Laboratory Assessment of Patients with Systemic Mastocytosis

系统性肥大细胞增多症（SM）是一种异质性疾病，其特征是肥大细胞在组织内的病理性积聚。大多数成年患者被归类为惰性系统性肥大细胞增多症（ISM），通常转化为侵袭性状态的风险较低，并且不被认为会影响寿命。然而，一部分侵袭性系统性肥大细胞增多症（ASM）患者预后不良，需要进行细胞减灭治疗。 在肥大细胞增殖性疾病患者的发炎组织和受影响器官和组织中观察到肥大细胞负荷增加。然而，正常肥大细胞参与宿主防御，因此需要优先靶向克隆扩增肥大细胞的方法。我们发现，哺乳动物靶雷帕霉素复合物1（mTORC 1）和2（mTORC 2）的肿瘤和发展中的未成熟肥大细胞相比，他们的终末分化的同行上调。在肥大细胞增生患者的骨髓单个核细胞中也观察到mTOR mRNA升高。通过遗传和药理学操作选择性抑制mTORC 1和mTORC 2显示，虽然mTORC 1可能有助于肥大细胞存活，但mTORC 2仅对肿瘤性和分裂的未成熟肥大细胞的稳态至关重要。在增殖肥大细胞中mTORC 2下调的细胞抑制作用被确定为通过抑制细胞周期进程。由于观察到mTORC 2在分化的、非增殖的成熟肥大细胞的稳态中几乎不起作用，因此这些数据提供了采用靶向方法选择性抑制mTORC 2以有效减少与炎症和肥大细胞增殖障碍相关的肥大细胞增殖，同时使正常分化的肥大细胞基本上不受影响的基本原理。 在系统性肥大细胞增多症患者中经常观察到受体酪氨酸激酶KIT（最显著的是KIT D816 V）的活化突变。因此，抑制KIT已成为治疗这种疾病的主要焦点。我们研究了一种新的抑制方法。利用合理的药物设计，我们针对KIT的开关口袋（SP），它调节其催化构象。检查了由此鉴定的两种SP抑制剂DP-2976和DP-4851对肿瘤性肥大细胞增殖和肥大细胞活化的影响。在转染的293 T细胞、HMC 1.1和1.2人肥大细胞系以及干细胞因子（SCF）激活的CD 34+衍生的人肥大细胞中，这些化合物阻断了野生型和KIT D816 V（也进行了检查）激活的自磷酸化。这两种抑制剂诱导肿瘤性肥大细胞系凋亡，并降低肥大细胞增多症患者的原代骨髓肥大细胞的存活率。此外，SP抑制剂更有选择性地阻断SCF增强FcRI介导的脱粒。总体而言，SP抑制剂代表了KIT抑制的创新机制，其对KIT D816 V肿瘤性肥大细胞增殖和SCF增强的肥大细胞活化的双重抑制可提供显著的治疗益处。

Systemic mastocytosis (SM) is a heterogeneous disorder characterized by the pathological accumulation of mast cells within tissues. The majority of adult patients are classified with indolent systemic mastocytosis (ISM), which generally carries a low risk of transformation to an aggressive state and is not thought to affect lifespan. However, a subset of patients with aggressive systemic mastocytosis (ASM) have a poor prognosis and will require cytoreductive therapy. Increased mast cell burden is observed in the inflamed tissues and affected organs and tissues of patients with mast cell proliferative disorders. However, normal mast cells participate in host defense, so approaches to preferentially target clonally expanding mast cells are needed. We found that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) are up-regulated in neoplastic and developing immature mast cells compared with their terminally differentiated counterparts. Elevated mTOR mRNA was also observed in bone marrow mononuclear cells of patients exhibiting mast-cell hyperplasia. Selective inhibition of mTORC1 and mTORC2 through genetic and pharmacologic manipulation revealed that, whereas mTORC1 may contribute to mast-cell survival, mTORC2 was only critical for homeostasis of neoplastic and dividing immature mast cells. The cytostatic effect of mTORC2 down-regulation in proliferating mast cells was determined to be via inhibition of cell-cycle progression. Because mTORC2 was observed to play little role in the homeostasis of differentiated, nonproliferating, mature mast cells, these data provide a rationale for adopting a targeted approaching selectively inhibiting mTORC2 to effectively reduce the proliferation of mast cells associated with inflammation and disorders of mast cell proliferation while leaving normal differentiated mast cells largely unaffected. Acvating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. We investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT, which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild-type and, where also examined, KIT D816V activation was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34+-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF-enhanced mast cell activation may provide significant therapeutic benefits.