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

描述（由申请人提供）：B细胞系急性淋巴细胞白血病（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患者的治愈率一般较高。然而，具有所谓的费城染色体（Ph）编码致癌BCR- ABL1激酶的ALL亚群预后特别差。Ph+ ALL通常用BCR-ABL1激酶抑制剂如伊马替尼治疗。然而，对伊马替尼的治疗反应并不持久，在仅仅几个月的潜伏期后，Ph+ ALL细胞就会产生耐药性，ALL复发。值得注意的是，致癌性BCR-ABL1激酶不仅在Ph+ ALL中表达（主要是p190 BCR-ABL1），而且在bbb95%的慢性髓性白血病（CML，主要是p210 BCR-ABL1）中也表达。与Ph+ ALL相比，伊马替尼长期治疗CML是有效的，耐药性很少发生。然而，在CML患者的一个亚组中，疾病进展为B淋巴细胞危象（CML- lbc），其中治疗反应与Ph+ ALL一样短暂。在大多数情况下，Ph+ ALL和CML-LBC对伊马替尼的获得性耐药可归因于BCR-ABL1激酶结构域内的体细胞突变，这损害了伊马替尼的疗效。在本提案的初步实验中，我们发现AID在bcr - abl1驱动的白血病（Ph ALL和CML-LBC）的B细胞系+克隆中特异性表达。在这些细胞中，AID作为一个突变体发挥作用，从而有助于在Ph+ ALL和CML-LBC中观察到的典型耐药性。基于这些发现，我们的建议解决了以下问题：(1)AID如何导致Ph ALL的遗传不稳定性和耐药性（例如AID特异性缺失；目的1）；(2)AID在多大程度上促进了慢性粒细胞白血病向CML- lbc的进展(携带有利突变的B淋巴亚克隆的产物；目标2),(3)因素导致的异常表达援助在Ph值+和CML-LBC(目标3)+(4)以及是否AID-expressing克隆在Ph值和CML-LBC可以专门针对前体药物,酶活性的基础方法,利用援助(目标4)。加在一起,这四个目标将有助于阐明耐药性机制在Ph值和CML-LBC +提出一个新概念的靶向治疗Ph值和CML-LBC临床前评估。目的1:AID对Ph+ ALL遗传不稳定性的贡献：我们生成了bcr - abl1转化的B细胞系白血病细胞，根据其基因型，AID有三种表达水平，即AID- /-，内源性AID和强制AID-过表达。我们将这些白血病细胞注射到同源小鼠受体中，并将通过比较基因组杂交（CGH）分析来比较发展中的白血病克隆，以确定艾滋病特异性缺失。缺失断点将通过FISH分析验证，并映射到与艾滋病相关的体细胞超突变热点。我们将比较在BCR-ABL1 p190转基因小鼠中发生的Aid-/-和Aid-野生型白血病的伊马替尼耐药性的发展。目的2：艾滋病诱导的突变对CML进展为淋巴细胞危象的贡献：为了阐明AID在多大程度上促进了CML进展为淋巴细胞危象，我们将采取两种方法。(1) p210 BCR-ABL1转化造血干细胞（HSC）可诱导cml样白血病，随后发展为B淋巴细胞危象。在Aid-/-背景下研究在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Aid-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Scl-BCR-ABL1 p210转基因小鼠与Aid-Cre报告菌株杂交，该菌株携带YFP，前带有loxp -侧翼的Stop卡带。Aid在这些细胞中的表达将导致永久性的YFP基因标记。基于yfp标记，该小鼠模型将表明B淋巴亚克隆的生长是否需要在CML克隆进化过程中至少一个时间点表达Aid。目的3：在bcr - abl1驱动的白血病中调节Aid表达的因素的鉴定：我们观察到来自同一患者的原代Ph+ ALL细胞中Aid表达有很大差异。这些发现表明，除了均匀表达的BCR-ABL1和B细胞特异性转录因子外，其他aids调节因子仅在白血病人群的一个子集中表达。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aids -调节因子。目的4：以前药为基础靶向表达AID的Ph+ ALL细胞：AID可将单体脱氧胞苷脱胺为脱氧尿嘧啶。我们假设AID同样可以激活单体前药单体5- fc （Ancobon(R)）及其衍生物5- dfcr和卡培他滨（Xeloda(R)）进入细胞毒性代谢物5-氟尿嘧啶（5- fu）。利用AID在Ph+ ALL和CML- lbc细胞中的酶促活性，我们将利用5-FC、5-DFCR和卡培他滨靶向表达AID的细胞，特异性靶向Ph+ ALL和CML淋巴母细胞危象中表达AID的细胞。公共卫生相关性：费城染色体（Ph）编码致癌的BCR-ABL1激酶，该激酶驱动两种类型的白血病：急性淋巴细胞白血病（Ph+ ALL）源自转化的B淋巴细胞，慢性髓性白血病（CML）源自髓性细胞，否则会发展成巨噬细胞或单核细胞。虽然Ph+ ALL在一开始就代表了一种快速进展的疾病，但CML的病程通常在多年内保持稳定，仅在晚期（即所谓的“blast crisis”）才显示出快速进展。导致从慢性期进展到爆炸危象的原因在很大程度上是未知的。随着BCR-ABL1激酶抑制剂伊马替尼的发现，Ph+ ALL和CML的治疗已经发生了革命性的变化。然而，尽管两种白血病类型携带相同的遗传异常，伊马替尼治疗的结果却截然不同：尽管伊马替尼在治疗慢性期CML中非常有效，但对于Ph+ ALL或原细胞危象CML患者，治疗成功只是短暂的。在这些患者中，白血病平均在4个月后复发，并且由于获得额外的突变而通常具有耐药性。因此，了解潜在的突变机制及其潜在的抑制作用对于进一步改善Ph+ ALL和CML的治疗策略至关重要。我们小组最近的工作表明，在Ph+ ALL和母细胞危象（而不是慢性期CML）中，致癌性BCR-ABL1激酶诱导了一种称为AID（激活诱导胞苷脱氨酶）的突变酶的表达。在Ph+ ALL和原细胞危象CML中赋予耐药性的突变确实可以用AID酶的活性来解释。进一步的实验表明，在艾滋病阴性慢性期CML细胞中，AID的工程化表达引入了与艾滋病阳性Ph+ ALL和CML细胞危象患者产生耐药性相同的突变。基于这些观察结果，我们提出了四个系列的实验来解决以下问题：(1)AID酶是否需要在aids阳性Ph+ ALL中产生耐药性？为了验证这一假设，我们将研究携带aids基因缺失的小鼠bcr - abl1诱导的白血病细胞是否不能产生耐药性。(2) AID酶是否在慢性粒细胞白血病慢性期进入母细胞危象的过程中起关键作用？慢性期慢性粒细胞白血病可以治疗多年非常成功，而细胞危象代表了疾病的最后和往往致命的阶段。(3)在Ph+ ALL和blast危象CML中诱导AID酶异常表达的因素有哪些？这些因素的识别可能有助于了解如何阻止这种有害的突变酶的表达。(4)是否有可能利用AID的酶活性靶向表达AID的细胞？对于这种方法，我们建议使用一种前体药物，这种药物本身没有作用，但在艾滋病介导的转化过程中会变得有毒。鉴于表达艾滋病的细胞比其他细胞更有可能产生耐药性，我们提出了一种针对表达艾滋病的白血病细胞的治疗方法。

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. PUBLIC HEALTH RELEVANCE: The Philadelphia chromosome (Ph) encodes the oncogenic BCR-ABL1 kinase, which drives two types of leukemia: Acute lymphoblastic leukemia (Ph+ ALL) is derived from a transformed B lymphocyte and chronic myeloid leukemia (CML) originates from myeloid cells that would otherwise develop into macrophages or monocytes. While Ph+ ALL represents a rapidly progressive disease already at the outset, the course of CML is typically stable over many years and only shows rapid progression in the terminal stage, the so-called "blast crisis". The reasons leading to progression from chronic phase into blast crisis are largely unknown. The treatment of both Ph+ ALL and CML has been revolutionized by the discovery of the BCR-ABL1 kinase-inhibitor Imatinib. However, even though both leukemia types carry the same genetic abnormality, the outcome of Imatinib-treatment is strikingly different: whereas Imatinib is very effective in the treatment of chronic phase CML, treatment success is only transient for patients with Ph+ ALL or blast crisis CML. In these patients, the leukemia recurs after 4 months on average and is typically drug-resistant owing to the acquisition of additional mutations. Therefore, the understanding of the underlying mutation mechanism and its potential inhibition appears to be critical for further improvement of treatment strategies of Ph+ ALL and CML. Recent work by our group demonstrated that the oncogenic BCR-ABL1 kinase in Ph+ ALL and blast crisis, but not chronic phase CML, induces expression of a mutator enzyme, termed AID (Activation-induced Cytidine Deaminase). The mutations that confer drug-resistance in Ph+ ALL and blast crisis CML can indeed be explained by activity of the AID enzyme. Additional experiments showed that engineered expression of AID in AID-negative chronic phase CML cells introduces the same mutations that cause drug-resistance in patients with AID-positive Ph+ ALL and CML blast crisis. Based on these observations, we propose four series of experiments to address the following questions: (1) Is the AID enzyme required for drug-resistance in AID-positive Ph+ ALL? To test this hypothesis, we will investigate whether BCR-ABL1-induced leukemia cells from mice carrying a deletion of the AID-gene fail to develop drug-resistance. (2) Does the AID enzyme play a critical role in the progression of chronic phase CML into blast crisis? Chronic phase CML can be treated very successfully for many years, whereas blast crisis represents a final and often fatal stage of the disease. (3) Which are the factors that induce aberrant expression of the AID enzyme in Ph+ ALL and blast crisis CML? The identification of such factors will likely help to understand how expression of this deleterious mutator enzyme can be prevented. (4) Is it possible to target AID-expressing cells by taking advantage of the enzymatic activity of AID? For this approach, we propose to use a precursor-drug that has no effect as such but will become toxic upon AID-mediated conversion. Given that AID-expressing cells are more likely to be drug-resistant than others, we propose a treatment approach that is focused on the AID-expressing leukemia cells.