Laboratory Assessment of Patients with Systemic Mastocytosis

系统性肥大细胞增多症(SM)是一种以肥大细胞在组织内病理性堆积为特征的异质性疾病。大多数成年患者被归类为惰性系统性肥大细胞增多症(ISM)，这种疾病通常转化为攻击性状态的风险较低，而且被认为不会影响寿命。然而，侵袭性系统性肥大细胞增多症(ASM)的一部分患者预后较差，需要细胞减少性治疗。 在肥大细胞增生性疾病患者的炎症组织和受影响的器官和组织中观察到肥大细胞负荷增加。然而，正常的肥大细胞参与宿主防御，因此需要优先针对克隆性扩增的肥大细胞的方法。我们发现，哺乳动物靶标雷帕霉素复合体1(MTORC1)和2(MTORC2)在肿瘤和发育中的未成熟肥大细胞中比它们的末端分化肥大细胞上调。在肥大细胞增生症患者的骨髓单个核细胞中也观察到mTOR mRNA的升高。通过基因和药物操作选择性地抑制mTORC1和mTORC2表明，虽然mTORC1可能有助于肥大细胞的存活，但mTORC2只对肿瘤和分裂的未成熟肥大细胞的动态平衡起关键作用。MTORC2下调对增殖肥大细胞的细胞抑制作用是通过抑制细胞周期进程实现的。由于mTORC2在分化的、非增殖的、成熟的肥大细胞的动态平衡中发挥的作用很小，这些数据为采用靶向方法选择性抑制mTORC2有效地减少与炎症相关的肥大细胞的增殖和肥大细胞增殖障碍提供了理论基础，同时使正常分化的肥大细胞基本不受影响。 在全身性肥大细胞增多症患者中，通常可以观察到受体酪氨酸激酶试剂盒中的激活突变，最明显的是Kit D816V。因此，对KIT的抑制一直是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，我们针对试剂盒的开关口袋(SP)，它调节其催化构象。研究了两种SP抑制剂DP-2976和DP-4851对肿瘤肥大细胞增殖和肥大细胞活化的影响。在293T细胞、HMC 1.1和1.2人肥大细胞系中，以及在干细胞因子(SCF)激活的CD34+来源的人肥大细胞中，KIT D816V的野生型和Kit D816V的自磷酸化被这些化合物阻断。这两种抑制剂都能诱导肿瘤肥大细胞系的凋亡，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抑制剂更选择性地阻断了FcRI介导的脱颗粒的SCF增强。总体而言，SP抑制剂代表了KIT抑制的创新机制，其对KIT D816V肿瘤肥大细胞增殖的双重抑制和SCF增强的肥大细胞激活可能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.