Inflammation as determinant of clonal selection in MPN progression

背景资料。我们目前对bcr/Abl阴性骨髓增殖性肿瘤发病机制的认识 (MPN)涉及针对JAK2、MPL或CALR的限制性MPN驱动程序突变的识别，以及 炎性微环境，其特征是细胞因子和 趋化因子。然而，炎症在MPN进展中的作用尚未得到解决。同样， 导致MPN炎性微环境的原因尚未明确。有趣的是， 最近观察到的不确定电位克隆性造血(CHIP)表现出累积性突变 这与衰老有关，强烈表明炎症在克隆选择和疾病进展中发挥了作用。 初步结果。我们已经建立了一种慢性炎症的动物模型，在该模型中Notch的丢失 BM内皮细胞(Tie2CreerRBPJKO小鼠)中的信号诱导microRNA miR-155上调， 增加的核因子-kB信号和促炎细胞因子在8-12个月内导致MPN样病。 重要的是，这种微环境炎症环境极大地加速了造血细胞的扩张。 含有JAK2V617F或TET2-/-突变。假设。我们提出了一个模型，在该模型中：a)长期暴露 将JAK2V616F或TET2-/-造血细胞移植到慢性炎症性BM利基将有利于克隆扩增， 获得更多突变和选择侵袭性克隆导致MPN进展为 骨髓纤维化或/和AML；b)miR-155上调在产生 推动MPN进展的“恶性”炎症。目标。为了检验这些假设，我们提出：(1) 确定造血细胞暴露于炎性BM利基是否会导致克隆选择和 JAK2V617F和TET2/驱动MPN体内模型的疾病进展；(2)确定 MiR-155与核因子-kB在促进MPN进展中的关系；(3)鉴定炎性细胞 人类骨髓微环境中的信号与MPN进展相关。战略。为此，我们将 联合使用Tie2CreerRBPJKO小鼠作为慢性炎症的基因控制动物模型 在移植环境中使用JAK2V616F或TET2-/-型号。此外，kB-RAS-/-和miR155-/-小鼠将用于 阐述miR-155和核因子-kB在疾病进展中的作用。一种慢病毒-DNA联合条码系统 带有荧光的蛋白质将被用来跟踪体内的克隆进化。这一方法将得到以下补充 整个外显子组测序以识别突变，并通过活体显微镜观察克隆相互作用 有明确的BM利基市场。最后，将测试一种新的抗miR-155治疗方法的有效性，以及 将对MPN BM标本进行分析，以确定与疾病进展相关的炎症特征。 关联性。我们相信，这项工作的完成将对理解和 MPN的管理，为早期治疗干预抑制慢性疾病提供了坚实的理论基础 炎症，从而阻止MPN的进展。

Background. Our current understanding of the mechanisms for Bcr/Abl-negative myeloprolifereative neoplasia (MPN) involves recognition of restricted MPN driver mutations targeting JAK2, MPL, or CALR, and of an inflammatory microenvironment, characterized by heightened expression and secretion of cytokines and chemokines. However, the contribution of inflammation to MPN progression has not been addressed. Similarly, the causes that lead to an inflammatory microenvironment in MPN have not been defined. Interestingly, the recent observation that clonal hematopoiesis of indeterminate potential (CHIP) exhibit accumulating mutations that correlate with aging, strongly suggests a role for inflammation in clonal selection and disease progression. Preliminary Results. We have generated an animal model of chronic inflammation in which loss of Notch signaling in the BM endothelium (Tie2CreERRBPJKO mice) induces upregulation of the microRNA miR-155, increased NF-kB signaling and pro-inflammatory cytokines resulting in a MPN-like disease within 8-12 months. Importantly, this microenvironmental inflammatory milieu greatly accelerates expansion of hematopoietic cells containing JAK2V617F or TET2-/- mutations. Hypothesis. We propose a model in which: a) prolonged exposure of JAK2V616F or TET2-/- hematopoietic cells to a chronic inflammatory BM niche will favor clonal expansion, acquisition of additional mutations and selection of aggressive clones leading to MPN progression into myelofibrosis or/and AML; b) upregulation of miR-155 plays a pivotal role in generating a vicious cycle of “malignant” inflammation driving MPN progression. Aims. To test these hypotheses we propose: (1) To determine whether exposure of hematopoietic cells to an inflammatory BM niche leads to clonal selection and disease progression in in vivo models of JAK2V617F and TET2-/- driven MPN; (2) To define the functional relationship between miR-155 and NF-kB in promoting MPN progression, and; (3) To identify an inflammatory signature in the human BM microenvironment correlating with MPN progression. Strategy. To this end, we will use Tie2CreERRBPJKO mice as a genetically-controlled animal model of chronic inflammation, in combination with JAK2V616F or TET2-/- models in transplant settings. In addition, kB-Ras-/- and miR155-/- mice will be used to address the role of miR-155 and NF-kB in disease progression. A lentiviral-DNA barcoding system combined with fluorescent proteins will be used to follow clonal evolution in vivo. This approach will be complemented by whole exome sequencing to identify mutations, and by intravital microscopy, to visualize clones interactions with defined BM niches. Finally, the efficacy of a novel anti-miR-155 therapeutic approach will be tested, and MPN BM specimens will be analyzed to identify inflammatory signatures correlating with disease progression. Relevance. We believe that accomplishment of this work will have impact on the understanding and management of MPN, providing a solid rationale for early therapeutic intervention to suppress chronic inflammation and thus, preventing MPN progression.