The role of ICSBP in the pathogenesis of chronic myeloid leukemia

描述（由申请人提供）：干扰素一致序列结合蛋白（Icsbp或Irf8）是一种具有白血病抑制功能的转录因子。我们确定了一个icsbp靶基因集，该基因集富含控制Fas和/或ßcatenin活性的基因。这是一个有趣的发现，因为Icsbp表达降低、fas耐药和ßcatenin活性增加与慢性髓性白血病（CML）的不良预后和疾病进展相关。白血病干细胞（LSC）对Fas诱导的凋亡不敏感与CML耐药的发生有关，但与Fas或FasL表达降低无关。在CML中，ßcatenin活性升高先于细胞危像（BC），但与Wnt表达或CTNNB1转录无关。我们确定Icsbp抑制编码Fap1的基因；Fas抑制蛋白。我们在Bcr-abl+细胞中发现了Icsbp/ fap1依赖性fas耐药。Fap1也与Apc相互作用，我们在这些细胞中发现了Icsbp/Fap1/ gsk3b依赖性ßcatenin稳定。我们发现GAS2是另一个Icsbp靶基因。Gas2抑制calpain；一种蛋白酶，底物包括ßcatenin， Stat5和Xiap。我们发现Bcr-abl+或Icsbp-/-小鼠骨髓细胞中ßcatenin的Gas2/calpain依赖性增加。Stat5和Xiap在Icsbp-/-细胞中也以Gas2/calpain依赖的方式增加。我们发现Stat5以依赖于Gas2/calpain的方式抑制IRF8启动子。虽然酪氨酸激酶抑制剂（TKI）在大多数CML患者中诱导缓解，但LSC亚群在治疗期间持续存在，阻止TKI治愈。这些研究的假设是Fap1、calpain及其相关通路是消除持续性CML-LSC群体的合理治疗靶点；防止出现明显的耐药性和/或BC。这一假设将通过3个目标来实现；目的1：明确calpain和Fap1在CML TKI耐药发展中的作用。我们将利用原代小鼠骨髓细胞和人CML骨髓样本，在体外研究Fap1和calpain在Fas抗性中的作用。我们将在小鼠CML骨髓移植模型中研究靶向Fap1或calpain对TKI耐药性的影响。目的2：确定calpain和Fap1的合作是否促进CML中的BC。将在体外使用小鼠骨髓细胞和人CML样本研究Fap1和calpain in对ßcatenin活性的调节。靶向Fap1或calpain对BC的影响将在小鼠CML模型中进行体内研究。目的3：探讨calpain活化对CML中icsbp表达的影响。bcr -abl依赖性Stat5激活对Icsbp表达的调节将在骨髓细胞系和原代小鼠骨髓细胞中进行研究。将在小鼠CML模型中研究靶向钙蛋白酶增加Icsbp的方法。这些研究的目的是确定导致LSC持久性的分子机制，从而确定CML中TKI耐药性和/或BC。靶向这些机制可能通过消除LSC来治愈CML。

DESCRIPTION (provided by applicant): The Interferon Consensus Sequence Binding Protein (Icsbp or Irf8) is a transcription factor that functions as a leukemia-suppressor. We identified an Icsbp-target-gene set that is enriched for genes that control Fas and/or ßcatenin activity. This is of interest, because decreased expression of Icsbp, Fas-resistance and increased ßcatenin activity are associated with poor prognosis and disease progression in chronic myeloid leukemia (CML). Insensitivity of leukemia stem cells (LSC) to Fas-induced apoptosis is associated with development of drug resistance in CML, but does not correlate with decreased expression of Fas or FasL. Increased ßcatenin activity precedes blast crisis (BC) in CML, but does not correlate with Wnt expression or CTNNB1 transcription. We determined that Icsbp represses the gene encoding Fap1; a Fas inhibitory protein. We found Icsbp/Fap1-dependent Fas-resistance in Bcr-abl+ cells. Fap1 also interacts with Apc, and we found Icsbp/Fap1/Gsk3b-dependent ßcatenin stabilization in these cells. We identified GAS2 as another Icsbp- target-gene. Gas2 inhibits calpain; a protease with substrates that include ßcatenin, Stat5 and Xiap. We found a Gas2/calpain-dependent increase in ßcatenin in Bcr-abl+ or Icsbp-/- murine bone marrow cells. Stat5 and Xiap are also increased in Icsbp-/- cells in a Gas2/calpain-dependent manner. Xiap contribute to Fas- resistance, and we found that Stat5 represses the IRF8 promoter in a Gas2/calpain-dependent manner. Although tyrosine kinase inhibitors (TKI) induce remission in the majority of CML patients, an LSC sub- population persists during treatment, preventing cure with TKIs. The hypothesis of these studies is that Fap1, calpain and related pathways are rational therapeutic targets to abolish the persistent CML-LSC population; preventing emergence of overt drug-resistance and/or BC. This hypothesis will be pursued by 3 Aims; AIM 1: Define roles of calpain and Fap1 in development of TKI resistance in CML. The roles of Fap1 and calpain in Fas resistance will be studied in vitro using primary murine bone marrow cells and human CML bone marrow samples. The impact of targeting Fap1 or calpain on development of TKI resistance will be investigated in vivo in a murine CML bone marrow transplant model. AIM 2: Determine if cooperation between calpain and Fap1 facilitates BC in CML. Regulation of ßcatenin activity by Fap1 and calpain in will be studied in vitro using murine bone marrow cells and human CML samples. The impact of targeting Fap1 or calpain on BC will be studied in vivo in a murine CML model. AIM 3: Investigate the impact of calpain activation on Icsbp-expression in CML. Regulation of Icsbp expression by Bcr-abl-dependent Stat5 activation will be studied in myeloid cell lines and primary murine bone marrow cells. Targeting calpain to increase Icsbp will be investigated in vivo in murine CML models. The goal of these studies is to identify molecular mechanisms that lead to LSC persistence, and therefore TKI resistance and/or BC in CML. Targeting these mechanisms might cure CML by abolishing the LSC.