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

尤文氏肉瘤是一种与特征性染色体易位相关的骨和软组织侵袭性癌症。为了提高治愈率，需要新的治疗方法来根除能够重新启动肿瘤生长的微转移细胞。在之前的工作中，我们发现神经节苷脂GD2，一种间充质间质细胞标记物，在尤文氏肉瘤细胞表面以不同密度过表达。表面GD2可以通过GD2重定向免疫效应细胞特异性清除尤文氏肉瘤细胞，从而为靶向治疗开辟了新的途径。GD2hi和GD2low Ewing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。我们进一步获得了证据，表明GD2在尤文氏肉瘤细胞中的表达受到干扰表观遗传基因调控的物质的操纵。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，GD2在Ewing肉瘤中的表达受表观遗传调控，我们将在体外和体内系统地评估HDAC抑制剂和去甲基化剂对Ewing肉瘤中GD2和GD3S表达的影响。此外，我们将研究GD2表达在尤文氏肉瘤中的生物学作用和相关性。GD2low和GD2hi亚群将通过细胞分选从活检新启动的肿瘤细胞培养中分离出来。体外研究将涉及与遗传干细胞重编程相关的标记物和转录因子的表达，在不依赖锚定条件下的菌落的产生，以及通过从菌落中连续复制单细胞来进行自我更新。将通过将限制每个亚群细胞剂量的异种移植到免疫缺陷小鼠中来比较致瘤性。疾病传播的模式和程度将在系统性疾病异种移植模型中进行比较。为了确定GD2表达对肉瘤细胞功能的直接影响，在抑制GD2合成限速酶GD3S后，将重复进行关键实验。临床相关性将旨在建立GD2hi表达作为不同尤文氏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Ewing肉瘤具有协同作用的假设，我们将在全身性疾病模型中评估联合策略的抗肿瘤活性。总之，这些实验将探讨GD2表达在间质恶性肿瘤中的功能意义，并为尤因肉瘤和其他表达GD2的恶性肿瘤的细胞和表观遗传联合治疗的临床翻译奠定临床前前提。

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.