Exploring the oncogenic potential of human APOBEC3 cytosine deaminases

 描述(申请人提供)：癌症是由DNA核苷酸损伤引起的躯体获得性点突变和DNA双链断裂(DSB)引起的基因组重排引起的。这些DNA损伤的病因尚不清楚，但已知增加DNA损伤率和DSB频率的细胞过程可诱导损伤启动转化。在恶性肿瘤中发展的突变和基因组重排也可能导致对特定化疗药物耐药或敏感的克隆性群体。识别能够产生DNA损伤并推动恶性进展的细胞酶，将为开发治疗耐药癌症的新药提供靶点。人类基因组编码七种APOBEC3(HA3)酶，通常会导致突变和DSB。这些HA3酶和相关的AID蛋白组成了一个脱氨酶家族，在单链DNA底物中将胞嘧啶转化为尿嘧啶。APOBEC3蛋白(hA3A至hA3H)突变病毒基因组，从而赋予抗病毒功能。AID诱导B淋巴细胞免疫球蛋白基因突变和DSB，以产生抗体多样性，从而保护人类免受病原体的侵袭。越来越多的证据表明，“非靶点”的APOBEC3/AID活性诱导突变和基因组重排，从而推动肿瘤的启动、进展和耐药性，但也可能为新的癌症治疗策略提供机会。这项建议的目的是评估HA3表达对(I)小鼠模型中的增殖、基因组完整性和恶性转化以及(Ii)获得人类癌细胞的耐药性和敏感性的影响。我们重点关注hA3A和hA3B，因为这些活性的胞嘧啶脱氨酶在人类细胞中过度表达时会导致基因组中的突变和DSB。HA3A蛋白在造血细胞中表达，并可被病毒感染和干扰素诱导至高水平，而hA3B蛋白广泛表达，但在许多人类癌症中上调。我们的假设是，hA3A或hA3B的持续表达会导致宿主细胞基因组的脱氨化，诱导致癌突变表型和/或耐药癌症的发展。我们将通过评估hA3A和hA3B表达对小鼠造血细胞的影响(目标1)以及hA3A和hA3B导致耐药癌细胞的能力，同时也使癌细胞对DNA修复因子的抑制敏感(目标2)来验证我们的假设。拟议研究的理由是，个人 在缺乏内源性蛋白的细胞中表达hA3A或hA3B将揭示HA3蛋白如何参与人类癌症的启动和耐药。这些目的的结果将揭示hA3A和hA3B在肿瘤启动、进展或获得治疗耐药性方面的促肿瘤活性的程度，并将表明抑制HA3蛋白活性可能限制致瘤突变和耐药性的获得。

 DESCRIPTION (provided by applicant): Cancer is driven by somatically acquired point mutations resulting from DNA nucleotide damage, and genomic rearrangements arising from DNA double strand breaks (DSBs). The etiology of these DNA lesions remains unclear, but it is known that cellular processes that increase rates of DNA damage and frequencies of DSBs can induce lesions that initiate transformation. Mutations and genomic rearrangements that develop in a malignancy may also result in clonal populations that are either resistant or sensitive to particular chemotherapeutic agents. Identifying cellular enzymes capable of generating DNA lesions that drive malignant progression will suggest targets for development of novel drugs to treat therapy-resistant cancers. The human genome encodes seven APOBEC3 (hA3) enzymes that normally induce mutations and DSBs. These hA3 enzymes, and the related AID protein, constitute a family of deaminases that convert cytosines to uracils in single-stranded DNA substrates. The APOBEC3 proteins (hA3A through hA3H) mutate viral genomes and thereby confer antiviral functions. AID induces mutations and DSBs in immunoglobulin loci in B lymphocytes to generate antibody diversity and thereby protect humans from pathogens. Accumulating evidence suggests that "off-target" APOBEC3/AID activities induce mutations and genomic rearrangements that drive initiation, progression, and drug-resistance of tumors, but may also present opportunities for new cancer treatment strategies. The objectives of this proposal are to assess the impact of hA3 expression on (i) proliferation, genome integrity, and malignant transformation in mouse models and (ii) acquisition of drug resistance and sensitivity in human cancer cells. We focus on hA3A and hA3B since these active cytosine deaminases have been shown to cause mutations and DSBs in the genome when over-expressed in human cells. The hA3A protein is expressed in hematopoietic cells and can be induced to high levels by virus infection and interferon, while hA3B is broadly expressed but is upregulated in a number of human cancers. Our hypothesis is that sustained expression of hA3A or hA3B results in deamination of the host cellular genome, inducing a cancer- promoting mutator phenotype and/or development of drug-resistant cancers. We will test our hypothesis by assessing the impact of hA3A and hA3B expression on mouse hematopoietic cells (Aim 1) and the ability of hA3A and hA3B to cause drug-resistant cancer cells, yet also render cancer cells sensitive to inhibition of DNA repair factors (Aim 2). The rationale for the proposed studies is that individual expression of hA3A or hA3B in cells that lack endogenous protein will reveal insights into how hA3 proteins contribute to initiation and drug- resistance of human cancers. The outcomes of these Aims will reveal the extent to which hA3A and hA3B confer pro-oncogenic activities in tumor initiation, progression, or acquisition of therapy resistance, and will suggest that inhibitig hA3 protein activities may limit acquisition of tumorigenic mutations and drug resistance.