The role of CEACAM1 in oncogenic B-cell receptor signaling and immunotherapy in mantle cell lymphoma

摘要 套细胞淋巴瘤（MCL）占所有非霍奇金淋巴瘤（NHL）的6-8%。尽管金额不小 尽管其他NHL的治疗已经取得了进展，但MCL仍然是一种无法治愈的淋巴瘤， 这是未知的。MCL患者预后差，中位总生存期为3-5年。 标准治疗包括伊曲替尼，一种B细胞受体（BCR）近端酪氨酸的小分子抑制剂， 激酶BTK。然而，三分之一的MCL患者对药物没有反应。即使是最初的伊布替尼- 敏感患者总是会产生耐药性;然而，现在人们已经了解了伊布替尼耐药性的机制 清楚由于对DLBCL和CLL中致癌BCR信号传导的机制性洞察使得DLBCL和CLL的发展成为可能， 高效的治疗方法，这一建议将解决致癌BCR的机制， MCL中的信号。在结合全基因组CRISPR-Cas9文库的整合功能分析中， 在表达谱和BCR信号转导研究中，我们发现CEACAM 1是一个重要的 在MCL中是必需的，但在正常B细胞或其它B细胞中不是必需的致癌BCR信号传导组分 恶性肿瘤。CEACAM 1作为一种跨膜蛋白，在活化的淋巴细胞表面表达 并在其胞质尾区携带两个基于免疫受体酪氨酸的抑制基序（ITIM）。由于 通过将抑制性磷酸酶SHP 1募集到ITIM，CEACAM 1作为T细胞增殖的调节因子发挥作用。 尽管TCR在T细胞中的功能是TCR受体（TCR）信号传导，但是其在正常B细胞和MCL中的功能尚不清楚。 出乎意料的是，我们的初步数据显示CEACAM 1功能诱导BCR信号转导的净增加， 导致体外和体内MCL细胞的存活和增殖增加。我们的机械研究 表明CEACAM 1将肌动蛋白结合蛋白细丝蛋白A募集到质膜上 抗原结合后，BCR-近端激酶林恩被激活。此外，超- 共聚焦显微镜显示CEACAM 1促进肌动蛋白细胞骨架的重组， BCR交联后的网络。利用City of Hope的临床级细胞治疗设施， 我们设计并验证了一种新的CEACAM 1嵌合抗原受体（CAR）， 人类T细胞CAR-T细胞在消除CEACAM 1 + MCL方面具有高度活性，但缺乏针对CEACAM 1 + MCL的反应性。 其他细胞类型。基于我们发现CEACAM 1是MCL中关键的BCR信号组分， CEACAM 1 CAR-T细胞的成功开发，我们假设CEACAM 1作为一种 致癌BCR活性的中心驱动因子，代表MCL的新治疗靶点。以下 具体目标将测试和完善基于CEACAM 1的MCL治疗的概念：目标1）定义 CEACAM 1在MCL中致癌BCR信号传导中的机制作用;目的2）剖析CEACAM 1在MCL中的作用， MCL的新遗传小鼠模型;目的3）在MCL中验证靶向CEACAM 1的CAR T细胞策略 难治性MCL这些研究的结果有望提供：1）关于中央 CEACAM 1在致癌BCR信号传导和MCL中的作用，以及2）新的CEACAM 1的临床前验证。 难治性MCL中靶向CEACAM 1的免疫策略。

Abstract Mantle cell lymphoma (MCL) accounts for 6-8% of all non-Hodgkin lymphomas (NHLs). While substantial therapeutic advances have been achieved for other NHLs, MCL remains an incurable lymphoma, the reason of which is not known. MCL patients have a dismal prognosis with a median overall survival of 3-5 years. Standard of care includes ibrutinib, a small molecule inhibitor of the B-cell receptor (BCR)-proximal tyrosine kinase BTK. However, one-third of MCL patients do not respond to the drug. Even initially ibrutinib- sensitive patients invariably develop resistance; however, the mechanisms of ibrutinib-resistance are now clear. Since mechanistic insight into oncogenic BCR signaling in DLBCL and CLL enabled the development of highly effective treatment approaches, this proposal will address the mechanisms of oncogenic BCR- signaling in MCL. In an integrated functional analysis combining a genome-wide CRISPR-Cas9 library, gene expression profiling and BCR signal transduction studies, we have uncovered CEACAM1 as a central component of oncogenic BCR signaling that is essential in MCL but not in normal B cells or other B-cell malignancies. As a transmembrane protein, CEACAM1 is expressed on the surface of activated lymphocytes and carries two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) on its cytoplasmic tail. Owing to recruitment of the inhibitory phosphatase SHP1 to the ITIMs, CEACAM1 functions as a regulator of T-cell receptor (TCR) signaling in T cells, however, its function in normal B cells and MCL is not known. Unexpectedly, our preliminary data showed that CEACAM1 function induced a net increase of BCR signaling, leading to increased survival and proliferation of MCL cells in vitro and in vivo. Our mechanistic studies revealed that CEACAM1 recruited the actin-binding protein filamin A to the plasma membrane microdomains and activated the BCR-proximal kinase LYN after antigen engagement. Furthermore, super- resolution confocal microscopy revealed that CEACAM1 promoted reorganization of the actin cytoskeletal network following BCR cross-linking. Leveraging the clinical grade Cell Therapeutics Facility at City of Hope, we designed and validated a novel CEACAM1 chimeric antigen receptor (CAR) engineered in primary human T cells. The CAR-T cells were highly active in eliminating CEACAM1+ MCL but lacked reactivity against other cell types. Based on our discovery of CEACAM1 as a critical BCR signaling component in MCL and the successful development of CEACAM1 CAR-T cells, we hypothesize that CEACAM1 functions as a central driver of oncogenic BCR activity and represents a novel therapeutic target in MCL. The following specific aims will test and refine the concept of CEACAM1-based therapies for MCL: Aim 1) Define the mechanistic role of CEACAM1 in oncogenic BCR signaling in MCL; Aim 2) Dissect the role of CEACAM1 in new genetic mouse models for MCL; Aim 3) Validation of CAR T-cell strategies targeting CEACAM1 in refractory MCL. Results from the proposed studies are expected to provide: 1) new information on the central role of CEACAM1 in oncogenic BCR signaling and MCL, and 2) pre-clinical validation of novel immunotherapeutic strategies targeting CEACAM1 in refractory MCL.