pediatric OS patients. Podxl is a cell surface transmembrane glycoprotein belonging to the CD34 family. The functional role of Podxl in tumorigenesis is largely unknown, but it has been demonstrated to promote cancer cell invasion and migration and to enhance metastatic potential. Podxl is upregulated on a variety of human tumors, facilitates disease progression, and is a promising target for immunotherapy as an approach to blocking metastatic disease. Accordingly, in collaboration for the Centre for Drug Research and Development, Kelly McNagny and his collaborators have developed a novel panel of monoclonal antibodies to human Podxl and have explored their utility in suppressing tumor cell growth in vitro and in vivo. To define the relevance of Podxl in the biology of metastasis, we examined Podxl expression in different OS cell lines with different metastatic profiles. Then, we studied the importance of Podxl in migration and invasion processes and in in vitro 3D cell system formation. To assess Podxl’s role in metastatic progression we performed the Pulmonary Metastasis Assay (PuMA). PuMA is an ex vivo lung explant and closed cell culture system that permits to study the biology of lung colonization by fluorescence microscopy. With PuMA model, we assessed the effects of antimetastatic therapeutics over time. In the present study, we found that suppression of Podxl profoundly impairs cell proliferation, migration, invasion properties, and tumorsphere formation in OS cells in vitro. With PuMA model, we found that Podxl is required for the progression of metastasis disease. These data tend to validate podocalyxin as a regulator of tumor progression and a novel therapeutic target in OS. It is necessary to perform in vivo experiments to further explore the functions of Podxl and the effect of the podocalyxin-specific monoclonal antibody (coupled or not to a toxic payload) to suppress the metastasis progression in our model. Citation Format: Anne-Chloe Dhez, Irene Paassen, Poul Sorensen. Podxl as therapeutic target for metastasis [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A31.
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.
Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We pefrormed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p< 0.05).Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.
Introduction: Poly(ADP-ribose) polymerase (PARP) plays a major role in DNA repair and PARP inhibitors (PARPi) have shown promise in pre-clinical studies for the treatment of Ewing sarcoma (ES). While a clinical trial using olaparib as a single agent failed to show significant response against ES, combination therapies with PARPi have emerged as an area of interest. Deacetylation of histones, controlled by histone deacetylases (HDACs) is a key regulatory event in DNA repair and inhibition of HDACs has been shown to reduce ES tumor growth in vitro and in vivo. PARP inhibition combined with HDAC inhibition has demonstrated enhanced efficacy in pre-clinical studies in various tumor indications, and a clinical trial of olaparib and vorinostat combination therapy against metastatic breast cancer is currently ongoing. However, combination therapies can be limited in clinical utility due to overlapping toxicities and different pharmacokinetic profiles. Here, we report the efficacy of a novel bifunctional small-molecule compound, kt-3283, designed to have both PARP and HDAC inhibitory activities. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte® S3 system and CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by western blot, immunofluorescence, and comet assay. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module and inhibition of metastases was assessed in a PUMA ES mouse model. Results and discussion: Kt-3283 showed potent inhibition of PARP1/2 activity and PAR synthesis with IC50 values comparable to olaparib. Kt-3283 also showed inhibition of HDACs with an IC50 value in the low µM range. Cell survival EC50 values for the compound were also superior to those of olaparib and vorinostat in ES cell lines. Cell cycle and DNA damage analyses indicated S/G2/M cell cycle arrest and strong DNA damage upon treatment with kt-3283 at lower concentration range compared to olaparib and vorinostat. This compound also exhibited potent inhibition of 3D spheroid growth of ES cells with low µM EC50 values, and inhibited metastatic growth in a PUMA mouse model. Conclusion: Kt-3283 shows potent inhibition of PARP1/2 and HDAC activities. It induces S and G2/M cell cycle arrest and DNA damage, and inhibits 3D spheroid growth and metastatic potential of ES cells. Further investigation of this bifunctional single-molecule inhibitor may offer a novel treatment opportunity for ES and other solid tumors with limited responses to PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Jay Joshi, Fariba Ghaidi, Michael M. Lizardo, Taras Shyp, John Langlands, Dennis Brown, Jeffrey Bacha, Poul Sorensen, Wang Shen, Mads Daugaard. A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6194.
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