B cell receptor induced autophagy and endocytosis in Chronic Lymphocytic Leukaemia

慢性淋巴细胞白血病（CLL）是欧洲和美国最常见的成人白血病，目前无法治愈。CLL主要分为两个亚组，惰性（M-CLL）和侵袭性（U-CLL）疾病，其特征是其IGHV突变状态。这种疾病是通过B细胞受体（BCR）的激活来驱动的，针对这一途径的抑制剂正在彻底改变这种疾病，但这些药物每天服用，不能治愈，而且对这些药物的耐药性已经出现。因此，迫切需要新的策略或挽救疗法来治疗这种疾病。因此，如果我们要确定新的治疗靶点，更好地了解BCR信号通路是必不可少的。Steele小组已经证明，与健康供体（HD）细胞相比，CLL样本中与自噬相关的标志物，如LC3BII和GABARAPL2的基础表达升高（p= 0.009和p= 0.013）。自噬是一种已知的促进肿瘤发生的过程。此外，与M-CLL相比，U-CLL样本中LC3BII的表达仍然更高（p= 0.02）。新发现表明，使用头固定（BI）或可溶性抗igm激活CLL细胞可以以一种时间依赖性的方式进一步增加LC3BII的表达，这与细胞通过BCR发出信号的能力相关。此外，与M-CLL （p= 0.004）或HD （p= 0.023）细胞相比，bcr诱导的U-CLL样本中LC3BII表达的增加更大。用BI抗IgM孵育18-24h的CLL细胞也能吸收涂有抗IgM的2.8微米的珠粒，但不吸收同型对照，其过程未知。这些小珠的大小表明网格蛋白介导的内吞作用不太可能。然而，已知一种称为lc3相关吞噬（LAP）的机制在参与受体介导的信号通路（如FcR）的细胞外病原体摄取中起作用，但尚未描述sIgM。LAP过程不同于自噬，但仍然需要LC3BII的发生。自噬和LAP都与MHCII受体的抗原加工和递呈有关，而自噬本身也参与MHCI受体的抗原加工和递呈。因此，这个项目的目标是：1。在可溶性和BI-anti-IgM治疗后，确定哪些信号通路参与bcr诱导的自噬和LAP， 2)。2 .确定CLL细胞和正常B细胞中自噬和LAP如何参与抗原向MHCII的递呈；确定这些途径在HD和CLL细胞之间的差异，以便我们可以靶向治疗这些途径。该博士奖学金将汇集几位重要学者的专业知识，并将b细胞恶性肿瘤、细胞信号、新疗法和成像技术等领域的研究优势联系起来，这些领域包括医学（斯蒂尔博士、克雷格教授和詹姆斯博士）、生物科学（Tumbarello博士）、国防科学和技术实验室（DSTL、波顿索尔兹伯里、卡罗琳罗兰博士）和克里克（图泽博士）。该项目将提高我们对自噬和LC3相关吞噬（LAP）途径如何调节CLL发病机制的理解，从而为患者确定新的治疗方法或治疗策略。

Skills Priority Alignment: Advanced TherapeuticsChronic Lymphocytic Leukaemia (CLL) is the most prevalent adult leukaemia in Europe and the United States and is currently incurable. CLL is largely split into two subgroups, as indolent (M-CLL) and an aggressive (U-CLL) disease characterised by their IGHV mutational status. The disease is driven through activation of the B cell receptor (BCR) and inhibitors that target this pathway are revolutionising this disease, but these agents are taken daily, not curative and resistance to these drugs is already emerging. Therefore novel strategies or salvage therapies to treat this disease are urgently required.Consequently, a better understanding of the BCR signalling pathways is essential if we are to identify novel therapeutic targets. The Steele group has demonstrated that basal expression of markers such as LC3BII and GABARAPL2 which are associated with autophagy, a process known to promote tumorigenesis, is elevated in CLL samples compared to healthy donor (HD) cells (p=.009 & p=.013 respectively). Moreover, LC3BII expression is greater still in U-CLL samples compared to M-CLL (p=.02). It has been demonstrated the novel finding that activating CLL cells using bead immobilised (BI) or soluble anti-IgM could further augment LC3BII expression in a time dependent manner and this correlated with the cells ability to signal via the BCR. Moreover, the BCR-induced increase in LC3BII expression was greater in U-CLL samples compared to M-CLL (p=.004) or HD (p=.023) cells. CLL cells incubated with BI anti-IgM for 18-24h were also able to take up the beads (2.8 micrometre) coated with anti- IgM, but not the isotype control, by an unknown process. The size of these beads suggests that clathrin-mediated endocytosis was unlikely. However, a mechanism called LC3-associated phagocytosis (LAP) is known to plays a role in the uptake of extracellular pathogens that engage receptor mediated signalling pathway such as FcR but has not been described for sIgM. The LAP process is distinct from autophagy but still requires LC3BII to occur. Both autophagy and LAP are associated with antigen processing and presentation by the MHCII receptor, whereas autophagy alone is also involved in antigen processing for presentation by the MHCI receptor. Therefore, the aims of this project are to 1.) Determine which signalling pathways are involved in BCR-induced autophagy and LAP following treatment with soluble and BI-anti-IgM, 2.) Determine how autophagy and LAP are involved in antigen presentation to MHCII in CLL and normal B cells and 3.) Determine how these pathways differ between HD and CLL cells so that we can target these pathways therapeutically.This PhD studentship will bring together expertise from several key academics and link established research strengths in B-cell malignancies, cell signalling, novel therapeutics and imaging technologies between medicine (Dr Steele, Prof Cragg and Dr James), biosciences (Dr Tumbarello) and Defence science and technology laboratories (DSTL, Porton Salisbury, Dr Caroline Rowland) and the Crick (Dr Tooze). This project will improved our understanding of how autophagy and LC3 associated phagocytosis (LAP) pathways regulate CLL pathogenesis, leading to the identification of novel therapies or treatment strategies for patients.