RIP1/RIP3-Calpain-Stat3 and NF-kappa B pathways in AML pathogenesis and treatment

RIP1/RIP3-Calpain-Stat3 和 NF-kappa B 通路在 AML 发病机制和治疗中的作用

• 批准号: 9922242
• 负责人: Jiwang Zhang
• 金额: $ 33.95万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922242
• 关键词: Acute Myelocytic Leukemia; Alternative Splicing; Animal Model; Apoptosis; Attenuated; Bortezomib; C-terminal; Calpain; Cells; Chemicals; Cleaved cell; Clinical; Dominant-Negative Mutation; Drug resistance; Electron Transport; Family; Gene Expression; Genetic Transcription; Growth; Half-Life; Hematopoietic stem cells; Human; Inflammatory; Interleukin-1 Receptors; Interleukin-1 beta; Leukemic Cell; MLL gene; Malignant Neoplasms; Mediating; Mitochondria; Molecular; Mutation; NF-kappa B; NPM1 gene; Necrosis; Normal tissue morphology; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Phosphotransferases; Play; Production; Proteasome Inhibitor; Protein Isoforms; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; RIPK1 gene; RIPK3 gene; RNA Splicing; Receptor Activation; Receptor Inhibition; Reporter; Reporting; Repression; Role; Signal Pathway; Signal Transduction; Stat3 Signaling Pathway; TNF gene; Testing; Time; Transactivation; Treatment Protocols; Xenograft Model; Xenograft procedure; acute myeloid leukemia cell; cancer therapy; chemotherapy; cytokine; dosage; improved; in vivo; inhibitor/antagonist; leukemia; leukemia treatment; leukemic stem cell; member; novel; outcome forecast; self-renewal; targeted treatment; transcription factor; tumor; tumor growth; tumorigenesis

Both NF-κB and Stat3 are abnormally activated in leukemic blasts and are implicated in drug-resistance and poor prognosis, suggesting they could be potential targets for therapy. We found that inactivation of both NF-κB and Stat3 signaling pathways synergistically represses self-renewal and drug-resistance in leukemia stem cells (LSCs), suggesting a compensatory role for these two pathways in the pathogenesis of leukemia. Stat3α and Stat3β are two major splicing isoforms. Active Stat3α promotes tumor growth by regulating target gene expression (functions as a transcription factor) and controlling mitochondrial production of ATP and ROS (functions as a regulator of the electron transport chain), while Stat3β lacks a transactivation domain and functions as a dominant-negative to Stat3α. All currently used inhibitors of Stat3 only repress its transcriptional activity without taking consideration of its mitochondrial activity, which might explain why these inhibitors failed to repress leukemia in patients. It was reported that induction of the switch from Stat3α to Stat3β provides a better tumor repressive effect than inhibition of both isoforms. We found that we can induce such a switch by inhibiting the serine/threonine-protein kinases receptor-interacting protein kinase 1 (Rip1) and Rip3. Rip3 and NF-κB are parallel downstream signaling pathways of Rip1, mediating cytokine-induced kinase- dependent and -independent activities of Rip1. We found that a moderate level of activation of Rip1-Rip3 kinase signaling exists in acute myeloid leukemia (AML) cells with MLL1-rearrangement (MLL-r) or NPM1 mutation (NPM1c+). Rip1-Rip3 signaling plays distinct roles in normal hematopoietic stem/progenitor cells（HSPCs）and AML cells. In HSPCs, Rip1-Rip3 signaling mediates TNFα and IL1β-induced necroptosis, while in AML cells, the moderate activation of such signaling is required for maintaining the levels of Stat3α by inhibition of calpain (CAPN), a family of proteolytic enzymes. CAPN reduces Stat3α and enhances Stat3β by specifically cleaving Stat3α protein and also SFRS5, a splice regulator for alternative splicing for Stat3α. Inhibition of Rip1-Rip3 kinase signaling results in depletion of Stat3α and an increase of Stat3β. Our study suggested that, as with co-inhibition of Stat3 and NF-κB, co-inhibition of Rip1-Rip3 signaling and NF-κB also compromises self-renewal of LSCs and sensitizes AML to standard chemotherapy. We want to test our novel combination treatment regimen in primary human AML cells using xenograft models. We also intend to elucidate the molecular mechanisms by which Stat3 and NF-κB regulate self-renewal and drug-resistance in LSCs as well as the molecular mechanism by which Rip3 signaling regulates CAPN-dependent Stat3 isoform switch. The expected results of this study will allow us to determine whether combinations of currently known inhibitors of Rip1/Rip3 and NF-kB signaling could improve treatment for MLL-r and NPM1c+ AML when combined with standard chemotherapy. The mechanistic studies will provide detailed information allowing us to more effectively target the Rip3-CAPN-Stat3 pathway to treat AML.

NF-κB和Stat 3在白血病原始细胞中异常激活并与耐药性有关 预后差，提示它们可能是治疗的潜在靶点。我们发现， NF-κB和Stat 3信号通路协同抑制白血病干细胞自我更新和耐药 细胞（LSC），表明这两个途径在白血病发病机制中的代偿作用。 Stat 3 α和Stat 3 β是两种主要的剪接异构体。活性Stat 3 α通过调节肿瘤生长 靶向基因表达（作为转录因子发挥作用）并控制线粒体产生ATP， ROS（作为电子传递链的调节剂），而Stat 3 β缺乏反式激活结构域， 对Stat 3 α起显性负调控作用。所有目前使用的Stat 3抑制剂仅抑制其转录 活性而不考虑其线粒体活性，这可能解释了为什么这些抑制剂失败 来抑制白血病患者。据报道，诱导从Stat 3 α到Stat 3 β的转换提供了一种新的途径。 比抑制两种亚型更好的肿瘤抑制效果。我们发现，我们可以诱导这样的开关， 抑制丝氨酸/苏氨酸蛋白激酶受体相互作用蛋白激酶1（Rip 1）和Rip 3。 Rip 3和NF-κB是Rip 1的下游信号传导通路，介导酪氨酸诱导的激酶- Rip 1的依赖和独立活动。我们发现Rip 1-Rip 3激酶的中等水平激活 在具有MLL 1重排（MLL-r）或NPM 1突变的急性髓性白血病（AML）细胞中存在信号传导 （NPM1c+）。Rip 1-Rip 3信号传导在正常造血干/祖细胞（HSPC）中起着不同的作用， AML细胞。在HSPC中，Rip 1-Rip 3信号转导介导TNFα和IL 1 β诱导的坏死性凋亡，而在AML细胞中，Rip 1-Rip 3信号转导介导TNF α和IL 1 β诱导的坏死性凋亡。 通过抑制钙蛋白酶来维持Stat 3 α的水平需要这种信号传导的适度激活 （CAPN），蛋白水解酶家族。CAPN通过特异性切割Stat 3 α和Stat 3 β来降低Stat 3 α和增强Stat 3 β Stat 3 α蛋白和SFRS 5，一种Stat 3 α选择性剪接的剪接调节因子。Rip 1-Rip 3激酶的抑制 信号转导导致Stat 3 α的耗竭和Stat 3 β的增加。我们的研究表明，与共同抑制一样， Stat 3和NF-κB的共同抑制，Rip 1-Rip 3信号传导和NF-κB的共同抑制也损害了LSC的自我更新， 使AML对标准化疗敏感。我们想在原发性肝癌中测试我们的新型联合治疗方案， 使用异种移植模型的人AML细胞。我们还打算阐明Stat 3 NF-κ B和NF-κB调节LSC的自我更新和耐药性，以及其分子机制 Rip 3信号调节CAPN依赖性Stat 3亚型开关。 这项研究的预期结果将使我们能够确定目前已知的 Rip 1/Rip 3和NF-kB信号传导抑制剂联合使用可改善MLL-r和NPM 1c + AML的治疗 进行标准化疗机制研究将提供详细的信息，使我们能够更多地 有效靶向Rip 3-CAPN-Stat 3通路以治疗AML。