AML1/Runx1 Is a Cytoplasmic Attenuator of NF-Kb Signaling: Implication in Pathogenesis and Targeted Therapy of AML1-Related Leukemia.
AML1/Runx1 Is a Cytoplasmic Attenuator of NF-Kb Signaling: Implication in Pathogenesis and Targeted Therapy of AML1-Related Leukemia.
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DOI：
10.1182/blood.v114.22.1962.1962
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发表时间：
2009-11
期刊：
影响因子：
20.3
通讯作者：
M. Nakagawa;M. Shimabe;Nahoko Nishimoto;N. Watanabe-Okochi;M. Ichikawa;Y. Nannya;Y. Imai;M. Kurokawa
中科院分区：
文献类型：
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作者：
M. Nakagawa;M. Shimabe;Nahoko Nishimoto;N. Watanabe-Okochi;M. Ichikawa;Y. Nannya;Y. Imai;M. Kurokawa
简介：AML1/Runx1是人类白血病染色体异常最常见的靶点之一。在白血病或骨髓增生异常综合征（MDS）患者中也有由点突变引起的AML1功能损伤的报道。然而，AML1功能损伤导致白血病发生的分子基础尚不清楚。在本研究中，我们阐明了AML1缺失引起的信号通路失调。结果：我们利用Cre-ER系统比较了AML1条件缺失与正常KSL细胞的基因表达谱，以寻找AML1的直接靶点。利用分子特征数据库（MSigDB）进行基因集富集分析（GSEA），明确了NF-kB靶基因在AML1缺陷细胞中的表达增强。此外，NF-kB抑制剂可减弱AML1条件敲除（cKO）小鼠骨髓细胞的集落形成活性。这些数据表明，在AML1缺陷小鼠的干细胞/祖细胞中NF-kB信号通路异常激活。NF-kB是一种转录因子，参与许多生理现象，包括增殖、生存和炎症。由于有报道称NF-kB信号的失调控激活导致了包括血液系统恶性肿瘤在内的许多类型的肿瘤，我们假设aml1介导的NF-kB信号抑制缺失导致造血细胞的恶性转化。NF-kB的主要成分之一p65与IkB一起在细胞质中保持稳定状态。一旦收到细胞表面受体如TNF-a受体的刺激信号，IkB被IKK复合物磷酸化，随后通过泛素-蛋白酶体途径降解，导致p65的核易位和NF-kB靶基因的反激活。首先，我们发现AML1抑制p65的核易位，并且在AML1缺陷细胞中p65的核定位增强，这被NF-kB抑制剂取消。此外，AML1抑制p65 536丝氨酸的磷酸化，这对其激活是重要的。我们发现AML1与IKK复合物物理相互作用，从而抑制其激酶活性，这解释了AML1抑制NF-kB信号传导的机制基础。AML1抑制IKK激酶活性可抑制p65核易位和NF-kB靶基因的激活。接下来，我们研究了白血病相关的AML1突变体如何影响NF-kB信号传导。值得注意的是，在MDS中发现的AML1 D171N突变体既没有抑制p65的核易位，也没有减弱IKK复合物的激酶活性。t白血病中产生的AML1/ETO也获得了类似的结果（8;21）。与另一种白血病相关融合蛋白MLL/ENL永生化的小鼠骨髓细胞相比，AML1/ETO永生化的小鼠骨髓细胞显示p65的核定位增强。事实上，AML1/ETO永生化细胞对NF-kB抑制剂介导的生长抑制更敏感，这表明NF-kB信号在AML1/ETO转化中起着关键作用。为了验证AML1/ETO在人造血细胞中激活NF-kB信号，我们分析了Valk等人在硅芯片上报道的基因表达数据。我们发现NF-kB信号在aml1相关白血病患者中明显激活。这些结果表明，AML1功能损伤诱导的NF-kB信号异常激活可能通过增殖信号促进白血病的发展。结论：我们发现AML1是NF-kB信号通路的细胞质衰减剂。由遗传破坏引起的AML1功能损伤通过改变IKK的动力学活性导致NF-kB信号的明显激活。这种异常激活可能在aml1相关白血病和MDS的发病机制中起核心作用。因此，NF-kB信号是aml1相关血液病分子靶向治疗的有吸引力的候选者之一。披露：无相关利益冲突需要申报。
Abstract 1962 Poster Board I-985 Introduction: AML1/Runx1 is one of the most frequent targets of chromosomal abnormalities in human leukemia. Functional impairment of AML1 caused by point mutation is also reported in patients with leukemia or myelodysplastic syndrome (MDS). However, molecular basis for leukemogenesis caused by functional impairment of AML1 is still elusive. In this study, we clarified the deregulated signaling pathway induced by loss of AML1. Results: To find the direct target of AML1, we compared gene expression profile between AML1-conditionally deleted and normal KSL cells using Cre-ER system. Gene set enrichment analysis (GSEA) using molecular signature database (MSigDB) clarified enhanced expression of NF-kB target genes in AML1 deficient cells. In addition, NF-kB inhibitor attenuated the enhanced colony forming activity of bone marrow cells from AML1 conditional knockout (cKO) mice. These data indicate the aberrant activation of NF-kB signaling pathway in stem/progenitor cells of AML1 deficient mice. NF-kB is a transcription factor which is involved in many physiological phenomena including proliferation, survival, and inflammation. Because deregulated activation of NF-kB signaling has been reported to be responsible for many types of tumors including hematological malignancies, we assumed that lack of AML1-mediated suppression of NF-kB signaling lead to malignant transformation of hematopoietic cells. p65, one of the major components of NF-kB stays in cytoplasm with IkB in a steady state. Once receiving stimulating signals from cell surface receptors such as TNF-a receptor, IkB is phosphorylated by IKK complex and subsequently degraded through the ubiquitin-proteasome pathway, resulting in nuclear translocation of p65 and transactivation of NF-kB target genes. First, we found that AML1 inhibits nuclear translocation of p65 and that nuclear localization of p65 is enhanced in AML1 deficient cells, which is cancelled by NF-kB inhibitors. In addition, AML1 inhibited p65 phosphorylation at serine 536, which is important for its activation. We found that AML1 physically interacts with IKK complex and thus suppresses its kinase activity, which accounts for a mechanistic basis for inhibition of NF-kB signaling by AML1. Suppression of IKK kinase activity by AML1 results in inhibition of both nuclear translocation of p65 and activation of NF-kB target genes. Next, we examined how leukemia-related AML1 mutants affect NF-kB signaling. Remarkably, AML1 D171N mutant found in MDS neither inhibited nuclear translocation of p65 nor attenuated the kinase activity of IKK complex. Similar results were obtained with AML1/ETO generated in leukemia with t(8;21). Mouse bone marrow cells immortalized by AML1/ETO showed enhanced nuclear localization of p65 compared with those immortalized by MLL/ENL, another leukemia-related fusion protein. Indeed, AML1/ETO immortalized cells are more sensitive to NF-kB inhibitor-mediated growth suppression, indicating a critical role of NF-kB signaling in transformation by AML1/ETO. To verify the activation of NF-kB signaling by AML1/ETO in human hematopoietic cells, we analyzed the gene expression data reported by Valk et al. in silico. We found that NF-kB signaling is distinctly activated in AML1-related leukemia patients. These results suggest that aberrant activation of NF-kB signaling induced by functional impairment of AML1 may contribute to the development of leukemia via proliferation signals. Conclusions: We found that AML1 is a cytoplasmic attenuator of NF-kB signaling pathway. Functional impairment of AML1 caused by genetic disruption results in distinct activation of NF-kB signaling by altering IKK kinetic activity. This aberrant activation may play a central role in pathogenesis of AML1-related leukemia and MDS. Therefore, NF-kB signaling is one of the attractive candidates for molecular targeted therapy against AML1-related hematological disorders. Disclosures: No relevant conflicts of interest to declare.