Ganglioside GD2 as a target for novel combined therapies with chimeric antigen receptor-modified T cells in Ewing sarcomas

尤文肉瘤是一种侵袭性的骨和软组织肿瘤，与特征性染色体易位有关。为了提高治愈率，需要根除能够重新启动肿瘤生长的微转移细胞的新疗法。在以前的工作中，我们发现，神经节苷脂GD2，间充质基质细胞标记物，在尤文肉瘤细胞的表面上以不同的密度过表达。表面GD2允许通过GD2重定向的免疫效应细胞特异性地消除尤文肉瘤细胞，从而为治疗靶向开辟了新途径。GD2hi和GD2low尤文肉瘤亚群的功能比较显示，GD2在这种癌症中的表达是动态的，并与特征性细胞表型相关。我们进一步获得的证据表明，GD2在尤文肉瘤细胞中的表达受到操纵的代理人干扰表观遗传基因调控。在这里，我们探索了GD2作为这种癌症创新疗法的特定靶点的潜力。为了证实我们的发现，即GD2在尤文肉瘤中的表达受到表观遗传调控，我们将系统地评估HDAC抑制剂和去甲基化剂对体外和体内尤文肉瘤培养物中GD2和GD3S表达的影响。此外，我们将研究GD2表达在尤文肉瘤中的生物学作用和相关性。将通过细胞分选从活组织检查新诱导的肿瘤细胞培养物中分离GD2low和GD2hi亚群。体外研究将解决与遗传干细胞重编程相关的标志物和转录因子的表达，在锚定非依赖性条件下产生集落，以及通过从集落中连续重新接种单细胞进行自我更新。将通过将每个亚群的限制细胞剂量异种移植到免疫缺陷小鼠中来比较致瘤性。将在系统性疾病异种移植模型中比较疾病传播的模式和程度。为了确定GD2表达对肉瘤细胞功能的直接影响，将在抑制GD2合成的限速酶GD3S后重复关键实验。临床相关性旨在建立GD2hi表达作为不同尤文肉瘤亚型的生物标志物。为了探索表观遗传调节剂和GD2靶向策略对尤文肉瘤具有协同活性的假设，将在我们的全身性疾病模型中评估组合策略的抗肿瘤活性。总之，这些实验将探索GD2表达在间叶恶性肿瘤中的功能意义，并建立尤文肉瘤和其他表达GD2的恶性肿瘤中细胞和表观遗传联合治疗的临床转化的临床前先决条件。

Ewing sarcomas are aggressive cancers of bone and soft tissues associated with a characteristic chromosomal translocation. To increase cure rates, novel therapies are needed that eradicate micrometastatic cells capable to reinitiate tumor growth. In previous work, we found that the ganglioside GD2, a mesenchymal stroma cell marker, is overexpressed at variable densities on the surface of Ewing sarcoma cells. Surface GD2 allowed to specifically eliminate Ewing sarcoma cells by GD2-redirected immune effector cells and thus opens a new avenue for therapeutic targeting. Functional comparisons of GD2hi and GD2low Ewing sarcoma subpopulations revealed that GD2 expression in this cancer is dynamic and associated with a characteristic cellular phenotype. We further obtained evidence that GD2 expression in Ewing sarcoma cells is subject to manipulation by agents interfering with epigenetic gene regulation. Here, we explore the potential of GD2 to serve as a specific target for innovative therapies in this cancer. To substantiate our finding that GD2 expression in Ewing sarcoma is subject to epigenetic regulation, we will systematically assess the effect of HDAC inhibitors and demethylating agents on expression of GD2 and GD3S in Ewing sarcoma cultures in vitro and in vivo. Moreover, we will investigate the biological role and relevance of GD2 expression in Ewing sarcoma. GD2low and GD2hi subpopulations will be isolated by cell sorting from tumor cell cultures newly initated from biopsies. In vitro studies will address expression of markers and transcription factors associated with genetic stem cell reprogramming, generation of colonies under anchorage-independent conditions, and self-renewal by sequential replating of single cells from colonies. Tumorigenicity will be compared by xenografting limiting cell doses of each subpopulation into immunodeficient mice. The pattern and extent of disease dissemination will be compared in a systemic disease xenograft model. To establish the direct effects of GD2 expression on sarcoma cell functionality, key experiments will be repeated following inhibition of the rate-limiting enzyme of GD2 synthesis, GD3S. Clinical correlations will aim to establish GD2hi expression as a biomarker of distinct Ewing sarcoma subtypes. To explore the hypothesis that epigenetic modulators and GD2 targeting strategies have synergistic activity against Ewing sarcomas, the antitumor activity of the combination strategy will be assessed in our systemic disease model. Together, these experiments will explore the functional significance of GD2 expression in a mesenchymal malignancy and establish the preclinical prerequisites for clinical translation of combined cellular and epigenetic therapy in Ewing sarcoma and other GD2-expressing malignancies.