Preclinical Models Of Osteosarcoma Research Articles

Oncolytic Viral Therapy in Osteosarcoma.

Primary bone malignancies, including osteosarcoma (OS), are rare but aggressive. Current OS treatment, involving surgical resection and chemotherapy, has improved survival for non-metastatic cases but remains ineffective for recurrent or metastatic OS. Oncolytic viral therapy (OVT) is a promising alternative, using naturally occurring or genetically modified viruses to selectively target and lyse cancer cells and induce a robust immune response against remaining OS cells. Various oncolytic viruses (OVs), such as adenovirus, herpes simplex virus, and measles virus, have demonstrated efficacy in preclinical OS models. Combining OVT with other therapeutics, such as chemotherapy or immunotherapy, may further improve outcomes. Despite these advances, challenges in reliability of preclinical models, safety, delivery, and immune response must be addressed to optimize OVT for clinical use. Future research should focus on refining delivery methods, exploring combination treatments, and clinical trials to ensure OVT's efficacy and safety for OS. Overall, OVT represents a novel approach with the potential to drastically improve survival outcomes for patients with OS.

Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model.

Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma.

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αβ T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, having innate antitumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allocompatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Rα-IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared with unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together, the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control.

Retraction: Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Retraction: Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

DESIGN AND DEVELOPMENT OF A CUSTOM PLATFORM TO GROW AND CHARACTERIZE OSTEOSARCOMA SPHEROIDS

Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research.Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms.To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.

A chimeric human/dog-DNA vaccine against CSPG4 induces immunity with therapeutic potential in comparative preclinical models of osteosarcoma

The high mortality rate of osteosarcoma (OSA) patients highlights the requirement of alternative strategies. The young age of patients, as well as the rarity and aggressiveness of the disease, limits opportunities for the robust testing of novel therapies, suggesting the need for valuable preclinical systems. Having previously shown the overexpression of the chondroitin sulfate proteoglycan (CSPG)4 in OSA, herein the functional consequences of its downmodulation in human OSA cells were evaluated invitro, with a significant impairment of cell proliferation, migration, and osteosphere generation. The potential of a chimeric human/dog (HuDo)-CSPG4 DNA vaccine was explored in translational comparative OSA models, including human xenograft mouse models and canine patients affected by spontaneous OSA. The adoptive transfer of HuDo-CSPG4 vaccine-induced CD8+ Tcells and sera in immunodeficient human OSA-bearing mice delayed tumor growth and metastasis development. HuDo-CSPG4 vaccination resulted safe and effective in inducing anti-CSPG4 immunity in OSA-affected dogs, which displayed prolonged survival as compared to controls. Finally, HuDo-CSPG4 was also able to induce a cytotoxic response in a human surrogate setting invitro. On the basis of these results and the high predictive value of spontaneous OSA in dogs, this study paves the way for a possible translation of this approach to humans.

Abstract LB329: A potent and selective small molecule inhibitor of CSF-1R ABSK021 demonstrates strong efficacy in preclinical models of osteosarcoma

Abstract Introduction: Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgery combined with multimodal chemotherapy remains as the standard treatment for osteosarcoma patients. However, patients with osteosarcoma metastasis have a five-year survival rate of less than 30%, and their long-term outcomes have not improved over the last 30 years. CSF-1/CSF-1R signaling is crucial for the survival, function, proliferation and differentiation of myeloid lineage cells, including osteoclasts and monocytes/macrophages. Targeting CSF-1R either on tumor cells or tumor-associated macrophages has been reported to limit osteosarcoma progression in preclinical models. ABSK021, an oral, highly potent and selective small molecule inhibitor of CSF-1R discovered by Abbisko, has entered into Phase I trial (NCT04192344) and showed significant anti-tumor activity and favorable safety profile in patients with advanced tenosynovial giant cell tumor. Here, we conducted a series of preclinical in vitro and in vivo experiments, as well as human osteosarcoma profiling, to investigate the treatment potential of ABSK021 for osteosarcoma patients. Methods: Cellular potency of ABSK021 was evaluated by Celltiter-Glo assay in osteosarcoma cell lines and CSF-1R expression was evaluated by western blot. In vivo efficacy studies were conducted in murine osteosarcoma models and pharmacodynamics changes were measured. CSF-1R IHC staining was conducted in tissue microarray samples from osteosarcoma patients. Results: ABSK021 potently inhibited the proliferation of K7M2, a murine osteosarcoma cell line with high expression of CSF-1R. In animal, ABSK021 showed strong anti-tumor activity on K7M2 syngeneic model and SA4094 osteosarcoma PDX model without causing significant body weight loss. Pharmacodynamics analysis displayed robust inhibition of macrophage infiltration and CSF-1R signaling by ABSK021, confirming its effective target engagement in vivo. Parallel IHC analysis of human tissue microarray samples revealed high prevalence of positive CSF-1R staining in osteosarcoma patients. Conclusion: Taken together, these results demonstrate that ABSK021 has strong inhibition of CSF-1R activity and corresponding anti-tumor activity in preclinical osteosarcoma models. High prevalence of CSF-1R expression was also found in osteosarcoma patients, suggesting great potential of utilizing ABSK021 as a novel therapy to treat osteosarcoma patients in clinic. Citation Format: Nannan Zhang, Cheng Dai, Jiacheng Zhang, Shuqun Yang, Jie Wang, Jie Zhang, Manqi Liu, Zhui Chen. A potent and selective small molecule inhibitor of CSF-1R ABSK021 demonstrates strong efficacy in preclinical models of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB329.

MAPK-interacting kinases inhibition by eFT508 overcomes chemoresistance in preclinical model of osteosarcoma.

The MAPK-interacting kinases 1 and 2 (MNK1/2) have generated increasing interest as therapeutic targets for many cancers with little known in osteosarcoma. This study evaluated the efficacy of eFT508, a highly selective inhibitor of MNK1/2, as single drug alone and in combination with paclitaxel in preclinical models of osteosarcoma. EFT508 is active against multiple osteosarcoma cell lines via inhibiting growth, survival and migration. It also demonstrates anti-osteosarcoma selectivity with much less toxicity on normal osteoblastic than osteosarcoma cells. Consistent with in vitro findings, eFT508 at non-toxic dose significantly arrested tumor growth in mice throughout the whole duration of treatment. Mechanistically, eEFT508 is highly effective in blocking eIF4E phosphorylation and eIF4E-mediated protein translation. Combination index shows that eFT508 and paclitaxel is synergistic in osteosarcoma cells. Our findings highlight the therapeutic value of MNK1/2 inhibition and suggest eFT508 as a promising candidate for the treatment of osteosarcoma.

Generation and characterization of patient-derived xenografts from patients with osteosarcoma

BackgroundStandard therapy of osteosarcoma (OS) rests on cytotoxic regimes, which dramatically limits the further improvement in the prognosis of patients with OS. Preclinical models of OS are extremely urgent to break this impasse. Materials and MethodsHere, we describe our experience of developing patient-derived tumor xenografts (PDXs) using surgical samples from patients with OS. All the xenografts growing subcutaneously in mice will be identified pathologically and genetically. Furthermore, we explored the consistency of the drug sensitivity between the PDX models and corresponding patients against specific regimens. ResultsWe generated nine OS PDXs from 21 surgical resections of primary OS tumors, with an engraftment rate of 42.9 %. Morphological and immunohistochemical (SATB2, MDM2, CDK4, and Ki67) studies and whole-exome sequencing showed a remarkable similarity between the patient’s tumor and corresponding PDX, which was maintained over several passages in mice. A therapeutic combination of Pirarubicin and Cisplatin was tested against two OS PDX models, in which the corresponding patient was insensitive and sensitive to the regimen, respectively. ConclusionThe panel of OS PDX models faithfully mirrored morphologic and genetic features of OS, representing reliable models to test therapeutic approaches.

Insight into the interplay between mitochondria-regulated cell death and energetic metabolism in osteosarcoma.

Osteosarcoma (OS) is a pediatric malignant bone tumor that predominantly affects adolescent and young adults. It has high risk for relapse and over the last four decades no improvement of prognosis was achieved. It is therefore crucial to identify new drug candidates for OS treatment to combat drug resistance, limit relapse, and stop metastatic spread. Two acquired hallmarks of cancer cells, mitochondria-related regulated cell death (RCD) and metabolism are intimately connected. Both have been shown to be dysregulated in OS, making them attractive targets for novel treatment. Promising OS treatment strategies focus on promoting RCD by targeting key molecular actors in metabolic reprogramming. The exact interplay in OS, however, has not been systematically analyzed. We therefore review these aspects by synthesizing current knowledge in apoptosis, ferroptosis, necroptosis, pyroptosis, and autophagy in OS. Additionally, we outline an overview of mitochondrial function and metabolic profiles in different preclinical OS models. Finally, we discuss the mechanism of action of two novel molecule combinations currently investigated in active clinical trials: metformin and the combination of ADI-PEG20, Docetaxel and Gemcitabine.

Abstract 3062: Drug repurposing of β-blocker propranolol and hemostatic compound desmopressin in osteosarcoma: Preclinical antitumor activity on 2D and 3D cell growth, chemotaxis and xenograft progression

Abstract Introduction. Osteosarcoma (OS) is the most common malignant primary bone tumor in children and young adults, with alarmingly elevated mortality rates, especially in developing countries. OS patients bear highly invasive and vascularized tumors, with limited response to standard of care (SoC) therapies, and are in urgent need of novel therapeutic strategies. Propranolol (PPN) is a non-selective β-adrenergic receptor (β-AR) antagonist originally used in the treatment of diverse heart diseases. On the other hand, desmopressin (dDAVP) is a hemostatic drug that acts as a selective agonist for the vasopressin type-2 receptor (AVPR2) present in blood microvessels and several tumor types. Given that β-ARs and AVPR2 signalling regulates many cellular processes involved in the initiation and progression of cancer, multiple efforts have been made to repurpose PPN and dDAVP in indications such as breast and colorectal cancer, melanoma and angiosarcoma, among others. Considering the unsatisfied clinical needs of OS, the objective of this work was to evaluate the in vitro/in vivo antitumoral activity of PPN and dDAVP on highly aggressive preclinical models of OS. Materials and methods. The human OS cell lines MG-63 and U2-OS were used for in vitro or in vivo experiments. Target expression was assessed by qPCR and immunohistochemistry. Sensitivity to PPN or dDAVP was evaluated by in vitro 72 h proliferation, 7 d clonogenic growth, 3D spheroid formation, transwell chemotaxis assays and MG-63 xenograft progression in nude mice. In addition, PPN was evaluated as monotherapy or in combination with SoC chemotherapy. Results. PPN (10-100 µM) and dDAVP (0.1-10 µM) drastically reduced clonogenic and 3D growth, migration, mitotic index and proliferation of β-AR and AVPR2-expressing OS cells (p<0.05). Synergistic antitumoral effects were observed after combining PPN with cisplatin or methotrexate, in vitro and in vivo (CI<1). In animals bearing growing OS s.c. xenografts sustained treatment during 4 weeks with PPN (10 mg/kg i.p. daily) or dDAVP (12 µg/kg i.v. three times a week) markedly abrogated tumor progression, exhibiting modulation of local tumor aggressiveness and reducing final tumor burden by 25 or 45%, respectively (p<0.01). Conclusions. Taking into account PPN and dDAVP robust antitumoral effects, β-ARs antagonization or agonist activation of AVPR2 are achievable and interesting therapeutic approaches to modulate OS tumor aggressiveness. Both agents could be proposed as coadyuvant agents for treating OS, not only in combination with chemotherapy but also administered during the perioperative setting. Citation Format: Luisina Solerno, Natasha Sobol, Rocío Rodriguez, Marina Pifano, Giselle Ripoll, Hernan Farina, Liliana Vasquez, Daniel F. Alonso, Juan Garona. Drug repurposing of β-blocker propranolol and hemostatic compound desmopressin in osteosarcoma: Preclinical antitumor activity on 2D and 3D cell growth, chemotaxis and xenograft progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3062.

Dose-response effect of eribulin in preclinical models of osteosarcoma by the pediatric preclinical testing consortium.

The pediatric preclinical testing program previously demonstrated activity of eribulin in osteosarcoma patient-derived xenograft (PDX) models. The phase 2 trial in patients with relapsed osteosarcoma failed to meet response endpoints. Eribulin was evaluated in the original and an expanded set of PDX models and tested at multiple dose levels and schedules to evaluate dose-response. Maximal response was observed at the highest dose, consistent with prior results. The alternative schedule generated similar responses. We demonstrate steep dose-response for eribulin in osteosarcoma PDX models, implying that any deviation from achievement of effective concentrations may have a significant impact on activity.

Oncolytic Viruses and Their Potential as a Therapeutic Opportunity in Osteosarcoma.

Osteosarcoma remains an unmet medical need. Oncolytic viruses are gaining traction as novel cancer therapeutics. These viruses are either naturally nonpathogenic or engineered to be safe by specific genetic deletions yet retain the ability to infect and kill human cancer cells and elicit anticancer immunity. Some versions are being specifically designed and tested in patients with osteosarcoma, though due to their generalized mechanism of action most are being tested in patients across a broad range of cancer types. The activity of these viruses is impacted not only by the susceptibility of tumor cells to infection but also by the tumor microenvironment (TME) and by tumor immunogenicity. Here we review the field of oncolytic viruses with a particular emphasis on highlighting any available data in preclinical osteosarcoma models or in patients with osteosarcoma. While in general the viruses have been shown safe to administer to patients by a variety of routes, their therapeutic efficacy to date has been limited. Given the low rate of adverse events and the likely absence of long-term side effects, the utility of oncolytic viruses will most likely be realized when used in combination with other agents.

Antitumor activity of pazopanib (P) and trametinib (T) in preclinical models of osteosarcoma (OS).

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

Oral administration of edelfosine encapsulated lipid nanoparticles causes regression of lung metastases in pre-clinical models of osteosarcoma

Osteosarcoma (OS) is the most frequent paediatric bone cancer, responsible for 9% of all cancer-related deaths in children. In this paper, a new strategy based on delivering edelfosine (ET) in lipid nanoparticles (LN) was explored in order to target the primary tumour and eliminate metastases. The in vitro and in vivo efficacy of the free drug, drug loaded into lipid nanoparticles (ET-LN) and doxorubicin (DOX) against osteosarcoma (OS) cells was analysed. ET and ET-LN decreased the growth of OS cells in vitro in a time- and dose-dependent manner. Interestingly, the uptake of ET and ET-LN was lower when OS cells were pre-treated with DOX. In vivo studies revealed that ET and ET-LN slowed down the primary tumour growth in two OS models. However, the combination of both drugs showed no additional anti-tumour effect. Importantly, ET-LN successfully prevented the metastatic spread of OS cells from the primary tumour to the lungs. On the whole, ET-LN are a promising candidate for OS chemotherapy.

Abstract PR13: Pivotal role of interleukin 23 in osteosarcoma development and its link with glutamate metabotropic 4: Identification of novel therapeutic targets for osteosarcoma

Abstract Objective: The 5-year survival of patients with metastatic or recurrent osteosarcoma is less than 25%. There is an urgent need to identify new treatment options for this cancer. Osteosarcomas represent a promising indication for strategies that target the immune system (1). Here we identify interleukin 23 (IL23), an inflammatory cytokine, as a potential therapeutic target in osteosarcoma. Our interest in IL23 is reinforced by the recent genome-wide association study, where a locus at rs1906953 at 6p21.3 in the glutamate metabotropic receptor 4 (Grm4) gene was identified as having the strongest association with genetic susceptibility to osteosarcoma in humans (2). GRM4 activation has been associated with autoimmune diseases through suppression of inflammatory cytokines, including IL23 (3). Whether GRM4 plays an immune modulatory role in cancer development is unknown. Methods: We screened a panel of mouse genotypes deficient in immune-related genes or cells to identify those genotypes that would predispose or protect from the development of osteosarcoma using a radiation model of cancer. IL23 expression was examined in mouse and human osteosarcomas using in situ hybridization. We investigated targeting IL23 alone and in combination with chemotherapy in a preclinical transplant model of osteosarcoma. The role of Grm4 and its association with IL23 was investigated using Grm4 knockout mice and Grm4 agonists. Results: IL23 knockout mice were strikingly protected from the development of osteosarcomas with 80% of animals disease free at 2 years of age compared to tumors in 100% of wild-type mice (P<0.0001). This suggests that IL23 is oncogenic in cancer development. In both mouse and human osteosarcomas infiltrating dendritic cells were found to express this cytokine, contributing to an inflammatory immune environment. Antagonist blocking IL23 could suppress tumor growth and in preliminary studies combining IL23 antagonists with doxorubicin further synergized to suppress tumor growth. GRM4 knockout mice were found to have significantly enhanced IL23 expression compared to wild-type mice. In vitro agonists to Grm4 were found to down-regulate IL23 and are being explored further in preclinical models of osteosarcoma. Conclusion: We find that blocking IL23 suppresses the growth of primary osteosarcomas. Blocking IL23 may have more profound roles in suppressing metastatic disease and is currently being studied. These findings are important, as Ustekinumab a neutralizing antibody to IL23, is FDA approved for the treatment of plaque psoriasis. This antibody targets IL-12 p40, which blocks both IL12 and IL23, but more specific monoclonal antibodies to human IL23p19 are in development for autoimmune diseases and could be repurposed for the treatment of cancer. In addition, agonists targeting GRM4 are in development and we will further investigate whether these have utility in osteosarcoma. References Kansara M et al. Translational biology of osteosarcoma. Nat Rev Cancer 2014;14:722-35; doi:10.1038/nrc3838. Jiang C et al. GRM4 gene polymorphism is associated with susceptibility and prognosis of osteosarcoma in a Chinese Han population. Med Oncol 2014;31:50. Fallarino F et al. Metabotropic glutamate receptor-4 modulates adaptive immunity and restrains neuroinflammation. Nat Med 2010;16:897-902; doi:10.1038/nm.2183. This abstract is also being presented as Poster B30. Citation Format: Maya Kansara, Puiyi Pang, Nina Sulkowski, Michele Teng, Mark Smyth, David Thomas. Pivotal role of interleukin 23 in osteosarcoma development and its link with glutamate metabotropic 4: Identification of novel therapeutic targets for osteosarcoma [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 PR13.

Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Osteosarcoma is the most frequently occurring bone cancer in children and adolescents. Unfortunately, treatment failures are common. Eribulin is a synthetic microtubule inhibitor that has demonstrated activity in preclinical osteosarcoma models. The effects of eribulin were evaluated in two human osteosarcoma cell lines as well as in eribulin-sensitive and -resistant osteosarcoma xenograft tumors of the Pediatric Preclinical Testing Program (PPTP) by characterizing cell viability, microtubule destabilization, mitotic arrest and mechanism of cell death. Eribulin demonstrated cytotoxic activity in vitro, through promotion of microtubule dynamic instability, arrest of cells in the G2/M phase, mitotic catastrophe and cell death. The microtubule-destabilizing protein stathmin-1 (STMN1) was coimmunoprecipitated with the cyclin-dependent kinase inhibitor p27 indicating that these cytoplasmic complexes can protect cells from the microtubule destabilizing effect of eribulin. Increased tumoral expression of P-glycoprotein (P-gp) and TUBB3 were also associated with lower drug sensitivity. In summary, eribulin successfully blocked cells in G2/M phase but interfered with mitochondria activity to inhibit proteins involved in apoptosis. Understanding the complex and inter-related mechanisms involved in the overall drug response to eribulin may help in the design of therapeutic strategies that enhance drug activity and improve benefits of eribulin in pediatric patients with osteosarcoma.

The anti-cancer activity of the mTORC1/2 dual inhibitor XL388 in preclinical osteosarcoma models.

In the present study, we investigated the activity of XL388, a novel mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor, in preclinical osteosarcoma (OS) models. XL388 was cytotoxic, cytostatic and pro-apoptotic to multiple established OS cell lines and primary human OS cells. XL388 blocked mTORC1/2 activation and downregulated cyclin D1/B1 expressions in OS cells, leaving AKT Thr-308 phosphorylation un-affected. Intriguingly, AKT1 T308A mutation potentiated XL388-induced cytotoxicity in OS cells. XL388 activated cytoprotective autophagy in OS cells. Autophagy inhibition, either pharmacologically or genetically, augmented XL388-induced anti-OS activity. Further, XL388 oral administration inhibited U2OS xenografts growth in severe combined immuno-deficient (SCID) mice. Such activity was enhanced with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). Similarly, Beclin-1-silenced U2OS xenografts were remarkably more sensitive to XL388. Thus, concurrent blockage of mTORC1/2 with XL388 may have therapeutic value for OS.

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Dual mTORC1/2 inhibition by INK-128 results in antitumor activity in preclinical models of osteosarcoma

Existing evidence has shown that mammalian target of rapamycin (mTOR) overactivation is an important contributor of osteosarcoma (OS) progression. Here, we studied the potential anti-OS activity of a potent mTOR kinase inhibitor: INK-128 (MLN0128). We demonstrated that INK-128 induced potent cytotoxic effects against several human OS cell lines (U2OS, MG-63 and SaOs-2), yet same INK-128 treatment was safe (non-cytotoxic) to OB-6 human osteoblastic cells and MLO-Y4 human osteocytic cells. INK-128 induced caspase-dependent apoptosis in OS cells, but not in MLO-Y4/OB-6 cells. The caspase-3 specific inhibitor (z-DVED-fmk) or the pan caspase inhibitor (z-VAD-fmk) dramatically attenuated INK-128-exerted cytotoxicity against OS cells. Molecularly, INK-128 inhibited activation of mTORC1 (S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 phosphorylation), without affecting AKT Thr-308 phosphorylation in U2OS cells. Significantly, AKT inhibition by MK-2206 (an AKT inhibitor), or AKT1/2 stable knockdown by targeted-shRNA, remarkably sensitized INK-128-induced activity in OS cells. In vivo, oral administration of INK-128 potently inhibited U2OS xenograft growth in severe combined immuno-deficient (SCID) mice. mTORC1/2 activation in xenograft tumors was also suppressed with INK-128 administration. In summary, we show that INK-128 exerts potent anti-OS activity in vitro and in vivo. INK-128 might be further investigated as a novel anti-OS agent.