Molecular Mechanisms of Disease Progression in Myeloid Malignancy

描述（由申请人提供）： 干扰素共有序列结合蛋白（Icsbp）是一种干扰素调节转录因子，其作为肿瘤抑制因子（也称为Irf 8）发挥作用。在这个Merit Review项目中，我们使用高通量筛选方法来鉴定介导肿瘤抑制活性的ICSBP靶基因。我们确定了一个ICSBP靶基因集，该基因集富含控制Fas诱导的细胞凋亡和/或bcatenin活性的基因。这是令人感兴趣的，因为减少Icsbp表达，Fas耐药，和增加bcatenin活性与慢性粒细胞白血病（CML）预后不良。癌症/白血病干细胞（CSC/LSC）对Fas诱导的凋亡不敏感与CML耐药的发展有关，但与Fas/FasL降低无关。增加？CML-CSC/LSC中的连环蛋白活性先于急变（BC）的进展，但与Wnt表达或CTNNB 1转录无关。靶向Bcr-abl（CML癌基因）的酪氨酸激酶抑制剂（TKI）在大多数患者中诱导缓解。然而，CSC/LSC人群在缓解期间扩大，阻碍了TKI的治愈。假设CSC/LSC持续性是由于Fas和/或bcatenin的失调。 在上一个资助期间，我们确定了ICSBP靶基因，解释了CML中Fas耐药和bcatenin活性增加。我们发现，Icsbp抑制PTPN 13;编码Fap 1的基因。Fap 1与Fas相互作用并抑制Fas，我们发现在表达Bcr-abl的细胞中存在Icsbp/Fap 1依赖的Fas抗性。Fap 1也与Apc相互作用，我们发现在这些细胞中Gsk 3b被抑制，bcatenin被稳定。我们确定GAS 2是另一个相关的ICSBP靶基因。Gas 2抑制钙蛋白酶;一种丝氨酸蛋白酶，其底物包括bcate-nin、Stat 3、Stat 5和Xip。我们发现在Bcr-abl+或Icsbp-/-小鼠骨髓细胞中钙蛋白酶活性的Gas 2依赖性降低和bcatenin的增加。Stat 3、Stat 5和Xip蛋白在Icsbp-/-细胞中也以Gas 2/钙蛋白酶依赖性方式增加。有趣的是，我们发现Stat 5以Gas 2/钙蛋白酶依赖的方式抑制IRF 8启动子。Xip抑制半胱天冬酶3，有助于Fas抗性。我们鉴定了编码Nore 1的基因RASSF 5作为另一个ICSBP靶基因。Nore 1激活Mst 1;一种促进半胱天冬酶切割并因此促进Fas诱导的细胞凋亡的激酶。 这些研究的假设是，肿瘤抑制因子Icsbp的表达减少导致Fas抗性和bcatenin活性增加。我们还假设ICSBP靶基因或同源通路将是合理的治疗靶点，以防止CML的明显耐药和进展为BC。这一假设将通过三个目标来实现：目标1：定义Nore 1和Xip的ICSBP依赖性表达在CML-LSC扩增中的作用。将在CML-LSC中研究Nore 1a或Xip对Fas抗性的贡献以及与Fap 1的协同作用。将确定靶向这些途径对体内CML鼠模型中LSC扩增的影响。目的2：确定ICsbp是否通过抑制CML-LSC中的Gsk 3或增加Stat 3而促进疾病进展。如上所述，将在体外和体内研究Gsk 3和Stat 3对bcatenin活性的贡献。将在体内CML鼠模型中研究靶向这些途径对疾病进展的影响。目的3：研究Stat 5和Aml 1在IRF 8转录和白血病发生中的作用。将在髓性白血病细胞系和原代鼠骨髓细胞的研究中研究Bcr-abl依赖性Stat 5激活对IRF 8转录的调控。将在体外和体内的鼠模型中研究靶向钙蛋白酶降低Stat 5蛋白稳定性的作用，从而增加Icsbp表达。 这些研究的目的是确定Icsbp肿瘤抑制活性的分子机制。靶向这些机制可能通过消除CSC/LSC而导致CML的治愈，并建议治疗具有Fas和bcatenin失调的其他形式的癌症的方法。

DESCRIPTION (provided by applicant): The Interferon Consensus Sequence Binding Protein (Icsbp) is an interferon regulatory transcription factor that functions as a tumor-suppressor (also referred to as Irf8). During this Merit Review project, we used high throughput screening approaches to identify Icsbp-target-genes that mediate tumor-suppressor activity. We identified an Icsbp-target-gene set that is enriched for genes which control Fas-induced apoptosis and/or bcatenin activity. This is of interest, because decreased Icsbp-expression, Fas-resistance, and increased bcatenin activity are associated with poor prognosis in chronic myeloid leukemia (CML). Insensitivity of cancer/leukemia stem cells (CSC/LSC) to Fas-induced apoptosis is associated with development of drug resistance in CML, but does not correlate with decreased Fas/FasL. Increased ¿catenin activity in CML- CSC/LSC precedes progression to blast crisis (BC), but does not correlate with Wnt expression or CTNNB1 transcription. Tyrosine kinase inhibitors (TKI) that target Bcr-abl (the CML oncogene) induce remission in the majority of patients. However, the CSC/LSC population expands during remission, preventing cure with TKIs. CSC/LSC persistence is hypothesized to be due to dysregulation of Fas and/or bcatenin. During the previous funding period, we identified Icsbp-target-genes that explain Fas-resistance and increased bcatenin activity in CML. We found that Icsbp represses PTPN13; the gene encoding Fap1. Fap1 interacts with and inhibits Fas, and we found Icsbp/Fap1-dependent Fas-resistance in cells expressing Bcr-abl. Fap1 also interacts with Apc, and we found inhibition of Gsk3b and stabilization of bcatenin in these cells. We identified GAS2 as another relevant Icsbp-target-gene. Gas2 inhibits calpain; a serine protease with substrates that include bcate-nin, Stat3, Stat5 and Xiap. We found a Gas2-dependent decrease in calpain activity and increase in bcatenin in Bcr-abl+ or Icsbp-/- murine bone marrow cells. Stat3, Stat5 and Xiap proteins are also increased in Icsbp-/- cells in a Gas2/calpain-dependent manner. Interestingly, we found that Stat5 represses the IRF8 promoter in a Gas2/calpain-dependent manner. Xiap inhibits caspase 3, contributing to Fas-resistance. We identified RASSF5, the gene encoding Nore1, as another Icsbp-target-gene. Nore1 activates Mst1; a kinase that facilitates caspase cleavage and therefore Fas-induced apoptosis. The hypothesis of these studies is that decreased expression of the tumor-suppressor Icsbp results in Fas-resistance and increased bcatenin activity. We also hypothesize that Icsbp-target-genes or cognate pathways would be rational therapeutic targets to prevent overt drug resistance and progression to BC in CML. This hypotheses will be pursued through three Aims; AIM 1: Define the role of Icsbp-dependent expression of Nore1 and Xiap in CML-LSC expansion. The contribution of Nore1a or Xiap to Fas-resistance and cooperation with Fap1 will be studied in CML-LSC. The impact of targeting these pathways on LSC expansion in an in vivo CML murine model will be determined. AIM 2: Determine if Icsbp contributes to disease progression by inhibiting Gsk3¿ or increasing Stat3 in CML-LSC. Contribution of Gsk3¿ and Stat3 to bcatenin activity will be studied in vitro and in vivo, as above. The effect of targeting these pathways on disease progression will be studied in an in vivo CML murine model. AIM 3: Identify the roles of Stat5 and Aml1 in IRF8 transcription and leukemogenesis. IRF8 transcriptional regulation by Bcr-abl-dependent Stat5 activation will be studied in studies with myeloid leukemia cell lines and with primary murine bone marrow cells. The role of targeting calpain to decrease Stat5 protein stability, and thereby increase Icsbp expression, will be investigated in murine models in vitro and in vivo. The goal of these studies is to identify molecular mechanisms for Icsbp tumor suppressor activity. Targeting these mechanisms may lead to cure in CML by abolishing the CSC/LSC, and suggest therapeutic approaches to other forms of cancer with dysregulation of Fas and bcatenin.