The role of ICSBP in the pathogenesis of chronic myeloid leukemia

描述(申请人提供)：干扰素共识序列结合蛋白(IcsBP或IRF8)是一种转录因子，具有抑制白血病的功能。我们确定了一个IcsBP靶基因集，该基因集富含控制Fas和/或？连环蛋白活性的基因。这一点很有意义，因为IcsBP、Fas-抗性的表达降低和çcatenin活性的增加与慢性髓系白血病(CML)的不良预后和疾病进展有关。白血病干细胞(LSC)对Fas诱导的细胞凋亡不敏感与CML耐药的发生有关，但与Fas或FasL表达降低无关。在慢性粒细胞白血病发生急变期(BC)之前，B连环蛋白活性升高，但与Wnt表达或CTNNB1转录无关。我们确定IcsBP抑制编码Fap1的基因；Fap1是一种Fas抑制蛋白。我们在BCR-ABL+细胞中发现了IcsBP/Fap1依赖的Fas抵抗。FAP1也与APC相互作用，我们在这些细胞中发现了IcsBP/Fap1/Gsk3b依赖的？catenin稳定。我们鉴定Gas2是另一个IcsBP靶基因。Gas2抑制钙蛋白酶，这是一种底物包括？连环蛋白、STAT5和XIAP的蛋白酶。我们发现，在BCR-ABL+或IcsBP-/-小鼠骨髓细胞中，Gas2/calain依赖的çcatenin表达增加。在IcsBP-/-细胞中，Stat5和XIAP也以依赖于Gas2/calain的方式增加。XIAP参与Fas耐药，我们发现Stat5以Gas2/calain依赖的方式抑制IRF8启动子。尽管酪氨酸激酶抑制剂(TKI)可使大多数CML患者缓解，但LSC亚群在治疗过程中持续存在，阻碍了TKI的治愈。这些研究的假设是Fap1、calain和相关的通路是合理的治疗靶点，可以消除持续的CML-LSC群体，防止出现明显的耐药和/或BC。这一假说将通过三个目标来实现：目标1：确定Calain和Fap1在慢性粒细胞白血病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calain在Fas耐药中的作用。靶向Fap1或calain对TKI耐药发展的影响将在小鼠CML骨髓移植模型中进行体内研究。目的2：确定Calain和Fap1之间的协同作用是否促进慢性粒细胞白血病的BC。我们将使用小鼠骨髓细胞和人类慢性粒细胞白血病样本在体外研究Fap1和Calain对？连环蛋白活性的调节。靶向Fap1或calain对BC的影响将在小鼠CML模型中进行活体研究。目的：探讨钙蛋白酶激活对慢性粒细胞白血病IcsBP基因表达的影响。BCR-ABL依赖的Stat5激活对IcsBP表达的调节将在髓系细胞和原代小鼠骨髓细胞中进行研究。靶向Calain以增加IcsBP将在小鼠CML模型中进行体内研究。这些研究的目的是确定导致慢性粒细胞白血病中LSC持续存在，从而导致TKI抵抗和/或BC的分子机制。针对这些机制，可能会通过废除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.