Novel Pathways for Bcr-Abl transformation

描述（由申请人提供）：大量动物研究表明融合蛋白p210 Bcr-Abl是慢性髓性白血病（CML）的病原体，靶向该分子的药理学抑制剂（如伊马替尼）在许多患者中产生有效、持久的反应。伊马替尼靶向Abl序列中含有的酪氨酸激酶活性，该活性被认为是CML背后的主要驱动力。尽管伊马替尼的成功验证了合理的药物设计在癌症治疗中，两个临床问题正在出现在治疗CML。首先，患者通过获得激酶结构域内的突变对伊马替尼产生耐药性（获得性耐药性）。其次，可能更成问题的是，超过95%的患者即使在伊马替尼治疗12个月后仍有p210 Bcr-Abl阳性细胞（最小残留疾病）。获得性耐药和微小残留病是否在机制上相关尚不清楚，但这两个问题都强调需要开发不同形式的互补抑制剂，以靶向p210 Bcr-Abl的其他功能。尽管p210 Bcr- abl的致瘤特性主要归因于abl编码的激酶活性，但许多研究已经确定，在Bcr序列中也存在支持致癌活性所需的结构域。虽然特征不明确，但这些结构域很有趣，因为它们代表了药理学干预的未开发目标。在之前的资助期内，我们的实验室一直在探索Bcr编码活性的表征将确定p210 Bcr- abl抑制的可行新靶点的假设。现在已经确定了三种这样的活动。首先，已经确定在Bcr中被自抑制的RhoGEF结构域在p210 Bcr- abl中被组成性激活。在细胞和动物模型中，p210 Bcr-Abl中消除该活性的突变表现出降低的转化活性，这表明该结构域可能是CML所必需的。其次，已经确定Bcr是内体分选途径的调节成分，并且造血细胞中p210 Bcr- abl的过表达会破坏该途径。这代表了p210 Bcr-Abl生物学的一个全新的、完全未被探索的方面，无疑将与CML相关。最后，已经确定Bcr具有核功能，通过核功能可以调节蛋白质表达水平。这包括已知与CML有关的c-Myc癌蛋白。Bcr-Abl通过与XPB结合来调节c- Myc，在细胞和动物模型中，这种相互作用的破坏也与转化活性降低有关。在这一持续的应用中，这些活性将在造血细胞的背景下进行表征，并将确定它们是否是抑制p210 Bcr-Abl的可行靶点。在这项分析中，产生了p210 Bcr-Abl突变体，这些突变体在这些活动中受损，并且可以在基于细胞和动物的CML模型中进行评估。公共卫生相关性：伊马替尼是一种药理学抑制剂，可引起许多慢性髓性白血病患者的临床缓解。然而，伊马替尼控制而不是治愈这种疾病，许多患者对这种治疗产生了耐药性。因此，本建议的重点是开发可用于补充伊马替尼治疗的新方法和抑制剂。

DESCRIPTION (provided by applicant): Numerous animal studies have demonstrated that the fusion protein p210 Bcr-Abl is the causative agent of Chronic Myelogenous Leukemia (CML), and pharmacological inhibitors (such as imatinib) that target this molecule produce effective, durable responses in many patients. Imatinib targets a tyrosine kinase activity contained within the Abl sequences that is considered to be the principle driving force behind CML. Although the success of imatinib has validated rational drug design in cancer therapy, two clinical issues are emerging in the treatment of CML. First, patients are developing resistance to imatinib by acquiring mutations within the kinase domain (acquired resistance). Second, and probably more problematic, over 95% of patients still have p210 Bcr-Abl positive cells, even after 12 months of imatinib treatment (minimal residual disease). Whether or not acquired resistance and minimal residual disease are mechanistically related is unclear, but both problems highlight the need to develop complementary inhibitors, with different modalities, that can target additional functions of p210 Bcr-Abl. Although the tumorigenic properties of p210 Bcr-Abl can be primarily attributed to the Abl-encoded kinase activity, numerous studies have determined that there are also domains present within the Bcr sequences that are required to support oncogenic activity. Although poorly characterized, these domains are intriguing since they represent unexploited targets for pharmacological intervention. In the previous funding period, our laboratory as been exploring the hypothesis that the characterization of Bcr- encoded activities will identify viable new targets for p210 Bcr-Abl inhibition. Three such activities have now been identified. First, it has been determined that the RhoGEF domain, which is autoinhibited in Bcr, is constitutively activated in p210 Bcr-Abl. Mutations that eliminate this activity in p210 Bcr-Abl exhibit diminished transforming activity in cell- and animal-based models suggesting that this domain may be required for CML. Second, it has been determined that Bcr is a regulatory component of the endosomal sorting pathway, and that this pathway is disrupted by overexpression of p210 Bcr-Abl in hematopoietic cells. This represents a novel, and completely unexplored aspect of p210 Bcr-Abl biology, which undoubtedly will be relevant to CML. Finally, it has been determined that Bcr has a nuclear function through which it can regulate levels of protein expression. This includes the c-Myc oncoprotein which is known to contribute to CML. Bcr-Abl regulates c- Myc through binding to XPB, and disruption of this interaction is also associated with diminished transforming activity in cell- and animal-based models. In this continuation application these activities will be characterized in the context of hematopoietic cells, and it will be determined whether they are viable targets for p210 Bcr-Abl inhibition. For this analysis, p210 Bcr-Abl mutants have been generated that are impaired in these activities, and which can be evaluated in cell- and animal-based models for CML. PUBLIC HEALTH RELEVANCE: Imatinib is a pharmacological inhibitor that causes clinical remission in many patients with Chronic Myelogenous Leukemia. However, imatinib controls rather than cures the disease, and many patients develop resistance to the treatment. Thus, the focus of this proposal is the development of novel approaches and inhibitors that can be used to complement imatinib treatment.