Transponson-based screens for genes involved in acute lymphoblastic Leukemia

描述（由申请人提供）：Ebf1、Pax5和Ikaros这三种转录因子的突变，形成了启动B细胞发育所需的相互关联的基因网络，在B- all祖细胞中经常发生突变。同样，转录因子STAT5在B-ALL中也经常被激活。重要的是，Ebf1/Pax5/Ikaros网络的突变，尤其是Ikaros，已被证明与人类ALL的不良预后相关；我们和其他人已经观察到ALL中STAT5激活与不良预后之间的类似相关性。然而，究竟是Ebf1Pax5/Ikaros网络中的突变或STAT5激活驱动了转化，还是仅仅是这一过程中的乘客，目前尚不清楚。我们最近通过将表达STAT5b组成活性形式（称为STAT5b - ca）的小鼠与Ebf1或Pax5杂合的小鼠杂交，获得了对这个问题的重要见解。Stat5b-CA小鼠患ALL的外显率很低（1-2%），而Pax5和Ebf1小鼠没有患ALL的倾向。相比之下，100%的Stat5b-CA x Pax5和Stat5b-CA x Ebf1小鼠分别在出生后55天和108天的平均发病年龄发生ALL。重要的是，Stat5b-CA x Ebf1和Stat5b-CA x Pax5白血病分别没有丢失Ebf1或Pax5的另一个等位基因。我们的假设是，构成Ebf1/Pax5/Ikaros网络的基因不作为经典的肿瘤抑制基因，而是作为一个综合肿瘤抑制基因网络的一部分发挥作用。在这个模型中，网络中任何基因的任何两个等位基因的丢失，而不是同一基因的两个等位基因的丢失，都足以导致肿瘤抑制功能的丧失。我们进一步提出STAT5激活需要与Ebf1/Pax5/Ikaros网络中的功能缺失突变配对以启动转化。在这项拨款申请中，我们将使用基于睡美人转座子的诱变策略来测试这些假设。此外，我们将测试人类ALL中Ebf1/Pax5/Ikaros网络的缺陷是否与STAT5信号的增加相关。最后，我们还将使用睡美人基因筛选来鉴定与Ebf1/Pax5/Ikaros网络缺陷和STAT5激活协同启动和促进祖细胞B-ALL的其他基因。这些研究将为了解STAT5和Ebf1/Pax5/Ikaros网络在B-ALL发病中的作用以及鉴定在B-ALL转化过程中发挥关键作用的其他基因提供重要的见解。

DESCRIPTION (provided by applicant): Mutations in Ebf1, Pax5 and Ikaros, three transcription factors that form an interrelated gene network required to initiate B cell development, are frequently mutated in progenitor B-ALL. Likewise, the transcription factor STAT5 is frequently activated in B-ALL. Importantly, mutations in the Ebf1/Pax5/Ikaros network, most notably for Ikaros, have been shown to correlate with poor outcome in human ALL; we and others have observed a similar correlation between STAT5 activation in ALL and poor prognosis. However, whether mutations in the Ebf1Pax5/Ikaros network, or STAT5 activation, drive transformation or are merely passengers in this process remains unclear. We recently obtained an important insight to this question by crossing mice expressing a constitutively active form of STAT5b (called Stat5b-CA) to mice heterozygous for Ebf1 or Pax5. Stat5b-CA mice develop ALL with very low penetrance (1-2%) while Pax5 and Ebf1 mice show no predisposition to develop ALL. In contrast, 100% of Stat5b-CA x Pax5 and Stat5b-CA x Ebf1 mice developed ALL with an average age of onset of 55 and 108 days after birth, respectively. Importantly, Stat5b-CA x Ebf1 and Stat5b-CA x Pax5 leukemias did not lose the other allele of either Ebf1 or Pax5, respectively. Our hypothesis is that genes that make up the Ebf1/Pax5/Ikaros network do not act as classical tumor suppressor genes but rather function as part of an integrated tumor suppressor gene network. In this model loss of any two alleles of any of the genes in the network, as opposed to both alleles of the same gene is sufficient for loss of tumor suppressor function. We further propose that STAT5 activation is needed to pair with loss-of-function mutations in the Ebf1/Pax5/Ikaros network to initiate transformation. In this grant application, we will test these hypotheses using a Sleeping beauty transposon-based mutagenesis strategy. In addition, we will test whether defects in the Ebf1/Pax5/Ikaros network in human ALL correlate with increased STAT5 signaling. Finally, we will also use the Sleeping Beauty genetic screen to identify other genes that cooperate with defects in the Ebf1/Pax5/Ikaros network and STAT5 activation to initiate and promote progenitor B-ALL. These studies should provide important insights into the role that STAT5 and the Ebf1/Pax5/Ikaros network play in causing B-ALL as well as identify additional genes that play key roles in the process of transformation.