Molecular Mechanisms of Disease Progression in Myeloid Malignancy

描述(由申请人提供)： 干扰素一致序列结合蛋白(IcsBP)是一种干扰素调节性转录因子，具有肿瘤抑制因子(也称为IRF8)的功能。在这个优点回顾项目中，我们使用高通量筛选方法来识别介导肿瘤抑制活性的IcsBP靶基因。我们发现了一个IcsBP靶基因集，它富含控制Fas诱导的细胞凋亡和/或bcatenin活性的基因。这一点很有意义，因为IcsBP表达降低、Fas抵抗和bcatenin活性增加与慢性髓系白血病(CML)预后不良有关。肿瘤/白血病干细胞(CSC/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/FasL的降低无关。CML-CSC/LSC中连接素活性升高先于急变期(BC)进展，但与Wnt表达或CTNNB1转录无关。针对BCR-ABL(慢性粒细胞白血病癌基因)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人口在缓解期扩大，阻止了TKI的治愈。CSC/LSC的持续性被认为是由于Fas和/或bcatenin的失调所致。在之前的资助期间，我们发现了解释慢性粒细胞白血病Fas耐药和bcatenin活性增加的IcsBP靶基因。我们发现IcsBP抑制编码Fap1的PTPN13基因。FAP1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/FAP1依赖的Fas抵抗。FAP1还与APC相互作用，我们发现在这些细胞中Gsk3b受到抑制，bcatenin得到稳定。我们鉴定Gas2是另一个与IcsBP相关的靶基因。GAS2抑制钙蛋白酶，这是一种丝氨酸蛋白酶，底物包括bate-nin、STAT3、Stat5和XIAP。我们发现，在bcr-abl+或IcsBP-/-小鼠骨髓细胞中，依赖于Gas2的calain活性降低，bcatenin增加。在IcsBP-/-细胞中，STAT3、Stat5和XIAP蛋白也以依赖于Gas2/calain的方式增加。有趣的是，我们发现Stat5以Gas2/calain依赖的方式抑制IRF8启动子。XIAP抑制caspase3，促进Fas耐药。我们鉴定了编码Nore1的基因RASSF5是另一个IcsBP靶基因。Nore1激活Mst1；Mst1是一种促进半胱氨酸天冬氨酸氨基转移酶(Caspase)裂解的激酶，因此Fas诱导细胞凋亡。这些研究的假设是，肿瘤抑制基因IcsBP的表达减少会导致Fas抵抗和bcatenin活性增加。我们还假设IcsBP靶基因或同源通路将是防止CML显性耐药和进展为BC的合理治疗靶点。这一假设将通过三个目标来实现：目标1：确定依赖于IcsBP的NORE1和XIAP表达在CML-LSC扩展中的作用。Nore1a或XIAP在Fas耐药中的作用以及与Fap1的协同作用将在CML-LSC中进行研究。靶向这些通路对在活体CML小鼠模型中LSC扩张的影响将被确定。目的2：确定IcsBP在CML-LSC中是否通过抑制GSK3或增加STAT3而促进疾病的进展。GSK3和STAT3对bcatenin活性的贡献将在体外和体内进行研究，如上所述。靶向这些通路对疾病进展的影响将在体内CML小鼠模型中进行研究。目的3：探讨Stat5和Aml1在IRF8转录和白血病发生中的作用。将在髓系白血病细胞系和原代小鼠骨髓细胞的研究中研究依赖于BCR-ABL的Stat5激活对IRF8转录调节的影响。靶向calain降低Stat5蛋白稳定性，从而增加IcsBP表达的作用，将在体外和体内模型中进行研究。这些研究的目的是确定IcsBP抑制肿瘤活性的分子机制。针对这些机制，可能会通过取消CSC/LSC来治愈CML，并建议对Fas和bcatenin调节失调的其他形式的癌症进行治疗。

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.