Function of tyrosine kinases Lyn and Btk in high-risk CLL

提出的项目是基于一个前提，即新型激酶抑制剂的主要作用涉及CLL微环境细胞中的靶标，而不是白血病细胞。使用Lyn敲除小鼠或化学特异性抑制Btk突变体的实验表明，Lyn和Btk在直接杀死或恶性B细胞凋亡中对激酶失活的贡献相对较小。与之形成鲜明对比的是，在微环境细胞中抑制这两种激酶，特别是在巨噬细胞中，似乎可以抑制CLL细胞的生长并增强CLL细胞的凋亡。这些发现为CLL患者观察到的Btk和PI3K抑制剂缺乏直接细胞杀伤作用提供了解释，CLL患者在治疗期间白血病细胞从淋巴器官重新分布到外周血中长达数月。综上所述，有越来越多的证据表明，间接（微环境）效应比直接效应更能说明这类药物的治疗效果。这些新型抑制剂作用的确切机制尚不完全清楚。特别是，激酶抑制剂在具有高度基因组不稳定性（复杂核型）和DNA损伤反应（DDR）频繁功能障碍（TP53功能障碍）的高风险CLL中的治疗机制尚不清楚。这些抑制剂的临床疗效表明，高风险CLL细胞仍然依赖于微环境支持。在初步实验中，我们已经看到了Lyn激酶和CLL中增强的DDR之间的功能联系。基于这些观察结果，第二个资助期的总体目标是在分子水平上阐明激酶（Lyn, Btk）的作用，这些激酶似乎对高危CLL的生长至关重要。为此，我们将探讨这些酪氨酸激酶和CLL细胞中DDR的相互作用。此外，我们将阐明这些激酶在高风险、TP53功能失调的CLL细胞与微环境之间的细胞间对话中的作用。在四个具体目标中，我们将解决以下问题：探讨微环境细胞对高危CLL发生的支持作用。2. 研究在CLL细胞中自主介导的DDR调节。3. 利用化学遗传学工具探究ddr缺陷CLL细胞中btk介导的微环境相互作用。4. 检测高风险（del(17p)，复杂核型）CLL在途径靶向抑制剂治疗下的克隆进化。这些实验不仅将为这些激酶及其抑制剂在CLL中的作用机制提供必要的见解，而且还将为治疗干预提供新的靶点。

The proposed project is based on the premise that major effects of novel kinase inhibitors involve targets in cells of the CLL microenvironment rather than in leukemic cells. Experiments using Lyn knock out mice, or chemical, specific inhibition of Btk mutants, show a relatively modest contribution of kinase inactivation in the direct killing or for the apoptosis of malignant B cells, both for Lyn and Btk. In stark contrast, inhibition of both kinases in microenvironmental cells, in particular in macrophages, seems to inhibit CLL growth and enhance apoptosis of CLL cells. These findings provide an explanation for the lack of direct cell killing effects of both Btk and PI3K inhibitors observed in CLL patients, where leukemic cells are redistributed from lymphoid organs to the peripheral blood for months during treatment. Taken together, there is cumulating evidence that indirect (microenvironmental) effects rather than direct effects account for the therapeutic benefit of this class of agents.The precise mechanisms of action of these effects of novel inhibitors are not completely understood. In particular, the therapeutic mechanisms of kinase inhibitors in high-risk CLL, with a high degree of genomic instability (complex karyotype) and the frequent dysfunction of the DNA damage response (DDR) (TP53 dysfunction), are poorly understood. The clinical efficacy of these inhibitors suggests that high-risk CLL cells remain dependent on microenvironmental support. In preliminary experiments, we have seen a functional link between Lyn kinase and an enhanced DDR in CLL. Based on these observations, the overall aim of the second funding period is to clarify on a molecular level the role of kinases (Lyn, Btk) that seem essential for CLL growth in high-risk CLL. To this end, we will explore the interactions of these tyrosine kinases and the DDR in CLL cells. Moreover, we will clarify the role of these kinases for the intercellular dialogue between CLL cells and the microenvironment in high-risk, TP53 dysfunctional CLL. In four specific aims we will address the following questions: 1. Investigate Lyn-mediated support from microenvironmental cells to the development of high-risk CLL. 2. Investigate the autonomous Lyn-mediated modulation of the DDR in CLL cells. 3. Interrogate Btk-mediated microenvironmental interactions in DDR-defective CLL cells with the use of chemico-genetic tools. 4. Test the clonal evolution of high-risk (del(17p), complex karyotype) CLL under treatment with pathway targeted inhibitors. These experiments will not only provide essential insights into the mechanisms of action of these kinases and their inhibitors in CLL, but also provide novel targets for therapeutic intervention.