RUNX-fusion Target Genes in Normal and Leukemic Hematopoiesis

描述（由申请人提供）：RUNX 1/CBF β是正常造血的关键调节因子，常参与白血病。涉及CBF家族的重排约占25%的新发髓性白血病和22%的小儿淋巴母细胞白血病。该研究的总体目标是了解由RUNX 1/CBFbeta转录因子的白血病相关易位融合蛋白决定的基因表达，并评估这些受调控基因在正常和白血病干细胞自我更新、存活和分化中的作用。我们将关注的融合蛋白是编码AML 1-ETO的t（8;21）、编码AML 1-EVI 1的t（3;21）和编码CBFbeta-MYH 11的inv 16/t（16;16）。这些将通过慢病毒转导在人HSPC中表达。对照将包括缺乏转基因的慢病毒以及表达RUNX 1的慢病毒。将使用表达AML 1-ETO或CBFp-MYH 11的人HSPC验证差异表达的靶基因，这些人HSPC由于癌基因表达而获得增强的自我更新特性。还将分析来自表达RUNX融合癌基因的AML患者的临床样本中已识别基因的表达（目的1）。将通过过表达被融合蛋白抑制的基因，以及相反地通过敲低在融合蛋白表达后上调的那些基因，使用表达这些致癌基因并在体外自我更新的人HSPC的慢病毒转导，评价鉴定的靶基因对HSPC功能的特异性贡献。将对原代人HSPC进行类似的转导，以确定靶基因对正常干细胞功能的贡献（目的2）。了解这些融合蛋白对自我更新过程的调节可以深入了解干细胞自我更新的正常机制。

DESCRIPTION (provided by applicant): RUNX1/CBFbeta is a key regulator of normal hematopoiesis that is frequently involved in leukemia. Rearrangements involving the CBF family account for approximately 25% of de novo myeloid leukemias and 22% of pediatric lymphoblastic leukemias. The overall objective of the proposed study is to understand gene expression that is determined by leukemia-associated translocation fusion proteins of the RUNX1/CBFbeta transcription factor, and to evaluate the role of these regulated genes in normal and leukemic stem cell self-renewal, survival and differentiation. The fusion proteins we will focus on are the t(8;21) encoding AML1-ETO, t(3;21) encoding AML1-EVI1, and the inv16/t(16;16) encoding CBFbeta- MYH11. These will be expressed in human HSPC by lentiviral transduction. Controls will include a lentivirus lacking a transgene as well as a lentivirus expressing RUNX1. Differentially expressed target genes will be validated using human HSPC expressing either AML1-ETO or CBFp-MYH11 that have acquired enhanced self-renewal properties due to the oncogene expression. Clinical samples from AML patients expressing a RUNX-fusion oncogene will also be analyzed for expression of the identified genes (Aim 1). The specific contribution of identified target genes to HSPC function will be evaluated by over-expressing genes that are repressed by the fusion proteins, and conversely by knocking-down those genes that become upregulated upon fusion protein expression, using lentiviral transduction of the human HSPC that express these oncogenes and that self-renew in vitro. Primary human HSPC will be similarly transduced to determine the contribution of the targeted gene to normal stem cell function (Aim 2). Understanding the modulation of the self-renewal process by these fusion proteins could give insight into the normal mechanisms of self-renewal employed by the stem cell.