Signaling Pathways and Therapeutic Targeting of Leukemic Cells

描述(由申请人提供)： 靶向BCR-ABL的特异性激酶抑制剂已经导致了显著的临床进步，并极大地改变了CML和Ph+ALL患者的预后。尽管如此，白血病细胞耐药性的出现引起了严重的关切，需要开发克服这种耐药性的方法。同样重要的是要注意，现在有证据表明，TKIs不能消除白血病起始干细胞(LIC)，即使在完全缓解的敏感病例中也是如此。因此，靶向bcr-abl下游的细胞通路，绕过bcr-abl水平的耐药性，可能为治疗这类Ph+白血病和消除LICs提供重要的临床途径。我们实验室的工作已经证实，在Ph+白血病中，mTOR途径被解除调控，并增加了这种放松调控导致白血病细胞耐药出现的可能性。我们已经提供了证据证明在BCR-ABL转化的细胞中存在两个功能不同的mTORC1复合体，即雷帕霉素敏感(RS)和雷帕霉素不敏感(RI)mTORC1，并表明RI-mTORC1在调节促进白血病细胞增殖的癌基因蛋白的mRNA翻译中发挥关键作用。我们还证实了mTORC2复合体的形成，它们的激活对白血病细胞和原发Ph+白血病前体细胞的生存至关重要。使用mTORC1和mTORC2的一种独特的双重催化抑制剂OSI-027，我们已经建立了靶向这种复合体对CML患者原始白血病前体和表达T315I bcr-abl突变的细胞的有效抑制作用。在针对Ph+细胞中AMPK代谢感受器通路的平行努力中，我们发现AMPK诱导剂抑制RI-mTORC1复合体，从而产生强大的抗白血病作用。这类药物还可以克服表达难治性bcr-abl突变的细胞的耐药性，例如T315I。这些发现提出了未来通过直接靶向RI-mTORC1和mTORC2复合体和/或通过参与AMPK来克服CML和Ph+ALL耐药性的翻译方法的前景。目前的建议是一种系统的方法，以确定Ph+白血病mTOR通路放松调控的机制，并确定可以作为治疗靶点的下游效应因子。此外，它还涉及确定导致白血病细胞耐药的反馈途径的研究，并使用针对这些途径的药物来消除Ph+白血病中的LICs。具体目标1将剖析BCR-ABL调节的信号事件，这些信号事件控制mTORC2和RI-mTORC1复合体，并将定义AMPK调节在这一过程中的调节作用。具体目标2将确定mTORC1和mTORC2复合体的下游效应物，并将系统地确定靶向不同的效应物在产生抗白血病反应中的相关性。特异性目标3将使用TKI敏感和耐药(T315I-BCR-ABL)CML的CML小鼠模型来检测mTORC1/2靶向剂的体内抗白血病特性。最后，特指目标4将系统地研究OSI-027和AMPK激活剂对大量CML和Ph+ALL患者原代细胞的影响，以及它们对白血病起始干细胞(LIC)存活的影响。它还将检查负反馈通路的激活，并将定义具有不同反馈环路调节器的双重mTORC2/mTORC1制剂的组合效果，以促进LIC的消除。总之，这些研究将促进我们对bcr-abl介导的白血病发生机制的理解，并为未来的临床翻译工作提供理论基础，包括使用双重mTORC1/2催化抑制剂和/或AMPK激活剂治疗耐药的CML和Ph+ALL。

DESCRIPTION (provided by applicant): Targeting BCR-ABL with specific kinase inhibitors has resulted in remarkable clinical advances and has dramatically changed the outcome of patients with CML and Ph+ ALL. Despite that, the emergence of leukemic cell resistance raises serious concerns and the need for the development of approaches to overcome this resistance. It is also important to note that there is now evidence that, TKIs do not eliminate leukemia initiating stem cells (LICs), even in sensitive cases where complete remission is achieved. Thus, efforts to target cellular pathways down-stream of BCR-ABL, circumventing resistance at the BCR-ABL level, may provide an important clinical approach for the treatment of such Ph+ leukemias and the elimination of LICs. Work from our laboratory has established that the mTOR pathway is deregulated in Ph+ leukemias and has raised the possibility that such deregulation contributes to the emergence of leukemic cell resistance. We have provided evidence on the existence of two functionally distinct mTORC1 complexes in BCR-ABL transformed cells, rapamycin-sensitive (RS) and rapamycin-insensitive (RI) mTORC1, and shown that RI-mTORC1 plays critical roles in the regulation of mRNA translation for oncogenic proteins that promote leukemic cell proliferation. We have also established that mTORC2 complexes are formed and their activation is important for survival of leukemia cells and primary Ph+ leukemic precursors. Using a unique dual catalytic inhibitor of mTORC1 and mTORC2, OSI-027, we have established that targeting such complexes results in potent suppressive effects on primitive leukemic precursors from CML patients and cells expressing the T315I BCR- ABL mutation. In parallel efforts to target the AMPK metabolic-sensor pathway in Ph+ cells, we found that AMPK inducers suppress RI-mTORC1 complexes, resulting in potent antileukemic effects. Such agents also overcome resistance in cells expressing refractory BCR-ABL mutations, such as T315I. These findings raise the prospect of future translational approaches to overcome resistance in CML and Ph+ ALL by directly targeting RI-mTORC1 and mTORC2 complexes and/or by engaging AMPK. The current proposal is a systematic approach to define the mechanisms of deregulation of mTOR pathways in Ph+ leukemias and to identify downstream effectors that could be therapeutically targeted. In addition it involves studies to identify feedback pathways that account for leukemic cell resistance and to use agents that target such pathways to eliminate LICs in Ph+ leukemias. Specific aim 1 will dissect BCR-ABL-regulated signaling events that control mTORC2 and RI-mTORC1 complexes and will define the regulatory effects of AMPK modulation in the process. Specific aim 2 will identify downstream effectors of mTORC1 and mTORC2 complexes and will systematically define the relevance of targeting distinct effectors in the generation of antileukemic responses. Specific aim 3 will employ CML mouse models for TKI sensitive and resistant (T315I- BCR-ABL) CML to examine the in vivo antileukemic properties of mTORC1/2 targeting agents. Finally, specific aim 4 will systematically study the effects of OSI-027 and AMPK activators on primary cells from a large number of patients with CML and Ph+ ALL and their effects on survival of leukemia initiating stem cells (LICs). It will also examine the activaion of negative feedback pathways and will define the effects of combinations of dual mTORC2/mTORC1 agents with different modulators of feedback loops, to promote elimination of LICs. Altogether, these studies should advance our understanding of the mechanisms of BCR-ABL-mediated leukemogenesis and provide the rationale for future clinical-translational efforts involving the use of dual mTORC1/2 catalytic inhibitors and/or AMPK activators for the treatment of resistant CML and Ph+ ALL.