MOLECULAR INHIBITION OF BCR-ABL TYROSINE KINASE BY BCR SEQUENCES

Bcr-Abl癌蛋白是费城的主要致病因素 染色体（Ph）相关白血病。激活的酪氨酸激酶， Bcr-Abl癌蛋白是其致癌的主要驱动力， 活动我们已经证明了Bcr [Bcr（64-413）]的删除形式， 包括Bcr的Abl SH 2结合结构域，降低了Bcr的表达。 细胞内c-Abl和Bcr-Abl的磷酸酪氨酸含量，并抑制 体外Bcr-A自身磷酸化活性。类似地，Bcr肽 Ser 354的磷酸化在体外阻断c-Abl和Bcr-Abl激酶 而未磷酸化的肽没有抑制性。Bcr（64-413）为 也抵抗由活化的c-Abl或 重要的是，Bcr（64-413）干扰BCR-Abl的生长 表达细胞系。我们的研究结果表明，Abl SH 2结合 磷酸丝氨酸形式的Bcr结构域抑制Bcr-Abl癌蛋白， 但是Bcr这个结构域的酪氨酸磷酸化逆转了其 Bcr-Abl的抑制作用。这些发现的结果形成了 治疗慢性期CML患者的新策略的基础。具体 目的是：1）确定Bcr（64-413）的最佳形式， 表达Bcr-Abl的人造血细胞逆转 Bcr-Abl诱导的生长/凋亡作用。这些构建体将被 与仅载体、BCR（1-413）和S354 A BCR（64-413）转染相比， 细胞这些研究将使用转染和阳性对照进行。 用四环素阻遏物启动子系统进行选择。2)构建 BCR（64-413）重组复制缺陷型腺病毒，用于插入 将BCR抑制剂序列导入Bcr-Abl阳性和阴性细胞系。 3)检测重组缺陷型Bcr腺病毒在 用源自CML的细胞系编程的SCID/NOD SCID小鼠系统 患者我们计划测试Bcr（64-413）对患者细胞系的影响 在SCID小鼠模型中生长。我们计划感染K562细胞， 用Ad 5 CMV BCR（64-413）感染细胞，以确定其对生长的影响 这些患者细胞系在适当的SCID模型中的特性 与编码不相关基因的腺病毒制剂（抗- 有义C-CAM）。最近的研究结果表明，Ad 5 CMV BCR（64-413）诱导了 感染2天的K562细胞死亡率为75%。的细胞杀伤 作用是特异性的BCR序列，因为Ad 5 CMV反义CCAM 不影响细胞生长或诱导细胞死亡。4)测试的影响 重组复制缺陷型BCR（64-413）腺病毒感染 CML慢性期患者的人骨髓制备物，Ph- ALL阳性骨髓正常这些影响将与 阴性对照腺病毒。细胞死亡（50%）已被诱导在一个低的 来自未经治疗的CML患者的高密度骨髓制备物。我们预计 Bcr（64-413）可抑制Ph阳性菌落的生长。第五章） 研究来源于以下的合成肽的抑制作用： Bcr（64-413）作为Bcr-Abl表达细胞抑制剂的用途 和骨髓样本病毒介导的 引入Bcr（64-413）或来自Bcr该区域的可溶性肽 将可用于包括在治疗方案中， 慢性期CML患者。

The Bcr-Abl oncoprotein is the primary causative factor in Philadelphia chromosome (Ph) associated leukemias. The activated tyrosine kinase of the Bcr-Abl oncoprotein is the primary driving force behind its oncogenic activity. We have shown that a deleted form of Bcr [Bcr(64-413)], encompassing the Abl SH2 binding domains of Bcr, reduced the phosphotyrosine content of c-Abl and Bcr-Abl within cells, and inhibited Bcr-A autophosphorylation activity in vitro. Similarly, a Bcr peptide phosphorylated on Ser 354 blocked the c-Abl and Bcr-Abl kinases in vitro whereas the unphosphorylated peptide was not inhibitory. Bcr(64-413) was also resistant to tyrosine phosphorylation by either activated c-Abl or Bcr-Abl. Importantly, Bcr(64-413) interfered with the growth of BCR-Abl expressing cell liners. Our findings indicate that the Abl SH2 binding domain of Bcr in the phospho-serine form inhibits the Bcr-Abl oncoprotein, but that tyrosine phosphorylation of this domain of Bcr reverse its inhibitory effects of Bcr-Abl. The results of these findings form the basis of a new strategy to treat chronic phase CML patients. The specific aims are: 1) Determine the optimum form of Bcr(64-413) will be tested in human hemopoietic cells expressing Bcr-Abl for their ability to reverse growth/apoptosis effects induced by Bcr-Abl. These constructs will be compared to vector only, BCR(1-413), and S354A BCR(64-413) transfected cells. These studies will be done using transfection and positive selection with the tetracycline repressor promoter system. 2) Construct a BCR(64-413) recombinant, replication-defective adenovirus for insertion of the BCR inhibitor sequence into Bcr-Abl positive and negative cell lines. 3) Test the effectiveness of a recombinant defective Bcr adenovirus in SCID/NOD SCID mouse systems programmed with cell lines derived from CML patients. We plan to test the effects of Bcr(64-413) on patient cell lines growing in a SCID mouse model. We plan to infect K562 cells and KBM-5 cells with Ad5 CMV BCR(64-413) to determine its effects on the growth properties of these patient cell lines in the appropriate SCID model compared to an adenovirus preparation encoding a unrelated gene (anti- sense C-CAM). Recent findings indicate that Ad5 CMV BCR(64-413) induced cell death in 75% in K562 cells infected for 2 days. The cell killing effects was specific to BCR sequences because Ad5 CMV anti-sense C CAM did not affect cell growth or induce cell death. 4) Test the effects of recombinant, replication defective BCR(64-413) adenovirus infection in human bone marrow preparations from CML chronic phase patients, Ph- positive ALL, and normal bone marrow. These effects will be compared to negative control adenoviruses. Cell death (50%) has been induced in a low density marrow preparation from an untreated CML patient. We anticipate that Bcr(64-413) will inhibit growth of Ph-positive colonies. 5) Investigate the inhibitory effects of synthetic peptides derived from Bcr(64-413) for their utility as inhibitors of Bcr-Abl expressing cell lines and marrow samples. Positive results from either virus-mediated introduction of Bcr(64-413) or soluble peptides from this region of Bcr will be useful for inclusion in therapeutic protocols for treatment of chronic phase CML patients.