Aberrant glycolysis as a driver of mutant HSPC expansion in clonal hematopoiesis

项目摘要/摘要 这项建议的目的是确定靶向克隆造血(CH)的新方法。中国是 特点是选择性扩增含有造血干细胞和祖细胞(HSPC)的克隆 TET2和DNMT3A等基因突变。这些HSPC继而产生致病突变的髓系细胞 这可能会导致几种与衰老相关的并发症，包括心血管疾病(CVD)和各种原因 死亡率。突变HSPC池的扩大也可能增加血液系统恶性肿瘤的风险。 在老年人和既往有遗传毒性暴露、吸烟的个人中，CH的患病率显著升高 病史和/或慢性炎症性疾病。这些情况与长期心烦意乱有关 生理动态平衡，以高度炎症为特征。理解机制(S)推动 突变型HSPC的选择性扩增对于优先选择可以抑制CH的治疗靶点至关重要。 我们应用的一个中心前提是CH产生于炎症性疾病之间有针对性的相互作用 信号和改变的新陈代谢程序，支持能量需求，从而优先 CHHSPC的扩容。利用小鼠作为代表人类CH主要特征的模型，我们的初步研究 资料显示，CHHSPC表现出转录因子HIF-1异常糖酵解水平升高 与野生型HSPC相比，其代谢和ATP产量增加。我们发现炎性细胞因子， 尤其是IL-1能显著增强CHHSPC的增殖、糖酵解代谢和HIF-1活性。令人惊讶的是， NLRP3炎性小体抑制剂OLT-1177对CH小鼠的治疗作用 IL-1能有效抑制CH.我们假设CH是一种相互依赖的机制的结果，在这种机制中 NLRP3介导的IL-1产生增强HIF-1及其下游糖酵解活性以支持CHHSPC 扩张。我们认为，NLRP3的抑制破坏了这一回路，限制了CH HSPC的扩张。 为了解决这一机制，我们提出了两个具体目标：1)我们将表征 TET2/、TET2/和DNMT3aR878H/CH HSPC的体内质谱学和流式细胞术研究 新陈代谢和葡萄糖流量分析。我们还将确定CH HSPC对HIF-1的依赖程度 和糖酵解的能量需求；2)我们将评估NLRP3在促进异常方面的需求 CHHSPC的糖酵解活性和/或优先扩张。我们将使用分子遗传学的方法来 探讨HIF-1和NLRP3调节CHHSPC代谢及促进其代谢的作用和机制 体内扩张。使用我们的非条件性骨髓移植小鼠模型，我们将验证我们的 OLT-1177抑制NLRP3的药理作用机制及NLRP3是否阻断 抑制HSPC的扩张、异常代谢活动和炎性免疫细胞的聚集 心脏和其他组织。这项工作有可能将CH重新定位为一种具有治疗靶向的代谢 表型，显著改善了老年人等CH高发人群的健康状况。

PROJECT SUMMARY / ABSTRACT The objective of this proposal is to identify new approaches for targeting clonal hematopoiesis (CH). CH is characterized by selective expansion of hematopoietic stem and progenitor cell (HSPC) clones harboring mutations in genes such as TET2 and DNMT3A. These HSPC in turn produce pathogenic mutant myeloid cells that can contribute to several aging-related co-morbidities including cardiovascular disease (CVD) and all-cause mortality. An expanded mutant HSPC pool may also contribute to increased risk of hematological malignancies. CH prevalence is significantly elevated in the elderly and individuals with prior genotoxic exposures, smoking history, and/or chronic inflammatory disease. These conditions are associated with chronically perturbed physiological homeostasis, characterized by hyper-inflammation. Understanding the mechanism(s) promoting the selective expansion of mutant HSPC is crucial for prioritizing therapeutic targets that can suppress CH. A central premise of our application is that that CH arises from a targetable interplay between inflammatory signals and altered metabolic programming that supports the energetic needs and thereby the preferential expansion of CH HSPC. Using the mouse as a model representing key features of human CH, our preliminary data show that CH HSPC exhibit increased levels of the transcription factor Hif-1 aberrant glycolytic metabolism and increased ATP production relative to wild-type HSPC. We find that inflammatory cytokines, particularly IL-1 strongly potentiates CH HSPC expansion, glycolytic metabolism and Hif-1 activity. Strikingly, treatment of CH mice with OLT-1177, an NLRP3 inflammasome inhibitor that prevents cleavage and activation of IL-1 potently suppresses CH. We hypothesize that CH is the result of an interdependent mechanism in which NLRP3-mediated IL-1 production potentiates Hif-1 and downstream glycolytic activity to support CH HSPC expansion. We propose that NLRP3 inhibition disrupts this circuit, limiting expansion of CH HSPC. To address the mechanism, we propose two Specific Aims: 1) we will characterize the metabolic features of Tet2/, Tet2+/ and Dnmt3aR878H/+ CH HSPC using in vivo mass spectrometry- and flow cytometry-based analyses of metabolism and glucose flux. We will also identify the extent to which CH HSPC rely upon Hif-1 and glycolysis for their energetic needs; 2) we will evaluate the requirement for NLRP3 in promoting aberrant glycolytic activity and/or preferential expansion of CH HSPC. We will use molecular genetics approaches to assess the role and mechanism of Hif-1 and NLRP3 in regulating CH HSPC metabolism and promoting their expansion in vivo. Using our non-conditioned adoptive BM transfer mouse model of CH, we will validate our mechanism using pharmacological inhibition of NLRP3 with OLT-1177 and establish whether NLRP3 blockade suppresses HSPC expansion, aberrant metabolic activity and accumulation of inflammatory immune cells in the heart and other tissues. This work has the potential to re-frame CH as a therapeutically targetable metabolic phenotype, significantly improving health among groups with high prevalence of CH such as the elderly.