Transponson-based screens for genes involved in acute lymphoblastic Leukemia

描述（由申请方提供）：Ebf 1、Pax 5和Ikaros（形成启动B细胞发育所需的相关基因网络的三种转录因子）突变在B-ALL祖细胞中经常发生突变。同样，转录因子STAT 5在B-ALL中经常被激活。重要的是，Ebf 1/Pax 5/Ikaros网络中的突变，尤其是Ikaros，已被证明与人类ALL的不良预后相关;我们和其他人观察到ALL中STAT 5激活与不良预后之间存在类似的相关性。然而，Ebf 1 Pax 5/Ikaros网络中的突变或STAT 5激活是否驱动转化或仅仅是这一过程中的乘客仍不清楚。我们最近通过将表达组成型活性形式的STAT 5 b（称为Stat 5 b-CA）的小鼠与Ebf 1或Pax 5杂合子小鼠杂交，获得了对这个问题的重要见解。Stat 5 b-CA小鼠发生ALL的机率非常低（1-2%），而Pax 5和Ebf 1小鼠没有显示出发生ALL的倾向。相比之下，100%的Stat 5 b-CA x Pax 5和Stat 5 b-CA x Ebf 1小鼠发生ALL，平均发病年龄分别为出生后55天和108天。重要的是，Stat 5 b-CA x Ebf 1和Stat 5 b-CA x Pax 5白血病分别没有丢失Ebf 1或Pax 5的其他等位基因。我们的假设是，组成Ebf 1/Pax 5/Ikaros网络的基因不作为经典的肿瘤抑制基因，而是作为一个整合的肿瘤抑制基因网络的一部分发挥作用。在该模型中，与相同基因的两个等位基因相反，网络中任何基因的任何两个等位基因的丢失足以导致肿瘤抑制功能的丢失。我们进一步提出，STAT 5激活需要与Ebf 1/Pax 5/Ikaros网络中的功能缺失突变配对，以启动转化。在本申请中，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Ebf 1/Pax 5/Ikaros网络的缺陷是否与STAT 5信号传导增加相关。最后，我们还将使用睡美人遗传筛查来识别与Ebf 1/Pax 5/Ikaros网络缺陷和STAT 5激活合作启动和促进祖细胞B-ALL的其他基因。这些研究应该为STAT 5和Ebf 1/Pax 5/Ikaros网络在导致B-ALL中的作用提供重要的见解，并确定在转化过程中发挥关键作用的其他基因。

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.