ANTI-NEOPLASTIC EFFECTS OF GAMMA-SECRETASE INHIBITORS

γ-分泌酶是一种多亚基膜蛋白酶，催化许多I型膜蛋白的调节切割。这些包括Notch受体，其需要γ-分泌酶裂解以激活。已经提出γ-分泌酶抑制剂作为表达Notch受体的癌症，特别是T细胞急性淋巴细胞白血病（T-ALL）的治疗剂，其中最近发现频繁的激活Notch-1突变。我们的实验室正在探索药理学和遗传学策略，以抑制Notch信号转导治疗肿瘤性疾病。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。Osborne和Golde博士合作，我们研究了胸腺细胞和外周T细胞中的Notch信号传导，并将其与我们在T-ALL的恶性T细胞和其他癌细胞中观察到的信号传导进行了比较。有证据表明，Notch-1激活胸腺细胞、外周T细胞、白血病T细胞、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NF-κ B。这些观察结果表明，Notch-1触发激活NF-κ B的保守途径。我们已经确定，该途径始于切割的Notch-1和两种关键蛋白激酶IKK和PI 3 K之间的γ-分泌酶依赖性复合物的形成，导致AKT介导的IKK和NF-κ B活化。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设。我们将在T-ALL细胞中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（分别防止NF-κ B活化或拮抗其作用的药物）组合。在与Kast、Osborne和Golde博士的合作中，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，这些药物的全身给药可能具有免疫抑制作用，并在与它们一起使用时损害癌症疫苗的功效。因此，重要的是确定γ-分泌酶抑制剂在已建立的癌症疫苗模型中的作用，并确定联合化疗-免疫治疗的最佳给药方案。为了解决这些问题，我们再次与Osborne、Golde和Kast博士合作。 我们的假设是，γ-分泌酶抑制剂如LY 411，575（LY）将在体外和体内对T-ALL细胞和其他Notch表达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-κ B活化。我们进一步假设，蛋白酶体抑制剂硼替佐米，它阻断NF-κ B的活化，和糖皮质激素，拮抗NF-κ B和导致Notch-1降解，将协同LY在T-ALL细胞。我们假设LY的免疫抑制作用可以通过在免疫应答的引发阶段之后定时给药来与宫颈癌疫苗一起使用时规避。 为了验证我们的假设，我们提出了以下具体目标：1。确定LY单独给药和与临床相关药物联合给药在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-κ B。2.确定LY单独给药和与根据机制选择的临床相关化疗药物联合给药对T-ALL异种移植模型的体内作用。3.使用完善的肿瘤疫苗接种模型，确定LY对癌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.