PROTEIN KINASE C BASED ANTILEUKEMIC THERAPY

说明：（申请人的摘要）本申请的目的 是确定蛋白激酶C（PKC）的激活是否是一种可行的 抗白血病治疗策略。急性髓性白血病（AML）和 骨髓增生异常（MDS）表现为骨髓衰竭， 部分或完全阻断造血细胞的成熟 元素蛋白激酶C（PKC）家族的钙和 磷脂依赖性丝氨酸-苏氨酸激酶起关键作用 在单核细胞参与的细胞外信号转导中， 分化佛波酯12-0-十四酰基-佛波醇-13- 醋酸（TPA），一种有效的成熟诱导剂，沿着单核细胞 谱系，结合并激活PKC。申请人证明， 全反式维甲酸（ATRA）对TPA耐药HL-60细胞的治疗作用 亚系（HL- 525）显著增加了相对较低的组成性 在该变体中发现的PKCb mRNA和蛋白水平。ATRA- 介导的PKC活性和PKCb基因产物的增加与 随着以粘附为特征的TPA响应性的获得， 非特异性酯酶染色和c-fms基因表达。他 证明苔藓抑素1（一种非肿瘤促进化合物， 在纳摩尔浓度下激活PKC）处理ATRA引发的 HL-60和U-937细胞也导致特征的显著增强 单核细胞的分化。因此，组合 ATRA和苔藓抑素1的联合应用可能适用于特征为 骨髓分化阻滞。此应用程序将建立在 申请人通过定义PKC亚型的初步观察结果 视黄酸会使其急剧增加， 在几种白血病细胞系中被TPA或苔藓抑素1激活， 人白血病细胞（特定目标1）。下游元件直接 与相关PKC亚型的相互作用将通过以下方法确定： 谷胱甘肽-S-转移酶融合构建体（特异性目的2）。到 为了确保他在白血病细胞系上的发现具有普遍性， 将测试视黄酸/苔藓抑素1组合对人类的 白血病在免疫缺陷小鼠中繁殖（特异性目标3）。最后， 他将进行全反式维甲酸的第二阶段试验， 与苔藓抑素1联合治疗MDS和难治性AML患者， 将治疗反应与评估生物学效应的研究相关联 包括来自治疗患者的血清诱导 在体外和体内的分化（具体目标4）。

DESCRIPTION: (Applicant's Abstract) The objective of this application is to determine whether activation of protein kinase C (PKC) is a viable strategy for anti-leukemic therapy. Acute myeloid leukemia (AML) and myelodysplasia (MDS) are manifested by bone marrow failure resulting from a partial or complete block in maturation of hematopoietic elements. The protein kinase C (PKC) family of calcium and phospholipid-dependent serine-threonine kinases plays a critical role in transducing extracellular signals involved in monocytic differentiation. The phorbol ester 12-0-tetradecanoyl-phorbol-13- acetate (TPA), a potent inducer of maturation along the monocytic lineage, binds to and activates PKC. The applicant demonstrated that all-trans retinoic acid (ATRA) treatment of the TPA-resistant HL-60 cell subline (HL- 525) markedly increases the relatively low constitutive levels of PKCb mRNA and protein found in this variant. The ATRA- mediated increase in PKC activity and PKCb gene product is associated with the acquisition of TPA responsiveness characterized by adherence, non-specific esterase staining, and c-fms gene expression. He has demonstrated that bryostatin 1 (a non-tumor promoting compound which activates PKC at nanomolar concentrations) treatment of ATRA- primed HL-60 and U-937 cells also leads to a marked augmentation of features of monocytic differentiation in these cells. Therefore, the combination of ATRA and bryostatin 1 may be useful in diseases characterized by a block in myeloid differentiation. This application will build upon the applicant's preliminary observations by defining the PKC isoform(s) which are critically increased by retinoic acid and subsequently activated by TPA or bryostatin 1 in several leukemia cell lines and human leukemic cells (Specific Aim 1). Downstream elements directly interacting with the relevant PKC isoform will be determined by means of glutathione-S-transferase fusion constructs (Specific Aim 2). To insure that his findings with leukemic cell lines are generalizable, he will test the retinoic acid/bryostatin 1 combination against human leukemias propagated in immunodeficient mice (Specific Aim 3). Finally, he will conduct a phase II trial of all trans retinoic acid in combination with bryostatin 1 in patients with MDS and refractory AML, correlating therapeutic response with studies to assess biologic effects of therapy including the ability of sera from treated patients to induce differentiation in vitro and in vivo (Specific Aim 4).