Defining the mechanisms of B7-H3 overexpression and role in neuroblastoma metastasis and immune evasion

项目摘要 神经母细胞瘤是一种起源于发育中的交感神经系统的癌症，是最常见的 诊断为儿童颅外实体瘤。尽管有密集的化疗方案， 手术、放射治疗和免疫治疗，高危神经母细胞瘤患者的五年存活率仍然 只有50%。神经母细胞瘤的低肿瘤突变负担对靶向治疗的发展提出了挑战。 和免疫疗法，然而，通过用单抗靶向GD2已经取得了适度的成功 抗体治疗，神经母细胞瘤的免疫治疗策略前景看好 接近。到目前为止，GD2靶向治疗的成功一直受到阻碍，因为 靶上/肿瘤外的毒性，因为GD2也表达在疼痛纤维上，而抗原丢失是一种 治疗抵抗。因此，发现新的治疗靶点的需求尚未得到满足。 神经母细胞瘤。B7-H3由CD276基因编码，是B7家族中的一种1型跨膜蛋白。 免疫调节蛋白在许多成人和儿童癌症中高度表达，包括 神经母细胞瘤。除了与免疫抑制有关外，B7-H3还可能介导肿瘤的迁移 和转移。针对B7-H3的几种免疫治疗策略的临床前成功，包括 CAR-T细胞和抗体药物结合物，提示B7-H3是一种靶向肿瘤相关抗原，与 几项正在进行或计划进行的儿科临床试验。因此，了解肿瘤的致癌功能是至关重要的。 B7-H3及其表达如何被调节以预测治疗耐药的机制。我们的中央 假设B7-H3促进神经母细胞瘤转移和免疫逃避，其表达是 受肿瘤微环境衍生的细胞因子和神经母细胞瘤特异性转录因子的调节。我们的 初步数据显示，在神经母细胞瘤细胞系中使用CRISPRi敲除B7-H3基因可抑制细胞 扩散。此外，芯片测序数据确定了B7-H3处的MYC和MYCN结合区域 启动子提示MYC转录因子在调节B7-H3表达中的潜在作用。B7-H3 随着神经母细胞瘤细胞株上调B7-H3的表达，炎性细胞因子也可能调节其表达 肿瘤坏死因子-α或转化生长因子-β暴露后的表达。最后，重组人B7-H3抑制T细胞激活， 肿瘤坏死因子-α和干扰素-γ。鉴于T细胞，揭示B7-H3如何促进神经母细胞瘤的免疫逃避是至关重要的 以及正在进行临床测试的基于NK细胞的免疫疗法。我们认为B7-H3是一种多功能的 作为神经母细胞瘤治疗靶点的蛋白质。此NRSA F31将定义 神经母细胞瘤依赖B7-H3的转移和免疫逃避的机制，同时还定义了 过度表达的机制，为未来的B7-H3靶向治疗提供信息，并最终改善 高危神经母细胞瘤患者。

Project Summary Neuroblastoma is a cancer derived from the developing sympathetic nervous system and is the most commonly diagnosed extracranial solid tumor of childhood. Despite an intensive treatment regimen of chemotherapy, surgery, radiation, and immunotherapy, the five-year survival rate of high-risk neuroblastoma patients remains at only 50%. The low tumor mutational burden of neuroblastoma has challenged the development of targeted- and immuno-therapies, however moderate success has been achieved by targeting GD2 with monoclonal antibody therapy, credentialing immunotherapeutic treatment strategies for neuroblastoma as a promising approach. The success of GD2-targeted therapy to date has been hindered due to debilitating side effects from on-target/off-tumor toxicity since GD2 is also expressed on pain fibers, and antigen loss as a mechanism of therapy resistance. Therefore, there is an unmet need for the discovery of new therapeutic targets in neuroblastoma. B7-H3, encoded by the CD276 gene, is a type 1 transmembrane protein in the B7 family of immunoregulatory proteins and is highly expressed in many adult and pediatric cancers, including neuroblastoma. In addition to being implicated in immunoinhibition, B7-H3 may also mediate tumor migration and metastasis. Preclinical success of several immunotherapeutic strategies directed toward B7-H3, including CAR-T cells and antibody drug conjugates, suggest that B7-H3 is a targetable tumor-associated antigen with several pediatric clinical trials ongoing or planned. Therefore, it is critical to understand the oncogenic functions of B7-H3 and how its expression is regulated to anticipate mechanisms of therapy resistance. Our central hypothesis is that B7-H3 promotes neuroblastoma metastasis and immune evasion, and its expression is regulated by tumor microenvironment-derived cytokines and neuroblastoma-specific transcription factors. Our preliminary data shows that B7-H3 knockdown using CRISPRi in neuroblastoma cell lines inhibits cellular proliferation. Additionally, ChIP-sequencing data identifies regions of MYC and MYCN binding at the B7-H3 promoter indicating a potential role of the MYC transcription factors in regulating B7-H3 expression. B7-H3 expression may also be regulated by inflammatory cytokines, as neuroblastoma cell lines upregulate B7-H3 expression following TNF-α or TGF-β exposure. Finally, recombinant human B7-H3 inhibits T cell activation, TNF-α, and IFN-y. Uncovering how B7-H3 promotes immune evasion in neuroblastoma is crucial given the T and NK cell-based immunotherapies undergoing clinical testing. We propose that B7-H3 is a multifunctional protein that serves as a promising therapeutic target in neuroblastoma. This NRSA F31 will define the mechanisms of neuroblastoma dependance on B7-H3 for metastasis and immune evasion, while also defining mechanisms of overexpression, to inform future B7-H3-targeting therapies and ultimately improve outcomes for patients with high-risk neuroblastoma.