Apoptosis in myelofibrosis with myeloid metaplasia

描述(申请人提供)：骨髓纤维化伴髓系化生是一种克隆性造血干细胞疾病，导致进行性细胞减少、脾肿大、母细胞性转化和死亡。目前还没有广泛适用的治疗方法。MMM的发病机制目前尚不清楚。在与慢性粒细胞白血病和真性红细胞增多症相关的骨髓增生性疾病中，已发现正常的细胞凋亡过程中存在缺陷。我们已经证明，MMM患者的粒细胞中的凋亡(自发的、血清剥夺和肿瘤坏死因子-α诱导的)是定量减少的。我们还观察到，在缺乏必要的细胞因子促红细胞生成素的情况下，MMM患者的红系前体细胞可以在体外培养。在真性红细胞增多症中，细胞因子非依赖性生长的特征是由于Bclxl(Bcl2家族中的一种抗凋亡成员)的过度表达。我们认为，我们在MMM中观察到的细胞凋亡减少可能与细胞因子超敏有关，并可能与抗凋亡通路Bcl2或Akt途径有关。我们假设MMM的粒细胞中的细胞凋亡是失调的，这是异常克隆中相应缺陷的反映。在这份赠款申请中，我们建议： 1.比较MMM患者和对照组的凋亡蛋白和调节因子的基线水平。将在一系列MMM患者和正常对照中评估凋亡蛋白(半胱氨酸天冬氨酸氨基转移酶)和调节因子(LAP‘s、Bcl2家族成员)的基线水平。 2.通过细胞系统和非细胞系统评价凋亡刺激对MMM中性粒细胞caspase激活的调节作用。从MMM患者和对照组分离的中性粒细胞将受到不同的凋亡刺激，以描绘哪条凋亡途径被异常调控。随后的实验将同时使用免疫印迹和胞浆caspase激活试验来确定哪些caspase和调节因子对MMM中性粒细胞中出现的凋亡缺陷负责。 3.评价磷脂酰肌醇3-激酶通路在骨髓瘤髓系祖细胞细胞因子非依赖性生长中的作用。髓系集落的细胞因子非依赖性生长将在一系列MMM患者中得到证实。随后的实验将描述磷脂酰肌醇-3激酶通路在细胞凋亡抵抗和细胞因子非依赖性集落生长中的作用。 这些目标的成功实现将为治疗MMM患者和潜在相关的慢性髓系疾病患者提供靶向抗骨髓增殖治疗的科学基础。

DESCRIPTION (provided by applicant): Myelofibrosis with myeloid metaplasia is a clonal hematopoietic stem cell disorder that results in progressive cytopenias, splenomegaly, blastic transformation, and death. No broadly applicable therapy is available. The pathogenetic mechanism of MMM is currently unknown. A defect in the normal process of apoptosis has been demonstrated in the related myeloproliferative disorders of chronic myeloid leukemia and polycythemia vera. We have shown that apoptosis (spontaneous, serum deprivation, and TNF-alpha induced) is quantitively diminished in the granulocytes of patients with MMM. We have also observed that erythroid precursors from MMM patients can be grown in vitro in the absence of the prerequisite cytokine erythropoietin. Cytokine independent growth has been characterized in polycythemia Vera to arise from over-expression of Bcl-XL (an anti-apoptotic member of the Bcl-2 family). We believe the diminished apoptosis we have observed in MMM may be linked to cytokine hypersensitivity and, potentially, to the anti-apoptotic pathways of Bcl-2 or the Akt pathway. We hypothesize that apoptosis is dysregulated in granulocytes in MMM, and this is a reflection of the corresponding defect in the aberrant clone. In this grant application we propose to: 1.Compare baseline levels of apoptotic proteins and regulators across the spectrum of MMM patients and controls. Baseline levels of apoptotic proteins (caspases), and regulators (lAP's, Bcl-2 family members) will be assessed across a spectrum of MMM patients and normal controls. 2. Evaluate the regulation of caspase activation in MMM neutrophils subjected to apoptotic stimuli through both cellular and cell free systems. Isolated neutrophils from MMM patients and controls will be subjected to various apoptotic stimuli to delineate which pathway of apoptosis is aberrantly regulated. Subsequent experiments will use both immunoblotting and a cytosol caspase activation assay to determine which caspases and regulators are responsible for the apoptotic defect seen in MMM neutrophils. 3. Evaluate the role of the phosphatidylinositol 3- kinase pathway on cytokine independent growth in myeloid progenitors in MMM. Cytokine independent growth of myeloid colonies will be confirmed across a spectrum of MMM patients. Subsequent experiments will delineate the role of the phosphatidylinositol-3 kinase pathway in both apoptosis resistance and cytokine independent colony growth. Successful accomplishments of these goals will provide the scientific basis for targeted anti-myeloproliferative therapy for the treatment of patients suffering from MMM and potentially related chronic myeloid disorders.