Role of Early B Cell Factor-1 (Ebf1) in replication initiation of normal B cells and in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL).

B细胞是白色血细胞，可产生抗体以消除感染。这些细胞在骨髓中从祖细胞发育而来，它们在受控的环境下生长、繁殖和死亡。早期B细胞因子1（Ebf 1）是一种对B细胞发育至关重要的基因，在高比例的急性淋巴细胞白血病（ALL）患者中发现了该基因的突变。通过对小鼠中该基因的遗传失活，我们发现不表达Ebf 1的B细胞不能启动DNA复制。为了维持真核生物基因组的完整性，DNA必须在细胞分裂发生前精确复制一次。一个被称为复制许可的过程确保染色体在每个细胞周期只复制一次。许可的起始点，潜在的复制起始点是通常使用的起始点数量的十倍，以确保如果一些起始点失败，另一个附近的起始点接管，以确保整个DNA长度被复制。我们假设，在白血病前Ebf 1突变细胞的DNA复制许可过程被干扰，但监视机制，这是所谓的复制许可检查点，防止DNA复制过程开始，直到细胞积累足够数量的许可的起源。如果监视机制不起作用，细胞可以在DNA复制完成之前开始分裂。幸运的是，含有未复制DNA的细胞在细胞分裂开始之前，在细胞周期的S或G2期被随后的检查点识别和消除。在某些白血病细胞中，甚至这第二种安全机制也被破坏了，我们相信在正常的前体B细胞中，已知的致癌基因c-myB控制着这一过程。目前的提议旨在找出Ebf 1是如何指导B细胞开始复制它们的DNA的。在我们的模型中，Ebf 1的丢失代表了癌变的第一阶段，复制许可检查点的丢失代表了第二阶段。在检查点缺陷的癌细胞中抑制复制许可过程是一种重要的治疗可能性。正常细胞对这种抑制剂的反应是可逆的生长停滞，但检查点缺陷的癌细胞继续复制并由于S/G2检查点的活性而死亡。Cdc 7丝氨酸/苏氨酸激酶在来源许可中起重要作用，并且已证明Cdc 7的抑制可选择性地诱导恶性细胞中的细胞死亡。我们证明，Cdc 7抑制模仿的影响Ebf 1突变的小鼠模型的ALL。我们能够根据小鼠白血病细胞对Cdc 7抑制的反应将其分为两种不同类型。有趣的是，Cdc 7抑制的细胞周期阻滞与细胞死亡反应预测了来自Ebf 1条件性敲除小鼠的ALL细胞中Ebf 1丢失的类似反应，在该小鼠中，我们可以以诱导的方式删除Ebf 1基因。此外，我们观察到相同的两种反应类型的细胞治疗与格列卫，著名的激酶抑制剂，显着改善BCR-ABL 1（或费城染色体）阳性白血病的临床结果。拟议的研究旨在更好地了解复制许可检查点如何在正常B细胞中工作，为什么它在ALL中受到干扰，最后，我们计划发现生物标志物，白血病细胞的可测量参数，这些参数将预测这种药物在人类ALL细胞中的疗效，从而为白血病患者提供未来的个性化治疗。

B cells are white blood cells that produce antibodies to eliminate infections. These cells develop in the bone marrow from progenitor cells, and they grow, multiply and die under controlled circumstances. In Acute Lymphoid Leukaemia, the fine balance between these processes becomes unstable.Early B Cell Factor 1(Ebf1) is a gene that is crucial for the development of B cells, and mutations in this gene are found in a high proportion of Acute Lymphocytic Leukaemia (ALL) patients.By genetic inactivation of this gene in mice, we have found that B cells which do not express Ebf1 were unable to initiate DNA-replication. To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. Licensed origins, potential starting points of replication are in tenfold excess over the number of origins normally used, to make sure that if some origins fail to fire, another nearby origin takes over to ensure that the entire length of DNA is duplicated. We hypothesise that in pre-leukaemic Ebf1-mutant cells the DNA-replication licensing process is disturbed, but the surveillance machinery, which is called a replication licensing checkpoint, prevents the DNA-replication process to start until cells accumulate the sufficient number of licensed origins. If the surveillance mechanism is not working, the cells can start dividing before the duplication of their DNA is finished. Fortunately, cells that contain unreplicated DNA are recognised and eliminated by a subsequent checkpoint in the S or G2 phase of the cell cycle, before the onset of cell-division. In some leukaemic cells even this second safety mechanism is destroyed, and we believe that in normal progenitor B cells the known oncogen, c-myb controls this process. The current proposal aims to find out how exactly Ebf1 instructs the B cells to start to replicate their DNA. In our model, loss of Ebf1 represents the first stage of carcinogenesis, loss of the replication licensing checkpoint represents the second stage. It is an important therapeutic possibility to inhibit the replication licensing process in checkpoint-deficient cancer cells. Normal cells respond to such inhibitors with reversible growth arrest, but checkpoint-deficient cancer cells proceed with replication and die due to the activity of the S/G2 checkpoint. The Cdc7 serine/threonine kinase plays an essential role in origin licensing, and inhibition of Cdc7 has been demonstrated to selectively induce cell death in malignant cells. We demonstrated that Cdc7 inhibition mimicks the effect of Ebf1-mutagenesis in a mouse model of ALL. We were able to categorise our murine leukaemic cells to two distinct types based on their response to Cdc7 inhibition. Interestingly, the cell cycle arrest versus cell death response to Cdc7 inhibition predicted a similar response to the loss of Ebf1 in ALL cells derived from our Ebf1 conditional knockout mouse, in which we could delete the Ebf1 gene in an inducible manner. Furthermore, we observed the same two response types upon treatment of cells with Gleevec, the famous kinase inhibitor that dramatically improved the clinical outcome of BCR-ABL1 (or Philadelphia chromosome) positive leukaemias. The proposed research aims at the better understanding how the replication licensing checkpoint works in normal B cells, why it is disturbed in ALL, and finally, we plan to discover biomarkers, measurable parameters of leukaemia cells that would predict the efficacy of this drug in human ALL cells, leading to future personalised therapy in leukaemia patients.