Aberrant Monocytic Differentiation Induced by Gain-of-Function Shp2 Mutants

描述（申请人提供）：努南综合征（NS）是一种常见的（1/1500 - 2500活产）常染色体显性遗传疾病，由PTPN 11（50%）、SOS 1（20%）或KRAS（<5%）突变引起，特征为面部畸形、先天性心脏畸形、骨骼异常，以及多种血液学异常，包括易患青少年粒单核细胞白血病（JMML）。虽然在NS中观察到的异常是多种多样的，但几种并发症可归因于巨噬细胞或单核细胞衍生细胞的功能或数量增加。NS患者中巨噬细胞过度产生的最极端和致命的例子是儿童白血病JMML。JMML的临床特征在于骨髓单核细胞的过度产生和造血祖细胞对粒细胞-巨噬细胞集落刺激因子（GM-CSF）的体外超敏表型。除了GM-CSF刺激的过度增殖和磷酸化Erk水平升高外，我们发现激活Shp 2突变（Shp 2 E76 K和Shp 2D 61 Y）促进向单核细胞分化的转变，以其他骨髓谱系为代价，与JMML患者中观察到的疾病表型一致。这种向单核细胞谱系的偏斜分化与c-Jun增加和GATA 2表达降低相关。基于这些发现，我们假设激活Shp 2诱导的Ras超活化改变了转录谱，导致以替代性髓系为代价增强单核细胞分化。从机制上讲，我们认为Ras过度活化产生组成型c-Jun表达，与PU.1合作，允许过度的单核细胞分化和减少GATA 2表达。本申请的目的是1）描述JMML相关Shp 2功能获得性突变是否以JNK依赖性或非JNK依赖性方式诱导c-Jun表达并促进异常单核细胞分化; 2）研究组蛋白脱乙酰酶抑制剂阿司他丁A降低c-Jun和PU.1并重新激活GATA 2和C/EBP的潜力。在突变体Shp 2表达造血祖细胞中的表达;和3）检查与正常对照相比JMML的人样品中的造血特异性转录因子水平。我们预计，这些研究的结果将导致新的治疗策略，在JMML，一个致命的儿童白血病和努南综合征的并发症。公共卫生相关性：这项名为“功能获得性Shp 2突变体诱导的异常单核细胞分化”的提案关注的是一种特别致命的儿童髓性白血病，称为青少年粒单核细胞白血病（JMML）。JMML是一种5岁以下儿童的致命性白血病，其特征是单核细胞大量过量产生。不幸的是，JMML对化疗有抵抗力，大多数受折磨的儿童由于恶性单核细胞和巨噬细胞的器官浸润而死于疾病，最终以出血和感染告终。本提案中概述的实验旨在确定增强单核细胞过度产生的分子机制，以描绘JMML治疗干预的新靶点。

DESCRIPTION (provided by applicant): Noonan syndrome (NS) is a common (1 in 1500 to 2500 live births) autosomal-dominant disorder caused by mutations in PTPN11 (50%), SOS1 (20%), or KRAS (<5%) and is characterized by dysmorphic facial features, congenital heart malformations, skeletal anomalies, and a variety of hematologic abnormalities including a predisposition to juvenile myelomonocytic leukemia (JMML). Although the anomalies observed in NS are diverse, several of the complications can be attributed to the increased function or number of macrophages or monocyte-derived cells. The most extreme and lethal example of macrophage overproduction in NS patients is the childhood leukemia, JMML. JMML is characterized clinically by overproduction of myelomonocytic cells and by the in vitro phenotype of hematopoietic progenitor hypersensitivity to granulocyte-macrophage colony- stimulating factor (GM-CSF). In addition to GM-CSF-stimulated hyperproliferation and elevated levels of phospho-Erk, we found that activating Shp2 mutations (Shp2E76K and Shp2D61Y) promote a shift toward monocytic differentiation at the expense of other myeloid lineages, consistent with the disease phenotype observed in JMML patients. This skewed differentiation toward the monocytic lineage is associated with increased c-Jun and decreased GATA2 expression. Based on these findings, we hypothesize that activating Shp2-induced Ras hyperactivation alters the transcriptional profile leading to enhanced monocytic differentiation at the expense of alternative myeloid lineages. Mechanistically, we propose that Ras hyperactivation produces constitutive c-Jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression. The objectives of this application are to 1) delineate if JMML associated Shp2 gain-of-function mutations induce c-Jun expression and promote aberrant monocytic differentiation in a JNK-dependent or a JNK-independent manner; 2) investigate the potential of trichostatin A, a histone deacetylase inhibitor, to reduce c-Jun and PU.1 and to re-activate GATA2 and C/EBP? expression in mutant Shp2-expressing hematopoietic progenitors; and 3) examine hematopoietic- specific transcription factor levels in human samples of JMML compared to normal controls. We anticipate that results of these studies will lead to novel therapeutic strategies in JMML, a lethal childhood leukemia and complication of Noonan syndrome. PUBLIC HEALTH RELEVANCE: This proposal, "Aberrant Monocytic Differentiation Induced by Gain-of-Function Shp2 Mutants" focuses on a particularly lethal form of childhood myeloid leukemia called juvenile myelomonocytic leukemia (JMML). JMML is a lethal leukemia of children less than 5 years of age characterized by massive overproduction of monocytic cells. Unfortunately, JMML is resistant to chemotherapy and most afflicted children succumb to disease due to organ infiltration with malignant monocytes and macrophages, ending terminally in bleeding and infection. Experiments outlined in this proposal are directed at identifying the molecular mechanisms that enhance monocyte overproduction in order to delineate novel targets for therapeutic intervention in JMML.