Panel Of Osteosarcoma Cell Lines Research Articles

Abstract 3912: ATM and PARP combined inhibition demonstrate synergistic antitumor efficacy in osteosarcoma models

Abstract Signatures of BRCAness are found in many osteosarcoma (OS) tumors, driving an interest in PARP inhibition (PARPi) for OS treatment. Although PARPi have shown limited efficacy as OS monotherapy, rational combination strategies have not yet been explored for this disease. We performed a genome-scale loss-of-function CRISPR-Cas9 screen in two OS cell lines in the presence and absence of olaparib, a small molecule PARPi. ATM knockout (KO) scored highly as a potent synergizer of PARPi across both cell lines. Here we report this screen finding as well as in vitro validation of PARPi and ATM inhibition (ATMi) synergy in OS models. OS cell lines SAOS2 and CAL72 were infected with the Avana genome-scale lentiviral CRISPR library, targeting 20,000 genes with 4 uniquely barcoded guides (sgRNAs) per gene. After infection and antibiotic selection, cells were grown in an IC40 dose of olaparib or matched control for 18 days. Following DNA sequencing of barcodes and rigorous quality control, sgRNA dropout hits were calculated against the control arm. ATM KO sensitizing to PARPi was first validated utilizing a CRISPR-based genetic approach. ATM KO in CAL72 and SAOS2 was performed via lentiviral infection. After KO confirmation by western immunoblotting (WB), ATM KO and control KO cells were treated with PARPi in a dose range of 19.5 nM-10 µM. ATP-based and Incucyte assays were used to quantify differential cell viability. Secondly, the combination of a small molecule ATMi and a PARPi was studied in a panel of OS cell lines. Synergistic cytotoxicity was quantified using the Zero Interaction Potency (ZIP) model. DNA damage levels after PARPi and ATMi (by chemical inhibition and genetic KO) were assessed by WB for γH2AX, a marker of DNA double-stranded breaks (DSBs). In our genome-scale CRISPR screens, ATM arose as a top druggable hit across both lines. Reassuringly, PARP1 KO scored as a top resistance mediator to PARPi, confirming biologic relevance of the screen and supporting the known PARP-trapping mechanism of these drugs in OS. At low-throughput, ATM KO lines trended toward increased sensitivity to PARPi in short-term viability assays. Combined chemical inhibition yielded highly synergistic ZIP scores across OS cell lines; synergy was achieved with both drugs in the low nanomolar range. γH2AX levels were substantially increased in the setting of combined PARPi and ATMi (via chemical inhibition or genetic KO) as compared to inactivation of either PARP or ATM alone, indicating increased presence of DNA DSBs. In conclusion, these data demonstrate synergy of ATM and PARP inhibition, providing promise for their potential combined use in the treatment of OS. Accumulation of DNA DSBs in response to PARP and ATM inactivation suggests a shared role in DNA damage repair as the molecular basis of synergism. Ongoing studies are investigating anti-tumor potential in in vivo OS models and exploring the mechanistic impact of the combination in OS tumors. Citation Format: Sona N. Kocinsky, Janeala J. Morsby, William C. Wright, Monika Wierdl, Caroline S. Wechsler, Gabriela Alexe, Paul Geeleher, Kimberly Stegmaier, Lillian M. Guenther. ATM and PARP combined inhibition demonstrate synergistic antitumor efficacy in osteosarcoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3912.

Abemaciclib is synergistic with doxorubicin in osteosarcoma pre-clinical models via inhibition of CDK4/6-Cyclin D-Rb pathway.

Osteosarcoma is a prevalent type of bone tumor in children and adolescents, with limited treatment and poor prognosis. Abemaciclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), is approved for the treatment of advanced breast cancer as single agent therapy and is currently under investigation in clinical trials for the treatment of several solid tumors. The efficacy of abemaciclib was determined using osteosarcoma cellular assays and xenograft mouse model. The combination studies were performed based on the Chou-Talalay method. Immunoblotting analysis was performed to determine the underlying mechanisms of abemaciclib in osteosarcoma cell lines. Abemaciclib potently inhibits growth, anchorage-independent colony formation and survival of a panel of osteosarcoma cell lines, with IC50 range from 90 nM to >20 μM. In addition, the combination of abemaciclib and doxorubicin is synergistic and antagonistic in abemaciclib-sensitive (IC50<1 μM) and abemaciclib-resistant (IC50 >1 μM), respectively. Abemaciclib inhibits tumor formation and growth in a dose-dependent manner without causing significant drug toxicity in mice. The combination of abemaciclib and doxorubicin results in much greater efficacy than doxorubicin alone in inhibiting tumor growth throughout the whole treatment duration. Abemaciclib acts on osteosarcoma via suppressing CDK4/6-Cyclin D-Rb pathway. Our pre-clinical evidence provides a rationale of initializing clinical trial of investigating the efficacy of abemaciclib in combination with doxorubicin in osteosarcoma patients. Our work also highlights the therapeutic value of CDK4/6 inhibition in osteosarcoma with proper function of Rb.

Abstract 84: Inhibition of Hedgehog signalling in ligand dependent tumours

Abstract Hedgehog (Hh) signalling is evolutionarily conserved pathway that is critical during development and maintenance of stem cells in adults. Activation of the Hedgehog pathway can occur through either ligand-dependent or ligand-independent mechanisms. While mutations in key Hh signalling components (PTCH1, SUFU, SMO, GLI1) result in ligand-dependent signalling and constitutive activation in some cancers, the vast majority of cancers in which Hh signalling is implicated do not have pathway mutations and are therefore driven upstream at the level of Hh ligand. Despite this, there are no biomarkers that can predict which tumours are dependent on Hh ligand and are likely to be responsive to Hh pathway inhibition. Using a panel of osteosarcoma cell lines derived from either radiation-induced or conditional Trp53 and Rb1 genetic inactivation murine models of osteosarcoma, we observed a dramatic increase in Gli1 mRNA, a downstream target of the Hh pathway, in response to Hh ligand stimulation in Trp53 and Rb1 deficient cells. This was accompanied by a significant increase in primary cilia frequency, a critical organelle for Hh pathway activation. Importantly, Hh response to ligand was abolished using the SMO inhibitor LDE225. Using osteosarcoma allograft models, we showed that LDE225-mediated inhibition of the Hh pathway resulted in reduced tumour growth and increased survival in ciliated Trp53 and Rb1-deficient allografts but no response in a non-ciliated Trp53 and Rb1 wildtype-allograft. The sustained clinical use of SMO inhibitors ultimately leads to development of resistance. We next evaluated the efficacy of Itraconazole, a SMO inhibitor previously demonstrated to be effective in Hh-therapy resistant ligand-independent tumour models, in our ligand-dependent osteosarcoma models. We show that Itraconazole inhibits Hh pathway activation in response to ligand leading to reduced Gli1 mRNA in vitro, and reduced tumour growth and increased survival in vivo. Collectively, this data implicates Trp53, Rb1 and primary cilia as a potential biomarker of Hh ligand responsiveness in osteosarcoma and response to Hh pathway inhibition by SMO inhibitors LDE225 and Itraconazole. Citation Format: Vijesh G. Vaghjiani, Catherine Cochrane, Neil Watkins, Stuart Mudge, Jason E. Cain. Inhibition of Hedgehog signalling in ligand dependent tumours [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 84.

3357 TL1 Team Approach to Using a Combination of Ganglioside 2 and 3 as an Immunoaffinity Target for Circulating Osteosarcoma Cell Detection

OBJECTIVES/SPECIFIC AIMS: The objective of our collaboration is to develop a strong trans-disciplinary team consisting of microfluidics engineers, cancer biologists, and clinicians, to identify a universal marker to detect circulating osteosarcoma cells (COC) using microfluidic devices. Our goals are 3 fold: 1) Identify cell surface markers unique to osteosarcoma (OS) for COC isolation, 2) Develop a Geometrically Enhanced Mixing (GEM) device to isolate COCs, and 3) Evaluate the efficacy of GEM device to detect COCs in patients with OS. The long term goal of this collaboration is to utilize this cell detection approach to evaluate treatment efficacy and correlate the presence of circulating osteosarcoma cells with metastatic incidence. METHODS/STUDY POPULATION: In this phase of our study, we have identified an abundant and conserved cell surface marker across a panel of OS cell lines. Flow cytometry was used to evaluate the relative expression of Epithelial Cell Adhesion Molecule (EpCAM), and Ganglioside 2 or/and 3 (GD2/3) on a panel of OS cell lines. An antibody coated GEM microfluidic device is used to affirm the efficacy of GD2/3 to capture COCs. Further capture studies will be conducted using OS cell spiked blood samples. Analysis of variance (ANOVA) will be used to determine any significant difference in capture efficiency between EpCAM, GD2/3 cell surface markers. RESULTS/ANTICIPATED RESULTS: Our results demonstrate that EpCAM is not a suitable marker for COC detection. Results from our flow cytometry studies demonstrate that GD2/3 expression is significantly higher than EpCAM expression, across all OS cell lines within our panel. The cell capture efficiency strongly correlates with the cell surface expression data obtained from flow cytometry analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: OS is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. At diagnosis, 80% of patients will present with metastasis, however only 20% of these cases are clinically detectable. Innovative strategies to identify patients at risk of metastasis would allow for stratification of intervention therapies. Liquid biopsies are a novel alternative to current diagnostic imaging systems to monitor metastatic incidence and treatment efficacy. The detection of circulating tumor cells (CTCs) through routine blood sampling has the potential to be used clinically for earlier detection, monitoring the treatment of metastatic cancers and surveying the effect of therapeutic interventions on metastasis. To date, the majority of the studies on CTCs have evaluated their presence in carcinomas. Although sarcomas are rare, they generally have a poorer prognosis. This study will address one of the unmet medical needs in the field of CTC detection; the identification of cell surface OS makers to improve binding specificity, increase purity, and maintain a high capture efficiency.

Abstract B38: Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy

Abstract Introduction: Osteosarcoma is the most common pediatric high-grade bone tumor. Although multimodal therapeutic approaches have significantly improved patient survival to more than 60%, the prognosis for patients with metastatic or relapsed disease remains dismal—an urgent unmet need. Its genomic complexity and higher mutational burden compared to other pediatric cancers should provide neoantigens as potential targets for T cell-based immunotherapy; however, these tumors are in general “cold,” with insufficient or inactive tumor-infiltrating lymphocytes. Ganglioside GD2 is a tumor-associated surface antigen expressed in a broad spectrum of pediatric malignancies, including neuroblastoma, brain tumors, Ewing’s sarcoma, rhabdomyosarcoma, and osteosarcoma, while being restricted in normal tissues. Another promising target, HER2 or ErbB2, is overexpressed in many aggressive malignancies, including pediatric medulloblastoma, nephroblastoma, osteosarcoma, and desmoplastic small round cell tumor (DSRT). Fully humanized tetravalent bispecific antibodies (BsAb) specific for human GD2 and CD3 (hu3F8-BsAb) or HER2 and CD3 (HER2-BsAb), built on an IgG(L)-scFv platform, have been developed (Oncoimmunology 2017; Can Immunol Res 2015). They induce rapid and quantitative T-cell homing to tumors, mediating potent T-cell dependent cytotoxicity (TDCC) against GD2 or HER2 expressing tumor cells, effecting cures of xenografts in SCID mice. In this study, we evaluated the in vitro and in vivo antitumor properties of these BsAbs in the treatment of osteosarcoma. Methods: Hu3F8-BsAb and HER2-BsAb were tested both in vitro and in vivo against a panel of osteosarcoma cell lines (RG143B, U2OS, CRL1427, HOS, and SaOS2). Using these cell lines, FACS analysis for surface antigen (GD2 and HER2) expression (MFI), sensitivity to TDCC (EC50), and antitumor activity in vivo were evaluated. Two in vivo tumor models with different effector routes were used to simulate different clinical situations: 1) subcutaneous (sc) tumor cells/sc effector peripheral blood mononuclear cells (PBMCs), and 2) sc tumor cells/intravenous (iv) PBMCs. In vivo tumor responses were measured by Peira TM900 imaging device or by bioluminescence. Results: The majority of osteosarcoma cell lines express GD2 and HER2 on their surface, and their in vitro sensitivity to TDCC (EC50) was inversely correlated with MFI of their respective antigen. Both hu3F8-BsAb and HER2-BsAb mediated potent TDCC against osteosarcoma cell lines. In vivo, both hu3F8-BsAb and HER2-BsAb exerted a significant antitumor effect compared to control BsAb (P=0.001). Conclusions: Hu3F8-BsAb and HER2-BsAb induced strong TDCC and had significant antitumor effect against osteosarcoma cell lines both in vitro and in vivo. Considering the limited therapeutic options currently available in advanced osteosarcomas, these results support their further clinical development as potential T cell-based immunotherapeutics. Citation Format: Jeong A. Park, Hong Xu, Irene Cheung, Nai-Kong V. Cheung. Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B38.

Abstract 1634: BRCA-like phenotype constitutes hallmark of osteosarcoma

Abstract Background: Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which limits treatment options in patients who do not respond to standard chemotherapy. Methods: We sequenced 104 exomes and low-coverage genomes from 14 time-series consisting of chemotherapy-naïve biopsies, resection specimens, locally recurring tumours and/or distant metastases. We then identified cancer driver mutations, signatures of mutation processes and reconciled clonal structures of tumours with phylogenetic trees, thus building and evolutionary history of each tumour. Where possible, we searched for the evidence of selection acting on the tumours and molecular hallmarks that could be therapeutically exploited. Results: Interrogating genomic data from time series, we identified pathogenic mutations in at least 10 cancer genes, but there was no universal cancer driver event present in the majority of tumours at any given time point. However, &amp;gt;80% of osteosarcomas exhibited specific combination of single-base substitutions, LOH and large-scale genome instability characteristic of BRCA1/2-deficient tumours. In osteosarcoma, "BRCAness" developed recurrently through independent BRCA1/2 mutations and mutations in their binding partners such as PALB2 or CHEK2. We therefore investigated the sensitivity of a panel of osteosarcoma cell lines to the poly(ADP)-ribose polymerase inhibitor talazoparib. In combination with temozolomide, talazoparib led to the to good response of osteosarcoma cell lines, activation of BAX and BAK, loss of mitochondrial membrane potential, DNA fragmentation and caspase-dependent cell death. Conclusion: The acquisition of a BRCA-like phenotype in osteosarcoma has implications for the development of novel treatment strategies using PARP inhibitors alone, or together with standard chemotherapy. Citation Format: Michal Kovac, Sebastian Ribi, Claudia Blattmann, Michaela Nathrath, Daniel Baumhoer. BRCA-like phenotype constitutes hallmark of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1634.

2090 TL1 team approach to osteosarcoma cell detection

OBJECTIVES/SPECIFIC AIMS: The objective of our collaboration is to develop a strong transdisciplinary team consisting of microfluidics engineers, cancer biologists, and clinicians, to identify cell surface markers capable of detecting circulating osteosarcoma cells (COC) using microfluidic devices. Our goals are 3-fold: (1) Identify cell surface markers unique to osteosarcoma (OS) for COC isolation, (2) develop a Geometrically Enhanced Mixing (GEM) device to isolate COCs, and (3) Evaluate the efficacy of GEM device to detect COCs in OS patients under treatment. The long-term goal is to utilize this cell detection approach to correlate the presence of COC with metastatic incidence. METHODS/STUDY POPULATION: To identify a marker to capture COCs we are utilizing flow cytometry and microfluidic capture devices. Flow cytometry will be used to evaluate the relative expression of epithelial cell adhesion molecule (EpCAM), CD45, cell surface vimentin (CSV), insulin-like growth factor 2 (IGF2R), interleukin 11 receptor subunit alpha (IL-11Rɑ), ganglioside 2 (GD2), and receptor activator of nuclear factor κ-B (RANK) on a panel of OS cell lines. These cell surface markers were selected based on an extensive review of OS cell surface markers. OS cell capture efficacy will be assessed by passaging a known concentration of OS cells through a GEM microfluidic device coated with antibodies targeting the selected marker, as indicated by flow cytometry. Once captured, COCs on the device will be analyzed and the capture efficiency for the indicated marker will be measured. ANOVA will be used to determine any significant difference in capture efficiency between marker types. Once an optimal marker or panel of markers has been selected we will conduct capture studies using OS cell spiked blood samples followed by clinical samples obtained from OS patients. In clinical samples, COC detection will be validated using the FDA approved triple immunocytochemistry technical definition of a circulating tumor cell (CTC). This will enable COCs to be differentiated from the normal whole blood cell population by selecting for CD45−, EpCAM+, and cytokeratin+ cells. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown that on our microfluidic device, EpCAM, a marker commonly used to identify circulating tumor cells in other cancer settings, has a poor capture efficiency (15.9%+7.7%) for HU09 OS cells while the same setup with EpCAM has a capture efficiency of 56.9%+2.7% for BXPc-3 pancreatic cells. We therefore anticipate our flow cytometry studies to show a low expression of EpCAM and CD45 for OS cell lines, while showing a moderate to high expression of CSV, IGF2R, IL-11Rɑ, GD2, and RANK. We expect to show a 60%–80% capture efficiency for markers selected for COC capture. Currently, CSV and GD2 are particularly promising as markers based on previously published studies. DISCUSSION/SIGNIFICANCE OF IMPACT: OS is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. At diagnosis 80% of patients will present with metastasis, however only 20% of these cases are clinically detectable. Innovative strategies to identify patients at risk of metastasis would allow for stratification of intervention therapies. Currently, tumor recurrence and metastasis are primarily dependent on diagnostic-imaging modalities such as computerized tomography or positron emission tomography scans. Unfortunately, these imaging modalities can only detect tumor masses of significant size (106 tumor cells). Liquid biopsies are a novel alternative to current diagnostic imaging systems to monitor metastatic incidence and treatment efficacy. The detection of CTCs through routine blood sampling has the potential to be used clinically for earlier detection, monitoring the treatment of metastatic cancers and surveying the effect of therapeutic interventions on metastasis. To date, the majority of the studies on CTCs have evaluated their presence in carcinomas. Although sarcomas are rare, they generally have a poor prognosis. This study will address one of the unmet medical needs in the field of CTC detection; the identification of cell surface OS makers to improve binding specificity, increase purity, and maintain a high capture efficiency. This phase of our proposal will evaluate the most abundant and conserved markers across a panel of OS cell lines. Once a marker or panel of markers is selected, we will begin to develop a microfluidic device that can be used clinically to detect CTCs in this disease setting.

Abstract A06: BRCA-like phenotype as a hallmark of osteosarcoma's converging evolution

Abstract Background: Osteosarcomas are aggressive bone tumors with a high degree of genetic heterogeneity, which has historically limited treatment options in patients who do not respond to standard chemotherapy. Methods: We sequenced exomes and genomes of 32 pretherapeutic osteosarcomas, 6 time-series consisting of chemotherapy-naïve biopsies, resection specimens, recurring tumors and/or metastases, and 15 unpaired metastases from (mainly) poor responders. We then interrogated signatures of mutation processes in cancer genomes and reconciled clonal maps with phylogenetic trees, thus building an evolutionary history of each tumor. Where possible, we searched for evidence of selection acting on the tumors and hallmarks that could be therapeutically exploited. Results: We identified pathogenic mutations in at least 14 cancer drivers, some of which were formerly unknown in the context of osteosarcoma. However, there was no single cancer driver gene that could explain the majority of cases. Instead, &amp;gt;80% of osteosarcomas exhibited a specific combination of single-base substitutions, LOH, or large-scale genome instability characteristic of BRCA1/2-deficient tumors. The BRCA-like phenotype developed through parallel evolution by deleterious mutations affecting the BRCA1/2 genes and their binding partners such as PALB2 or CHEK2. We therefore investigated the sensitivity of a panel of osteosarcoma cell lines to the poly(ADP)-ribose polymerase inhibitor talazoparib alone and in combination with chemotherapeutic drugs. We identified an association between homologous recombination repair deficiency and the response of osteosarcoma cell lines to talazoparib. In combination with temozolomide, talazoparib led to the activation of BAX and BAK, loss of mitochondrial membrane potential, DNA fragmentation, and caspase-dependent cell death. Conclusion: The acquisition of a BRCA-like phenotype through converging evolution has important implications for the development of novel treatment strategies in osteosarcoma using PARP inhibitors alone or together with standard chemotherapy. Citation Format: Michal Kovac, Sebastian Ribi, Claudia Blattmann, Michaela Nathrath, Daniel Baumhoer. BRCA-like phenotype as a hallmark of osteosarcoma's converging evolution [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 A06.

Abstract 5072: Inhibition of EZH2 as a therapeutic strategy for osteosarcoma

Abstract Introduction: Osteosarcoma is the most common primary malignant bone tumor. About 30% of osteosarcoma patients experience relapse and die of their disease. We previously demonstrated that IL-11Rα is overexpressed in primary and metastatic osteosarcoma and plays a role in the development of metastases. Our recent microarray data from KRIB, SJSA1, and LM7 IL-11Rα shRNA cells show decreased expression of several components of polycomb repressive complex 2 (PRC2). EZH2 is the catalytic subunit of PRC2 and blocks transcription of numerous tumor suppressors’ genes. EZH2 has been widely implicated in cancer and inhibition of its catalytic activity has recently emerged as a novel approach to treat cancers. Purpose: To explore the effects and mechanisms of inhibition of EZH2 on osteosarcoma metastasis. Inhibition was tested using GSK126, a novel small-molecule EZH2 inhibitor that competes with methyl group donor S-adenosyl-methionine in a highly selective fashion. Methods: Proliferation assays were performed on a panel of osteosarcoma cell lines subjected to increasing concentrations (0.0625 μm to 20 μm) of GSK126 for 3 days. The effects of IL-11 in PRC1 complex histone H3 methylation (H3K27) were studied by Western blotting. For in vivo experiments, we used an orthotopic metastatic model. Luciferase-labeled SJSA1 osteosarcoma cells were injected into the tibias of 4- to 5-week-old nude mice. After 2 weeks, mice were treated with vehicle or GSK126 three times per week intraperitoneally at 0.1 ml per 20 g of body weight in 20% captisol for 48 days. The tumor-containing legs were amputated when tumor volume reached 2 cm. Primary tumors were measured with calipers, and tumorigenesis and metastasis were evaluated by luciferase imaging. After 4 more weeks of treatment, all the mice were killed and their lungs collected. Immunohistochemical study for EZH2 (1:200, D2C9, Cell Signaling) was performed on tissue microarrays composed of 200 formalin-fixed decalcified human OS specimens: 141 primary and 59 metastatic. Intensity and extent of tumoral labeling were evaluated. Results: EZH2 expression was seen in 79/200 OS specimens (40%) with 54 specimens (27%) with moderate to strong labeling. In the majority of cases, when staining was present, more than 50% of labeling was seen. More metastatic specimens (24/59, 41%) had moderate to strong labeling than primary resection specimens (30/141, 21%). In vitro, SJSA1, HOS and CCH-OS-D osteosarcoma cell lines were sensitive to EZH2 inhibition (IC50= 3.576- 6.450uM). In vivo, mice treated with GSK126 developed significantly fewer osteosarcoma lung metastases than control mice injected with vehicle alone (P&amp;lt;0.01). Conclusion: GSK126, a potent small-molecule EZH2 inhibitor, inhibits the development of osteosarcoma lung metastases in vivo. Thus, GSK126 may be considered as a novel anticancer drug candidate for osteosarcoma. Citation Format: Eswaran Devarajan, Wei-lin Wang, Jen-Wei Tsai, Andrew Futreal, Valerae O. Lewis. Inhibition of EZH2 as a therapeutic strategy for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5072. doi:10.1158/1538-7445.AM2017-5072

MiR-205 suppresses the proliferative and migratory capacity of human osteosarcoma MG-63 cells by targeting VEGFA

BackgroundOsteosarcoma (OS) is the most common primary bone malignancy in children and young adults. MiR-205 has been reported to be negatively correlated with the proliferation and metastasis of many types of cancer, while its effects on the malignant phenotype of OS are unclear.MethodsUsing TaqMan RT polymerase chain reaction analysis, we firstly explored the expression of miR-205 in a panel of OS cell lines. As the expression of miR-205 was significantly decreased in these cell lines, we sought to compensate for its loss by transfection of exogenous miR-205 mimic into MG-63 cells. To further understand the role of miR-205 in OS, we investigated the effects of miR-205 on the proliferation, migration, and invasion of MG-63 cells, and further explored the mechanisms that might be involved.ResultsWe found that miR-205 was consistently suppressed in OS cells when compared with the normal human osteoblast (NHOst) cell line. Restored expression of miR-205 in the OS (MG-63) cell line significantly inhibited cell proliferation, migration, and invasion. Moreover, bioinformatic prediction suggested that vascular endothelial growth factor A (VEGFA) was the target oncogene for miR-205 in OS cells. Further quantitative RT polymerase chain reaction and Western blot assays identified that overexpression of miR-205 suppressed expression of VEGFA mRNA and protein. Restored expression of VEGFA in MG-63 cells previously treated with miR-205 mimic could partially abolish miR-205-mediated suppression of proliferation and invasion of these cells.ConclusionCollectively, these data suggest that miR-205 might function as a tumor suppressor in OS by, at least partially, targeting VEGFA.

Abstract 1750: Preclinical efficacy of Hsp90 inhibition by using PF-04942847 in osteosarcoma

Abstract This work was supported by foundation ARC and INSERM, France. Introduction and Objective: Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants the development of new strategies to improve overall patient survival. Among new therapeutic approaches, Heat shock protein (HSP) 90 is a molecular chaperone involved in the maturation and stability of various oncogenic proteins leading to tumor cells survival. In this study, we assessed the in vitro and in vivo antitumor properties of a novel synthetic HSP90 inhibitor, PF-04942847 both in vitro and in vivo in osteosarcoma. Methods: The effects of the HSP90 inhibitor were evaluated in vitro on U2OS, SaOS2, MNNG/HOS, KHOS, MG63, G292, CAL72, SJSA1 and 143B cell growth and apoptosis. The signaling pathways were analyzed by western blotting. The consequence of HSP90 therapy in vivo was evaluated in athymic mice bearing MNNG/HOS xenografts. The effect of PF-04942847 on osteoclastogenesis was assessed in vitro on purified human CD14+ monocytes. Results: In our panel of osteosarcoma cell lines, PF-04942847 inhibited cell growth in a dose-dependent manner (IC50 ± 50nM) and induced apoptosis with an increase of sub-G1 fraction and PARP cleavage. These biologic events were accompanied by decreased expression of Akt, Erk, MYC, c-MET, STAT-3, and modulation of HSP expression. When administered orally 3 times/week (25mg/kg) to mice bearing osteosarcoma tumors, PF-04942847 significantly inhibited tumor growth by 40% and prolonged survival compared to controls. Moreover, in contrast to 17-AAG, the novel PF-04942847 compound inhibits osteoclast differentiation in vitro. Conclusions: Targeting HSP90 using PF-04942847 inhibited osteosarcoma tumor growth in vitro and in vivo and appears to inhibit osteoclastogenesis. All these data provides a strong rationale for clinical evaluation of PF-04942847 in osteosarcoma. Citation Format: Marc Baud'Huin, Benjamin Ory, Franck Verrecchia, Dominique Heymann, Francoise Redini, Francois Lamoureux. Preclinical efficacy of Hsp90 inhibition by using PF-04942847 in osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1750. doi:10.1158/1538-7445.AM2015-1750

Abstract A53: Metadherin functions as a laminin receptor that is essential for metastasis and is associated with poor survival in osteosarcoma

Abstract Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. In order to improve the clinical outcomes for patients with poor prognosis, it is urgent to find new therapeutic targets to block metastasis in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. To evaluate the expression of MTDH in primary and metastatic lesions of osteosarcoma, two tissue microarrays containing patient-derived primary and metastatic tumor specimens were examined by immunohistochemical staining with anti-MTDH antibody. We also examined MTDH in a cDNA array expression database made from pretreatment diagnostic biopsies of high-grade osteosarcoma patients to further assess the correlation between MTDH expression and clinical outcome. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. In addition, we developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction and to identify the extracellular binding partner for cell surface MTDH, a series of adhesion assays were performed, followed by bidirectional co-immunoprecipitation. We have demonstrated that MTDH is upregulated in human osteosarcoma cell lines and patient-derived specimens compared with normal human osteoblasts. Overexpression of MTDH is more profound in metastatic lesions compared to primary tumors and is correlated with poor clinical outcomes in osteosarcoma patients. MTDH knockdown and blockade of cell surface MTDH significantly reduced migration and invasion in osteosarcoma cells. In the in vivo experiments, downregulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Both in vitro and in vivo studies confirmed the critical role of MTDH in the invasive and metastatic capacity of osteosarcoma cells. More importantly, we have identified the significance of cell surface localization of MTDH in mediating osteosarcoma motility and invasiveness. We showed that MTDH exists as a type II membrane protein in osteosarcoma cells and its expression on cell surface is facilitating cell invasion by means of modulating cell adhesion to the ECM through interaction with laminin. In total, these observations establish MTDH as a promising target for therapeutic interventions in metastatic osteosarcoma. The novel connection between MTDH and extracellular laminin also establishes a new paradigm for the function of MTDH in mediating tumor cell metastasis. Citation Format: Limin Zhu, Pingyu Zhang, Yanwen Yang, Adrianna S. Buford, Wei-Lien Wang, Dafydd G. Thomas, Dennis P.M. Hughes. Metadherin functions as a laminin receptor that is essential for metastasis and is associated with poor survival in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A53.

Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells.

The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.

Abstract 2683: Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma.

Abstract Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines, and used immunohistochemistry to confirm expression in patient-derived material. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. We also developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction, a series of adhesion assays were performed to assess the adhesion ability of osteosarcoma cells with or without MTDH inhibition to major components of ECM. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed increased MTDH expression. MTDH knockdown significantly reduced migration and invasion in osteosarcoma cells. Blockade of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness. In the in vivo experiments, down-regulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Immunohistochemical analysis of tumors samples isolated from the mice showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether, these data indicated an important role for Metadherin in osteosarcoma metastasis. Further, knockdown of MTDH by shRNA and inhibition of cell surface MTDH by anti-MTDH antibodies both profoundly decreased cell adhesion to matrigel and specific ECM proteins, which suggested that MTDH, particularly the proportion localized at the cell surface, may be involved in the interaction between tumor cells and the non-cellular matrix proteins that constitute tissue stroma. The interaction will be confirmed by future experiments. By identifying the ECM protein binding partners for MTDH, we may establish a new paradigm for the function of MTDH in mediating tumor cell migration and invasion in osteosarcoma. Citation Format: Limin Zhu, Adrianna Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2013-2683

Abstract B67: Metadherin mediates osteosarcoma invasion and metastasis

Abstract Osteosarcoma is the most common type of bone malignancy in children. It is a highly invasive mesenchymal tumor in which metastasis accounts for the vast majority of mortality and morbidity in patients. Understanding the mechanisms controlling metastasis in osteosarcoma and developing novel therapeutic options targeting metastasis are essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays a key role in mediating metastasis in a variety of human cancers. This study examined the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its oncogenic activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines. Patient-derived osteosarcoma samples were also examined for MTDH expression using immunohistochemistry. In parallel experiments we used MTDH-specific shRNA to down-regulate endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using migration assays and matrigel invasion assays. We also developed an osteosarcoma orthotopic xenograft model of NOD/SCID/IL2Rγ-deficient mice through intratibial injection of osteosarcoma cells with or without MTDH knockdown to study the relationship between MTDH expression and pulmonary metastatic potential of osteosarcoma cells. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed elevated MTDH expression as shown by western blot, qPCR and flow cytometry analyses. MTDH knockdown reduced migration and decreased invasion in matrigel. Inhibition of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness in vitro. In an orthotopic xenograft mouse model using CCHD cells, inhibition of MTDH delayed primary tumor growth and prohibited pulmonary metastasis. All mice in the control group developed lung metastases while fewer than 20% of the mice in the MTDH-knockdown group showed sign of metastasis at six weeks after inoculation. MTDH knockdown also reduced mean lung weight and mean number of pulmonary metastasis. Immunohistochemical analysis of tumor samples showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether these studies suggested that MTDH plays a critical role in promoting pulmonary metastasis of osteosarcoma and may serve as an attractive target for therapeutic intervention. Citation Format: Limin Zhu, Adrianna S. Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates osteosarcoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B67.

Targeting JNK-interacting protein 1 (JIP1) sensitises osteosarcoma to doxorubicin

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Despite aggressive therapy, survival outcomes remain unsatisfactory, especially for patients with metastatic disease or patients with a poor chemotherapy response. Chemoresistance contributes to treatment failure. To increase the efficacy of conventional chemotherapy, essential survival pathways should be targeted concomitantly. Here, we performed a loss-of-function siRNA screen of the human kinome in SaOS-2 cells to identify critical survival kinases after doxorubicin treatment. Gene silencing of JNK-interacting-protein-1 (JIP1) elicited the most potent sensitisation to doxorubicin. This candidate was further explored as potential target for chemosensitisation in OS. A panel of OS cell lines and human primary osteoblasts was examined for sensitisation to doxorubicin using small molecule JIP1-inhibitor BI-78D3. JIP1 expression and JIP1-inhibitor effects on JNK-signalling were investigated by Western blot analysis. JIP1 expression in human OS tumours was assessed by immunohistochemistry on tissue micro arrays. BI-78D3 blocked JNK-signalling and sensitised three out of four tested OS cell lines, but not healthy osteoblasts, to treatment with doxorubicin. Combination treatment increased the induction of apoptosis. JIP1 was found to be expressed in two-thirds of human primary OS tissue samples. Patients with JIP1 positive tumours showed a trend to inferior overall survival. Collectively, JIP1 appears a clinically relevant novel target in OS to enhance the efficacy of doxorubicin treatment by means of RNA interference or pharmacological inhibition.

Phage Display–Derived Peptides for Osteosarcoma Imaging

Osteosarcoma represents the most common malignant primary bone tumor in childhood; however, the survival rate has remained unchanged for the past 20 years. To improve existing diagnosis and treatment methods and broaden the spectrum of imaging agents that can be used for early detection and assessment of tumor response to therapy, we performed a phage display-based screening for peptide sequences that bind specifically to osteosarcoma cells. From the Ph.D.-12 phage display peptide library composed of 2.7 x 10(9) different displayed peptides, one peptide was enriched after four rounds of in vitro selection in 143B osteosarcoma tumor cells with 293T human embryonic kidney cells as a control. Both the peptide and the phage clone displaying the peptide were conjugated with fluorescent dyes for in vitro cell and ex vivo tumor tissue stainings. The peptide was further labeled with (18)F for positron emission tomography imaging studies. Cell uptake and efflux and ex vivo biodistribution were also done with (18)F-labeled osteosarcoma specific peptide. ASGALSPSRLDT was the dominant sequence isolated from biopanning and named as OSP-1. OSP-1 shares a significant homology with heparinase II/III family protein, which binds and reacts with heparan sulfate proteoglycans. The fluorescence staining showed that FITC-OSP-1-phage or Cy5.5-OSP-1 had high binding with a panel of osteosarcoma cell lines, much less binding with UM-SCC1 human head and neck squamous cell carcinoma cells, and almost no binding with 293T cells, whereas the scrambled peptide OSP-S had virtually no binding to all the cell lines. (18)F-OSP-1 had significantly higher accumulation in 143B tumor cells both in vitro and in vivo than (18)F-OSP-S. (18)F-OSP-1 also had higher uptake in 143B tumors than in UM-SCC-1 tumors. Our data suggest that OSP-1 peptide is osteosarcoma specific, and the binding site of OSP-1 might be related to heparan sulfate proteoglycans. Appropriately labeled OSP-1 peptide has the potential to serve as a novel probe for osteosarcoma imaging.