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.
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