B cell receptor induced autophagy and endocytosis in Chronic Lymphocytic Leukaemia

技能优先调整：高级治疗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，目前是无法治愈的。慢性淋巴细胞性白血病主要分为两个亚型，以IGHV突变状态为特征的惰性(M-CLL)和侵袭性(U-CLL)疾病。这种疾病是通过激活B细胞受体(BCR)来驱动的，针对这一途径的抑制剂正在使这种疾病发生革命性的变化，但这些药物每天服用，并不能治愈，而且已经出现了对这些药物的耐药性。因此，迫切需要新的策略或挽救疗法来治疗这种疾病。因此，如果我们要寻找新的治疗靶点，对BCR信号通路的更好理解是至关重要的。斯蒂尔小组已经证明，与自噬相关的标记物，如LC3BII和GABARAPL2，在CLL样本中的基础表达高于健康的供体(HD)细胞(分别为p=0.009和p=0.013)。自噬是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的表达仍更强(p=0.02)。已经证明，用珠子固定化(BI)或可溶性抗IgM激活CLL细胞可以以时间依赖的方式进一步增强LC3BII的表达，这与细胞通过BCR发出信号的能力有关。此外，与M-CLL(p=0.004)或HD(p=0.023)细胞相比，U-CLL样本中BCR诱导的LC3BII表达的增加更大。CLL细胞与BI抗-IgM孵育18-24小时后，也能以未知的过程摄取包被抗-IgM的微珠(2.8微米)，但不能摄取同型对照。这些珠子的大小表明，网状蛋白介导的内吞作用不太可能。然而，一种称为LC3相关吞噬细胞作用(LAP)的机制已知在参与受体介导的信号通路(如FCR)的细胞外病原体的摄取中发挥作用，但尚未对SIGM进行描述。LAP过程与自噬不同，但仍需要发生LC3BII。自噬和LAP都与MHCII受体的抗原处理和提呈有关，而自噬本身也参与了MHCI受体提呈的抗原处理。因此，本项目的目标是1。)确定哪些信号通路参与BCR诱导的自噬和在可溶性和BI-抗IgM处理后的LAP，2。确定自噬和LAP如何参与CLL和正常B细胞向MHCII递呈抗原。这个博士生项目将汇集几个关键学者的专业知识，并将医学(Steele博士、Cragg教授和James博士)、生物科学(Tumbarello博士)和国防科学和技术实验室(DSTL、Porton Salisbury、Caroline Rowland博士)和克里克(Tooze博士)之间在B细胞恶性肿瘤、细胞信号、新疗法和成像技术方面的既定研究优势联系起来。这个项目将提高我们对自噬和LC3相关吞噬(LAP)途径如何调控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.