ANTI-NEOPLASTIC EFFECTS OF GAMMA-SECRETASE INHIBITORS

γ -分泌酶是一种多亚基膜蛋白酶，可催化多种I型膜蛋白的调节裂解。其中包括Notch受体，它需要γ分泌酶裂解才能激活。γ -分泌酶抑制剂已被提议作为表达Notch受体的癌症的治疗药物，特别是t细胞急性淋巴细胞白血病（T-ALL），最近发现频繁激活Notch-1突变。我们的实验室正在探索抑制Notch信号治疗肿瘤疾病的药理学和遗传学策略。作为这项工作的一部分，除了关注癌细胞杀伤机制外，我们还在探索潜在的毒性和规避它们的策略。在与博士合作。Osborne和Golde，我们研究了胸腺细胞和外周t细胞中的Notch信号，并将其与我们在T-ALL恶性t细胞和其他癌细胞中观察到的进行了比较。有证据表明，Notch-1激活胸腺细胞、外周t细胞、白血病t细胞、小鼠红白血病细胞（MEL）、原发性角化细胞（KC）、宫颈癌细胞和神经元中的NF-KB。这些观察结果表明，Notch-1触发了一个激活NF-KB的保守通路。我们已经确定这一途径始于γ -分泌酶依赖的切割Notch-1和两个关键蛋白激酶（IKK和PI3K）之间复合物的形成，导致akt介导的IKK和NF-KB活化。这表明抑制Notch激活的药物和抑制NF-KB的药物可能协同作用，我们的初步数据支持这一假设。我们将在T-ALL细胞的药理学和机制实验中测试该模型，使用γ -分泌酶抑制剂与蛋白酶体抑制剂和糖皮质激素联合使用，分别阻止NF-KB激活或拮抗其作用。在与博士合作。最后，Osborne和Golde我们已经确定γ分泌酶抑制剂阻断树突状细胞分化和t细胞活化。因此，当与癌症疫苗一起使用时，这些药物的全身管理可能会产生免疫抑制作用，并损害癌症疫苗的功效。因此，确定γ -分泌酶抑制剂在已建立的癌症疫苗模型中的作用，并确定化疗-免疫联合治疗的最佳给药方案是很重要的。为了解决这些问题，我们再次与dr。奥斯本、戈尔德和卡斯特。

Gamma-Secretase is a multi-subunit membrane protease that catalyzes the regulated cleavage of numerous type I membrane proteins. These include Notch receptors, which require gamma-secretase cleavage for activation. Gamma-Secretase inhibitors have been proposed as therapeutic agents in cancers expressing Notch receptors, particularly T-cell acute lymphoblastic leukemia (T-ALL), where frequent activating Notch-1 mutations have been recently discovered. Our laboratory is exploring pharmacological and genetic strategies to inhibit Notch signaling for the treatment of neoplastic disorders. As a part of this effort, in addition to focusing on cancer cell killing mechanisms, we are exploring potential toxicities and strategies to circumvent them. In collaboration with Drs. Osborne and Golde, we have studied Notch signaling in thymocytes and peripheral T-cells and compared it to what we observe in malignant T-cells from T-ALL and in other cancer cells. Evidence indicates that Notch-1 activates NF-KB in thymocytes, peripheral T-cells, leukemic T-cells, murine erythroleukemia cells (MEL), primary keratinocytes (KC), cervical cancer cells and neurons. These observations suggest that Notch-1 triggers a conserved pathway that activates NF-KB. We have determined that this pathway begins with the gamma-secretase-dependent formation of complexes between cleaved Notch-1 and two key protein kinases: IKK and PI3K, resulting in AKT-mediated IKK and NF-KB activation. This implies that drugs that inhibit Notch activation and drugs that inhibit NF-KB may synergize, and our preliminary data support this hypothesis. We will test this model in pharmacological and mechanistic experiments in T-ALL cells, using gamma-secretase inhibitors in combination with proteasome inhibitors and with glucocorticoids, drugs that prevent NF-KB activation or antagonize its effects respectively. In collaboration with Drs. Kast, Osborne and Golde we have determined that gamma-secretase inhibitors block dendritic cell differentiation and T-cell activation. Therefore, it is possible that systemic administration of these drugs will have immunosuppressive effects and compromise the efficacy of cancer vaccines when used together with them. Therefore, it is important to determine the effects of gamma-secretase inhibitors in established cancer vaccine models and identify the best administration regimens for combined chemo-immunotherapy. To address these issues, we have once again teamed up with Drs. Osborne, Golde and Kast. Our Hypothesis is that gamma-secretase inhibitors such as LY411,575 (LY) will have chemotherapeutic effects in T-ALL cells and other Notch-expressing cancer cells in vitro and in vivo, mainly through inhibition of Notch-mediated activation of NF-KB via the new pathway we discovered. We further hypothesize that proteasome inhibitor bortezomib, which blocks NF-KB activation, and glucocorticoids, which antagonize NF-KB and cause Notch-1 degradation, will synergize with LY in T-ALL cells. We postulate that immunosuppressive effects of LY can be circumvented when used together with cervical cancer vaccines by timing its administration after the priming phase of the immune response. To test our hypothesis, we propose the following Specific Aims: 1. To establish if the in vitro effects and mechanism of action of LY alone and in combination with clinically relevant agents in T-ALL cell lines. Mechanistic studies will focus on AKT and NF-KB. 2. To determine the in vivo effects of LY, alone and in combination with clinically relevant chemotherapeutic agents chosen based on mechanism, on xenograft models of T-ALL. 3. To determine the in vivo effects of LY on the immune response against cancer cells, using well-established models of tumor vaccination.