Signaling Pathways and Therapeutic Targeting of Leukemic Cells

描述（由申请人提供）： 使用特异性激酶抑制剂靶向BCR-ABL已取得显著的临床进展，并显著改变了CML和Ph+ ALL患者的结局。尽管如此，白血病细胞耐药性的出现引起了人们的严重担忧，并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，TKI不能消除白血病起始干细胞（LIC），即使在达到完全缓解的敏感病例中也是如此。因此，努力靶向BCR-ABL下游的细胞通路，避免BCR-ABL水平的耐药性，可能为治疗此类Ph+白血病和消除LIC提供重要的临床方法。我们实验室的工作已经确定，mTOR通路在Ph+白血病中是失调的，并提出了这种失调有助于白血病细胞耐药性出现的可能性。我们已经提供了证据证明BCR-ABL转化细胞中存在两种功能不同的mTORC 1复合物，雷帕霉素敏感（RS）和雷帕霉素不敏感（RI）mTORC 1，并表明RI-mTORC 1在调节促进白血病细胞增殖的致癌蛋白的mRNA翻译中起着关键作用。我们还确定了mTORC 2复合物的形成，它们的激活对于白血病细胞和原发性Ph+白血病前体的存活是重要的。使用mTORC 1和mTORC 2的独特双重催化抑制剂OSI-027，我们已经确定靶向这种复合物对来自CML患者的原始白血病前体和表达T315 I BCR-ABL突变的细胞产生有效的抑制作用。在平行的努力，目标AMPK代谢传感器途径在Ph+细胞，我们发现AMPK诱导剂抑制RI-mTORC 1复合物，导致有效的抗白血病作用。这些药物还能克服表达难治性BCR-ABL突变（如T315 I）的细胞的耐药性。这些发现提高了通过直接靶向RI-mTORC 1和mTORC 2复合物和/或通过接合AMPK来克服CML和Ph+ ALL中的耐药性的未来翻译方法的前景。目前的建议是一个系统的方法来定义的mTOR通路在Ph+白血病的失调的机制，并确定下游效应，可以治疗靶向。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，以及使用靶向此类途径的药物消除Ph+白血病中的LIC。具体目标1将剖析控制mTORC 2和RI-mTORC 1复合物的BCR-ABL调节信号传导事件，并将定义AMPK调节在该过程中的调节作用。具体目标2将确定mTORC 1和mTORC 2复合物的下游效应子，并将系统地定义靶向不同效应子在产生抗白血病应答中的相关性。具体目标3将采用TKI敏感和耐药（T315 I-BCR-ABL）CML的CML小鼠模型，以检查mTORC 1/2靶向药物的体内抗白血病特性。最后，具体目标4将系统地研究OSI-027和AMPK激活剂对来自大量CML和Ph+ ALL患者的原代细胞的作用以及它们对白血病起始干细胞（LIC）存活的作用。它还将检查负反馈途径的激活，并将确定双重mTORC 2/mTORC 1药物与不同反馈环调节剂的组合的作用，以促进LIC的消除。总之，这些研究应该促进我们对BCR-ABL介导的白血病发生机制的理解，并为未来的临床转化工作提供理论基础，包括使用双重mTORC 1/2催化抑制剂和/或AMPK激活剂治疗耐药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.