原发性骨髓纤维化（primary myelofibrosis, PMF）是由异常造血干/祖细胞过度增殖和不典型分化而导致的慢性血液系统疾病，属于髓系骨髓增生性肿瘤（myeloproliferative neoplasm, MPN）的一种亚型。大约20%的PMF患者会转化成急性髓系白血病（acute myelogenous leukemia, AML），其整体中位生存期＜6年。90%的PMF患者携带Janus激酶（Janu kinase 2, JAK2）、促血小板生成素受体（Thrombopoietin receptor, TPOR；受体基因MPL）或钙网蛋白（Calreticulin, CALR）的驱动性基因突变。PMF的临床特征改变包括前体巨核细胞（Megakaryocyte, MK）分化异常和过度增殖、骨髓网状蛋白纤维化、髓外造血并伴有肝脾肿大等。这些异常分化的MK细胞在骨髓和脾等造血器官中过度增殖，并分泌多种细胞因子，从而促进PMF疾病的进展。因此，解析MK细胞分化的调控通路，有利于找到PMF疾病治疗的新靶点并依此开发相应的小分子抑制剂。

目前具有PMF潜在治愈的方法是同种异体造血干细胞移植（allogeneic hematopoietic stem cell transplant, AHSCT），然而，50%接受骨髓移植的患者会产生免疫排斥等并发症，甚至会走向死亡。小分子抑制剂的靶向治疗为PMF患者提供了有效的治疗方法，已经应用于临床的JAK抑制剂ruxolitinib明显降低PMF恶性细胞数量，但未能缓解患者驱动基因突变负荷和骨髓纤维化症状。靶向Aurora A激酶（Aurora Kinase A, AURKA）的诱导分化治疗能有效缓解PMF患者的骨髓纤维化和脾肿大症状。课题组前期筛选并确定了AURKA抑制剂Alisertib（MLN8237），它能显著诱导前体MK细胞分化和成熟，在PMF小鼠模型中，有效地缓解骨髓纤维化和脾肿大症状，然而，AURKA相关的造血干/祖细胞异常增殖和分化的调控通路仍未可知。

本研究通过蛋白互作网站（https://inbio-discover.com/index.html）的数据库分析系统筛查AURKA的互作蛋白，从中发现了哺乳动物转录因子Forkhead box P 1（FOXP1）。文献筛查发现AURKA通过下调FOXP1而降低抗凋亡蛋白Survivin的泛素化降解，从而与胃癌疾病进展和不良预后相关，并且FOPX1在细胞分化、细胞凋亡和免疫调节有关的基因转录调控中起重要作用。基于此，推测FOXP1是AURKA的下游重要调控因子之一，在PMF异常巨核细胞分化决定和PMF疾病进展中发挥重要作用。

本论文结合前期研究发现，FOXP1是AURKA下游重要调控因子之一，AURKA激酶抑制可上调FOXP1，且FOXP1在PMF患者外周血与PMF小鼠模型骨髓和脾中表达显著降低，据此推测PMF患者造血组织细胞中FOXP1的显著下调促进了PMF疾病进展。过表达FOXP1能够恢复RUNX1、GATA1的表达，进而促进前体巨核细胞系表面分化标志物CD41、CD42的表达，以及核多倍体的形成，诱导前体巨核细胞分化和成熟。本论文首次将FOXP1与MK前体细胞分化联系起来，并寻找其调控的下游靶点基因启动子的分子机制，揭示了FOXP1调控MK分化的分子转录调控机制。重要的是，本论文发现了小分子抑制剂lenalidomide可以上调细胞内FOXP1表达，它不仅能够抑制前体巨核细胞的增殖，也能通过上调FOXP1、RUNX1和GATA1关键巨核系分化的转录因子，促进巨核细胞分化，有可能成为PMF疾病诱导分化治疗的候选试剂。

Primary myelofibrosis (PMF) is a chronic hematologic disease characterized by excessive proliferation and atypical differentiation of aberrant hematopoietic stem/progenitor cells. It is a form of myeloproliferative neoplasm (MPN). About 20% of patients with PMF will transform into acute myelogenous leukemia (AML), with an overall median survival of <6 years. Driver mutations involving Janus kinase 2 (JAK2), thrombopoietin receptor (MPL) or calreticulin (CALR) in 90% of patients mediate constitutive JAK-STAT signaling. The clinical features of PMF include morphologically characteristic megakaryocyte proliferation, extramedullary hematopoiesis, anemia, marked hepatosplenomegaly, and constitutional symptoms. These aberrant immature MK cells will excessively proliferate in hematopoietic organs including bone marrow and spleen, secreting a plethora of pro-inflammatory cytokines that might promote reactive bone marrow fibrosis. Therefore, analyzing the regulatory pathways of MK cell differentiation is beneficial to find new targets for PMF disease therapy and design corresponding small molecule inhibitors.

Allogeneic hematopoietic stem cell transplant (AHSCT) is by far the first-line treatment of choice for high-risk PMF. However, half of all patients who have bone marrow transplants experience problems such as immunological rejection, which can result in death. Targeted therapy with small-molecule inhibitors provides an effective treatment for PMF patients. The JAK inhibitor ruxolitinib, which has been used in clinic, significantly reduces the number of malignant cells in PMF, but fails to alleviate the patient's driver gene mutation and myelofibrosis. Induction therapy targeting Aurora A kinase (AURKA) can effectively relieve the symptoms of myelofibrosis and splenomegaly in PMF patients. Our group identified the AURKA inhibitor Alisertib (MLN8237), which can significantly induce maturation of precursor MKs, and effectively alleviate myelofibrosis and splenomegaly in PMF mouse models. However, the regulatory mechanisms that control AURKA-related hematopoietic stem/progenitor cell aberrant proliferation and differentiation are unclear.

In this paper, we screened the interacting proteins of AURKA using the protein interaction website's database analysis system (https://inbio-discover.com/index.html), and discovered the mammalian transcription factor Forkhead box P 1 (FOXP1). AURKA induces ubiquitination and degradation of the anti-apoptotic protein Survivin by downregulating FOXP1, which is linked to gastric cancer disease progression and poor prognosis, according to a literature review. FOPX1 is also involved in gene transcription related to cell differentiation, death, and immunological regulation. Based on this, we hypothesize that FOXP1 is one of AURKA's major downstream regulators, which plays a key role in aberrant megakaryocyte differentiation and development of PMF disease.

Combined with previous studies, this paper found that FOXP1 is one of the important downstream regulators of AURKA, and AURKA kinase inhibition can up-regulate FOXP1, suggesting that the significant down-regulation of FOXP1 promotes PMF disease progression. Overexpression of FOXP1 can restore the expression of RUNX1 and GATA1, and then promote the expression of CD41 and CD42, as well as the formation of nuclear polyploidy, inducing the differentiation and maturation of precursor megakaryocytes. This paper links FOXP1 to MK precursor cell differentiation for the first time, revealing the molecular transcriptional regulatory mechanism of FOXP1 regulating MK differentiation. Importantly, we found that the small-molecule inhibitor lenalidomide upregulates intracellular FOXP1 expression, which not only inhibits the proliferation of precursor megakaryocytes, but also promotes megakaryocyte differentiation by upregulating FOXP1, RUNX1, and GATA1 key to megakaryocyte differentiation. It has the potential to be a candidate agent for the induction and differentiation therapy of PMF disease.