ANTI-NEOPLASTIC EFFECTS OF GAMMA-SECRETASE INHIBITORS

γ-分泌酶是一种多亚单位膜蛋白，可催化多种I型膜蛋白的受控裂解。其中包括Notch受体，它需要伽马分泌酶裂解才能激活。伽玛分泌酶抑制剂已被建议用于治疗表达Notch受体的癌症，特别是最近发现频繁激活Notch-1突变的T细胞急性淋巴细胞白血病(T-ALL)。我们的实验室正在探索抑制Notch信号的药理和遗传策略，以治疗肿瘤疾病。作为这项努力的一部分，除了关注癌细胞的杀伤机制外，我们还在探索潜在的毒性和规避它们的策略。在奥斯本和戈尔德博士的合作下，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和其他癌细胞中观察到的恶性T细胞中的信号进行了比较。有证据表明，Notch-1可激活胸腺细胞、外周血T细胞、白血病T细胞、小鼠红白血病细胞(MEL)、原代角质形成细胞(KC)、宫颈癌细胞和神经元中的核因子-KB。这些观察结果表明，Notch-1触发了一条激活核因子-KB的保守途径。我们已经确定，这一途径始于切割的Notch-1与两个关键蛋白激酶：IKK和PI3K之间依赖于伽马分泌酶的复合体的形成，导致AKT介导的IKK和NF-KB的激活。这意味着抑制Notch激活的药物和抑制NF-KB的药物可能具有协同作用，我们的初步数据支持这一假说。我们将在T-ALL细胞的药理学和机制实验中测试这一模型，分别使用伽马分泌酶抑制剂和蛋白酶体抑制剂以及糖皮质激素，这两种药物分别阻止核因子-KB激活或拮抗其影响。在与Kast、Osborne和Golde博士的合作中，我们确定了伽马分泌酶抑制剂阻止树突状细胞分化和T细胞激活。因此，当这些药物与它们一起使用时，全身给药可能会产生免疫抑制效应，并影响癌症疫苗的疗效。因此，在已建立的肿瘤疫苗模型中确定伽马分泌酶抑制剂的效果并确定联合化疗和免疫治疗的最佳给药方案是很重要的。为了解决这些问题，我们再次与奥斯本、戈尔德和卡斯特博士合作。 我们的假设是，伽玛分泌酶抑制剂，如LY411,575(LY)，在体内外对T-ALL细胞和其他表达Notch的癌细胞具有化疗作用，主要是通过我们发现的新途径抑制Notch介导的NF-KB的激活。我们进一步假设，在T-ALL细胞中，蛋白酶体抑制剂Bortezomib可以阻断NF-KB的激活，糖皮质激素可以拮抗NF-KB并导致Notch-1的降解，它们将与LY协同作用。我们推测，当LY与宫颈癌疫苗一起使用时，通过在免疫反应的启动阶段之后给药的时机，可以避免LY的免疫抑制效应。 为了验证我们的假设，我们提出了以下具体目标：1.确定LY单独和联合临床相关药物在T-ALL细胞系中的体外作用和作用机制。机制研究将集中在AKT和NF-KB上。2.观察LY单独及与临床相关化疗药物联合应用对T-ALL移植瘤模型的影响。3.采用成熟的肿瘤疫苗接种模型，确定LY对肿瘤细胞免疫应答的体内影响。

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.