Characterization of CEACAM1 as a novel therapeutic 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%，是一种侵袭性的淋巴瘤， 难治的B细胞恶性肿瘤MCL患者的预后很差，中位总生存期仅为 3-5由于频繁复发和对当代化疗的抵抗，尽管最近的治疗 FDA批准的靶向B细胞受体（BCR）信号分子的药物伊曲替尼的进展 布鲁顿的酪氨酸激酶（BTK），三分之一的MCL患者对伊替尼耐药，甚至对伊替尼敏感 病人最终会对药物产生抗药性。在伊布替尼治疗期间不敏感或进展的患者 治疗效果很差。因此，迫切需要更有效的伊曲替尼后治疗。 异常BCR信号在MCL中起着关键的致癌信号，但其潜在机制尚不清楚。 使用整合功能基因组学方法，结合全基因组CRISPR-Cas9筛选和基因 表达谱，我们的小组已经发现CEACAM 1（癌胚抗原相关细胞粘附 分子-1）作为MCL中BCR活性亢进的潜在贡献者。CEACAM 1是1型跨膜蛋白 在上皮细胞和活化的白细胞中由于选择性剪接而表达为不同的同种型。相比之下 与短同种型相比，CEACAM 1长同种型具有含有免疫受体的胞质尾区 基于酪氨酸的抑制基序（ITIM）已知负调节T细胞中的抗原受体信号传导。我们 发现与幼稚B细胞或其他B细胞相比，CEACAM 1在MCL中特异性过表达 恶性肿瘤。与我们的CRISPR筛选结果一致，CEACAM 1的消耗在多个MCL细胞中是有毒的。 细胞，但不是在非MCL淋巴瘤细胞。CEACAM 1缺失也显著降低了近端BCR信号传导 (e.g., SYK磷酸化）和BCR介导的Ca 2+信号传导。更重要的是，使用T细胞表达 CEACAM 1特异性嵌合抗原受体（CAR），我们表明这些CAR T细胞可以有效地 在体外消除MCL肿瘤，同时保留非MCL淋巴瘤。基于这些发现，我们假设 CEACAM 1是MCL中致癌BCR信号传导所必需的，因此是有效的治疗靶点。 恶性肿瘤我们将通过实现以下具体目标来测试假设：1）确定 CEACAM 1调节的BCR信号传导的潜在机制; 2）表征 CEACAM 1; 3）开发CAR T细胞策略以使用临床前患者来源的异种移植物在体内治疗MCL (PDX)模型这项研究的结果将改善我们对CEACAM 1在MCL中作用的有限认识 发病机制我们预计，该项目的成功完成将为以下方面提供相关的临床前数据： 未来的研究，以开发一种新的，有效的治疗MCL。

ABSTRACT Mantle cell lymphoma (MCL), accounting for 6-8% of all non-Hodgkin lymphomas (NHLs), is an aggressive and difficult-to-treat B-cell malignancy. MCL patients have a dismal prognosis with a median overall survival of only 3-5 years owing to frequent relapse and resistance to contemporary chemotherapy. Despite recent treatment advances with the FDA-approved drug ibrutinib, which targets the B-cell receptor (BCR) signaling molecule Bruton’s tyrosine kinase (BTK), one-third of MCL patients are ibrutinib-resistant, and even ibrutinib-sensitive patients eventually acquire resistance to the drug. Patients who are insensitive or progress during ibrutinib treatment have very poor outcomes. Thus, more effective post-ibrutinib treatments are urgently needed. Abnormal BCR signaling plays a key oncogenic signal in MCL, but the underlying mechanism is not known. Using an integrated functional genomics approach combining a genome-wide CRISPR-Cas9 screen and gene expression profiling, our group has uncovered CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule-1) as a potential contributor to BCR hyperactivity in MCL. CEACAM1 is a type 1 transmembrane protein expressed as different isoforms due to alternative splicing in epithelial cells and activated leukocytes. In contrast to the short isoform, the CEACAM1 long isoform possesses a cytoplasmic tail that contains the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to negatively regulate the antigen receptor signaling in T cells. We found that CEACAM1 is overexpressed specifically in MCL, as compared to naïve B cells or other B-cell malignancies. Consistent with our CRISPR screen results, depletion of CEACAM1 is toxic in multiple MCL cell lines, but not in non-MCL lymphoma cells. CEACAM1 depletion also markedly reduced proximal BCR signaling (e.g., SYK phosphorylation) and BCR-mediated Ca2+ signaling. More importantly, using T cells that express the CEACAM1-specific chimeric antigen receptor (CAR), we showed that these CAR T cells could effectively eliminate MCL tumors while sparing non-MCL lymphoma in vitro. On the basis of these findings, we hypothesize that CEACAM1 is essential for the oncogenic BCR signaling in MCL and thus a valid therapeutic target for this malignancy. We will test the hypothesis through accomplishing the following specific aims: 1) Determine the mechanisms underlying CEACAM1-regulated BCR signaling; 2) Characterize the MCL-specific expression of CEACAM1; 3) Develop a CAR T-cell strategy to treat MCL in vivo using pre-clinical patient-derived xenograft (PDX) models. Findings from this proposal will improve our limited knowledge of the role of CEACAM1 in MCL pathogenesis. We anticipate that successful completion of the project will provide relevant pre-clinical data for future studies towards developing a novel, effective therapy for MCL.