AID-mediated genetic instability in BCR-ABL1-transformed B cell lineage leukemia

描述(申请人提供)：B细胞系急性淋巴细胞性白血病(ALL)是儿童中最常见的恶性肿瘤，在成人中也很常见。与其他恶性肿瘤患者相比，ALL患者的治愈率总体上更高。然而，带有编码致癌BCR-ABL1激酶的所谓费城染色体(Ph)的ALL亚集预后特别差。PH+ALL通常使用BCR-ABL1激酶抑制剂，如伊马替尼。然而，伊马替尼的治疗反应并不持久，仅在几个月的潜伏期后，Ph+所有细胞都变得耐药，并且全部复发。值得注意的是，致癌的bcr-abl1激酶不仅在Ph+ALL(主要是p190 bcr-abl1)中表达，而且在95%的慢性髓系白血病(CML；主要是p210 bcr-abl1)中也有表达。与Ph+ALL相比，长期使用伊马替尼治疗CML是有效的，并且很少产生耐药性。然而，在CML的一部分患者中，疾病进展为B淋巴母细胞危象(CML-LBC)，在这种情况下，治疗反应与Ph+ALL一样短暂。在大多数情况下，Ph+ALL和CML-LBC对Imatinib的获得性耐药可归因于BCR-ABL1激动域的体细胞突变，这损害了Imatinib的疗效。在这一提议的初步实验中，我们发现AID特异性地表达在BCR-ABL1驱动的白血病(Ph ALL和CML-LBC)的B细胞系+克隆中。在这些细胞中，AID起突变因子的作用，从而导致在Ph+ALL和CML-LBC中观察到的典型耐药性。基于这些发现，我们的建议解决了+(1)AID如何导致Ph ALL的遗传不稳定性和耐药性的问题(例如，AID特异性缺失；AIM 1)，(2)AID在多大程度上促进了慢性期CML向CML-LBC的进展(携带有利突变的B淋巴系亚克隆的副产物；目的2)，(3)哪些因素导致AID在Ph+ALL和CML-LBC中的异常表达(Aim 3)，+(4)，以及在Ph ALL和CML-LBC中表达AID的克隆能否在利用AID的酶活性的前药基础上被特异性地靶向(Aim 4)。+这四个目标将有助于阐明Ph ALL和CML-LBC的耐药机制，并提出靶向治疗Ph ALL和CML-LBC的新概念，用于临床前评估。目的1：AID在Ph+ALL遗传不稳定性中的作用：我们建立了BCR-ABL1转化的B细胞系白血病细胞系，根据其基因型，AID有三种水平的表达，即AID-/-、内源性AID和强制AID-过表达。我们已经将这些白血病细胞注射到同基因小鼠受体中，并将通过比较基因组杂交(CGH)分析来比较发育中的白血病克隆，以确定AID特异性缺失。缺失断裂点将通过FISH分析进行验证，并定位到与艾滋病相关的体细胞高突变热点。我们将比较在BCR-ABL1p190转基因小鼠中发生的艾滋病/艾滋病和艾滋病野生型白血病中伊马替尼耐药性的发展。目的2：AID诱导的突变在CML发展为淋巴母细胞危象中的作用：为了阐明AID在CML发展为淋巴母细胞危象中的作用，我们将采取两种方法。(1)p210 bcr-abl1基因转化造血干细胞(HSC)可诱导CML样白血病，继而进展为B淋巴细胞危象。研究在HSC特异性scl启动子控制下表达p210 bcr-abl1的转基因小鼠，我们将研究辅助功能是否对B淋巴母细胞危机克隆的生长是必需的。其次，我们将把scl-bcr-abl1p210转基因小鼠与一个携带YFP的Aid-Cre报告株杂交，该报告株前面有一个loxP侧翼的Stop盒。在这些细胞中表达辅助基因将导致永久的YFP基因标记。基于YFP标记，这个小鼠模型将表明在CML的克隆进化过程中，B淋巴系亚克隆的生长是否至少在一个时间点需要AID的表达目的3：识别调节BCR-ABL1驱动的白血病中AID表达的因素：我们观察到同一患者的原代Ph+ALL细胞中AID的表达有很大差异。这些发现表明，除了同源表达的BCR-ABL1和B细胞特异性转录因子外，其他AID调节因子只在白血病人群的一小部分中表达。使用AID-GFP报告系统，我们将比较AIDHigh和AIDlow Ph+ALL细胞，以确定关键的AID调节因子。目的4：前药靶向表达AID的Ph+ALL细胞：AID可使单体脱氧胞苷脱氨基为脱氧尿嘧啶。我们假设AID同样可以将单体前体药物单体5-FC(Ancobon(R))及其衍生物5-DFCR和Capecitabine(Xeloda(R))激活为细胞毒性代谢物5-FU(5-FU)。利用AID在Ph+ALL和CML-LBC细胞中的酶活性，我们将使用5-FC、5-DFCR和卡培他滨靶向表达AID的细胞，以特异性靶向Ph+ALL和CML淋巴母细胞危象中表达AID的细胞。

DESCRIPTION (provided by applicant): B cell lineage acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children and is also common in adults. Compared to patients with other malignancies, cure rates for patients with ALL are in general higher. The ALL subset with the so-called Philadelphia chromosome (Ph) encoding the oncogenic BCR- ABL1 kinase, however, has a particularly poor prognosis. Ph+ ALL is typically treated with BCR-ABL1 kinase inhibitors such as Imatinib. The treatment response to Imatinib, however, is not durable and after a latency of only a few months, Ph+ ALL cells become drug-resistant and ALL relapses. Of note, the oncogenic BCR-ABL1 kinase is not only expressed in Ph+ ALL (mainly p190 BCR-ABL1) but also in >95% of cases of chronic myeloid leukemia (CML; mainly p210 BCR-ABL1). In contrast to Ph+ ALL, long-term treatment of CML with Imatinib is effective and resistance develops only rarely. In a subgroup of patients with CML, however, the disease progresses into B lymphoid blast crisis (CML-LBC), in which treatment responses are as short-lived as in Ph+ ALL. In most cases, acquired resistance to Imatinib in Ph+ ALL and CML-LBC can be attributed to somatic mutations within the BCR-ABL1 kinase domain, which compromise the efficacy of Imatinib. In preliminary experiments for this proposal, we show that AID is specifically expressed in B cell lineage + clones of BCR-ABL1-driven leukemia (Ph ALL and CML-LBC). In these cells, AID functions as a mutator and thereby contributes to the drug-resistance typically observed in Ph+ ALL and CML-LBC. Based on these findings, our proposal addresses the question of + (1) how AID contributes to genetic instability and drug-resistance in Ph ALL (e.g. AID-specific deletions; Aim 1), (2) to which extent AID contributes to the progression of chronic phase CML to CML-LBC (outgrowth of B lymphoid subclones that carry advantageous mutations; Aim 2), (3) which factors cause aberrant expression of AID in Ph+ ALL and CML-LBC (Aim 3), + (4) and whether AID-expressing clones in Ph ALL and CML-LBC can be specifically targeted in a prodrug- based approach that takes advantage of the enzymatic activity of AID (Aim 4). + Together, these four Aims will help to elucidate mechanisms of drug-resistance in Ph ALL and CML-LBC and + propose a novel concept of targeted treatment Ph ALL and CML-LBC for pre-clinical evaluation. Aim 1: Contribution of AID to genetic instability in Ph+ ALL: We have generated BCR-ABL1-transformed B cell lineage leukemia cells with three levels of AID expression based on their genotype, namely Aid-/-, endogenous AID and forced AID-overexpression. We have injected these leukemia cells into congenic mouse recipients and will compare the developing leukemia clones by comparative genomic hybridization (CGH) analysis to identify AID-specific deletions. Deletion breakpoints will be verified by FISH analysis and mapped to AID-related somatic hypermutation hot spots. We will compare development of Imatinib-resistance in Aid-/- and Aid-wildtype leukemias developing in BCR-ABL1 p190-transgenic mice. Aim 2: Contribution of AID-induced mutations to progression of CML into lymphoid blast crisis: To clarify to which extent AID contributes to the progression of CML into lymphoid blast crisis, we will take two approaches. (1) Transformation of hematopoietic stem cells (HSC) by p210 BCR-ABL1 induces CML-like leukemia with subsequent progression into B lymphoid blast crisis. Studying transgenic mice expressing p210 BCR-ABL1 under control of the HSC-specific Scl-promoter on an Aid-/- background, we will investigate whether Aid-function is required for the outgrowth of B lymphoid blast crisis clones. Second, we will cross Scl-BCR-ABL1 p210 transgenic mice with an Aid-Cre reporter strain that carries YFP preceded by a loxP-flanked Stop cassette. Expression of Aid in these cells will lead to permanent genetic labeling with YFP. Based on YFP-labeling, this mouse model will indicate whether or not outgrowth of B lymphoid subclones requires expression of Aid at least at one point in time during the clonal evolution of CML Aim 3: Identification of factors that regulate AID-expression in BCR-ABL1-driven leukemias: We observed that AID expression substantially varies among primary Ph+ ALL cells from the same patient. These findings suggest that besides homogenously expressed BCR-ABL1 and B cell-specific transcription factors, additional AID-regulatory factors are only expressed in a subset of the leukemia population. Using an Aid-GFP reporter system, we will compare AIDhigh and AIDlow Ph+ ALL cells to identify key AID-regulatory factors. Aim 4: Prodrug-based targeting of AID-expressing Ph+ ALL cells: AID can deaminate monomeric deoxycytidine to deoxyuracil. We hypothesize that AID can likewise activate the monomeric prodrugs monomeric 5-FC (Ancobon(R)) and its derivatives 5-DFCR and Capecitabine (Xeloda(R)) into the cytotoxic metabolite 5- fluorouracil (5-FU). Taking advantage of the enzymatic activity of AID in Ph+ ALL and CML-LBC cells, we will target AID-expressing cells using 5-FC, 5-DFCR and Capecitabine for specific targeting of AID-expressing cells in Ph+ ALL and CML lymphoid blast crisis.