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

BCR-ABL1 转化的 B 细胞系白血病中 AID 介导的遗传不稳定性

基本信息

批准号：
8444327
负责人：
Markus Müschen
金额：
$ 29.66万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-03-18 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8444327
关键词：
ABL1 gene Acute Lymphocytic Leukemia Address Adult B-Lymphocytes Blast Phase Cell Lineage Cells Child Chronic Myeloid Leukemia Chronic Phase Chronic-Phase Myeloid Leukemia Clonal Evolution Congenic Mice Data Deoxycytidine Development Disease Drug resistance Engineering Enzymes Flucytosine Fluorescent in Situ Hybridization Fluorouracil Genes Genetic Genotype Goals Health Hematopoietic stem cells Hot Spot Imatinib Immunoglobulin Class Switching Immunoglobulin Genes Immunoglobulin Somatic Hypermutation Immunoglobulin Switch Recombination In Vitro Induced Mutation Label Lead Life Lymphoid Malignant Neoplasms Maps Mediating Molecular Abnormality Mus Mutation Myeloid Cells Oncogenic Outcome PAX5 gene Patients Philadelphia Chromosome Phosphotransferases Play Population Prodrugs Progressive Disease Relapse Reporter Resistance Resistance development Role Series Somatic Mutation Staging Subgroup System Testing Time Transgenic Mice Work activation-induced cytidine deaminase base capecitabine comparative genomic hybridization cytotoxic in vivo kinase inhibitor leukemia macrophage monocyte mouse model novel outcome forecast overexpression paired box 5 protein (B-cell lineage specific activator)pre-clinical prevent promoter research clinical testing research study success transcription factor treatment response treatment strategy

项目摘要

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.

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患者的治愈率总体上更高。然而，具有编码致癌BCR-ABL 1激酶的所谓费城染色体（Ph）的ALL亚群具有特别差的预后。Ph+ ALL通常使用BCR-ABL 1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，仅在几个月的潜伏期后，Ph+ ALL细胞就变得耐药，ALL复发。值得注意的是，致癌BCR-ABL 1激酶不仅在Ph+ ALL（主要是p190 BCR-ABL 1）中表达，而且在>95%的慢性髓性白血病（CML;主要是p210 BCR-ABL 1）病例中表达。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，在一个CML患者亚组中，疾病进展为B淋巴母细胞危象（CML-LBC），治疗反应与Ph+ ALL一样短暂。在大多数情况下，Ph+ ALL和CML-LBC患者对伊马替尼的获得性耐药可归因于BCR-ABL 1激酶结构域内的体细胞突变，这损害了伊马替尼的疗效。在这个提议的初步实验中，我们表明AID在BCR-ABL 1驱动的白血病（Ph ALL和CML-LBC）的B细胞谱系+克隆中特异性表达。在这些细胞中，AID作为一种增变因子发挥作用，从而导致Ph+ ALL和CML-LBC中通常观察到的耐药性。基于这些发现，我们的建议解决了以下问题：（1）艾滋病如何导致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在何种程度上有助于慢性期CML向CML-LBC的进展（携带有利突变的B淋巴亚克隆的生长;目的2）、（3）哪些因素导致Ph+ ALL和CML-LBC中AID的异常表达（目的3），+（4）以及Ph ALL和CML-LBC中的AID表达克隆是否可以在前药中特异性靶向-该方法利用了AID的酶活性（目的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Ph ALL和CML-LBC靶向治疗的新概念，用于临床前评估。目标1：AID对Ph+ ALL遗传不稳定性的贡献：我们已经产生了BCR-ABL 1转化的B细胞系白血病细胞，根据其基因型具有三种AID表达水平，即Aid-/-、内源性AID和强制性AID过表达。我们已经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）分析来比较发育中的白血病克隆，以确定艾滋病特异性缺失。缺失断点将通过FISH分析验证，并映射到艾滋病相关的体细胞超突变热点。我们将比较在BCR-ABL 1 p190转基因小鼠中发生的Aid-/-和Aid-野生型白血病中伊马替尼耐药性的发生。目标二：艾滋病诱导的突变对慢性粒细胞白血病发展为淋巴母细胞危象的贡献：为了阐明艾滋病在何种程度上有助于慢性粒细胞白血病发展为淋巴母细胞危象，我们将采取两种方法。(1)通过p210 BCR-ABL 1转化造血干细胞（HSC）诱导CML样白血病，随后进展为B淋巴母细胞危象。研究在Aid-/-背景下HSC特异性Scl启动子控制下表达p210 BCR-ABL 1的转基因小鼠，我们将研究Aid功能是否是B淋巴母细胞危象克隆生长所必需的。其次，我们将Scl-BCR-ABL 1 p210转基因小鼠与Aid-Cre报告菌株杂交，该报告菌株携带YFP，前面是loxP侧翼的Stop盒。在这些细胞中表达Aid将导致YFP的永久遗传标记。基于YFP标记，该小鼠模型将指示B淋巴亚克隆的生长是否需要在CML克隆进化期间的至少一个时间点表达Aid目的3：鉴定在BCR-ABL 1驱动的白血病中调节Aid表达的因子：我们观察到同一患者的原发性Ph+ ALL细胞中Aid表达显著不同。这些发现表明，除了BCR-ABL 1和B细胞特异性转录因子的均质表达外，其他的AIDS调节因子仅在白血病人群的一个亚群中表达。使用Aid-GFP报告系统，我们将比较AIDhigh和AIDlow Ph+ ALL细胞，以确定关键的艾滋病调控因子。目的4：基于前药的AIDS表达Ph+ ALL细胞的靶向：AID可以将单体脱氧胞苷脱氨基为脱氧尿嘧啶。我们假设AID同样可以将单体前药单体5-FC（Ancobon（R））及其衍生物5-DFCR和卡培他滨（Xeloda（R））活化为细胞毒性代谢物5-氟尿嘧啶（5-FU）。利用AID在Ph+ ALL和CML-LBC细胞中的酶活性，我们将使用5-FC、5-DFCR和卡培他滨靶向表达AID的细胞，用于在Ph+ ALL和CML淋巴母细胞危象中特异性靶向表达AID的细胞。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemia.

DOI：
10.1016/j.ccr.2009.07.030
发表时间：
2009-09-08
期刊：
Cancer cell
影响因子：
50.3
作者：
Klemm L;Duy C;Iacobucci I;Kuchen S;von Levetzow G;Feldhahn N;Henke N;Li Z;Hoffmann TK;Kim YM;Hofmann WK;Jumaa H;Groffen J;Heisterkamp N;Martinelli G;Lieber MR;Casellas R;Müschen M
通讯作者：
Müschen M

PTEN opposes negative selection and enables oncogenic transformation of pre-B cells.