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STAT3在白血病爆炸中都被激活，并且在药物抗药性中暗示 预后不良，表明它们可能是治疗的潜在靶标。我们发现两者都失活 NF-κB和STAT3信号通路协同反映了白血病茎中的自我更新和抗药性 细胞（LSC），表明这两种途径在白血病的发病机理中具有补偿性作用。 STAT3α和STAT3β是两个主要的剪接同工型。主动STAT3α通过调节促进肿瘤生长 靶基因表达（充当转录因子），并控制ATP的线粒体产生 ROS（充当电子传输链的调节剂），而STAT3β缺乏反式激活结构域和 充当STAT3α的主要阴性。所有目前使用的STAT3抑制剂仅抑制其转录 活动而无需考虑其线粒体活动，这可能解释了为什么这些抑制剂失败 反映患者的白血病。据报道，从STAT3α到Stat3β的转换提供了 比两种同工型的抑制更好的肿瘤反射效应。我们发现我们可以通过 抑制丝氨酸/苏氨酸蛋白激酶受体相互作用蛋白激酶1（RIP1）和RIP3。 RIP3和NF-κB是RIP1的平行下游信号通路，介导细胞因子诱导的激酶 - RIP1的依赖和非依赖性活动。我们发现RIP1-RIP3激酶的现代激活水平 信号传导存在于具有MLL1重晶（MLL-R）或NPM1突变的急性髓样白血病（AML）细胞中 （NPM1C+）。 RIP1-RIP3信号传导在正常造血干/祖细胞（HSPC）和 AML细胞。在HSPC中，RIP1-RIP3信号传导介导TNFα和IL1β诱导的坏死性，而在AML细胞中， 通过抑制钙蛋白酶来维持STAT3α的水平需要中等的激活 （CAPN），一个蛋白水解酶家族。 CAPN通过特异性切割来降低STAT3α并增强STAT3β STAT3α蛋白以及SFRS5，是用于STAT3α的替代剪接的剪接调节剂。抑制RIP1-RIP3激酶 信号导致STAT3α的耗竭和STAT3β的增加。我们的研究表明，与共同抑制一样 STAT3和NF-κB，RIP1-RIP3信号和NF-κB的共抑制也损害了LSC的自我更新和 感觉AML对标准化疗。我们想测试我们的新型组合治疗方案 人类AML细胞使用色谱模型。我们还打算阐明STAT3的分子机制 NF-κB调节LSC中的自我更新和耐药性以及分子机制 RIP3信号传导调节CAPN依赖性STAT3同工型开关。 这项研究的预期结果将使我们能够确定当前已知的组合是否 当组合时，RIP1/RIP3和NF-KB信号传导的抑制剂可以改善MLL-R和NPM1C+ AML的处理 与标准化疗。机械研究将提供详细的信息，使我们能够更多 有效地靶向RIP3-CAPN-STAT3途径以治疗AML。