Apoptosis in myelofibrosis with myeloid metaplasia

描述（由申请人提供）：骨髓纤维化伴髓样化生是一种克隆性造血干细胞疾病，可导致进行性细胞减少、脾肿大、母细胞转化和死亡。目前尚无广泛适用的治疗方法。MMM的发病机制目前尚不清楚。在慢性髓性白血病和真性红细胞增多症等相关的骨髓增殖性疾病中，细胞凋亡的正常过程存在缺陷。我们已经证明，在MMM患者的粒细胞中，细胞凋亡（自发的、血清剥夺的和tnf - α诱导的）在数量上减少。我们还观察到，来自MMM患者的红细胞前体可以在体外生长，缺乏先决条件的细胞因子促红细胞生成素。在真性红细胞增多症中，细胞因子独立生长的特征是由Bcl-XL （Bcl-2家族的抗凋亡成员）的过度表达引起的。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过敏有关，并且可能与Bcl-2或Akt途径的抗凋亡途径有关。我们假设在MMM中，细胞凋亡在粒细胞中是失调的，这是异常克隆中相应缺陷的反映。在这项拨款申请中，我们建议：

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.