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细胞是产生抗体以消除感染的白细胞。这些细胞在骨髓中由祖细胞发育而来，它们在受控环境下生长、繁殖和死亡。在急性淋巴性白血病中，这些过程之间的微妙平衡变得不稳定。早期B细胞因子1（Ebf1）是一个对B细胞发育至关重要的基因，在急性淋巴细胞白血病（ALL）患者中发现该基因突变的比例很高。通过在小鼠中基因灭活该基因，我们发现不表达Ebf1的B细胞无法启动dna复制。在真核生物中，为了保持基因组的完整性，DNA必须在细胞分裂发生之前精确地复制一次。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。许可起始点，潜在的复制起始点比通常使用的起始点多十倍，以确保如果一些起始点不能激活，附近的另一个起始点会接管以确保整个DNA长度被复制。我们假设，在白血病前ebf1突变细胞中，dna复制许可过程受到干扰，但被称为复制许可检查点的监视机制阻止dna复制过程开始，直到细胞积累足够数量的许可起源。如果监视机制不起作用，细胞就会在DNA复制完成之前开始分裂。幸运的是，在细胞分裂开始之前，含有未复制DNA的细胞在细胞周期的S期或G2期被随后的检查点识别和清除。在一些白血病细胞中，甚至这第二种安全机制也被破坏了，我们认为在正常的祖B细胞中，已知的致癌原c-myb控制着这一过程。目前的提议旨在找出Ebf1究竟是如何指示B细胞开始复制它们的DNA的。在我们的模型中，Ebf1的缺失代表了癌变的第一阶段，复制许可检查点的缺失代表了第二阶段。抑制检查点缺陷癌细胞的复制许可过程是一种重要的治疗可能性。正常细胞对这些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。Cdc7丝氨酸/苏氨酸激酶在起源许可中起着至关重要的作用，Cdc7的抑制已被证明可以选择性地诱导恶性细胞死亡。我们证明了Cdc7抑制在ALL小鼠模型中模拟了ebf1诱变的效果。基于对Cdc7抑制的反应，我们能够将小鼠白血病细胞分为两种不同的类型。有趣的是，细胞周期阻滞与细胞死亡对Cdc7抑制的反应预测了Ebf1条件敲除小鼠衍生的所有细胞对Ebf1缺失的类似反应，在Ebf1条件敲除小鼠中，我们可以以诱导方式删除Ebf1基因。此外，我们观察到用格列卫（Gleevec）治疗细胞时出现了同样的两种反应类型，格列卫是一种著名的激酶抑制剂，可显著改善BCR-ABL1（或费城染色体）阳性白血病的临床结果。该研究旨在更好地了解复制许可检查点如何在正常B细胞中起作用，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，从而导致白血病患者未来的个性化治疗。

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.