Lawrence Pfeffer - US grants

cell population study; interferons; cell growth regulation; cytoskeleton;

Interferon-alpha (IFNalpha) is a multifunctional cytokine, which is clinically effective in the treatment of human malignancies, viral infections and auto-immune diseases. IFNalpha elicits pleiotropic biological effects by regulating gene expression through signals generated upon its binding to a surface receptor on target cells (IFNalphaR). The first cloned subunit of the IFNalphaR, IFNAR1, plays a decisive role as a IFN signal transducing subunit but does not directly bind IFNalpha. The IFNalpha-induced tyrosine phosphorylation of STAT transcription factors is crucial in activated protein kinase (ERK2) also contribute to IFNalpha action. Based on the applicant's recent studies, the hypothesis to be tested is that via the IFNalpha-induced binding of STAT3 to the IFNalphaR, STAT3 (a transcription factor regulation the acute phase response genes), couples IFNalphaR signaling to pathways involving PI-3 kinase (phosphatidylinositol-3' kinase, an upstream element in a serine kinase transduction cascade), NF-kappaB (nuclear factor-kappaB, a transcription factor for genes important in inflammation and preventing apoptosis), and serine kinases (PKC subspecies and ERK2). In Specific Aim 1, how STAT3 acts as an adapter to couple IFNalpha signaling pathways will be characterized. To be determined is if STAT3 activation (specifically via the docking domain in STAT3 for the p85 subunit of PI-3 kinase) is required for IFNalpha-induced: PI-3 kinase activation, PKC activation, ERK2 activation, gene expression, anti-viral action, and the anti- proliferative action. In Specific Aim 2, the role of PI-3 kinase arm of the STAT3-signaling pathway in IFNalpha action will be defined. The effect of expressing dominant negative and constitutively active PI-3 kinase constructs will be examined on IFNalpha-induced: serine phosphorylation of cellular substrates, PKC activation, ERK2 activation, DNA-binding activity, gene expression, anti-viral action, anti-proliferative action, and inhibition of programmed cell death. In specific Aim 3, the role of the NF-kappaB component of the STAT3-signaling pathway in IFNalpha action will be defined. To be determine are the mechanism for NF-kappaB activation, the interaction of STAT3 with NF-kappaB proteins, and the role of NF-kappaB activation on IFNalpha-induced: gene expression, anti-viral activity, anti-proliferative activity, inhibition of programmed cell death. The overall goal of these studies to determine how the activation of STAT3, PI-3 kinase and NF-kappaB are linked, and to determine their roles in effecting IFN's biological actions.

Understanding the molecular basis of interferon-alpha (IFN_) action is an important goal when one considers IFN's therapeutic potential in cancer, viral hepatitis, and multiple sclerosis, as well as its role as a model for understanding cytokine signal transduction. IFNot elicits its biological actions by regulating gene expression through the tyrosine phosphorylation and activation of members of the STAT (signal transducers and activators of transcription) protein family. The applicant found that STAT3, a transcription factor for acute phase response genes, is a critical element in IFN signaling and induction of IFN's biological actions. In addition, it was also found that IFN promotes cell survival by activating NF-KB (nuclear factor-v_B) through a serine kinase-dependent pathway involving PI-3K (phosphatidylinositol-3' kinase) and Akt, as well as STAT3. Based on these findings, the general hypothesis to be tested is that the IFN_x receptor integrates signaling pathways involving STAT3, PI-3K and NF-vJ3. In Specific Aim 1, the role of STAT3 as a transcription factor and an adapter protein for PI-3K will be defined. The proposed studies will determine which specific amino acid residues in STAT3 undergo IFN-dependent phosphorylation, the relationship of these phosphorylation events to the biologic actions of IFN, and which IFN-responsive genes are STAT3- regulated. In Specific Aim 2, the role of NF-vJ3 in IFNa action will be defined. The proposed studies will define the relationship between PI-3K/Akt-mediated phosphorylation events and the anti-apoptotic action of IFN, the roles of TRAFs (TNF receptor-associated factors) and NIK (NF-vJ3-inducing kinase) in IFN- induced NF-rJ3 activation, the role of NF-vJ3 in gene induction by IFN, and the role of the Iv33 kinase complex in IFN promoted NF-KB activation and cell survival. Despite advances made on the IFN_x signaling pathway, the mechanisms that underlie the induction of the different biological actions of IFN_x remain poorly understood. This proposal focuses on characterizing the molecular basis of signaling pathways as they relate to 1FN action on cell proliferation and survival.

The combination of pegylated interferon-alpha (IFNalpha) with the antiviral drug, ribavirin, is the current treatment of choice for hepatitis C (Hep C). However, the mechanism whereby the combination of IFNalpha with ribavirin causes a sustained virological response as determined by the clearance of Hep C in only a fraction of the patient population is unknown. Moreover, several studies have identified specific cohorts of patients that have a relatively low response to these therapeutic regimens. For example, our study performed at the Hepatitis C Cooperative Research Center at the University of Tennessee Health Science Center (UT-Hep C CRC) is consistent with several other studies and has established that the response rate of African-Americans is significantly lower than non-Hispanic whites. This finding is of major health concern since African-Americans account for approximately 22% of HCV-infected patients in the US. Therefore, these important issues will be addressed in this proposal: what is the molecular basis for the poor response of nonresponders (and African-Americans) to combination therapy, and what is the molecular basis for the ability of ribavirin to potentiate IFN's antiviral action in Hep C. To address these questions we have established skin fibroblast cell lines and Epstein Barr Virus (EBV)-immortalized B cell lines from patients infected with Hep C genotype-1 enrolled in the clinical trial of pegylated IFN with ribavirin at the UT-Hep C CRC. These human cell lines were derived from responders (R) and nonresponders (NR), and represent Caucasian (C) and African-American (AA) patients enrolled in the IFN-ribavirin trial. These cell lines represent key reagents to address the goal of this proposal, whether differences in the IFN system between responders versus nonresponders, and Caucasian (C) versus African-American (AA) patients, underlie the differential response to IFN-ribavirin therapy. In Specific Aim 1 we will determine whether there is a difference between non-responders and responders, and Caucasian and African-American patients, in IFN sensitivity at the levels of receptor interaction, signal transduction or IFN-induced gene expression. In Specific Aim 2 we will determine whether ribavirin enhances the clinical efficacy of IFN in Hep C by modulating IFN signal transduction, IFN-induced gene expression or induction of antiviral activity by IFN.

The combination of pegylated interferon-alpha (IFNalpha) with the antiviral drug, ribavirin, is the current treatment of choice for hepatitis C (Hep C). However, the mechanism whereby the combination of IFNalpha with ribavirin causes a sustained virological response as determined by the clearance of Hep C in only a fraction of the patient population is unknown. Moreover, several studies have identified specific cohorts of patients that have a relatively low response to these therapeutic regimens. For example, our study performed at the Hepatitis C Cooperative Research Center at the University of Tennessee Health Science Center (UT-Hep C CRC) is consistent with several other studies and has established that the response rate of African-Americans is significantly lower than non-Hispanic whites. This finding is of major health concern since African-Americans account for approximately 22% of HCV-infected patients in the US. Therefore, these important issues will be addressed in this proposal: what is the molecular basis for the poor response of nonresponders (and African-Americans) to combination therapy, and what is the molecular basis for the ability of ribavirin to potentiate IFN's antiviral action in Hep C. To address these questions we have established skin fibroblast cell lines and Epstein Barr Virus (EBV)-immortalized B cell lines from patients infected with Hep C genotype-1 enrolled in the clinical trial of pegylated IFN with ribavirin at the UT-Hep C CRC. These human cell lines were derived from responders (R) and nonresponders (NR), and represent Caucasian (C) and African-American (AA) patients enrolled in the IFN-ribavirin trial. These cell lines represent key reagents to address the goal of this proposal, whether differences in the IFN system between responders versus nonresponders, and Caucasian (C) versus African-American (AA) patients, underlie the differential response to IFN-ribavirin therapy. In Specific Aim 1 we will determine whether there is a difference between non-responders and responders, and Caucasian and African-American patients, in IFN sensitivity at the levels of receptor interaction, signal transduction or IFN-induced gene expression. In Specific Aim 2 we will determine whether ribavirin enhances the clinical efficacy of IFN in Hep C by modulating IFN signal transduction, IFN-induced gene expression or induction of antiviral activity by IFN.

DESCRIPTION (provided by applicant): Brain tumors are among the leading causes of cancer-related deaths in the United States, with glioblastoma multiforme (GBM) being one of the most aggressive and difficult subtypes to treat. In this proposal we plan to pursue a strategy to treat malignant glioma with the potent antitumor cytokine, interferon-beta (IFN2). Type I (1/2) interferons (IFNs) have long been recognized for their significant, pleiotropic anticancer activity. However, despite significant activity in preclinical models against a variety of tumor types, including gliomas, the antitumor efficacy of IFNs in clinical trials has been disappointing. A significant contributing factor includes the development of resistance to IFN-mediated cell death through downregulation of apoptotic pathways. We have established that nuclear factor kB (NFkB) promotes cell survival and suppresses the expression of a subset of IFN target genes that are likely effectors of IFN's antitumor activity. Unfortunately, not only is NFkB constitutively active in many cancers, including glioma, but it can also be activated by IFN itself. This finding suggests that the potent anticancer activity of IFN may be counterbalanced by NFkB activity. Based on these observations, we hypothesize that selective inhibition of NFkB will enhance the anticancer activity of IFN. We will perform a systematic and detailed evaluation of the role of NFkB in regulating the anticancer action of IFN2 in glioma cells. Based on the insights gained, we will select and test complementary agents that should provide synergistic antitumor activity with IFN. After confirming the synergy of these agents in vitro, we will test the effectiveness of combination therapy that includes IFN in relevant preclinical models of malignant glioma. The overriding goal of this project is to increase the antitumor activity of IFN through an improved understanding of IFN2's mechanism of action against glioma and the factors that work against it. In specific aim 1 we will examine the role of NFkB in suppressing the anticancer activity of IFN2 in gliomas. To test this, we will determine in glioma cell lines 1) the contribution of the classical NF:B pathway to constitutive and IFN-induced NF:B activity;2) the contribution of the alternative NF:B pathway to constitutive and IFN-induced NF:B activity;and 3) the effects of pharmacological and genetic NF:B inhibitors on IFN activity. Based on these findings, we will test the effects of clinically available NFkB inhibitors on IFN's anticancer activity in relevant, preclinical rodent models of malignant glioma. In specific aim 2 we will characterize the role of NFkB in regulating IFN target genes and the role of these target genes in the anticancer action of IFN2 in gliomas. To test this, we will: 1) characterize the induction of these genes in response to IFN;2) determine their importance in effecting the anticancer activity of IFN in vitro and in vivo and 3) translate these findings by testing the efficacy of appropriately selected combination therapy in relevant, preclinical models of malignant glioma. PUBLIC HEALTH RELEVANCE: Brain tumors are among the leading causes of cancer-related deaths in the United States, with glioblastoma multiforme (GBM) being one of the most aggressive and difficult subtypes to treat. Thus, the treatment of malignant glioma is a significant clinical problem for which new strategies are desperately needed. In this proposal we plan to pursue a strategy to treat malignant glioma with the potent antitumor cytokine, interferon beta (IFN2).