Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor that has a high propensity for metastases. Through osteoblast-specific alteration of p53 status, we developed a genetically ...engineered mouse model of localized and metastatic OS to gain an understanding into the molecular pathogenesis of OS. Microarray analysis of both localized tumors and metastatic tumors identified the downregulation of the naked cuticle homolog 2 (NKD2) gene, a negative regulator of Wnt signaling. Overexpression of NKD2 in metastatic human and mouse OS cells significantly decreases cell proliferation, migration and invasion ability in vitro and drastically diminishes OS tumor growth and metastasis in vivo, whereas downregulation enhances migratory and invasive potential. Evaluation of NKD2-overexpressing tumors revealed upregulation of tumor-suppressor genes and downregulation of molecules involved in blood vessel formation and cell migration. Furthermore, assessment of primary human OS revealed downregulation of NKD2 in metastatic and recurrent OS. Finally, we provide biological evidence that use of small-molecule inhibitors targeting the Wnt pathway can have therapeutic efficacy in decreasing metastatic properties in OS. Our studies provide compelling evidence that downregulation of NKD2 expression and alterations in associated regulated pathways have a significant role in driving OS tumor growth and metastasis.
The prognosis for metastatic sarcoma in children remains bleak. Cellular immunotherapy with chimeric antigen receptor T cells (CAR-T) can be a suitable approach for these hard-to-treat cancers. ...However, a paucity of relevant tumor-associated antigens exists in sarcoma, limiting their therapeutic potential. We describe the creation of a novel CAR-T against the surface protein endoglin (ENG, CD105), a TGF-β co-receptor, expresed on sarcomas. We identified ENG as a significant target of endogenous immune reactivity in an exceptional responder with metastatic sarcoma, after co-administration of HER2 CAR-T and lymphodepletion chemotherapy in our clinical study NCT00902044. We aim to target the broad expression of ENG on cancer cells, neovascular endothelial cells and cancer-associated fibroblasts in sarcomas using endoglin-directed CAR-T (ENG CAR-T).
To target ENG, we engineered iterations of second-generation CAR molecules combining the anti-ENG mAb c-SN6j, the intracellular signaling domain (ICD) derived from the CD28-, 41BB- or OX40-molecules and the CD3-ζ activating domain. Retroviral transduction was used to express the ENG CAR molecules on primary human T cells. The functional assessments for the ENG CAR-T included cytokine release, T-cell proliferation, impedance-based cytotoxicity, 3D spheroid disruption and anti-tumor efficacy in murine orthotopic xenograft models of sarcoma.
Co-culture of ENG CAR-T with sarcoma cell lines revealed the best outcomes with CD28-ICD-mediated signaling, displaying enhanced release of IFN-γ and IL-2, increased proliferation and robust cytotoxic responses in vitro. ENG CAR-T efficiently disrupted the formation of 3D spheroids in sarcoma lines, both independently and when co-cultured with fibroblast lines. In intratibial orthotopic models of osteosarcoma and Ewing sarcoma, as well as an intramuscular model of rhabdomyosarcoma, ENG-CART exhibited potent anti-tumor activity, extending survival rates in mice and delaying metastasis. In conclusion, the results suggest that the novel ENG CAR-T demonstrates anti-tumor activity in experimental models of advanced sarcomas
Ewing sarcoma (ES) is the second most common bone tumor in children and young adults. Unfortunately, there have been minimal recent advancements in improving patient outcomes, especially in ...metastatic and recurrent diseases. In this study, we investigated the biological role of p21-activated kinases (PAKs) in ES, and the ability to therapeutically target them in high-risk disease. Via informatics analysis, we established the inverse association of PAK1 and PAK4 expression with clinical stage and outcome in ES patients. Through expression knockdown and small-molecule inhibition of PAKs, utilizing FRAX-597, KPT-9274, and PF-3758309 in multiple ES cell lines and patient-derived xenograft models, we further explored the role of PAKs in ES tumor growth and metastatic capabilities. In vitro studies in several ES cell lines indicated that diminishing PAK1 and PAK4 expression reduces tumor cell viability, migratory, and invasive properties. In vivo studies using PAK4 inhibitors, KPT-9274 and PF-3758309 demonstrated significant inhibition of primary and metastatic tumor formation, while transcriptomic analysis of PAK4-inhibitor-treated tumors identified concomitant suppression of Notch, β-catenin, and hypoxia-mediated signatures. In addition, the analysis showed enrichment of anti-tumor immune regulatory mechanisms, including interferon (IFN)-ɣ and IFN-α responses. Altogether, our molecular and pre-clinical studies are the first to establish a critical role for PAKs in ES development and progression, and consequently as viable therapeutic targets for the treatment of high-risk ES in the near future.
Abstract
Background
The Wnt/β-catenin pathway is closely associated with osteosarcoma (OS) development and metastatic progression. We investigated the antitumor activity of Tegavivint, a novel ...β-catenin/transducin β-like protein 1 (TBL1) inhibitor, against OS employing in vitro, ex vivo, and in vivo cell line and patient-derived xenograft (PDX) models that recapitulate high risk disease.
Methods
The antitumor efficacy of Tegavivint was evaluated in vitro using established OS and PDX-derived cell lines. Use of an ex vivo three-dimensional pulmonary metastasis assay assessed targeting of β-catenin activity during micro- and macrometastatic development. The in vivo activity of Tegavivint was evaluated using chemoresistant and metastatic OS PDX models. Gene and protein expression were quantified by quantitative Reverse transcription polymerase chain reaction or immunoblot analysis. Bone integrity was determined via microCT. All statistical tests were two-sided.
Results
Tegavivint exhibited antiproliferative activity against OS cells in vitro and actively reduced micro- and macrometastatic development ex vivo. Multiple OS PDX tumors (n = 3), including paired patient primary and lung metastatic tumors with inherent chemoresistance, were suppressed by Tegavivint in vivo. We identified that metastatic lung OS cell lines (n = 2) exhibited increased stem cell signatures, including enhanced concomitant aldehyde dehydrogenase (ALDH1) and β-catenin expression and downstream activity, which were suppressed by Tegavivint (ALDH1: control group, mean relative mRNA expression = 1.00, 95% confidence interval CI = 0.68 to 1.22 vs Tegavivint group, mean = 0.011, 95% CI = 0.0012 to 0.056, P < .001; β-catenin: control group, mean relative mRNA expression = 1.00, 95% CI = 0.71 to 1.36 vs Tegavivint group, mean = 0.45, 95% CI = 0.36 to 0.52, P < .001). ALDH1high PDX-derived lung OS cells, which demonstrated enhanced metastatic potential compared with ALDHlow cells in vivo, were sensitive to Tegavivint. Toxicity studies revealed decreased bone density in male Tegavivint-treated mice (n = 4 mice per group).
Conclusions
Tegavivint is a promising therapeutic agent for advanced stages of OS via its targeting of the β-catenin/ALDH1 axis.
Rhabdomyosarcoma (RMS) is the most prevalent pediatric soft-tissue sarcoma. Multimodal treatment, including surgery and traditional chemotherapy with radiotherapy, has contributed to improvements in ...overall survival rates. However, patients with recurrent or metastatic disease have 5-year survival rates of less than 30%. One reason for the lack of therapeutic advancement is identification and targeting of critical signaling nodes. p21-activated kinases (PAK) are a family of serine/threonine kinases downstream of multiple critical tumorigenic receptor tyrosine kinase receptors and oncogenic regulators, including IGFR and RAS signaling, that significantly contribute to aggressive malignant phenotypes. Here, we report that RMS cell lines and tumors exhibit enhanced PAK4 expression levels and activity, which are further activated by growth factors involved in RMS development. Molecular perturbation of PAK4 in multiple RMS models
and
resulted in inhibition of RMS development and progression. Fusion-positive and -negative RMS models were sensitive to two PAK4 small-molecule inhibitors, PF-3758309 and KPT-9274, which elicited significant antitumor and antimetastatic potential in several primary and metastatic
models, including a relapsed RMS patient-derived xenograft model. Transcriptomic analysis of PAK4-targeted tumors revealed inhibition of the RAS-GTPase, Hedgehog, and Notch pathways, along with evidence of activation of antitumor immune response signatures. This PAK4-targeting gene signature showed prognostic significance for patients with sarcoma. Overall, our results show for the first time that PAK4 is a novel and viable therapeutic target for the treatment of high-risk RMS. SIGNIFICANCE: These data demonstrate a novel oncogenic role for PAK4 in rhabdomyosarcoma and show that targeting PAK4 activity is a promising viable therapeutic option for advanced rhabdomyosarcoma.
Osteosarcoma (OS) is the primary bone tumor in children and young adults. Currently, there are no reliable, noninvasive biologic markers to detect the presence or progression of disease, assess ...therapy response, or provide upfront prognostic insights. MicroRNAs (miRNAs) are evolutionarily conserved, stable, small noncoding RNA molecules that are key posttranscriptional regulators and are ideal candidates for circulating biomarker development due to their stability in plasma, ease of isolation, and the unique expressions associated with specific disease states. Using a qPCR‐based platform that analyzes more than 750 miRNAs, we analyzed control and diseased‐associated plasma from a genetically engineered mouse model of OS to identify a profile of four plasma miRNAs. Subsequent analysis of 40 human patient samples corroborated these results. We also identified disease‐specific endogenous reference plasma miRNAs for mouse and human studies. Specifically, we observed plasma miR‐205‐5p was decreased 2.68‐fold in mice with OS compared to control mice, whereas, miR‐214, and miR‐335‐5p were increased 2.37‐ and 2.69‐fold, respectively. In human samples, the same profile was seen with miR‐205‐5p decreased 1.75‐fold in patients with OS, whereas miR‐574‐3p, miR‐214, and miR‐335‐5p were increased 3.16‐, 8.31‐ and 2.52‐fold, respectively, compared to healthy controls. Furthermore, low plasma levels of miR‐214 in metastatic patients at time of diagnosis conveyed a significantly better overall survival. This is the first study to identify plasma miRNAs that could be used to prospectively identify disease, potentially monitor therapeutic efficacy and have prognostic implications for OS patients.
We use a genetically engineered mouse model of osteosarcoma (OS) to perform cross‐species identification and validation of plasma microRNAs that can be utilized as novel biomarkers for detection and responsiveness to therapy. Our study is also the first to identify a plasma microRNA with prognostic significance in OS. Therefore, these studies have identified novel noninvasive biomarkers for OS that have the potential to impact the care and management of patients with this highly aggressive disease.
Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while ...performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using EuII-based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and EuII. Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated EuII/III-containing probes. We studied three different EuII/III couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time (T1) and 19F signal of solutions containing different ratios of EuII- and EuIII-containing complexes was plotted against the percentage of EuII-containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.
Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while ...performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using Eu
-based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and Eu
. Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated Eu
-containing probes. We studied three different Eu
couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time (
) and
F signal of solutions containing different ratios of Eu
- and Eu
-containing complexes was plotted against the percentage of Eu
-containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.
Abstract
Osteosarcoma (OS) is the most common bone cancer in children and adolescents, and patients with metastatic disease still have extremely poor prognosis and no effective targeted therapy. It ...has been reported that the activation of Wnt/β-catenin pathway is closely associated with OS development and metastatic progression. Tegavivint (BC2059), a novel small molecule inhibitor of the Wnt/β-catenin pathway, has been reported to be active against multiple types of cancer cells in vitro, and anti-tumor efficacy has been published in animal models of acute myeloid leukemia and multiple myeloma. In this study, we investigated the antitumor activity of Tegavivint against metastatic OS both in vitro and in vivo using established human OS cell lines (143B, SaOS-2, LM7) and patient-derived xenograft (PDX) models. In vitro, Tegavivint effectively inhibited tumor cell survival in a dose-dependent manner in all established OS cell lines and OS PDX-derived cells. Subsequent in vivo studies using an orthotopic model of LM7 cells engrafted in the tibia of NSG mice demonstrated complete regression of the primary tumors in all treated mice as well as a significant reduction in lung metastasis by the treatment with Tegavivint. We further examined the activity of Tegavivint using a pair of PDX models derived from an inherently chemo-resistant OS patient at different disease stages: PDX63 was derived from the pre-treatment biopsy of the primary tumor, and PDX84 was derived from the relapsed metastatic lung lesion of the same patient after chemotherapy. The growth of subcutaneously engrafted PDX63 tumors was significantly suppressed by the treatment with Tegavivint alone and Tegavivint enhanced the antitumor activity of doxorubicin. Analysis of mRNA expression by qPCR in the tumor tissue demonstrated significant downregulation of critical genes by Tegavivint, including c-Myc and ALDH1, which is a potential marker for cancer stem cells. Furthermore, in an innovative lung metastasis model using PDX84 tumor-dissociated cells intravenously (IV) injected in NSG mice, we demonstrated that Tegavivint treatment markedly reduced the number of lung metastases. Finally, PDX84-derived cells were sorted into ALDH1-high and ALDH1-low populations. ALDH1-high cells showed higher expression of β-catenin and higher sensitivity to Tegavivint than the ALDH1-low cells in vitro and subsequent IV injection of ALDH1-high PDX84 or ALDH1-low PDX84 cells demonstrated significantly more lung lesions in the ALDH1-high group than the ALDH1-low group. The number of ALDH1-high derived lung metastases was significantly suppressed by Tegavivint. Taken together, our preclinical findings demonstrate that Tegavivint has promising therapeutic potential for primary and metastatic OS through the blockade of Wnt signaling /ALDH1 axis.
Citation Format: Motonari Nomura, Nino C. Rainusso, Ruolan Han, Jeff Larson, Ryan L. Shuck, Lyazat Kurenbekova, Jason T. Yustein. Tegavivint suppresses progression and metastasis of osteosarcoma via blockade of Wnt signaling/ALDH1 axis: Preclinical study of a novel Wnt/β-catenin pathway inhibitor 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 3186.
Abstract
Osteosarcoma (OS) is the most common bone tumor in pediatric patients, particularly in adolescents and young adults. Currently, no effective molecular targeted therapies are available for ...the OS, however, many OS patients possess genetically defined somatic DNA copy number alterations, including amplification of chromosome 8q24, which harbors the oncogene Myc, and correlates with a worse prognosis. To better understand the molecular pathogenesis, and identify targeted therapy for OS, a robust and reliable preclinical model is needed both for the primary and metastatic disease. We have developed a novel conditional, osteoblast-specific Myc knock-in murine model to understand the mechanisms that underlie the ability of Myc to drive the development and progression of OS and utilize it for the identification of Myc-dependent intrinsic and extrinsic molecular vulnerabilities. A tissue-specific Myc-in genetically engineered murine model was generated by crossing Col2.3-Cre; TP53Flox/+ mice with LSL-MycT58A mice and characterized integrating RNA-seq and Mass Spectrometry. Syngeneic murine OS cell lines were also generated and characterized. The molecular characterization of the GEMM model was compared with high and low Myc expressing human tumors using the OS-TARGET dataset. Differential gene expression in Myc-driven and non-driven-tumor tissue samples were quantified by RNA sequencing. The immune landscape of OS was evaluated using SymphonyFACS and IHC staining. Murine OS tumor type was confirmed by H&E staining. OS tumor was highly aggressive and metastatic in more than 60% of the mice. Myc expression was significantly higher both at the transcriptional and protein levels in the Myc-in tissue/cells compared to the non-Myc-driven model. Expression of 1479 genes (out of 13781 genes) were significantly altered in which 543 were upregulated and 936 were downregulated at transcriptional levels in Myc-in tumor tissue samples as compared to the low-Myc-driven OS tumor model analyzed by RNA sequencing. Myc-target-genes, splicing factor-related genes, E2F, G2M check-point, and DNA repair associated genes were significantly upregulated whereas the immune-related genes, oxidative phosphorylation, adipogenesis, and E2M transition genes were significantly downregulated when compared between two groups, which correlates to differential gene ontologies noted in high Myc human tumors. The total immune cell population, specifically the myeloid and macrophage populations were significantly diminished in the Myc-in tumor models, which could contribute to the poor prognosis in OS.Our murine model closely resembles human OS and will provide new opportunities for dissecting the molecular pathogenesis, identification of novel therapeutic strategies, and pre-clinical modeling for direct translational applications and targeting of Myc-driven OS.
Citation Format: Bikesh K. Nirala, Lyazat Kurenbekova, Ryan L. Shuck, Tajhal Patel, Kimal Rajapakshe, Jason T. Yustein. Development and characterization of a c-Myc-driven preclinical mouse model of osteosarcoma to investigate the tumor immune microenvironment abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1668.
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