The role of ICSBP in the pathogenesis of chronic myeloid leukemia

描述（申请人提供）：干扰素共有序列结合蛋白（Icsbp或Irf 8）是一种作为白血病抑制因子发挥功能的转录因子。我们发现了一个 ICSBP-靶基因集，富集控制Fas和/或？连环蛋白活性这是 感兴趣的，因为减少表达的Icsbp，Fas抗性和增加？连环蛋白活性与慢性粒细胞白血病（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。增加？在CML中，连环蛋白活性先于急变（BC），但与Wnt表达或CTNNB 1转录无关。我们确定ICSbp抑制编码Fas抑制蛋白Fap 1的基因。我们在Bcr-abl+细胞中发现了Icsbp/Fap 1依赖的Fas抗性。Fap 1也与Apc相互作用，我们发现Icsbp/Fap 1/Gsk 3b依赖？这些细胞中的连环蛋白稳定化。我们将GAS 2鉴定为另一个Icsbp靶基因。Gas 2抑制钙蛋白酶;蛋白酶与底物，包括？catenin、Stat 5和Xip。我们发现Gas 2/钙蛋白酶依赖性增加？Bcr-abl+或Icsbp-/-鼠骨髓细胞中的连环蛋白。Stat 5和Xip也以Gas 2/钙蛋白酶依赖性方式在Icsbp-/-细胞中增加。Xip参与Fas抗性，我们发现Stat 5以Gas 2/钙蛋白酶依赖性方式抑制IRF 8启动子。 尽管酪氨酸激酶抑制剂（TKI）可诱导大多数CML患者缓解，但LSC亚群在治疗期间持续存在，阻碍了TKI的治愈。这些研究的假设是Fap 1、钙蛋白酶和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个目的来实现;目的1：确定钙蛋白酶和Fap 1在CML中TKI耐药发展中的作用。将使用原代鼠骨髓细胞和人CML骨髓样品在体外研究Fap 1和钙蛋白酶在Fas抗性中的作用。将在小鼠CML骨髓移植模型中研究靶向Fap 1或钙蛋白酶对TKI耐药性发展的影响。目的2：确定钙蛋白酶和Fap 1之间的合作是否有助于CML中的BC。监管？将使用鼠骨髓细胞和人CML样品在体外研究Fap 1和钙蛋白酶的连环蛋白活性。将在鼠CML模型中体内研究靶向Fap 1或钙蛋白酶对BC的影响。目的3：研究钙蛋白酶激活对慢性粒细胞白血病（CML）ICSBP-1表达的影响。将在骨髓细胞系和原代鼠骨髓细胞中研究Bcr-abl依赖性Stat 5活化对Icsbp表达的调节。将在鼠CML模型中体内研究靶向钙蛋白酶以增加Icsbp。 这些研究的目的是确定导致LSC持续存在的分子机制，从而确定CML中TKI耐药和/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 ?catenin activity. This is of interest, because decreased expression of Icsbp, Fas-resistance and increased ?catenin 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 ?catenin 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 ?catenin stabilization in these cells. We identified GAS2 as another Icsbp- target-gene. Gas2 inhibits calpain; a protease with substrates that include ?catenin, Stat5 and Xiap. We found a Gas2/calpain-dependent increase in ?catenin 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 ?catenin 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.