Validation of the Fes Tyrosine Kinase as an Inhibitor Target in Multiple Myeloma

 描述(申请人提供)：c-fes原癌基因编码细胞质蛋白酪氨酸激酶fes，参与控制分化、存活、迁移和炎症的细胞信号级联反应。最近对人类多发性骨髓瘤(MM)细胞系进行的一项全基因组siRNA筛查发现，FES对其生长和存活至关重要。FES也是几种血管生成因子的共同信号介质，使FES成为MM和其他癌症抗血管生成治疗的潜在靶点。最近，我们发现了一类具有纳摩尔效力的小分子FeS激酶抑制剂。使用这些抑制剂，我们确定了FES激酶活性在小鼠骨髓巨噬细胞向破骨细胞分化中的新作用，表明抑制这一依赖FES的途径可能对MM相关的溶骨性骨丢失有临床意义。这些观察表明，抑制FES激酶活性在MM中可能有三个方面的好处：1)直接抑制骨髓瘤细胞的生长；2)阻断MM诱导的破骨细胞分化；3)抑制肿瘤血管生成。在这里，我们建议验证FES作为我们在MM的这些方面的抑制剂的靶点，具体目的如下：目的1：验证FES蛋白酪氨酸激酶是多发性骨髓瘤的肿瘤内在药物靶点的假设。尽管RNAi介导的FES表达下调可诱导几种人骨髓瘤细胞系的凋亡，但在多发性骨髓瘤中FES蛋白表达和激酶活性上调的频率尚未被探索。我们建议确定一组不同的人类多发性骨髓瘤细胞系、患者来源的多发性骨髓瘤细胞和骨髓瘤组织微阵列中FES的表达和活性。然后将测试MM细胞对我们的FeS抑制剂在细胞增殖和存活方面的敏感性。验证FeS作为抑制剂的靶点将涉及到通过异位表达工程耐药FeS来挽救抑制剂的敏感性 敏感细胞中的突变体。相反，在抑制剂不敏感、FES阴性的MM细胞中引入活性FES可能会使它们对这些化合物敏感。目的2：验证与骨髓瘤相关的骨溶解和血管生成需要FeS激酶活性的假说。最近，我们有了一个意想不到的发现，即多种类型的FeS激酶抑制剂可以有效地阻止小鼠原代骨髓巨噬细胞和细胞系向破骨细胞分化。我们建议测试我们的FES抑制剂抑制原代人巨噬细胞破骨细胞的形成，并在骨吸收试验中确定这些抑制剂是否也抑制其溶骨活性。此外，我们将使用我们的FeS抑制剂来探索FeS激酶活性在使用人脐静脉内皮细胞(HUVECs)的内皮细胞增殖、迁移和毛细血管形成中的作用。最后，我们将使用MM/HUVEC共培养系统评估FeS抑制MM细胞与血管内皮细胞黏附的后果。

 DESCRIPTION (provided by applicant): The c-fes proto-oncogene encodes the cytoplasmic protein-tyrosine kinase FES, which participates in cellular signaling cascades that govern differentiation, survival, migration and inflammation. A recent kinome-wide siRNA screen identified FES as essential for the growth and survival of human multiple myeloma (MM) cell lines. FES is also a common signaling mediator for several angiogenic factors, making FES a potential target for anti-angiogenic therapy in MM and other cancers. Recently, we reported the discovery of first-in-class small-molecule FES kinase inhibitors with nanomolar potency. Using these inhibitors, we established a new role for FES kinase activity in osteoclast differentiation from mouse bone marrow macrophages, suggesting that inhibition of this FES-dependent pathway may be of clinical benefit in osteolytic bone loss associated with MM. Together, these observations suggest that inhibition of FES kinase activity may have a three-pronged benefit in MM: 1) direct inhibition of myeloma tumor cell growth; 2) block in MM-driven osteoclast differentiation; 3) suppression of tumor angiogenesis. Here we propose to validate FES as the target for our inhibitors in each of these aspects of MM with the following Specific Aims: Aim 1: Test the hypothesis that the FES protein-tyrosine kinase is a tumor-intrinsic drug target in multiple myeloma. Although RNAi-mediated knockdown of FES expression induces apoptosis in several human myeloma cell lines, the frequency with which FES protein expression and kinase activity are upregulated in MM has not been explored. We propose to determine FES expression and activity profiles for a diverse panel of human MM cell lines, patient- derived MM cells and myeloma tissue microarrays. MM cells will then be tested for sensitivity to our FES inhibitors in terms of cell proliferation and survival. Validation of FES as the inhibitor target wll involve rescue of inhibitor sensitivity by ectopic expression of engineered inhibitor-resistant FES mutants in sensitive cells. Conversely, introduction of active FES into inhibitor-insensitive, FES-negative MM cells may render them sensitive to these compounds. Aim 2: Test the hypothesis that FES kinase activity is required for MM-associated osteolysis and angiogenesis. Recently we made the unexpected discovery that multiple classes of FES kinase inhibitors potently block osteoclast differentiation from primary mouse bone marrow macrophages and cell lines. We propose to test our Fes inhibitors for suppression of osteoclast formation from primary human macrophages, and determine whether the inhibitors also suppress their osteolytic activity in bone resorption assays. In addition, we will use our FES inhibitors to explore the role of FES kinase activity in endothelial cell proliferation, migration and capillary formation using human umbilical vein endothelial cells (HUVECs). Finally, we will evaluate the consequences of FES inhibition on MM cell adhesion to vascular endothelial cells using a MM/HUVEC co-culture system.