Role of IL-10R signaling in inflammation induced exhaustion of HSC and emergence of JAK2V617F clonal hematopoiesis

修订后的项目摘要 年龄和慢性炎症应激促使突变的骨髓干细胞的出现，这些突变可能导致特定的血液系统恶性肿瘤相关突变，允许HSC抵抗特定的应激源，可能被用于旨在中和恶性克隆的选择优势的治疗。我们最近发现，骨髓增生性肿瘤患者对抗炎细胞因子IL-10的反应减弱，从而导致炎性细胞因子肿瘤坏死因子α的持续产生。在小鼠JAK2V617F MPN模型中，药物阻断IL-10R信号增强了JAK2V617F突变细胞的选择优势。本项目的目的是确定当IL-10R被阻断时，JAK2V617F突变体HSC如何获得相对于野生型(WT)HSC的选择优势。我们的中心假设是，抑制的IL-10R信号会产生一种炎症状态，对WT产生负面影响，但不会对JAK2V617F突变的HSC产生负面影响，从而赋予JAK2V617F突变细胞选择性优势。此外，我们假设，抑制IL-10R信号是易患MPN的人的固有特征。在目标1中，我们将确定MPN家庭中IL-10耐药表型的流行率。我们发现MPN患者对IL-10无反应，我们称之为“IL-10抵抗表型”。然而，有MPN家族史的病例还没有被系统地评估其表型，也没有未受影响的家庭成员。我们将确定IL-10耐药表型在我们不断增长的MPN家庭登记中受影响和未受影响的成员中的流行率，目前登记有53个MPN家庭。在目标2中，我们将确定MPN中IL-10R信号钝化的遗传基础。我们将进行有针对性的DNA测序和基因表达谱分析(即RNA-SEQ、CHIP-SEQ和差异DNA甲基化分析)，以确定MPN中IL-10信号减弱的潜在机制。使用分选的外周血单核细胞，我们将使用Illumina Infinium MethylationEPIC芯片来确定MPN患者和IL-10信号钝化的未受影响的家庭成员是否存在IL-10R信号基因的差异甲基化。基因表达谱将并行执行，以确定甲基化模式是否准确预测基因表达。我们还将进行有针对性的DNA测序，重点关注细胞因子受体途径基因，并密切关注任何发现有差异甲基化和/或表达的基因，以确定MPN患者IL-10信号减弱的遗传基础。

Revised Project Summary Age and chronic inflammatory stress drive the emergence of mutant bone marrow stem cells that can lead to specific hematologic malignancy associated mutations allow HSC to resist specific stressors could be leveraged toward therapies aimed at neutralizing the selective advantage of the malignant clone. We have recently found that patients with myeloproliferative neoplasm (MPN) have a dampened response to the anti-inflammatory cytokine IL-10 and this leads to persistent production of the inflammatory cytokine TNFα. In a murine Jak2V617F MPN model, pharmacologic blockade of IL-10R signaling enhances the selective advantage of Jak2V617F mutant cells. The objective of this project is to define how JAK2V617F mutant HSC gain a selective advantage over wild-type (WT) HSC when IL-10R is blocked. Our central hypothesis is that dampened IL-10R signaling creates an inflammatory state that negatively affects WT but not JAK2V617F mutant HSC, thus endowing JAK2V617F mutant cells a selective advantage. Moreover, we hypothesize that dampened IL-10R signaling is an intrinsic feature of those predisposed to acquire MPN. In Aim 1 we will determine the prevalence of an IL-10 resistance phenotype among MPN families. We have found that MPN patients display an inability to respond to IL-10, which we term the “IL-10 resistance phenotype”. However, cases with a family history of MPN have not been systematically evaluated for the phenotype, nor have unaffected family members. We will determine the prevalence of the IL-10 resistance phenotype in affected and unaffected members of our growing MPN family registry which currently contains 53 MPN families. In Aim 2 we will identify the genetic basis for blunted IL-10R signaling in MPN. We will perform targeted DNA sequencing and gene expression profiling (i.e., RNA-seq, ChIP-seq, and differential DNA methylation analysis) to identify the underlying mechanism responsible for dampened IL-10 signaling in MPN. Using sorted peripheral blood monocytes we will use Illumina Infinium MethylationEPIC chips to determine if MPN patients and unaffected family members with blunted IL-10 signaling have differential methylation of IL-10R signaling genes. Gene expression profiling will be performed in parallel to determine if the methylation pattern accurately predicts gene expression. We will also perform targeted DNA sequencing, focusing on cytokine receptor pathway genes, and paying close attention to any genes found to have differential methylation and/or expression, to identify the genetic basis for blunted IL-10 signaling in MPN patients.