Impact of Disease Burden and Timing of Disease Initiation on the Response to Interferonalpha(IFNa) in MPN-associated myelofibrosis

聚乙二醇化干扰素α(IFNA)免疫疗法已被批准用于真性红细胞增多症(PV)患者的治疗，它可以诱导早期骨髓增生性肿瘤(MPN)患者的临床甚至分子缓解。然而，随着疾病的进展，患者对IFNA变得没有反应。此外，最近发现，导致MPN的JAK2V617F突变在儿童时期很早就出现，这表明从突变出现到MPN疾病的临床表现需要数十年的潜伏期。因此，我们假设a)JAK2突变克隆的增殖在疾病临床表现之前的很长一段时间内由免疫系统控制，b)存在早期IFNA治疗的机会之窗，以及c)克隆细胞负荷的某些阈值对于骨髓(BM)纤维化的发展非常重要。因此，在本项目中，我们将评估IFNA在MPN相关性骨髓纤维化(MF)中的应答机制，特别关注疾病负担和发病时机的影响。在CRU的第一个资助期，我们已经建立了体外IPSC和原代细胞单一和共培养系统，现在可以对骨髓炎症和纤维化以及对IFNA和其他MPN药物的反应进行建模。我们正在进行的体内实验将让我们了解骨髓基质细胞对IFNA治疗的总体反应。在当前提案的具体目标1中，我们将使用我们先前建立的体外和体内系统来评估诱导骨髓纤维化所需的最小MPN疾病负担。我们将使用这些来定义IFNA能够抵消MPN相关MF的疾病负担阈值。在特定的目标2中，我们将研究疾病开始的时间对MPN相关性骨髓纤维化发展的影响，以及IFNA干预这一早期过程的可能性。为了做到这一点，我们的目标是在一个可诱导的cre介导的JAK2V617F小鼠模型中研究发病年龄的影响。此外，还将评估IFNA的年龄相关作用。最后，在特定的目标3中，我们将研究微生物区系、调节性T细胞(Treg)和传染病在年龄相关的MF诱导易感性中的作用。上述实验将提高我们对获取MPN驱动程序突变后骨髓纤维化如何以及何时发展的理解，以及免疫系统年龄、微生物暴露或IFNA等因素可能如何影响这些过程。最终，这些结果有望使使用IFNA或针对免疫系统组件(如Treg)的患者开发出早期治疗甚至预防MPN和/或骨髓纤维化的策略。

Immunotherapy with pegylated interferon alpha (IFNa) is approved for the treatment of polycythemia vera (PV) patients and it can induce clinical and even molecular remission in patients with early myeloproliferative neoplasms (MPN). However, patients become unresponsive to IFNa upon disease progression. Also, it has recently become clear that the JAK2V617F mutation, which drives MPN, arises very early during childhood, suggesting a latency of several decades from emergence of the mutation until clinical manifestation of MPN disease. Thus, we hypothesize a) that proliferation of the JAK2-mutant clone is controlled by the immune system for a long time before clinical manifestation of disease, b) that there is a window of opportunity for early IFNa treatment, and c) that certain thresholds of the clonal cell burden are important for the development of bone marrow (BM) fibrosis. Therefore, in the present project, we will assess the mechanisms of IFNa responsiveness in MPN-associated myelofibrosis (MF), with a particular focus on the effects of disease burden and timing of disease initiation. During the first funding period of this CRU, we have established in vitro iPSC and primary cell mono- and co-culture systems, which now allow modeling of BM inflammation and fibrosis as well as response to IFNa and other MPN drugs. Our ongoing in vivo experiments will inform us about the overall responsiveness of BM stromal cells to IFNa treatment. In Specific Aim 1 of the current proposal, we will assess the minimal amount of MPN disease burden necessary to induce myelofibrosis, using our previously established in vitro and in vivo systems. We will use these to define the threshold of disease burden at which IFNa is able to counteract MPN-associated MF. In Specific Aim 2, we will investigate the effects of the timing of disease initiation on the development of MPN-associated myelofibrosis and the potential of IFNa to interfere with this early process. To do this, we aim to study the impact of age at disease initiation in an inducible cre-mediated JAK2V617F mouse model. Also, the age-dependent role of IFNa will be assessed. Finally, in Specific Aim 3, we will investigate the role of microbiota, regulatory T cells (Treg) and infectious disease in the age-dependent susceptibility to MF induction. The above-mentioned experiments will improve our understanding of how and when myelofibrosis develops after the acquisition of an MPN driver mutation and how factors such as age of the immune system, microbial exposure, or IFNa, may influence these processes. Ultimately, these results are expected to allow the development of strategies to treat at an early stage or even prevent MPN and/or myelofibrosis in patients using IFNa or targeting components of the immune system such as Treg.