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.

中文翻译：

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Tegavivint和β-Catenin/ ALDH轴在抗化疗和转移性骨肉瘤中的作用。

背景技术Wnt /β-catenin途径与骨肉瘤（OS）的发展和转移进程密切相关。我们使用体外，离体和体内细胞系以及概括了患者来源的异种移植（PDX）模型，研究了Tegavivint（一种新型的β-catenin/ transducinβ样蛋白1（TBL1）抑制剂）对OS的抗肿瘤活性。高危疾病。方法使用已建立的OS和PDX衍生的细胞系，体外评估Tegavivint的抗肿瘤功效。使用体外三维肺转移测定评估了微转移和大转移发展过程中β-catenin活性的靶向性。使用化学抗性和转移性OS PDX模型评估Tegavivint的体内活性。通过定量逆转录聚合酶链反应或免疫印迹分析定量基因和蛋白质表达。通过microCT确定骨完整性。所有统计检验都是两面的。结果Tegavivint在体外表现出对OS细胞的抗增殖活性，并在体外显着降低了微转移和大转移的发生。Tegavivint在体内可抑制多种OS PDX肿瘤（n = 3），包括成对的原发性和肺转移性肿瘤以及固有的化学耐药性。我们发现转移性肺OS细胞系（n = 2）表现出增加的干细胞标志，包括增强的醛脱氢酶（ALDH1）和β-catenin表达以及下游活性，这些被Tegavivint（ALDH1：对照组，平均相对mRNA抑制）抑制表达式= 1.00，与Tegavivint组相比，95％置信区间[CI] = 0.68至1.22，平均值= 0.011，95％CI = 0.0012至0.056，P <.001；β-catenin：对照组，平均相对mRNA表达= 1.00,95％CI = 0.71至1.36，而Tegavivint组，平均值= 0.45,95％CI = 0.36至0.52，P <.001）。与体内ALDHlow细胞相比，ALDH1高PDX衍生的肺OS细胞具有更高的转移潜力，对Tegavivint敏感。毒性研究表明，雄性经Tegavivint治疗的小鼠（每组n = 4只小鼠）的骨密度降低。结论Tegavivint通过靶向β-catenin/ ALDH1轴，是用于OS晚期的有前途的治疗剂。与Tegavivint组相比，36均值= 0.45，95％CI = 0.36至0.52，P <.001）。与体内ALDHlow细胞相比，ALDH1高PDX衍生的肺OS细胞具有更高的转移潜力，对Tegavivint敏感。毒性研究表明，雄性经Tegavivint治疗的小鼠（每组n = 4只小鼠）的骨密度降低。结论Tegavivint通过靶向β-catenin/ ALDH1轴，是用于OS晚期的有前途的治疗剂。与Tegavivint组相比，36均值= 0.45，95％CI = 0.36至0.52，P <.001）。与体内ALDHlow细胞相比，ALDH1高PDX衍生的肺OS细胞具有更高的转移潜力，对Tegavivint敏感。毒性研究表明，雄性经Tegavivint治疗的小鼠（每组n = 4只小鼠）的骨密度降低。结论Tegavivint通过靶向β-catenin/ ALDH1轴，是用于OS晚期的有前途的治疗剂。