Conditional Mouse Model of Alveolar Rhabdomyosarcoma

肺泡横纹肌肉瘤是一种侵袭性的儿童肌肉癌，发病率和死亡率都很高。85%的肺泡横纹肌肉瘤表现出一种常见的遗传改变，即易位介导的发育调节的Pax转录因子（Pax3或Pax7）与叉头转录因子Fkhr的融合。对肺泡横纹肌肉瘤发展的分子事件的理解将提高我们对患者进行基于风险的治疗分层和开发新疗法的能力。该项目的目的是研究胚胎后易位介导的Pax3与Fkhr的融合是否启动了参与肺泡横纹肌肉瘤发生的异常胚胎肌肉发育程序，并研究促进肺泡横纹肌肉瘤发生的致癌特性激活的早期和中间步骤是否按照可预测的顺序发生。本研究的具体目的是：(1)研究Pax3:Fkhr的基因组单拷贝数是否足以和必要地引起Pax3通路的异常激活和肺泡横纹肌肉瘤的发生；(2)研究遗传、转录或翻译是否对常见的致癌机制(如：(3)确定Cre重组酶介导的异源染色体间易位是否能够以足够的频率诱导肺泡横纹肌肉瘤的发生。对于第一个和第二个目标，将创建一个条件敲入的肺泡横纹肌肉瘤小鼠模型，该模型最初含有两个功能正常的Pax3等位基因。有条件的，位点特异性DNA重组酶Cre的体细胞表达将介导仅在骨骼肌中将单个Pax3等位基因转化为单个Pax3:Fkhr等位基因的基因组重排。动物将被连续处死，以分析携带Pax3:Fkhr细胞的细胞内变化序列。分析将在基因组、mRNA和蛋白质水平上进行，重点是肿瘤进展的已知标记物。为了达到最终目的，将使用cre介导的异源染色体重排，尝试在Pax3和Fkhr位点之间进行有条件的体细胞易位。Keller博士在Capecchi博士的指导下接受的研究培训以及NCI的支持将为Keller博士的独立研究生涯做好准备，研究儿童癌症的发展方面。最终，对儿童恶性肿瘤中失调发育途径的详细了解可能会提高我们治疗儿童癌症的能力。

Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer associated with significant morbidity and mortality. Eighty-five percent of alveolar rhabdomyosarcomas demonstrate a common genetic alteration, the translocation-mediated fusion of a developmentally regulated Pax transcription factor (either Pax3 or Pax7) to the Forkhead transcription factor, Fkhr. An understanding of the molecular events underlying the development of alveolar rhabdomyosarcomas will improve our ability to stratify patients to risk-based therapies and to develop new therapies. The goals of this project are to investigate whether the postembryonic, translocation-mediated fusion of Pax3 to Fkhr initiates an aberrant embryonic muscle development program which is involved in the genesis of alveolar rhabdomyosarcoma, and to investigate whether the early and intermediate steps in the activation of oncogenic properties contributing to the genesis of alveolar rhabdomyosarcoma occur in a predictable sequence. The specific aims of this proposal are (I) to examine whether a genomic single copy-number of Pax3:Fkhr is sufficient and necessary to cause both aberrant Pax3 pathway activation and the initiation of alveolar rhabdomyosarcoma, (II) to study whether genetic, transcriptional, or translational contributions to common oncogenic mechanisms (eg. insensitivity to antigrowth signals, evasion of apoptosis) occur in a predictable sequence in alveolar rhabdomyosarcomas, and (III) to determine whether Cre recombinase-mediated translocations between heterologous chromosomes can be induced somatically at a frequency sufficient for the initiation of alveolar rhabdomyosarcoma. For the first and second aims, a conditional, knock-in mouse model of alveolar rhabdomyosarcoma will be created which initially harbors two functionally normal Pax3 alleles. Conditional, somatic expression of the site-specific DNA recombinase Cre will mediate a genomic rearrangement converting a single Pax3 allele to a single Pax3:Fkhr allele in skeletal muscle only. Animals will be serially sacrificed to analyze the ensuing sequence of intracellular changes in cells harboring Pax3:Fkhr. Analysis will be performed at the genomic, mRNA, and protein level with an emphasis on known markers of tumor progression. For the final aim, a conditional, somatic translocation between the Pax3 and Fkhr loci will be attempted using Cre-mediated rearrangement of heterologous chromosomes. Dr. Keller's research training with Dr. Capecchi and the support of the NCI will prepare Dr. Keller for an independent research career investigating developmental aspects of pediatric cancer. Ultimately, a detailed knowledge of dysregulated developmental pathways in pediatric malignancies may improve our ability to treat childhood cancer.