Preet Chaudhary - US grants

Fas and the TRAIL receptors, DR4 and DR5, are apoptosis-inducing members of the Tumor Necrosis Factor Receptor (TNFR) family that play a key role in the regulation of immune response. In addition to their known role in the mediation of apoptosis, these receptors are also known to activate the NF-kappaB and JNK pathways. However, the signaling events involved in the activation of these pathways by the Fas and TRAIL receptors have not been characterized. We have discovered that caspase 8 and its homologs, caspase 10 and MRIT, are likely to play a major role in NF-kappaB and JNK activation via Fas and DR4/DR5. These properties of caspase 8 may also explain the recently discovered role of the FADD-caspase 8 pathway in T cell activation and proliferation. The overall objective of this proposal is to further characterize the role of caspase 8 and its homologs in NF-kappaB and JNK activation by Fas and DR4/DR5 and study its biological significance. This objective will be achieved through the following specific aims. In specific aim 1, cell lines deficient in caspase 8, or expressing its mutant constructs, will be used to confirm its role in NF-kappaB and JNK activation by Fas and DR4/DR5. In specific aim 2, the molecular interactions underlying the NF-kappaB and JNK activating abilities of caspase 8 and homologs will be characterized and any novel protein(s) involved in these processes identified. In specific aim 3, role of caspase 8- and its homologs-mediated NF-kappaB and JNK activation in the process of T cell activation and development will be studied by in vitro studies in cell lines and in vivo studies in transgenic animals. We hope that the above studies will not only clarify the role of caspase 8 and its homologs in NF-kappaB and JNK activation by the Fas and TRAIL receptors but also enhance our understanding of the molecular interactions involved in immune regulation.

Infection with the human herpes virus 8 (HHV8) has been linked to the occurrence of Kaposi's sarcoma (KS) and several lymphoproliferative disorders, such as primary effusion lymphoma (PEL), multi-centric Castleman's disease, angio-immunoblastic lymphadenopathy with dysproteinemia, and multiple myeloma. However, the exact mechanism of action of HHV8 in the pathogenesis of these disorders is still unclear. Although HHV8 has been found to encode homologs of several cellular oncogenes and growth factors, almost all of them lack expression in latently infected KS and PEL cells, thereby arguing against their casual role in the pathogenesis of these disorders. We have discovered that orf-K13, an HHV8-encoded vFLIP (viral FLICE inhibitory protein), is capable of blocking apoptosis induced by death receptors belonging to the Tumor Necrosis Factor Receptor (TNFR) family. More importantly, orf-K13 is capable of activating the NF-kappaB pathway, which has been previously implicated in the pathogenesis of EBV (Epstein Barr virus)- and HTLV1 (Human T cell Leukemia virus 1)- associated lymphoproliferative disorders. As orf-K13 is one of the few HHV8 encoded proteins which are expressed in latently infected KS and PEL cells, the above results make it an ideal candidate for causing the cellular transformation associated with infection by HHV8. The overall objective of this proposal is to test the above hypothesis using in vitro and in vivo models. In aim 1, biochemical and molecular characterization of the mechanisms underlying the NF-kappaB activating ability of orf-K13 will be carried out with the hope of identifying the interactions critical for this activity. In aim 2, biological consequences of orf-K13 mediated NF-kappaB will be studied and its effect on cellular activation, proliferation and transformation characterized. Aim 3 will focus on further characterization of the anti-apoptotic properties of orf-K13 and its biological consequences. In aim 4, transgenic approach will be used to study the in vivo role of orf-K13 in the pathogenesis of AIDS- related malignancies.

Hypohidrotic ectodermal dysplasias (HED) are characterized by the triad of signs consisting of sparse hair, abnormal or missing teeth and inability to sweat. Recently, mutations in EDAR, a novel receptor of the Tumor Necrosis Factor Receptor Family (ThFR) were shown to be responsible for autosomal dominant and recessive forms of HED suggesting an essential role of this receptor in the process of ectodermal differentiation. However, the signaling mechanism(s) by which EDAR leads to ectodermal differentiation have not been defined. We have recently discovered that EDAR is capable of activating the NF-kappaB, JNK and cell death pathways. The overall goal of this proposal is further characterization of the above signaling activities of EDAR so as to better understand its role in ectodermal differentiation and in the pathogenesis of ectodermal dysplasias. This goal will be achieved through the following specific aims. In aim 1, deletion and site-directed mutagenesis of the EDAR cytoplasmic domain will be undertaken to map the domains and residues critical for NF-kappaB, JNK and cell death activation. The results of these studies will be also correlated with the clinical phenotype of ectodermal dysplasias associated with various known human and mouse EDAR mutations so as to explain the clinical heterogeneity of these disorders. In aim 2, mechanism(s) of EDAR-induced NF-kappaB, JNK and cell death pathways will be explored by using dominant negative inhibitors of the-proteins known to be involved in the activation of these pathways via other members of the TNFR family. Finally, in aim 3, yeast two- hybrid approach will be used to isolate novel signaling proteins that interact with the cytoplasmic domain of EDAR and their role in its various signaling activities explored. We hope that these studies will lead to a better understanding of the signaling pathways utilized by EDAR and of the processes involved in skin and hair differentiation. Ultimately, these studies will also lead to a better understanding of the pathogenesis of hypohidrotic ectodermal dysplasias caused by defect in EDAR-signaling.

DESCRIPTION (provided by applicant): XEDAR, EDAR and TAJ are three recently isolated receptors of the TNFR family that are mainly expressed in ectodermal derivatives during embryonic development. We have characterized the signaling pathways activated by XEDAR and discovered that it binds to TRAF3 and TRAF6 in a ligand-dependent fashion and activates the NF-kappaB and JNK pathways. Although XEDAR does not possess a death domain, it also induces apoptosis via both caspase-dependent and independent mechanisms. The overall aim of this proposal is to identify the downstream genes involved in the activation of NF-kappaB, JNK and cell death pathways by XEDAR and to compare its signaling activities with those of EDAR and TAJ. We plan to achieve this goal through the following specific aims. In specific aims 1 we will try to identify the genes involved in the activation of NF-kappaB and JNK pathways by XEDAR and its homologs. Specific aim 2 will focus on delineating the proteins involved in induction of caspase-dependent and independent cell death via XEDAR. In specific aim 3, we will identify novel proteins which are involved in signaling via XEDAR and its homologs. We believe that the above studies will not only lead to a better understanding of the process of ectodermal differentiation and craniofacial development but also help to clarify the clinical heterogeneity of ectodermal dysplasias. In the long-term these studies may lead to better diagnosis and treatment of ectodermal dysplasias and craniofacial abnormalities.

DESCRIPTION (provided by applicant): Human herpes virus 8 (HHV8), also known as Kaposi's sarcoma associated herpes virus (K.SHV) is the most frequent cause of malignancy among AIDS patients. Infection with HHV8 has been linked to the occurrence of Kaposi's sarcoma (KS) and several lymphoproliferative disorders, such as primary effusion lymphoma (PEL) and multicentric Castleman's disease. However, the exact mechanism of action of HHV8 in the pathogenesis of these disorders is still unclear. We have discovered that K13, an HHV8-encoded vFLIP (viral FLICE inhibitory protein), possesses the unique abilities to activate the classical and alternative NF-KB pathways by interacting with different components of the 1KB kinase (IKK) complex. We have further demonstrated that K13 is an oncogene which mediates increased cellular proliferation, transformation, cytokine secretion and protection against growth factor withdrawal-induced apoptosis via NF-KB activation. The overall goal of this proposal is to further understand the mechanism of NF-KB activation by K13 and identify the downstream genes involved in its various biological activities. We plan to achieve this goal through the following specific aims. In aim 1, we will characterize the molecular interactions of K13 involved in classical and alternative NF-KB activation. In aim 2, we will characterize the role of post-translational modifications of K13 and IKK complex subunits in K13-induced classical and alternative NF-KB pathways. Aim 3 will focus on studying the role of nuclear translocation of K13 and IKK subunits in K13 induced NF- KB pathway. We hope that these studies will lead to the identification of genes and proteins that are critically involved in the pathogenesis of HHV8-associated diseases and, therefore, are ideal candidates for the development of molecularly targeted therapies.

Human herpes virus 8 (HHV8), also known as Kaposi's sarcoma associated herpes virus (KSHV) is the most frequent cause of malignancy among AIDS patients. Infection with HHV8 has been linked to the occurrence of Kaposi's sarcoma (KS) and several lymphoproliferative disorders, such as primary effusion lymphoma (PEL), multicentric Castleman's disease and immunoblastic/plasmablastic lymphomas. Because of underlying immunosuppression, HHV8-associated cancers have extremely poor prognosis when treated with conventional chemotherapy and there is urgent need for more effective and less toxic therapies for these disorders. However, the exact mechanism of action of HHV8 in the pathogenesis of these disorders is still unclear. We have discovered that K13,an HHV8-encoded vFLIP (viral FLICE inhibitory protein), possesses the unique abilities to activate the classical and alternative NF-KB pathways by interacting with different components of the kB kinase (IKK) complex. We have further demonstrated that K13 is an oncogene which mediates increased cellular proliferation, transformation, cytokine secretion and protection against growth factor withdrawal-induced apoptosis via NF-KB activation. Thus, we believe that K13 is a pivotal player in the pathogenesis of HHV8-associated lymphoproliferative disorders and an ideal candidate for development of molecularly targeted therapies. We have further discovered that arsenic trioxide (As203), a drug which is in clinical trials for a number of human cancers, is a potent inhibitor of K13-induced NF-KB activity. The primary goal of this proposal is to test the ability of As203 against HHV-8 associated malignancies when used alone and in combination with other agents. We plan to achieve this goal through the following specific aims. In aim 1, we will study the mechanism by which As203 blocks K13-induced NF-KB activation. In aim 2, we will study the role of NF-KB pathway in the pro-apoptotic and anti-proliferative activities of As203 against PEL cells. Finally, in aim 3 we will study the effect of As203 on in vitro and in vivo models of HHV8-associated malignancies. We hope that these studies will lead to the development of less toxic and more effective molecularly targeted agents for the treatment of HHV8-associated lymphoproliferative disorders.

[unreadable] DESCRIPTION (provided by applicant): Bone-marrow suppression is frequently seen in association with infection by human viruses, including the cytomegalovirus, Epstein-Barr virus, human herpes virus 6 and hepatitis B virus, and is an important cause of morbidity and mortality among post-transplant patients. Human herpes virus 8 (HHV8), also known as Kaposi's sarcoma associated herpes virus (KSHV), has been previously linked to the occurrence of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Recent studies have also linked infection with HHV-8 to bone marrow failure in solid-organ and stem cell transplant recipients. However, the exact mechanism of action of HHV8 in the pathogenesis of marrow failure is has not been characterized. We have discovered that K13, an HHV8-encoded vFLIP (viral FLICE inhibitory protein) is a strong activator of the NF-kB pathway, which has been previously linked to hematopoietic suppression. We have further demonstrated that K13 can upregulate the expression of cytokines with marrow suppressive properties. Thus, we believe that K13 may play a pivotal role in the pathogenesis of HHV8-associated marrow suppression and presents an ideal candidate for development of molecularly targeted therapies against this disorder. The primary goal of this application is to test the above hypotheses using in vitro and in vivo assays. In aim 1, we will express K13 in the marrow of transgenic mice and study its effect on hematopoieses. In aim 2, we will study the effect of K13 on the growth and proliferation of hematopoietic stem cells and progenitor cells. Finally, in aim 3, we will test the ability of inhibitors of K13-induced NF-?B activation and their downstream target genes to reverse its suppressive effect on hematopoieses. We believe that collectively these studies will not only lead to a better understanding of viral-induced marrow failure syndromes but will also provide the framework for future clinical evaluation of NF-kB inhibitors in these disorders. Bone marrow failure is frequently observed in patients undergoing bone marrow and solid-organ transplants. This study will lead to a better understanding of the mechanism(s) by which infection with viruses leads to bone marrow failure in transplant patients and provide new treatment strategies. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): PROJECT SUMMARY Kaposi's sarcoma associated herpes virus (KSHV), also known as Human herpes virus 8 (HHV8), is the most frequent cause of malignancy among AIDS patients. Infection with KSHV has been linked to the occurrence of Kaposi's sarcoma (KS) and several lymphoproliferative disorders including primary effusion lymphoma (PEL), multicentric Castleman's disease and immunoblastic/plasmablastic lymphomas. Because of underlying immunosuppression, KSHV-associated cancers have extremely poor prognosis when treated with conventional chemotherapy and there is urgent need for more effective and less toxic therapies for these disorders. However, the exact mechanism of action of KSHV in the pathogenesis of these disorders is still unclear. We have discovered that K13, a KSHV-encoded vFLIP (viral FLICE inhibitory protein), possesses the unique abilities to activate the classical and alternative NF-?B pathways by interacting with different components of the I?B kinase (IKK) complex. We have further demonstrated that K13 is an oncogene that mediates increased cellular proliferation, transformation, cytokine secretion and protection against growth factor withdrawal-induced apoptosis via NF?B activation. Thus, we believe that K13 is a pivotal player in the pathogenesis of KSHV-associated lymphoproliferative disorders and an ideal candidate for development of molecularly targeted therapies. The primary goal of this proposal is to develop a high throughput assay to isolate small molecule inhibitors of K13-induced NF?B activation and test their ability to block its biological activities using assays established in our laboratory. We hope that these studies will not only lead to a better understanding of the various biological functions of K13 but also to the identification of less toxic and more effective molecularly targeted agents for the treatment of KSHV-associated cancers. PROJECT NARRATIVE Infection with the Kaposi's sarcoma associated herpesvirus (KSHV) has been linked to a number of human cancers. The goal of this project is to develop a screening assay to identify inhibitors of K13, a key protein encoded by this virus. Such inhibitors will lead to a better understanding of the K13 protein in the pathogenesis of KSHV-associated cancers and to the development of more effective drugs for their treatment. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Human Herpesvirus 8 (HHV8, also known as KSHV) is one of the commonest causes of malignancies among young adults in parts of the world and has been associated with Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). The prognosis of patients with HHV8-associated lymphoproliferative disorders is extremely poor due to their immunocompromised status and there is an urgent need for less toxic therapies for the treatment of these malignancies. We have discovered that K13, a small protein encoded by HHV8, directly interacts with the NEMO/IKK3 subunit of the IkB kinase (IKK) complex to activate the NF-kB pathway and utilizes this pathway to promote cellular survival, proliferation, transformation and cytokine secretion. The above studies have established NF-kB pathway as an important therapeutic target for the treatment of HHV8-associated malignancies. However, since NF-kB pathway plays a key role in normal immune and inflammatory response, global inhibitors of this pathway are likely to lead to severe immunosuppression, thus limiting their potential clinical utility in HHV8-infected patients. To circumvent this problem, we propose to develop a high throughput screening (HTS) assay for isolating small molecule inhibitors of K13-NEMO interaction. It is hoped that such inhibitors will specifically block K13-induced NF-kB without interfering with the physiological activation of this pathway during normal immune and inflammatory response. Furthermore, specific inhibitors of K13-NEMO interaction will serve as useful pharmacological probes to understand the various biological activities of K13. PUBLIC HEALTH RELEVANCE: Infection with the Human Herpesvirus 8 (HHV8) has been linked to a number of human cancers. In this project, we propose to develop a high throughput screening assay for compounds that can block the interaction of K13, a small protein encoded by HHV8, with the cellular regulatory protein NEMO. It is hoped that such compounds will not only lead to a better understanding of the biological functions of K13 but also serve as lead compounds for the development of targeted therapies for HHV8-associated malignancies.

DESCRIPTION (provided by applicant): Infection with the Kaposi's sarcoma associated herpesvirus (KSHV) has been linked to the occurrence of Kaposi's sarcoma (KS) and several lymphoproliferative disorders, such as primary effusion lymphoma (PEL), multicentric Castleman's disease and immunoblastic/plasmablastic lymphomas. Due to underlying immunosuppression, KSHV-associated cancers have extremely poor prognosis when treated with conventional chemotherapy and there is urgent need for more effective and less toxic therapies for these disorders. Previous studies from our laboratory have shown that KSHV-encoded viral FLICE inhibitory protein (vFLIP) K13 is a powerful activator of the NF-kB pathway and plays a key role in the pathogenesis of KSHV-associated malignancies. K13 activates the NF-kB pathway by directly interacting with the NEMO/IKK3 subunit of the IkB kinase (IKK) complex and utilizes this pathway to promote cellular survival, proliferation, transformation and cytokine secretion. The above studies have established NF-kB pathway as an important therapeutic target for the treatment of KSHV-associated malignancies. However, since NF-kB pathway plays a key role in normal immune and inflammatory responses, global inhibitors of this pathway are likely to lead to severe immunosuppression, thus limiting their potential clinical utility in KSHV-infected patients. The overall goal of this proposal is to design cell-permeable helical peptides capable of blocking K13-NEMO interaction and to test their ability to block K13-induced NF-kB using in vitro and in vivo models developed in our laboratory. It is hoped that such peptides will specifically block K13-induced NF-kB without interfering with the physiological activation of this pathway during normal immune and inflammatory response. PUBLIC HEALTH RELEVANCE: Kaposi's sarcoma associated herpesvirus (KSHV) is the commonest cause of malignancies among patients with AIDS. The goal of this project is to develop peptides that can block the activity of K13, a small protein encoded by KSHV. It is hoped that such peptides will have utility for the development of targeted therapies for KSHV-associated malignancies.

? DESCRIPTION (provided by applicant): Human herpesvirus 8 (HHV8), also known as Kaposi's sarcoma associated herpesvirus (KSHV) is the most frequent cause of malignancy among AIDS patients. In addition to Kaposi's sarcoma, infection with HHV8 has been linked to the occurrence of primary effusion lymphoma (PEL) or body cavity lymphoma. Due to underlying immunosuppression, KSHV-associated cancers have extremely poor prognosis when treated with conventional chemotherapy and there is urgent need for more effective and less toxic therapies for these disorders. We and others have previously reported that NF-?B pathway is constitutively active in HHV8-infected cells primarily due to the activity of HHV8-encoded vFLIP (viral FLICE inhibitory protein) K13, which activates this pathway by interacting with and activating a ~700 kDa I?B kinase (IKK) complex. We have discovered that inhibitors of IKK? (IKK epsilon), an IKK related kinase, are selectively toxic to cells that are dependent on K13-induced NF-?B for their survival. Additionally, IKK? is up-regulated by K13 and physically interacts with it. Since IKK? knockout mice, in contrast to IKK? or IKK? knockout mice, are viable and fertile, our hypothesis is that IKK? may be a target of great clinical value for the treatment of KSHV associated PEL and KS. We plan to test this hypothesis through the following specific aims. In aim 1, we will delineate the role of IKK? in K13 and KSHV-induced NF-?B pathway. In aim 2, we will characterize the role of IKK? in the various biological activitis of K13 and KSHV. Finally, in aim 3, we will study the activity of IKK? inhibitors in KSHV-associated PEL and KS using in vitro and in vivo models. It is hoped that the above studies will not only clarify the role played by IKK? in K13- and KSHV- induced NF-?B but will also delineate its contribution to the pathogenesis of PEL of KS. Finally, these studies will lead to novel approaches for the treatment of PEL and KS.

? DESCRIPTION (provided by applicant): Human herpesvirus 8 (HHV8), also known as Kaposi's sarcoma associated herpesvirus (KSHV) is the most frequent cause of malignancy among AIDS patients. In addition to Kaposi's sarcoma, infection with HHV8 has been linked to the occurrence of primary effusion lymphoma (PEL) or body cavity lymphoma. Due to underlying immunosuppression, KSHV-associated cancers have extremely poor prognosis when treated with conventional chemotherapy and there is urgent need for more effective and less toxic therapies for these disorders. We and others have previously reported that NF-?B pathway is constitutively active in HHV8-infected cells primarily due to the activity of HHV8-encoded vFLIP (viral FLICE inhibitory protein) K13, which activates this pathway by interacting with and activating a ~700 kDa I?B kinase (IKK) complex. We have discovered that inhibitors of IKK? (IKK epsilon), an IKK related kinase, are selectively toxic to cells that are dependent on K13-induced NF-?B for their survival. Additionally, IKK? is up-regulated by K13 and physically interacts with it. Since IKK? knockout mice, in contrast to IKK? or IKK? knockout mice, are viable and fertile, our hypothesis is that IKK? may be a target of great clinical value for the treatment of KSHV associated PEL and KS. We plan to test this hypothesis through the following specific aims. In aim 1, we will delineate the role of IKK? in K13 and KSHV-induced NF-?B pathway. In aim 2, we will characterize the role of IKK? in the various biological activitis of K13 and KSHV. Finally, in aim 3, we will study the activity of IKK? inhibitors in KSHV-associated PEL and KS using in vitro and in vivo models. It is hoped that the above studies will not only clarify the role played by IKK? in K13- and KSHV- induced NF-?B but will also delineate its contribution to the pathogenesis of PEL of KS. Finally, these studies will lead to novel approaches for the treatment of PEL and KS.