Lyn kinase as a key regulator in the microenvironmental niche of B lymphoid tumors

以B细胞受体(BCR)相关激酶为靶点的小分子激酶抑制剂在治疗B淋巴样恶性肿瘤(如慢性淋巴细胞白血病(CLL))方面取得了巨大进展(Hallek等人，《柳叶刀》2018年)。随着这些抑制剂在常规治疗中的引入，有令人信服的证据表明，它们的疗效不仅取决于对B细胞自主功能的抑制。相反，这些抑制物的作用机制可以在很大程度上通过调节淋巴样肿瘤微环境(TME)中不同的细胞类型来解释(Nguyen，Niesen，Hallek，Leukhemia 2019)。最近，我们发现了BCR的中心信号成分Lyn Kinase对于创建亲恶性白血病微环境具有意想不到的、强大的功能作用(Nguyen等人，癌症细胞2016)。特别是，我们观察到Lyn激酶的存在是维持造血细胞和非造血细胞对白血病支持能力所必需的。此外，我们实验室的初步数据表明，Lyn缺乏导致TME中巨噬细胞和成纤维细胞蛋白质组的深刻变化。蛋白质组学图谱显示，Lyn诱导的这两种细胞类型的变化几乎没有重叠，这表明Lyn激活了白血病相关巨噬细胞和成纤维细胞中不同的、细胞类型特定的功能通路。该项目的中心假设是，非受体酪氨酸激酶，如LYN，在B细胞淋巴瘤的前肿瘤微环境中起着中心调节器的作用。因此，对淋巴组织TME中这些激酶功能的更好的机制理解将有助于设计改进的抗淋巴瘤疗法。在第一个目标中，我们将验证不同TME细胞中Lyn激酶调控网络中包括转录因子在内的新靶点的细胞类型特异性调节。同时，我们将探索LYN在成纤维细胞激活和诱导促癌表型中的潜在作用。在第二个目标中，我们将使用不同的体外和体内遗传工具来确定LYN的TME作用的结构信号域。在第三个目标中，我们将采用高含量、功能性阵列CRISPR基因敲除方法来确定有助于TME细胞支持癌症的新的治疗靶点。总之，拟议工作计划的总体目标是更深入地了解蛋白激酶与TME相关的功能，促进在临床试验中重新设计治疗应用，因为激酶抑制剂的可获得性很广。

Targeting B cell receptor (BCR)-associated kinases by small molecule kinase inhibitors has generated tremendous advances of the therapy of B lymphoid malignancies such as chronic lymphocytic leukemia (CLL) (Hallek et al., Lancet 2018). Following the introduction of these inhibitors in routine therapy, compelling evidence has emerged showing that their efficacy does not only rely on the inhibition of B cell-autonomous functions. Instead, the mechanism of action of these inhibitors can be explained to a large extent by the modulation of distinct cell types within the lymphoid tumor microenvironment (TME) (Nguyen, Niesen, Hallek, Leukemia 2019).Recently, we have discovered an unexpected, potent functional role of Lyn kinase, a central signaling component of the BCR, for the creation of a pro-malignant leukemia microenvironment (Nguyen et al., Cancer Cell 2016). Particularly, we observed that the presence of Lyn kinase is required to maintain the leukemia supporting capacity of both hematopoietic and non-hematopoietic cells. Moreover, preliminary data of our laboratory suggest that Lyn-deficiency leads to profound proteomic changes in macrophages and fibroblasts in the TME. The proteomic profiling revealed little overlap of Lyn-induced changes in the two cell types, suggesting that Lyn activates distinct, cell-type specific functional pathways in leukemia-associated macrophages and fibroblasts. The central hypothesis of this project is that non-receptor tyrosine kinases such as Lyn function as central modulators of the pro-neoplastic microenvironment for B cell lymphoma. Therefore, an improved mechanistic understanding of the function of these kinases in the lymphoid TME will be useful to design improved anti-lymphoma therapies. In the first aim, we will validate the cell type-specific modulation of novel targets including transcriptional factors within the regulatory network of Lyn kinase in different TME cells. In parallel, we will explore the potential role of Lyn in fibroblast activation and induction of a cancer-promoting phenotype. In the second aim, we will determine the structural signaling domains responsible for the TME action of Lyn using diverse in vitro and in vivo genetic tools. In the third aim, we will employ a high-content, functional arrayed CRISPR-knockout approach to identify new therapeutic targets contributing to the cancer supportive capacity of TME cells. Altogether, the overall aim of the proposed working plan is to provide a deeper understanding of the TME-related functions of protein kinases, fostering the redesigning of therapeutic applications in clinical trials, given the broad availability of kinase inhibitors.