JOURNAL OF THE NATIONAL CANCER INSTITUTE ONAL aN INSTITUTE FREER RE eee Third National AIDS Malignancy Conference 2000 Number 28 International Symposium on HIV, Leukemia, and Opportunistic Cancers Contents Introduction vi Ellen G. Feigal, Judith E. Karp Kaposi’s Sarcoma in South Africa 1 Freddy Sitas, Robert Newton Immunodeficiency, Immunosuppression, and Susceptibility to Neoplasms 5 Robert S. Schwartz Regulation of Neoplastic Angiogenesis 10 Isaiah J. Fidler Human Herpesvirus 8 K1-Associated Nuclear Factor-kappa B-Dependent Promoter Activity: 15 Role in Kaposi’s Sarcoma Inflammation? Felipe Samaniego, Shibani Pati, Judith E. Karp, Om Prakash, Debashish Bose Hematopoietic Stem Cells in HIV Disease 24 David T. Scadden, Hongmei Shen, Tao Cheng Regulation of Bcl2 Phosphorylation and Potential Significance for Leukemic Cell 30 Chemoresistance Xingming Deng, Steven M. Kornblau, Peter P. Ruvolo, W. Stratford May, Jr. Post-transplant Lymphoproliferative Disorders: Implications for Acquired Immunodeficiency 38 Syndrome-Associated Malignancies Lode J. Swinnen Acquired Immunodeficiency Syndrome-Related Kaposi’s Sarcoma Regression After Highly 44 Active Antiretroviral Therapy: Biologic Correlates of Clinical Outcome Anna Maria Cattelan, Maria Luisa Calabro, Paola Gasperini, Savina M. L. Aversa, Marisa Zanchetta, Francesco Meneghetti, Anita De Rossi, Luigi Chieco-Bianchi Papillomavirus-Like Particle Vaccines 50 John T. Schiller, Douglas R. Lowy Role of the National Cancer Institute in Acquired Immunodeficiency Syndrome-Related 35 Drug Discovery Edward A. Sausville, Robert H. Shoemaker @ Visit the Journal’s Web site at http://jnci.oupjournals.org/ @ ''PUSLIC HAL TH LIBRARY ''JOURNAL OF THE NATIONAL CANCER ‘MONOGRAPHS INSTITUTE Number 28 ISSN 0027-8874 ISBN 0-19-8509] 1-1 EDITORIAL BOARD ASSOCIATE EDITORS Frank M. Balis William J. Blot Peter M. Blumberg John D. Boice, Jr. Louise A. 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Tarone Sholom Wacholder David Schottenfeld Richard K. Severson Jerry W. Shay Debra T. Silverman Sandra M. Swain G. Marie Swanson Mario Sznol Raymond Taetle Peter R. Twentyman Regina G. Ziegler ''EDITORIAL STAFF Scientific Editors: Ellen Feigal, M.D. Judith E. Karp, M.D. Journal Managing Editor: W. Mark Leader Monograph Coordinator: Elizabeth Horowitz Manuscript Editors: Elaine Price Beck Janice Deal Joan O’Brien Rodriguez Elizabeth Horowitz Editorial Assistants: Annette C. Cook Joseph A. McCullough Amy L. Rodrigue MARKETING STAFF Marketing: Joy Cox Press Contact: Dan Eckstein* EDITORIAL POLICY: Manuscripts from key conferences dealing with cancer and closely related research fields, or a related group of papers on specific subjects of importance to cancer research, are considered for publication, with the understanding that they have not been published previously and are submitted exclusively to the Journal of the National Cancer Institute Monographs. 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E-mail: jnlorders @ oup-usa.org. © Oxford University Press *Contact by telephone at 301-986-1891, ext. 112. ''Third National AIDS Malignancy Conference Proceedings of a Conference Held at the National Institutes of Health Bethesda, Maryland May 26-27, 1999 Conference Sponsor National Cancer Institute International Symposium on HIV, Leukemia, and Opportunistic Cancers Proceedings of a Conference Held in Marrakech, Morocco May 23-28, 1999 Conference Sponsors International Association for Comparative Research on Leukemia and Related Disorders Harvard AIDS Institute Conference Co-sponsors Leukemia & Lymphoma Society of America International AIDS Society Pasteur Institute of Morocco 8» ''''Third National AIDS Malignancy Conference International Symposium on HIV, Leukemia, and Opportunistic Cancers Contents 2000 Number 28 Introduction Ellen G. Feigal, Judith E. Karp Kaposi’s Sarcoma in South Africa Freddy Sitas, Robert Newton Immunodeficiency, Immunosuppression, and Susceptibility to Neoplasms Robert S. Schwartz Regulation of Neoplastic Angiogenesis Isaiah J. Fidler Human Herpesvirus 8 K1-Associated Nuclear Factor-kappa B-Dependent Promoter Activity: Role in Kaposi’s Sarcoma Inflammation? Felipe Samaniego, Shibani Pati, Judith E. Karp, Om Prakash, Debashish Bose Hematopoietic Stem Cells in HIV Disease David T. Scadden, Hongmei Shen, Tao Cheng Regulation of Bcl2 Phosphorylation and Potential Significance for Leukemic Cell Chemoresistance Xingming Deng, Steven M. Kornblau, Peter P. Ruvolo, W. Stratford May, Jr. Post-transplant Lymphoproliferative Disorders: Implications for Acquired Immunodeficiency Syndrome-Associated Malignancies Lode J. Swinnen Acquired Immunodeficiency Syndrome-Related Kaposi’s Sarcoma Regression After Highly Active Antiretroviral Therapy: Biologic Correlates of Clinical Outcome Anna Maria Cattelan, Maria Luisa Calabro, Paola Gasperini, Savina M. L. Aversa, Marisa Zanchetta, Francesco Meneghetti, Anita De Rossi, Luigi Chieco-Bianchi Papillomavirus-Like Particle Vaccines John T. Schiller, Douglas R. Lowy Role of the National Cancer Institute in Acquired Immunodeficiency Syndrome-Related Drug Discovery Edward A. Sausville, Robert H. Shoemaker @ Visit the Journal’s Web site at http://jnci.oupjournals.org/ @ Vi 10 24 30 44 55 ''Introduction Ellen G. Feigal, Judith E. Karp Scientists, clinicians, health care workers, and community and patient advocates from around the world gathered in May 1999 at two major conferences focused on acquired immunode- ficiency syndrome (AIDS)-associated cancers, i.e., the Third National AIDS Malignancy Conference and the International Symposium on HIV, Leukemia, and Opportunistic Cancers. The Third National AIDS Malignancy Conference, sponsored by the National Cancer Institute, was held at the National Institutes of Health campus in Bethesda, MD. The International Sympo- sium on HIV, Leukemia, and Opportunistic Cancers, sponsored by the International Association for Comparative Research on Leukemia and Related Disorders (IACRLRD) and the Harvard AIDS Institute and cosponsored by the Leukemia Society of America, the International AIDS Society, and the Pasteur Insti- tute of Morocco, was held in Morocco. This monograph contains a selection of the papers presented at these two multidisciplinary meetings. AIDS, caused by the retrovirus known as human immunode- ficiency virus (HIV), was first recognized two decades ago. Since that time, scientists have made much progress in under- standing the etiology, pathogenesis, and pathophysiology of this devastating disease. The study of AIDS has generated important concepts that apply broadly to stem cell biology and immuno- biology. In turn, these basic scientific discoveries have served as a springboard for exciting therapeutic advances that hold great promise with respect to deterring, reversing, and, in some cases, preventing progressive HIV disease. AIDS remains a major killer throughout the world. A par- ticular challenge is the link to certain types of malignancies, driven at least in part by the prolongation of survival in the face of impaired immunity. The molecular and clinical dissection of these complex diseases is highly instructive with regard to set- tings where immunodeficiency promotes or permits tumorigen- esis—for instance, post-transplant lymphoproliferative disor- ders. The entire area of viral oncogenesis continues to be informed by AIDS-related malignancies, in particular the tumor- igenic roles of Epstein-Barr virus and human papillomaviruses, vi and the etiologic relationship of human herpesvirus 8 to dispa- rate malignancies such as Kaposi’s sarcoma (KS) and primary effusion lymphomas. In turn, these malignancies provide a piv- otal testing ground for antiviral and immunomodulatory strate- gies, including antiangiogenesis and cytokine-based approaches, adoptive immunotherapy, vaccine development, and gene-based strategies that could have a major impact on a broad spectrum of cancers. Ultimately, however, the major goal of AIDS research is to prevent progressive HIV infection and HIV-induced immuno- suppression. The impact of multitargeted, highly active antiret- roviral therapy is already being felt in terms of AIDS-associated KS. Moreover, the ability to preserve and, in some instances, to induce regeneration of a competent immune system stands to have a future impact on the incidence of virally induced lym- phoproliferative diseases and virally related epithelial tumors. Most importantly, the ability to deliver the current and future advances to the entire global population is essential to eradica- tion of the worldwide devastation that continues to result from AIDS infection. The next conference focused on AIDS malignancy is the Fifth International AIDS Malignancy Conference, which will take place on April 23-25, 2001, in Bethesda, MD. It will include sessions on human papillomavirus, Epstein-Barr virus, and Ka- posi’s sarcoma-associated herpesvirus. Registration and other conference information are available on the website http:// ctep.info.nih.gov/AIDSOncoResources. A summary of the Fourth International AIDS Malignancy Conference is available on the website http://hiv.medscape.com/conferences/malignancy 2000. Affiliations of authors: E.G. Feigal, Division of Cancer Treatment and Di- agnosis, National Cancer Institute,Bethesda, MD; J. E. Karp, Greenebaum Can- cer Center, University of Maryland, Baltimore. Correspondence to: Ellen G. Feigal, M.D., National Institutes of Health, Bldg. 31, Rm. 3A44, Bethesda, MD 20892. © Oxford University Press Journal of the National Cancer Institute Monographs No. 28, 2000 ''Kaposi’s Sarcoma in South Africa Freddy Sitas, Robert Newton Kaposi’s sarcoma was endemic in South Africa even before the advent of the human immunodeficiency virus (HIV). Be- tween 1988 and 1996, the incidence of Kaposi’s sarcoma in South Africa has risen at least threefold and continues to increase as the HIV epidemic grows. Research from South Africa has shown that infection with human herpesvirus 8 (HH V8) is associated with Kaposi’s sarcoma but not with any other major cancer site or type. In addition, the risk of Kaposi’s sarcoma increases with increasing antibody titer to HHV8, but, for a given titer, the risk is greater in HIV- seropositive compared with HIV-seronegative individuals. The age- and sex-standardized seroprevalence of HHV8 in black South African hospital patients was found to be slightly more than 30%; the seroprevalence of HHV8 in- creased with age and was similar in men and in women. The modes of transmission of HHV8 are yet to be fully eluci- dated. Limited evidence exists for sexual transmission in black South African adults, but mother-to-child and person- to-person transmission in childhood is also likely. Further- more, the seroprevalence of HHV8 decreases with increasing levels of education and is lower in whites than in blacks, suggesting that factors associated with poverty may be im- portant determinants of transmission. Future research should focus on risk factors for Kaposi’s sarcoma in HHV8- infected individuals, on determinants and mode of transmis- sion of HHV8, and on the elucidation of the effect of primary HHVS8 infection in adults and in children. [J Natl Cancer Inst Monogr 2000;28:1—4] Before the human immunodeficiency virus (HIV) epidemic, Kaposi’s sarcoma showed a greater geographic variation in in- cidence than almost any other cancer. It was as common in parts of sub-Saharan Africa, such as Uganda and eastern Zaire, as colon cancer is in Europe and the United States, representing up to 9% of all cancers in men (/-4). Narrow belts of relatively high incidence stretched westward across the former Zaire to the coast of Cameroon and southward down the Rift Valley into Malawi and parts of South Africa (Fig. 1) (4,5). Kaposi’s sar- coma was also endemic, although much rarer, in countries around the Mediterranean, particularly Italy, Greece, and the Middle East, but it was almost nonexistent elsewhere in the world, except in immigrants from those endemic countries (6— 8). In all of these areas, Kaposi’s sarcoma was considerably more common in men than in women (4). HIV AND Kaposi’s SARCOMA It was the appearance of aggressive forms of Kaposi’s sar- coma in the United States in the early 1980s that heralded the onset of the HIV epidemic in western countries. Although the incidence of Kaposi’s sarcoma has increased in populations at high risk of HIV in northern Europe and in the United States, it existed in the rest of these populations at such a low level before the onset of the epidemic that it still remains a relatively rare tumor (6,9). However, parts of Africa with a high prevalence of Journal of the National Cancer Institute Monographs No. 28, 2000 HIV and where Kaposi’s sarcoma was relatively common even before the era of acquired immunodeficiency syndrome (AIDS) have seen an explosion in the incidence of the disease. In the past 10-15 years, the incidence of Kaposi’s sarcoma has in- creased about 20-fold in Uganda and Zimbabwe, such that it is now the most common cancer in men and the second most common in women (/0,//). Similarly, between 1988 and 1996, the incidence of Kaposi’s sarcoma has risen at least threefold in South Africa and continues to increase as the HIV epidemic grows (/2). Data from the South African National Cancer Reg- istry show that, between 1992 and 1996, the incidence rates of Kaposi’s sarcoma have doubled in men but have increased about sevenfold in women, such that the sex ratio of 7:1 in males versus females in 1988 has now declined to only 2:1 (/2). The epidemiology of HIV-associated Kaposi’s sarcoma var- ies around the world, reflecting to a certain extent the situation that existed in the era before AIDS. For example, in a South African study (/3), the relative risk of Kaposi’s sarcoma in HIV-infected individuals, compared with HIV-uninfected indi- viduals, was 62 (95% confidence interval [CI] = 20-194). Al- though this is similar to results from elsewhere in Africa (/4,/5), it is an order of magnitude lower than would be expected from studies in, for example, the United States (5). This result simply reflects the fact that Kaposi’s sarcoma is endemic in Africa with a relatively high proportion of HIV-uninfected cases; thus, the absolute risk of developing Kaposi’s sarcoma among those in- dividuals who are co-infected with HIV and with human her- pesvirus 8 (HHV8) is probably about the same as in the United States. HHV8 Anpb Kaposi’s SARCOMA HHV8, a newly discovered human herpesvirus (/6), has been consistently associated with Kaposi’s sarcoma and is now con- sidered to be the principal cause of the disease (/7). Genomic sequences of HHV8 are present in tumor cells of Kaposi’s sar- coma lesions in virtually all subjects (78), and its presence, detected by polymerase chain reaction or serology in peripheral blood, predicts the subsequent development of the tumor (19,20). Furthermore, HHV8 is not a ubiquitous virus but is most prevalent in groups or populations at highest risk of developing Kaposi’s sarcoma, such as HIV-infected homosexual men in the United States and African populations in whom the tumor has long been endemic (4,2/,22). Affiliations of authors: F. Sitas, National Cancer Registry and Cancer Epide- miology Research Group, Department of Anatomical Pathology, South African Institute for Medical Research, University of the Witwatersrand, Johannesburg: R. Newton, Cancer Epidemiology Unit, Imperial Cancer Research Fund, The Radcliffe Infirmary, Oxford, U.K. Correspondence to: Freddy Sitas, D. Phil., National Cancer Registry and Cancer Epidemiology Research Group, Department of Anatomical Pathology, South African Institute for Medical Research, University of the Witwatersrand, P.O. Box 1038, Johannesburg, 2000, South Africa (e-mail: freddys @mail.saimr. wits.ac.za). See “Notes” following “References.” © Oxford University Press '' Kaposi’s sarcoma in Africa - pre-1980 Estimated cumulative incidence males aged 0-64 Rate per 1,000 ia] <0.5 0.5- a o Cook-Mozaffari, Newton and Beral (1998) Table 1. Relation of Kaposi’s sarcoma to fluorescent signal intensity (a measure of antibody titer) for HHV8, according to HIV serostatus* No. No. Fluorescent signal with without intensity for HHV8 Kaposi’s — Kaposi’s Odds ratio+ (and median titer) sarcoma sarcoma (95% confidence interval) HIV-1-seronegative subjects Absent (<1 : 100) 5 1990 1.0% Low (1 : 200) 2 665 1.5 (0.3-7.8) Medium (1 : 51 200) 4 331 6.2 (1.6-24.2) High (1 : 204 800) 2 131 12.0 (2.1-68.2) Test for trend x’, = 11.4; P = .0007 HIV-1-seropositive subjects Absent (50%) seroprevalence, do not develop KS (6-8). HHVS8 is a recently isolated gamma herpesvirus that is related to the tumorigenic viruses herpesvirus saimiri and Epstein-Barr virus. These viruses are associated with tumor induction, par- ticularly during immunosuppression. Their genomes contain nu- merous human oncogene homologues, such as cyclin D, immu- noreceptor tyrosine-based activation motif (ITAM)-bearing signaling proteins (see below), bcl-2, and cytokine homologues of monocyte inflammatory protein I (MIP-I) and MIP-II, thereby making them resourceful models for comparative studies of viral oncogenesis (9,/0). The unique hyperplastic features of KS lesions have led in- vestigators to conclude that these lesions are distinctly different from tumors of neoplastic cells. The early stages of KS are characterized by the presence of activated endothelial cells (ECs), inflammatory cell infiltration, spindle-shaped cells of vascular origin, and angiogenesis (//,/2). The spindle cell popu- lation is dominated by activated ECs and macrophages (/3,/4) that over time proliferate to become the predominant cell phe- notype. We have shown that inflammation is a feature of KS and that inflammatory cytokines are necessary for maintaining the spindle cell phenotype found in these lesions. ECs and mono- cytes are considered to be the progenitors of KS spindle cells because they acquire the spindle cell morphology, marker ex- pression, and functional features of KS spindle cells when ex- posed to inflammatory cytokines (tumor necrosis factor-a, in- terleukin [IL] 18, and interferon gamma) (/5—/7). Inflammatory cytokines induce ECs to produce (30-fold) more basic fibroblast growth factor and vascular endothelial growth factor that are essential for generating the angiogenic features of KS lesions (15,16,18,19). Inflammatory cytokines also render primary ECs with a capacity to induce angiogenic KS-like lesions when im- Affiliations of authors: F. Samaniego, Departments of Lymphoma/Myeloma and Clinical Cancer Prevention, The University of Texas M. D. Anderson Can- cer Center, Houston; S. Pati, D. Bose (Institute of Human Virology), J. E. Karp (The Greenebaum Cancer Center), University of Maryland, Baltimore; O. Prakash, Alton Ochsner Medical Foundation, New Orleans, LA. Correspondence to: Felipe Samaniego, M.D., Department of Lymphoma/ Myeloma and Clinical Cancer Prevention, The University of Texas M. D. Ander- son Cancer Center, Box 429, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail fsamaniego @ mdanderson.org). See “Notes” following “References.” © Oxford University Press ''planted in nude mice. Moreover, inflammatory cytokines also increase the level of HHV8 DNA in blood-derived cells (//,20). Taken together, inflammation plays a key role in the develop- ment of many of the features of KS, and it is possible that the local source for inflammation may stem from the expression of the HHV8 KI gene (see below). HIV-1 contributes directly to KS development through pro- duction of HIV-1 Tat (2/—23). Tat is essential for viral gene expression, yet it also exhibits the capacity to exit live cells, disseminates systemically, and enters other cells where it can activate latent HIV-1 and promoters of other genes (i.e., trans- forming growth factor-8 and tumor necrosis factor-a) (24). Mice made transgenic for tat express the protein in blood and show preferential KS tumor growth when inoculated with KS cells (21,25,26). Thus, Tat is a systemically distributed viral cytokine that can participate in inflammation and tumor promotion. Recipients of solid-organ transplants, especially renal trans- plant patients, are at increased risk of developing KS. It is be- lieved that immune stimulation from engrafted allogeneic tissue and challenges by microbes in the setting of drug-induced im- munosuppression provide the necessary conditions that would promote tissue inflammation. Multiple studies, including our current data, suggest that HHV8 infection may play a role in host cell activation and proliferation to produce KS lesions. Even though a minority (approximately 5%) of KS spindle cells express HHV8 lytic- phase genes, expression of selected HHV8 genes may influence host cells sufficiently to induce diffusable inflammatory cyto- kines in a paracrine manner. It is interesting that EC proliferation with HHV8 infection has been demonstrated by Flore et al. (27). Despite the fact that only 5% of the ECs were infected with HHV8, all cells, including uninfected cells, exhibited an ex- tended replicative life span beyond senescence with acquired telomerase activity, thereby indicating a potent paracrine effect of HHV8 (27). Also, HHV8 and ECs inoculated under the hu- man skin of a human skin—severe combined immunodeficiency (SCID) mouse chimera produced angiogenic lesions similar to human KS (28). Among HHV8 genes, KI is a promising candidate for medi- ating activation signal pathways based on its characteristic cy- toplasmic motif (29,30). K1 contains a cytoplasmic ITAM (37). ITAMs are contained in subunits of multiprotein complexes, B-cell receptor, and T-cell receptor that are critically involved in inflammatory responses (32). Thus, we anticipate K1 to signal in and activate inflammation-related pathways by mimicking the functions of host ITAM proteins (33). The ITAM of KI has been shown to transmit signals when this motif is tested as a chimeric protein fused to the extracellular domain of CD8 (3/). This construct, however, may not reliably reflect native K1 function because the vast majority of this fused protein is CD8. ITAMs can also exhibit paradoxical effects that clearly depend on the position on the polypeptide. One isolate of K1 (clade 3A) has been shown to stimulate nuclear factor of activated T-cell (NFAT)-dependent promoter activity (30). Be- cause ITAM-dependent signaling typically stimulates several types of promoters and because multiple isolates of K1 exist, we sought to examine |) whether other isolates of K1 are active in promoter activity, 2) whether K1 expression would stimulate promoters of other types, 3) whether K1 is expressed in KS tumor and cell lines, and 4) whether K1 cooperates with other factors in promoting inflammation of KS. 16 METHODS Cloning The open reading frame K1 of HHV8 was cloned by poly- merase chain reaction (PCR) from DNA of BC-3 cells (provided by E. Cesarman, Cornell Medical College, New York, NY) (34,35). Thirty-five cycles (95°C for 30 seconds, 58°C for 60 seconds, and 72°C for 90 seconds) were performed by PCR (/) with the use of the AmpliTaq Gold PCR Kit (The Perkin-Elmer Corp., Foster City, CA), 50 mM KCI, 10 mM Tris-HCI (pH 8.3), 1.5 mM MgCl, and 0.001% (wt/vol) gelatin and oligomers (5’- GACGGATCCAGACCTTGTTGGACATCCTG-3" and 5’- TTTTATGTAAAATACTCCAGCCCTAGGGTG-3’). Gel elec- trophoresis was performed, and a single prominent band of separated PCR products of approximately 1 kilobase (kb) was extracted from the gel and cloned into pCR2.1 with the use of TA cloning (Invitrogen Corp., Carlsbad, CA). The insert was sequenced. The BamHI fragment containing K1 was cloned into pSG5 and pCR3.1. To epitope tag K1, we generated a fusion construct in pCR3.1 by PCR with the use of pCR2.1K1 and with oligomers 5'’-CACAAGCTTCGCGAATTCATGTTCCTG- TATGTTGTTTGCAGTCTGG-3' and 5’'-GATATCCCCG- GATCCCTACAGATCTTCTTCAGAAATAAGTTTTTGT- TCGTACCAATCCACTGGTTGCGTA-3’ that directed the addition of the DNA coding for c-myc epitope that is recognized by monoclonal antibody 9E10: EQKLISEEDL. Constructs were sequenced to confirm DNA sequence. Cells and Transfection KS-1 and BC-3 cells are HHV8-infected lymphoma cells de- rived from primary effusion lymphoma (provided by H. P. Koef- fler, University of California at Los Angeles, and E. Cesarman) (36,37). They were propagated in RPMI-1640 medium with 15% fetal bovine serum and supplemented with | mM gluta- mine, penicillin G (100 U/mL), and streptomycin (100 mg/mL). Cos-1 (American Type Culture Collection, Manassas, VA) cells were selected for studying K1 expression because of their ease of transfection and their support of high-level plasmid-driven expression. KS Y-1 cells are transformed KS-derived cells iso- lated from an HIV-1-infected individual with KS (38). KS Y-1 cells exhibit characteristics of KS spindle cells and, like other KS-derived cell lines, lack HHV8 DNA. Cos-1 cells and KS Y-1 cells were transfected (4 x 10°) with the use of the FPugene Transfection Reagent (Boehringer Mannheim Biochemicals, In- dianapolis, IN), and, after 48 hours, 20 pg of cell extract was mixed with the luciferase assay reagent (Promega Corp., Madi- son, WI) and light emission was measured over a 15-second period on a luminometer (Turner Designs, Sunnyvale, CA). Be- cause cell stress and various stimuli (e.g., oxidation stress and high serum levels) may contribute to a nuclear factor-kappa B (NF-kB) transcriptional response, we optimized the assay con- ditions by minimizing nonspecific procedure-related cell activa- tion (in RPMI-1640 medium with 0.5% fetal bovine serum) and the minimal amount of plasmid DNA (1-2 jg) to elicit a nuclear transcription response. In other studies, transgene expression was accomplished by mixing 10’ cells with a total of 30 pg of total plasmid and electroporation of cells (0.28 V, 25 WF) on the Bio-Rad Electroporator (Bio-Rad Laboratories, Hercules, CA). Assays were done in triplicate, and the means were reported. A second plasmid, using simian virus 40 promoter to direct ex- pression of green fluorescent protein (pGreenLantern; Strata- Journal of the National Cancer Institute Monographs No. 28, 2000 ''gene, La Jolla, CA), was used to compare transfection efficien- cies. Reporter plasmids used in these studies were pAP-1-Luc and pNF«BLuc (Stratagene). Test plasmids pCR3.1K1, pCR3.1K I myc, and pSG5KI mye contained K1 from BC-3 cells. Control plasmid pFC-MEKK, which directs expression of ex- tracellular-regulated kinase kinase (MEKK), was used as a po- tent intracellular stimulus for NF-«B activation (Stratagene). To begin to define the pathway K1 uses in activation, we deter- mined whether NF-«B transcriptional activity was sensitive to the blocking effects of aspirin (1 mM) or cyclosporin (1 1M) that are known to abrogate NF-«B signaling (39). After transfection, cells were refed media with or without each inhibitor and incu- bated for another 24 hours. Whole-cell extracts were made, and 20 wg was added to the luciferase reagent and emissions were assayed on luminometer. Cells transfected with vector alone and cells transfected with pFC-MEKK served as negative and posi- tive controls, respectively. Immunofluorescence Staining and Cytokine Levels Immediately after transfection with pSGSKImyc or pSGS, cells were seeded on 12-well slides (Erie Scientific, Portsmouth, NH) and incubated in RPMI-1640 medium with 10% fetal bo- vine serum. The cells were washed, air-dried, fixed with acetone at —20°C, and stained by indirect immunofluorescence. The slides were dried and permeabilized with phosphate-buffered saline, 1% bovine serum albumin, 0.1% Tween, and 5% wt/vol sucrose. The cells were treated with primary antibody 9E10 (1: 100) and secondary antibody anti-mouse fluorescein isothio- cyanate-conjugated antibody (1:50) (mixed with Evans blue) each for 0.5 hour at 37°C. The cells were rehydrated, and anti- fade solution (Molecular Probes, Inc., Eugene, OR) was applied before sealing with coverslips. Representative cell fields were captured on 1600 film. Cells transfected with plasmid pSVK3- Bcatenin-myc were used as a positive control (D. Sussman, Uni- versity of Maryland). After transfection, cells were refed, and the medium was collected after 24 hours. The supernatants were handled with the use of plastic ware precoated with 0.1% bovine serum albumin in phosphate-buffered saline to avoid adherence of cytokines to surfaces. Supernatants were frozen at —80°C before measurement by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Reverse Transcription (RT)—PCR and Northern Blot Analysis Tissue from human KS was obtained after diagnostic evalu- ation was completed and after consent was signed, according to the policy of our Institutional Review Board. Tissue samples were snap-frozen in a dry ice/methanol bath, and total RNA was isolated with the use of Trizol according to the instructions of the manufacturer (Life Technologies, Inc. [GIBCO BRL], Gaithers- burg, MD). Cultured cell RNA was isolated with the use of the Trizol reagent. RT was performed with the Titan RT-PCR Kit (Boehringer Mannheim Biochemicals) with the use of avian my- eloblastosis virus, RT, a Taq/Pwo DNA polymerase blend, and 5'-TTTGTGCCCTAGAGTGAGTT-3" and 5'-TGACTGT- GTTTGATGGTTGT-3’. For northern blots, 15 pg of total-cell RNA was separated in 0.8% formaldehyde gel and transferred to a nylon membrane. The KI DNA sequence was labeled by the random primer method and was used for northern blot hybrid- ization at 42 °C, and washing was done at 45 °C in | x standard saline citrate/0.1% sodium dodecyl] sulfate (SDS). Journal of the National Cancer Institute Monographs No. 28, 2000 Immunoprecipitation/Immunoblot Analysis Cos-1 cells were electroporated with pSG5KImyc or with pSGS. After 48 hours, cell extracts were made in 1% Nonidet buffer (40). Immunoprecipitation was conducted with anti-myc antibody (1 wg) and rabbit anti-mouse antibody (Santa Cruz Biotech, Santa Cruz, CA, and ICN, Costa Mesa, CA), respec- tively, and protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) and tumbled for 12 hours at 4°C. Immunopre- cipitants were size-separated on an 8% SDS polyacrylamide gel and transferred to nitrocellulose. Blotting was carried out with anti-myc antibody (1 g/mL), and a duplicate filter was blotted with antiphosphotyrosine (4G10) (0.5 g/mL) (Upstate Biotech- nology, Lake Placid, NY), and the signal was read with the use of chemiluminescence (Amersham Pharmacia Biotech). Statistical Analysis Where appropriate, the means are shown with standard de- viation. Student’s ¢ test was applied to estimate the statistical significance of the mean differences (4/). All P values are two- sided. RESULTS To determine whether KI might play a role in the clinical manifestations of KS, we analyzed human KS for the presence of Kl RNA. RT-PCR was performed on total RNA isolated from a KS lesion from an HIV-1-infected individual. RT-PCR analysis showed a product of approximately 280 base pairs (bp), and the band was not generated by PCR after treatment of the template with ribonuclease (RNase) A (Fig. 1, A). The band remained after treatment of the templated RNA with deoxyribo- nuclease (DNase) I (not shown). RNA isolated from 12-O- tetradecanoylphorbol-13-acetate (TPA)-treated BC-3 cells (re- ported to contain approximately 40 viral genomes per cell during latency) used as a positive control showed a more intense signal. Thus, K1 is expressed in human KS, and TPA enhances its RNA levels in cell lines. These results, combined with recent evidence that HHV8-infected humans develop cytotoxic T-cell lympho- cyte responses targeting K1 (42), indicate that K1 is expressed at the protein level in the course of HHV8 infection. Because viral gene expression is anticipated to be tightly regulated, particularly in chronic viral infection, and viral tran- scripts are produced, we attempted to stimulate and characterize KI gene expression of latent HHV8-infected cells. Cellular RNA of TPA-treated KS-1 and BC-3 cells was subjected to northern blot analysis with the use of Kl DNA as the labeled probe. Transcripts of 1.3 and 3.6 kb were observed to be either induced or enhanced with TPA treatment (Fig. |, B). The smaller band corresponds to the length of a K1 transcript, whereas the higher molecular weight transcript likely represents a complex transcript encompassing K1 (43,44). Ethidium bro- mide staining of 28S ribosomal RNA served to indicate the relative levels of RNA loading. Thus, K1 is expressed in these cells, consistent with lytic-phase gene expression in vitro and by extrapolation during lytic-phase HHV8 expression in human KS. Of the HHV8 genes considered as candidates for mediating inflammation in KS, K1 is a promising candidate. The K1 pro- tein sequence exhibits a highly conserved cytoplasmic ITAM that is involved in inflammation (45,46). We cloned K1 (Gen- Bank accession No. AF170531) from the primary effusion lym- phoma-derived cell line BC-3, which is chronically infected with 17 '' A a _ Ee = o G9 mk“ 3 Eos 88 2 acre no treatment 300 200 RNase treatment B _ KS-1 BC-3 stimulation - 12 24 7 12 24 (hours) 5 ei ae ; 3.6 kb | 1.35 kb 288 Cc VR1 VR2 ™ ITAM | | | L[T [| YYSLHDLCTEDYTQPVDWY Y.X.x.L.X.x.X.X.X.X.X.Y.X%.x.L ITAM consensus Fig. 1. K1 from BC-3 cells was cloned and contains an immunoreceptor tyro- sine-based activation motif (ITAM) (YxxLxxxxxxxYxxL). A) Kaposi’s sarcoma (KS) tumor and cell lines express KI] RNA. Reverse transcription—polymerase chain reaction was performed on total RNA isolated from KS tumor. DNA was separated on agarose gel and stained with ethidium bromide. Treatment of RNA template with ribonuclease (RNase) prevented the synthesis of DNA product, whereas the treatment with deoxyribonuclease allowed synthesis of DNA prod- uct. B) Human herpesvirus 8-infected BC-3 and KS-1 cells express KI when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The smaller band corresponds to the transcript of K1, and the larger band is likely a multigene transcript that includes K1. C) K1 from BC-3 primary effusion lymphoma pro- tein sequence predicts a transmembrane protein with extracellular highly vari- able regions (VR1 and VR2), a transmembrane (TM), and a short cytoplasmic tail with an ITAM (YxxLxxxxxxxYxxL), which plays an essential role in host signaling pathways. The amino acid sequence is for clades A and C. bp = base pair; kb = kilobase. HHVS8 (see below). The predicted protein sequence from the K1 DNA from BC-3 cells reveals a transmembrane domain protein with an extracellular immunoglobulin light chain-like domain, two hypervariable regions (29), and a short cytoplasmic tail that contains an ITAM (YxxLxxxxxxxYxxL) (Fig. 1, C). Amino acid sequence shared by clades A and C is shown, and the ITAM is identified. The rightmost leucine (L) is substituted by proline (P) in KI. K1’s highly variable protein sequence can be classi- fied into several clades A-D (BC-3 is C); however, thus far, a clade-specific clinical entity or pathology has not emerged (29). K1 DNA from BC-3 cells was tagged at its cytoplasmic car- boxy terminus with a myc epitope that is targeted by 9E10 antibody. Also, to speed up the evaluation of KI function, we performed initial studies on Cos-1 cells because of their rela- tive ease of transfection and their reproducibly high level of expression of plasmids. These studies serve as the basis for further studies with KS-derived spindle cells (38). Expression of the tagged protein was confirmed by detection of KImyc protein in transfected cells (Fig. 2, A). Transfectants were stained with anti-myc and fluorescein isothiocyanate-conjugated secondary antibody, which localized activity to cell membrane in KI myc transfectants but showed no staining in vector trans- fectant controls (not shown). Transfection with a plasmid- directing expression of a cytosolic protein 8 catenin-myc showed activity in a different distribution and predominantly in dividing cells (Fig. 2, B). For the investigation of a possible role for the ITAM- containing HHV8 K1 protein in inducing inflammation, K1 was expressed in Cos-1 cells, and cell extracts were analyzed for NF-«B-dependent promoter activity (Fig. 3, A). Cells expressing KImyc showed NF-«B-driven luciferase activity of 19200 U and its vector control transfectant level of 4400 U (Fig. 3, A). Of interest, no cross-linking reagents were necessary for K1 to gen- erate NF-«B-driven expression. Cells expressing MEKK dis- played enhanced luciferase activity, whereas mock transfectants displayed luciferase activity comparable to vector transfectants (not shown). Transfection of plasmid-directing expression of green fluorescent protein showed equivalent cell transfection rates in K1 versus vector transfectants, indicating that the levels of transfection were similar regardless of the plasmid used. Transfection of cells with K1 that lacked a myc-tag produced similar NF-KB-dependent activation, indicating lack of interfer- ence from the myc epitope (not shown). To determine if another NF-«KB-related promoter could be activated by KI, we coexpressed K1 with plasmid containing luciferase driven by a promoter containing AP-1 sites. KI ex- pression showed a greater than twofold increase in AP-1- dependent luciferase activity (Fig. 3, B). To begin analyzing the effects of K1 expression in KS cells, we transfected pCR3.1KImyc into KS Y-1 cells along with the luciferase re- Fig. 2. K] appears to be a membrane-associated protein. A) Cos-1 cells trans- fected with pSG5K I myc and stained with the anti-myc and fluorescein isothio- cyanate anti-mouse antibodies show distribution of K1 with cell membrane. Cos-1 cells transfected with pSGS lack any fluorescence activity (not shown). The staining pattern of K1 is consistent with the distribution of protein associated with cytoplasmic membrane. B) Staining of Cos-1 cells transfected with a plas- mid expressing the cytosolic protein B catenin-myc displayed a different distri- bution in cells, which also displayed reduced cell spreading and was seen usually in dividing cells. Nuclei were visualized with Evans blue staining. Original magnification x400. Journal of the National Cancer Institute Monographs No. 28, 2000 '' 20000 15000 RLuc 10000 5000 control 800 600 RLuc 400 200 control K1 4000 | 3000 RLuc 2000 1000 control K1 Fig. 3. Expression of K1 stimulates nuclear factor-kappa B (NF-«B)-dependent transcription. Cos-1 cells were transfected with the use of pCR3.1KImyc or pCR3.1 with pNF-«BLuc and Fugene (1 wg DNA/10/ cells); 24 hours later, cell extracts were analyzed for luciferase activity. A) The results are the mean and standard deviation of each cell from three wells of six-well plates. Expression of green fluorescence protein indicated no differences in transfection efficiencies. Transfection with control positive pFCMEKK produced enhanced luciferase activity. B) Expression of K1 induces AP-1-dependent activity in Cos-1 cells. C) Transfection of KS Y-1 cells with pCR3.1K1myc and pNF-KBLuc was analyzed for luciferase activity. Bars indicate the means and standard deviation from different transfection pools. RLUC = relative light units concentration. porter construct. NF-«B-dependent luciferase activity in KI transfectants was three times the activity observed in vector (control) transfectants (Fig. 3, C). In all cases, K1-stimulated activity was consistently higher than vector transfectant controls. Thus, KI stimulates transcriptional activity at two promoter/ enhancer sites, AP-1 and NF-«B, and suggests that KS cells are competent to handle ITAM-related signaling. NF-«B plays a central role in the induction of numerous immunoregulatory responses, including expression of IL-1, IL- 2, IL-3, IL-6, IL-8, tumor necrosis factor-a, and interferon gamma. KS Y-1 cells transfected with K1 compared with vector transfectants showed | 1-fold higher levels of IL-12 and signifi- cantly higher levels of IL-6 and granulocyte—macrophage colony-stimulating factor (P<.05) (Table 1). K1-transfected Journal of the National Cancer Institute Monographs No. 28, 2000 Table 1. Cytokines secreted by K1 transfectants* Mean + standard deviation Cytokine Vector transfectants, pg/mL K1 transfectants, pg/mL GM-CSF 66 (+8.5) 171 (411.3) IL-6 1587 (+18) 1870 (+42) IL-8 32 (+28) 123 (+59) IL-12 12 (+1) 132 (+5) *Cells were transfected with pCR3.1KImyc or pCR3.1, and cell supernatants were collected at 24 hours. The cells’ conditioned media were analyzed for human cytokines by enzyme-linked immunosorbent assay (ELISA). Results are reported from Kaposi’s sarcoma (KS) Y-1 transfectants with the exception of the analysis of interleukin (IL)-8, which was done with Cos-1! cells with the use of ELISA for human IL-8. No statistically significant differences were noted in the levels of RANTES, monocyte inflammatory protein I (MIP-I), and MIP-II. The means of cytokine levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-8, and IL-12 of K1 transfectant supernatants were statisti- cally significant (each P<.05). Cos-1 cells also showed enhanced IL-8 secretion. These cyto- kines bear NF-kB-response elements in their promoters (47). This cytokine production was not a generalized effect because K1 expression did not induce elevation of other inflammatory cytokines, such as RANTES, MIP-I, or MIP-II. The cytokines induced by K1 are inducible by cell stimuli that operate through NF-«B (47), and the cytokines are also implicated in KS lesion formation (/5—/8,20,23). We have hypothesized that the inflammation in KS lesions is mediated by a few lytically active cells that possess pervasive inflammatory effects on other cells. To evaluate the possibility of these paracrine effects, we examined the cells’ conditioned media for their ability to promote NF-KB-dependent activation. Cos-1 cells incubated with conditioned media of K1 transfec- tants exhibited enhanced NF-«B-promoter activity, indicating that K1 mediates a paracrine effect (not shown). Taken together, the secretion of numerous cytokines and a second wave of NF- KB activation would support the development of diffuse tissue inflammation present in KS lesions. Activation pathways can be blocked by specific therapeutic anti-inflammatory or immunoregulatory agents, such as aspirin and cyclosporin (48,49). To determine whether aspirin or cyclo- sporin would block Kl-triggered activation, we treated pCR3.1KImyc and pCR3.1 transfectants with either aspirin or cyclosporin for 24 hours after transfection. K1 transfectants demonstrated enhanced NF-«B-dependent activity over buffer- treated vector transfectants (Fig. 4). Treatment of cells with aspirin blocked the K1-dependent promoter activity down to the activity of vector-alone transfectants, indicating selective block- age of inducible luciferase activity. Treatment of cells with cy- closporin also abrogated the K1-induced luciferase activity (Fig. 4). However, in contrast to aspirin treatment, the vector trans- fectants treated with cyclosporin also showed significantly lower luciferase activity, indicating that baseline activity was also af- fected. Thus, drugs that block inflammation and cell activation demonstrate the ability to block inflammation-related K1 signal- ing and further substantiate that K1 signals via host NF-«B- dependent pathways. Because expression of the cell membrane-associated K1 ac- tivates NF-«B in the absence of an added ligand and viral re- ceptors generally mimic activated host receptors, we surmised that KI from BC-3 might be constitutively activated. ITAM- 19 '' 4000 3000 RLuc 2000 1000 K1 ctrl K1 ctrl K1 ctrl buffer ctrl 1mM ASA 1uM CSA Fig. 4. Signaling of K1-triggered nuclear factor-kappa B (NF-KB) pathways can be blocked by reagents that block cell activation and inflammation. Cos-1 cells were transfected with pCR3.1KImyc or pCR3.1 with pNF-«BLuc and incubated with growth media containing aspirin (ASA) or cyclosporin (CSA). After 24 hours, cell extracts were made and the luciferase assay was performed. RLUC = relative light units concentration; ctrl = vector alone transfect units. receptor binding and/or aggregation is typically required for phosphorylation of ITAMs that constitutes an activated state. Therefore, we examined K1 for phosphorylation by combined immunoprecipitation/immunoblot analysis by transfecting Cos- | cells with pSGSKImyc and detection with anti-myc antibody. Our studies show that K1 appeared as a protein of approximately 47 kilodaltons with apparent lower molecular weight diffuse bands (Fig. 5). K1 can be glycosylated, and the lower molecular weight bands likely represent incompletely glycosylated forms of K1 (50). No apparent multimers of KI were observed in contrast to other studies (30,50). The bases for this difference may rest on the different clades of K1 or on the differences in methods of detection. Similar immunoprecipitation/immunoblot of B catenin-myc transfectants showed an expected band of B 5 gs 8 = > x Vector K1imyc kDa 62 — 51 — Retear ty ee 33. Fig. 5. K1 is expressed as a constitutively phosphorylated protein. Cos-1 cells were electroporated with pSGSKImyc or pSGS, and extracts were subjected to combined immunoprecipitation and reducing SDS-PAGE. Immunoblotting of filters were performed with anti-myc antibody and a duplicate filter blotted with anti-phosphotyrosine (anti-phosph) antibody. A 47-kilodalton (kDa) band is rec- ognized by anti-myc antibody as well as less intense and lower molecular weight forms. The antiphosphotyrosine (4G10) blotting shows phosphoprotein of 47 kDa. An anticipated band of approximately 80 kDa appeared on immunopre- cipitation/immunoblot of Cos-1 cells transfected with pSVK3-B catenin-myc (not shown). 20 catenin-myc, and vector-transfected Cos-1 cells lacked either of these bands (not shown). To determine whether the immunopre- cipitated KI myc was also phosphorylated, we blotted a duplicate filter with anti-phosphotyrosine antibody and noted the appear- ance of a phosphoprotein approximating the size of the largest K1 band (Fig. 5). There was no phosphorylation signal in the region immediately below the K1 band, even after extended exposure of the film. Of interest, only the high-molecular-weight KI band was phosphorylated (at the present level of detection), suggesting that only the completely processed K1 (glycosylated) is preferentially phosphorylated. This finding indicates that K1 that is myc-tagged can serve as a substrate for phosphorylation and is active in NF-«B-dependent transcription. Thus, even in the absence of a ligand, K1 is expressed as a phosphorylated protein, consistent with its being a constitutively activated pro- tein capable of signaling through ITAM-dependent pathways. Although HHV8 infection alone, without intercurrent ill- nesses, is sometimes associated with milder forms of KS, the presence of a second virus, i.e., HIV-1, usually dictates an ag- gressive clinical course of KS that parallels the activity of HIV- 1. HIV-1 appears to stimulate KS cell proliferation indirectly by inducing inflammatory cytokine production, and this production depends, in part, on NF-KB-dependent promoters (5/). To assess for a possible contribution of HIV-1 Tat to the ability of K1 to stimulate NF-«B-dependent transcription, we coexpressed K1 and Tat (52). Transfection of pCR3.1K1 or pCMVtat stimulated NF-«B-dependent luciferase activity of 490 and 380 arbitrary units, respectively, over vector-alone transfectants (Fig. 6). However, transfection with the combination of plasmids showed enhanced promoter activity in an additive fashion. Thus, in this model, two genes from disparate origins converge on NF-kB- dependent transcriptional activity in a cooperative fashion. In this regard, HIV-1 and HHV8 share signaling targets to bring about a cellular inflammatory response at a level not observed with each viral product acting alone. DISCUSSION The variable clinical course of KS and the histologic features of KS lesions suggest that the inflammatory process is central to the promotion of KS lesions. HHV8 may be the stimulus for 1200 900 RLuc 600 300 control HIV-1tat K1 — HIV-1 tat + K1 Fig. 6. K1 and human immunodeficiency virus type 1 (HIV-1) Tat cooperate in activating nuclear factor-kappa B (NF-«B)-dependent transcription. Cos-1 cells were cotransfected with plasmids encoding for both proteins. Each plasmid alone shows enhanced NF-KB-dependent promoter activity, and in combination the promoter activity was increased over single plasmid transfectants in an additive fashion. RLUC = relative light units concentration. Journal of the National Cancer Institute Monographs No. 28, 2000 ''inflammation and, to that end, we have presented data implicat- ing HHV8 KI in activating pathways that operate in cell acti- vation and inflammation. K1, like other I[TAM-containing pro- teins, may provide cells with a critical signal that ultimately determines cell activation, at least, in part, by inducing NF-KB- and AP-|-dependent promoter activity. The ITAM from KI can transmit signals when tested as a chimeric protein joined to the extracellular domain of a known receptor (CD8) (3/). Even though signaling through ITAM can transmit inhibitory signals that are dependent on context, K1 ITAM fused to CD8 positively stimulated signaling (3/,45, 53,54). More recently, full-length K1 from body cavity-based lymphoma-1 cells (clade A3) was shown to induce NFAT- dependent promoter activity (30,50). NFAT is another ITAM- dependent factor described in lymphocytes that regulates pro- moter-driven expression of proteins associated with inflammation and proliferation (55). Thus, K1 leads to activation of NFAT- and NF-«B-dependent promoter activities that, in turn, orchestrate the transcription of an array of proteins in- volved in cell activation and inflammation. KI is likely to activate pathways used by host cell ITAM- containing proteins. However, unlike the K1 protein product, which is constitutionally active, host cell ITAM-containing pro- teins generally reside in the resting state, and, on ligand binding or receptor aggregation, they undergo phosphorylation and be- come competent for signaling. ITAM-containing receptors in- clude subunits of the B-cell receptor, T-cell receptor, and Fe receptor, which contain one or more copies of ITAMs within their cytoplasmic tails (56). Specifically, these ITAM-bearing proteins include human proteins TCR-¢, CD36, and FceRI and viral proteins, such as HHV8 LMP2A-like protein, Hantavirus glycoprotein G1, bovine leukemia virus gp30, Epstein-Barr vi- rus LMP2A, and rhesus monkey rhadinovirus R1 (32,45,53,57— 61). NF-«B-dependent signaling is modulated by viral proteins as well as by ligands and drugs that affect inflammation and cell activation. HIV-1 Tat is shown to contribute to NF-KB- dependent promoter activity that is additive to that of K1. Be- cause HIV-1 infection substantially accelerates the aggressive course of KS, additive effects at NF-kB-dependent promoter activity may be one key mechanism in which HIV-1 and HHV8 converge to stimulate cell activation and inflammation and ac- count for an aggressive course of KS in HIV-1 infection (62). Although lymphocytes were first discovered and extensively studied for expression of ITAM receptors, other cells are known to harbor functional ITAM-bearing receptors as well. Indeed, monocytes and macrophages (MO/MC) contain ITAM- containing receptors, such as the immunoglobulin Fe receptors. In MO/MC, ligand binding activates Fe receptors to induce cell activation by expression of the IL-2 receptor and secretion of inflammatory cytokines (tumor necrosis factor-a and IL-12), which together can synergize in mounting a greatly amplified inflammatory response (63,64). The copious cytokines released in K1-expressing cells would predict that K1 expression in MO/ MC would mount a wave of cytokine secretion and would con- tribute toward an overall activation of MO/MC. In KS tissue, despite the fact that most cells contain HHV8 in the latent phase, some lytic viral replication does occur in cells that share markers of activated MO/MC and ECs (CD68) (65). These cells are expected to express K1 as part of their lytic-phase expression program that would be implicated in KS inflammation (Fig. 7). Journal of the National Cancer Institute Monographs No. 28, 2000 HHV8 lytic phase Endothelial Cells G@©@ contact inhibited OC QC Endothelial Cells -activated state -uninhibited -angiogenesis cytokine K4 release vessel Fig. 7. Expression of human herpesvirus 8 (HHV8) K1 stimulates signaling pathways involved in cell activation and cytokine production. Cell signaling through immunoreceptor tyrosine-based activation motif-containing proteins in- duces activation of the cells and also induces secretion of inflammatory cyto- kines. K1 expressed in lytically active cells would contribute to inflammation that includes secretion of inflammatory cytokines. Inflammatory cytokines in- duce endothelial cells to acquire features of Kaposi’s sarcoma spindle cells that include angiogenic factor production, expression of adhesion proteins, and a secondary wave of inflammatory cytokine production. MO/MC, in particular the subendothelial MO/MC, have been noted to express substantial levels of activation markers, and their location in the subendothelium suggests that they play piv- otal roles in translumenal trafficking. The small percentage of cells in KS lesions that undergo lytic-phase replication are anticipated to express K1 and to dic- tate the inflammatory status of KS tissue. Therapy targeting herpesvirus in humans leads to regression of KS and lowers the frequency of KS development (66). Antiviral therapy in experi- mental models has reduced the levels of HHV8 in human SCID mouse models infected with HHV8 (/4,33). In our cell model, we show that K1 stimulates NF-KB and that agents known to block inflammation or cell activation also block K1-mediated NF-«B-dependent promoter activity. Thus, the link between in- flammation and HHV8 gene regulation and replication appears as a central event to the development of KS. In summary, K1, which functions by mimicking the activity of host ITAM pro- teins, is likely to be a major trigger for cell activation and in- flammation in KS. REFERENCES (1) Moore PS, Chang Y. Detection of herpesvirus-like DNA sequences in Kaposi’s sarcoma in patients with and without HIV infection. N Engl J Med 1995;332:1181-5. (2) Nador RG, Cesarman E, Chadburn A, Dawson DB, Ansari MQ, Sald J, et al. 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J Virol 1999;73:5123-31. (62) Safai B, Johnson KG, Myskowski PL, Koziner B, Yang SY, Cunningham- Rundles S, et al. The natural history of Kaposi’s sarcoma in the acquired immunodeficiency syndrome. Ann Intern Med 1985;103:744—50. (63) Hoffmann T, Tripathi AK, Lee YL, Lizzio EF, Bonvini E. Stimulation of human monocytes by anti-CD3 monoclonal antibody: induction of inflam- matory mediator release via immobilization of Fe receptor by adsorbed immunoglobulin and T-lymphocytes. Inflammation 1992;16:571-85. (64) Chu J, Liu Y, Koretzky GA, Durden DL. SLP-76-Cbl-Grb2-She interac- tions in FeyRI signaling. Blood 1998;92:1697-706. (65) Blasig C, Zietz C, Haar B, Neipel F, Esser S, Brockmeyer NH, et al. Monocytes in Kaposi’s sarcoma lesions are productively infected by human herpesvirus 8. J Virol 1997;71:7963-8. (66) Martin DF, Kuppermann BD, Wolitz RA, Palestine AG, Li H, Robinson CA. Oral ganciclovir for patients with cytomegalovirus retinitis treated with a ganciclovir implant. Roche Ganciclovir Study Group. N Engl J Med 1999;340: 1063-70. NOTES Presented at the International Symposium on HIV, Leukemia, and Opportu- nistic Cancers. Supported by Public Health Service (PHS) grant KO8CA80815-01 (National Cancer Institute) to F. Samaniego from the National Institutes of Health, De- partment of Health and Human Services; and by a predoctoral Fellowship from the National Cancer Institute to S. Pati. ''Hematopoietic Stem Cells in HIV Disease David T. Scadden, Hongmei Shen, Tao Cheng The hematopoietic stem cell has long been hypothesized to be a target of human immunodeficiency virus type-1 (HIV) in- fection that limits the potential for compensatory immune cell production. Data have recently emerged documenting stem cell dysfunction in HIV disease and indicating that im- mune recovery from potent antiretroviral therapy is partly driven by new T-cell generation. Effects of HIV on stem cell physiology, however, appear to be indirect, as stem cells are highly resistant to HIV infection. Despite the presence of surface receptors for HIV, the hematopoietic stem cell is not infectible with HIV. However, stem transduction can be achieved with HIV constructs in which the envelope glyco- proteins have been replaced by vesicular stomatitis virus G protein. Therefore, hematopoietic stem cells are likely par- ticipants in HIV-related cytopenias, but they are spared di- rect infection and can serve as a resource for cellular thera- pies for AIDS. [J Natl Cancer Inst Monogr 2000;28:24—9] Human immunodeficiency virus (HIV) induces a multitude of alterations in the innate and adaptive immune system that are broad if not equally distributed among virtually every arm of host defense. Because T cells are but one of the cell types af- fected, the potential for HIV being a disease of stem cells as well as mature effector cells has long been hypothesized. If stem cell dysfunction or destruction plays an important role in HIV dis- ease, the implications are multiple and include limiting immune reconstitution and affecting the potential for stem cell-based gene therapy strategies. Possible mechanisms include direct virus infection or indirect effects by altering the cellular or cytokine milieu of the bone marrow microenvironment. The issue of direct infection will be addressed in a later section of this paper in which the marked resistance of stem cells to HIV-1 infection is discussed. The lack of infectibility of stem cells, however, is not synonymous with the lack of adverse effects of HIV because of direct interactions with the virus. The virus envelope glycoprotein, gp120, is ca- pable of interacting with the CXCR-4 and of inducing intracel- lular signaling events as manifest by calcium flux, kinase acti- vation, and even functional changes, such as chemotaxis of some cell types (/,2). Whether it may directly induce altered function of stem cells that express CXCR-4 is not clear, but data suggest that apoptosis may be induced (3). Alteration in the cellular and cytokine milieu of the bone marrow has been reported by a number of investigators who have demonstrated infection of bone marrow stromal elements and perturbation of either cytokine production or ability to sus- tain hematopoiesis (4-6). In Vivo EVALUATION OF STEM CELLS IN HIV DISEASE Stem cell functional defects in vivo have been demonstrated both in human studies or in animal models. In animal models in 24 which human hematopoietic tissue is engrafted in immunodefi- cient mice, reduced CD34* cells and/or decreased colony- forming capacity have been shown to occur after HIV infection (7,8). Efforts to define the stem cell pool in HIV-infected pa- tients have been less uniform in their conclusions, but an im- portant study (9) quantitating circulating CD34* cells after granulocyte colony-stimulating factor (G-CSF) mobilization has recently been concluded. These studies have shown that a de- cline of CD34* numbers is seen with more advanced HIV dis- ease. Patients with lower CD4 cells have lower concentrations of CD34* cells after G-CSF mobilization. The stem cell numbers that can be harvested from patients with CD4 counts below 200 cells/mm? may still be adequate for purposes of transplan- tation, but their concentration per milliliter of blood is demon- strably lower than those patients with more preserved immune function (9). CELL KINETICS IN IMMUNE DECLINE AND REGENERATION A causal relationship between lower CD4* cells and CD34* cells has been suggested by two recent lines of evidence. The first is the sequential analysis of stem cell and lymphocyte num- bers in the previously mentioned mouse models. These studies have indicated that, after acute HIV infection, there is a decline in primitive cell numbers and function that precedes the decline in thymocytes (8). The second is related to the changes seen as viral replication decreases after antiretroviral therapy. Reduced HIV replication is associated with an improvement in CD34* cells, myeloid colony-forming capacity, and CD4* T cells (/0). The increase in CD4* cells is not related to improved T-cell survival but rather appears to be due to increased rates of pro- duction (//). The initial increase in CD4* cells may be due to expansion of existing mature memory cells. However, with the use of immunophenotypic markers for naive cells and a recently developed polymerase chain reaction (PCR) method to quanti- tate cells recently emigrating from the thymus (/2), it is clear that de novo generation of T cells is occurring (/2—/4). Although it has not been rigorously shown that the T-cell regeneration reflects stem cell changes, it is apparent that hematopoiesis rather than improved cell survival times is the driving force of immune reconstitution after antiretroviral treatment. The im- provement in T-cell generation may then either reflect improved primitive cell function or number or the improved health of the tissue microenvironments in which the differentiation of primi- tive cells occurs. Affiliation of authors: D. T. Scadden, H. Shen, T. Cheng, AIDS Research Center and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston. Correspondence to: David T. Scadden, M.D., AIDS Research Center and Cancer Center, Massachusetts General Hospital, 149 13" St., Rm. 5212D, Bos- ton, MA 02129 (e-mail: scadden.david@mgh.harvard.edu). See “Notes” following “References.” © Oxford University Press Journal of the National Cancer Institute Monographs No. 28, 2000 ''THYMIC FUNCTION IN HIV DISEASE Disruption of thymic architecture has been well described with HIV infection (/5—/7), but recent data would suggest that the defects in thymic function are neither complete nor irrevers- ible. A radiographically detectable thymus is present in many HIV-infected patients (/8); and even in settings of severe dys- function, T-cell production is occurring. HIV-infected individu- als who have had thymectomies for myasthenia gravis have ongoing T-cell generation (/9). Also, in the setting of antiretro- viral therapy, thymic dysfunction appears to be at least partially reversible. Jn vivo models have shown improvement in T-cell generation from precursor populations both endogenous and ex- ogenous to the thymus with accompanying control of viremia (13,20). The potential of thymic function is greater than previ- ously thought, and abnormalities of function are likely to be at least partially reversible. Thus, the thymus is considered to be less likely to represent a significant limiting factor in determin- ing the extent to which T-cell numbers will improve after anti- retroviral therapy. Rather, the primitive cell pool or other dif- ferentiation regulators may provide the difference between those patients who improve to normal or near normal levels and those who do not. STEM CELL SUSCEPTIBILITY TO HIV-1 INFECTION The emphasis on the stem cell in the context of HIV disease is then shifting in several important ways. The first is recogni- tion that stem cells may indeed play a role in restricting immune restoration. The second is that their longevity presents stem cells as a potentially long-lived reservoir for HIV if infected. And the third is the greater potential for stem cells as a therapeutic tool in the context of gene therapy. If the thymus is not limiting, genetically protected stem cells may indeed be capable of pro- viding the substrate for T-cell generation. Crucial to each of these issues is the infectibility of the stem cell by either HIV-1 or lentivirus vectors derived from HIV-1. HIV RECEPTOR/CO-RECEPTOR EXPRESSION IN HEMATOPOIETIC CELL SUBSETS We recently completed studies in which cells representing particular stages of blood cell development were isolated by flow cytometric or functionally based systems (/4,2/—23). The stem cell population was isolated with the use of the previously described method of selectively killing more mature cells thereby enriching for a cytokine nonresponsive subset with stem-like characteristics. Cells were assessed for expression of CD4, the chemokine receptors (CCR-5 and CXCR-4), messen- ger RNA, and protein with the use of the techniques adapted for the small numbers of primary cells available from standard donations. Low levels of message for CD4 were detectable by reverse transcription (RT)—PCR in peripheral blood mono- nuclear cells (PBMCs), myelomonocytic cells (CD11b*), mature T cells (CD3*CD4"*), heterogeneous hematopoietic progenitor cells (CD34*), lineage-committed hematopoietic progenitor cells (CD34*CD38"*), primitive hematopoietic progenitor cells (CD34*CD38°), and stem cells (Go), but not in NIH3T3 control cells. Similarly, the message for CXCR-4 and CCR-5 was detectable in each hematopoietic cell type tested, although CCR-5 levels in CD34*CD38™ cells appeared to be lower Journal of the National Cancer Institute Monographs No. 28, 2000 as confirmed on multiple independent samples (n = 4). To more precisely define the presence of the receptor transcripts in stem cells, individual cells were isolated by micromanipulation, and single-cell RT-PCR profiles were generated as described previously (24,25). The cells consistently demonstrated detect- able CD4, CXCR-4, and CCR-5 messages compared with con- trol cells. The presence of protein produced from the receptor transcripts was assessed by specific antibody staining and, in- dependently for chemokine receptors, by calcium flux. Anti- CD4 staining analyzed by flow cytometry indicated CD4 ex- pression in subpopulations of CD34* cells similar to the findings reported by others (3,26—28). CCR-5- and CXCR-4-specific an- tibodies stained fractions of relevant CD34* cells with only minimal staining of CD34*CD38° cells with the use of the anti- CCR-5 antibody, consistent with the low transcript levels ob- served. Chemokine signaling, as measured by the generation of a calcium flux in cells bearing cognate receptors, was used as a functional assessment of chemokine receptors. Responsiveness of various subsets of CD34* cells to SDF-1 (ligand for the CXCR-4 receptor) and regulated-on-activation normal T cells expressed and secreted (RANTES), MIP-la, and MIP-1b (li- gands for the CCR-5 receptor) was measured on Indo-1-loaded cells with the use of the fluorescence-activated cell sorter analy- sis. NIH3T3 cells were used as a cell control, IP-10 (the ligand for CXCR-3) was used as a chemokine control, and measure- ments were made over time by using the cells before and after exposure to chemokine to establish a target-cell baseline control. Response to SDF-1 was substantial in all populations of CD34* cells, although increased response was noted in the CD34*CD38° cells, despite no difference in the frequency of CXCR-4 surface protein in that subfraction compared with CD34*CD38" cells. Similarly, the relationship between detect- able surface protein for CCR-5 and response to ligand was not direct. Despite low levels of message and surface CCR-5 in the CD34*CD38~ subset, calcium flux was approximately equiva- lent to other cell fractions when cognate ligands were applied. NIH3T3 cells did not demonstrate calcium flux, and IP-10 did not induce calcium flux except in a huCXCR-3-expressing cell line. The rarity of stem cells (Gp) precluded the routine use of flow cytometry, and, thus, we developed an immunomagnetic bead rosette assay (Fig. 1). This assay uses the binding of specific monoclonal antibodies to target epitopes on cells similar to im- munofluorescence assays. However, instead of fluorescein con- jugation, immunomagnetic bead conjugation was used as a means of enhancing the ability to detect antibody binding with the use of microscopy; the size of Dynal beads permitted ready enumeration of rosetted cells and had a low frequency of non- specific binding (0.7%-6%) when second-step alone or when irrelevant antibody-conjugated beads were used. Estimated frequency of CD4-, CXCR-4-, and CCR-5-expressing cells compared with bead alone or with irrelevant antibody- conjugated bead controls (specific-nonspecific binding) was 12.2%, 23.2%, and 23.6%, respectively. Large-scale stem cell preparation generated by pooling multiple independent marrow preparations permitted flow cytometry analysis of CD34* and CD4* cells and demonstrated that a high fraction of these cells co-expressed CXCR-4 and CCR-5, compared with isotype con- trol (Fig. 2). '' | Laat ' ee CD19-BMMC | CD4-BMMC | 7 > . a Count CCR-5-FITC CXCR-4-FITC Fig. 1. Analysis of protein expression on stem cells was performed by using an immunomagnetic bead rosetting assay. Immunomagnetic beads (dark circles) were assessed for their binding to target cells by two independent readers with the use of phase-contrast microscopy (original magnification x40). The upper panel demonstrates representative CD4-specific beads binding to Go stem cells. The middle panels demonstrate lack of CD19-bead binding to Go stem cells (negative control) or beads binding to peripheral blood lymphocytes (positive controls). Lower panel: G, cells selected for CD4 were pooled, and flow cy- tometrically analyzed for staining with either CXCR-4 or CCR-5 or isotype controls. Single-cell digital fluorescence imaging was used to docu- ment stem cell (Gy) calcium flux in response to chemokines. MIP-la, MIP-1b, and SDF-1 generated evidence of calcium flux (Fig. 2, B) and thereby confirmed the functional status of surface CCR-5 and CXCR-4 on stem cells. In contrast, IP-10 did not induce flux, whereas inducing calcium flux in huCXCR-3 trans- duced control cells. HIV-1 Co-RECEPTORS FUNCTION ON CD34* CELLS BUT NOT Gy STEM CELLS Definition of the functional characteristics of the co-receptor molecules was further pursued through exposure of cells to stocks of infectious HIV-1. Given the presence of identifiable receptors for M-tropic strains (with the use of CCR-5) and T- tropic strains (with the use of CXCR-4), appropriate virus en- velopes were used (HIV-1,,; and HIV-1,,,3 >, respectively). Following exposure to concentrated stocks of virus, infection was evaluated for 1) the presence of HIV DNA, indicating virus entry and RT, and 2) the production of HIV-1 p24 antigen after addition of highly infectible cell lines, indicating completion of a replicative virus life cycle and passage of the virus. Virus DNA was detectable at the level of a single cell diluted to 10 in titration experiments of ACH-2 cells that contained a single proviral copy per cell (data not shown). HIV-1 DNA was evident in all subsets of cells exposed to infectious, but not heat- inactivated virus, with the notable exception of the Gp stem cells (Fig. 3, A). Go cells independently isolated from independent normal donors (three are shown) were consistently negative for viral DNA. CD34*-cell fractions other than stem cells had de- tectable viral DNA that was inhibitable at a level of approxi- mately 50% by preincubation with cognate ligands for CCR-5, MIP-1la, MIP-1b, and RANTES (data not shown). The non-stem cell fractions also demonstrated productive completion of the virus life cycle by passage of HIV-1 to the readily infectible indicator cell lines PM-1 (Fig. 3, B) or H9 (data not shown) when HIV-1,, , or HIV-1,,,-2, respectively, were used. Prompt production and passage of the virus to PM-1 was noted from the relatively mature CD34*CD38* population of cells. Although CD34*CD38° cells had clearly identifiable virus DNA present, passage of infectious virions was very much delayed with p24 antigen detectable on co-cultivation with PM-1 only after prolonged periods (approximately 21 days). In no instance was p24 detectable on co-cultivation of indicator (PM-1 or H9) cells with virus-exposed Go cells, including experiments ex- tended out to 35 days. Fig. 2. Calcium flux in Gp stem cells after stimulation with MIP-la (A), MIP-1b (B), or SDF-1 (C) as assessed by laser- based digital fluorescence mi- Ratio croscopy with quantitative graphic depiction of the fluo- rescence increase over time in fields of approximately 10 cells each is shown. Figures 2, B and C reprinted with permission by the American Society for Mi- crobiology. 407 MIP-1a —> 3.54 4.0 Hy | 3.5 MIP-18 —» 8 8 8 8 Time (s) 26 Journal of the National Cancer Institute Monographs No. 28, 2000 '' A Q =) SK19 i ee - snex B 10000 -_- = 8000 + 4 Ee i _— mo 6000 + —+— H1/344+38+ & - 2 -34438- z+ 4000 + i —— 344+38+ N if —-60 O 2000 + ie 0 aad ° ° - = = N 8 Days Fig. 3. HIV-1 entry and production is subset specific in hematopoietic cells. A) M-tropic human immunodeficiency virus (HIV)-1, ;, infection in vitro detected by DNA polymerase chain reaction (PCR) in indicated cell types. Pools of approximately 200 cells were assessed for HIV-1 DNA. Three independent preparations from independent donors are shown for the Go subset of cells. Consistent results were obtained from independent replicates-quadruplicates of all lanes shown. B) HIV-1 virus production in CD34* subsets and Gg stem cells that were exposed to HIV-1,,,, for 24 hours, were extensively washed, and subsequently co-cultured with PMI cells as an indicator cell line. HIV-1 p24 assays were performed by enzyme-linked immunosorbent assay on culture su- pernatants at the indicated times. Similar results were obtained when identical experiments were performed by using HIV-1,,,, with H9 cells as the indicator cell line. Figure 3, A reprinted with permission by the American Society for Microbiology. In Vivo CORROBORATION THAT STEM CELLS ARE RESISTANT TO HIV-1 INFECTION To determine if stem cells were infected in vivo, bone marrow samples were obtained from HIV-1-infected patients with high levels of circulating virus and with low blood cell counts. With the use of multiple independent patient samples, HIV DNA was identified in PBMCs and bone marrow mononuclear cells (BMMC), but there was consistently no detectable HIV DNA in Gy cells (n = 7) (Fig. 4). STEM-CELL RESISTANCE MEDIATED AT THE LEVEL OF VIRUS FUSION AND ENTRY RT in quiescent cells may be incomplete and, in lymphocytes, may result in partial complementary DNA intermediates (29). To evaluate this possibility in stem cells, PCR primer pairs cor- responding to 5’ of the primer binding site and U3—U5 portions of the HIV-1 genome reverse transcribed even in quiescent lym- phocyte populations were used (labeled TC-1 in Fig. 5). To assess if the level of block to infection was at the receptor level or following receptor interaction, an HIV-1I green fluorescent protein (GFP)-encoding construct pseudotyped with either T- tropic (HXB2), M-tropic (YU-2), or dual tropic (89.6) HIV-1 envelopes were compared with the same virus construct pseu- dotyped with the vesicular stomatitis virus G (VSV G) protein. VSV G permits virus fusion with the cell membrane via mecha- nisms that bypass those mediated by CD4 and CCR-S (30). Only the VSV G-pseudotyped virus was capable of infecting the stem cell population (Fig. 5, A). No HIV DNA was detectable in stem cells when HIV-1 envelopes or heat-inactivated VSV G- envelope pseudotypes were used as assayed by either PCR or for GFP expression by fluorescence microscopy. HIV-1 envelope pseudotypes were capable of infecting Jurkat or primary mono- nuclear control cells. These data demonstrate that stem cells have a block to HIV-1 infection and that the level of the block is in the steps of viral-cell membrane fusion and entry. Steps downstream of these events are intact as evident from the VSV G-pseudotype infection. An independent stem cell purification process with the use of Rhodamine 123 and Hoechst 33342 staining as defined by others (22) was used to exclude the possibility that the selection method induced alterations in the ability of the stem cells to be infected. The exclusion of Rhodamine 123 and low-intensity staining with Hoechst 33342 in CD34* cells has been shown to associate with a population capable of functioning as stem cells in in vitro and in vivo experiments (3/). After exposure to the virus, the cells corresponding to a more mature population (bright/bright) acquired detectable HIV DNA, but stem cells (dim/dim) had neither late nor early RT products detectable (Fig. 5, B) identical to what was seen with Gp cells. Furthermore, these cells were readily infectible with the virus when the en- velope was VSV G. These data confirm the resistance of stem cells to HIV-1 infection and demonstrate that the block can be overcome if an alternative CXCR-4- and CCR-5-independent mechanism of envelope-cell membrane fusion is used. H,0 PBMC BMMC Go B-actin — tq iT kp —_ om | litle etn sa Fig. 4. Detection of human immunodeficiency virus (HIV)-1 genome in hema- topoietic cells from AIDS patients as assessed by HIV-1 DNA polymerase chain reaction (PCR) analysis on subsets of cells derived from individuals with ad- vanced HIV disease and cytopenia. Each lane represents cells from an indi- vidual patient. All Go lanes correspond to samples in the BMMC lanes with the Journal of the National Cancer Institute Monographs No. 28, 2000 exception of one patient in which an inadequate number of BMMC cells were obtained to proceed with Gy isolation. Peripheral blood mononuclear cell samples were obtained from independent patients with similar clinical profiles. Reprinted with permission by the American Society for Microbiology. '' A Gy Jurkat BMMC g _¢ on 5 a 9 & 9 Pobehe2isis bee Tcl — ¢ 666 «¢ p-actin — eee ee bee ~ ao B Ba-L VSV-G | | | | a a. PEGs Botte sis SK19 * e . ' e ret . “@ €& @ pacn O60 OHH 0 O46 Fig. 5. Stem cell block to human immunodeficiency virus (HIV)-1 infection regardless of HIV-1 tropism is confirmed in stem cells derived by independent methods and can be circumvented by pseudotyping in VSV G enveloped virus. A) Gy cells were exposed to recombinant HIV-1 pseudotyped in envelopes of M-tropic (YU,), T-tropic (HXB,), or dual tropic (89.6) specificity or in an envelope that contained VSV G. Cells were analyzed by DNA polymerase chain reaction (PCR) for early HIV transcripts. Heat-inactivated VSV G pseudotyped virus (HI/VSV-G) was used to control for virus infectivity. Jurkat and BMMC cells were used as positive control cell lines to control for HIV-1-enveloped virus infectivity. Lambda DNA (marker) or water alone (H,O) were used as PCR STEM CELLS AS THERAPEUTIC TOOLS The data presented here indicate that the hematopoietic stem cell is resistant to HIV-1 infection in vitro and in vivo despite receptor and co-receptor expression. The stem cell is, therefore, not a potential long-lived reservoir of virus and is an appropriate cell to consider in autologous gene therapy approaches to ac- quired immunodeficiency syndrome (AIDS). To the extent that this cell can be recovered from AIDS patients, it may be envi- sioned to be a virus-free cell type that may be transduced with anti-HIV constructs for possible immune reconstitution. The ability to transduce the stem cell with HIV-based constructs was also documented in this study but was restricted to constructs pseudotyped in VSV G. There appears to be no block to such constructs entering quiescent stem cells and, at least transiently, expressing a transgene. Integration of the transgene or durable expression was not tested in this study, but another report (32) has indicated that HIV-based vectors may indeed result in sus- tained transgene expression in transplanted hematopoietic cells. Thus, stem cells may ultimately be manipulated to serve as a component of therapies of the future designed to replace the damaged immune system of HIV-1-infected individuals. REFERENCES (1) lyengar S, Schwarts DH, Hildreth JE. T cell-tropic HIV gp120 mediates CD4 and CD8 cell chemotaxis through CXCR4 independent of CD4: im- plications for HIV pathogenesis. J Immunol 1999;162:6263-7. 28 controls. B) Cells isolated by either Rhodamine/Hoechst staining with bright/ bright (br/br), representing more differentiated cells, and dim/dim (d/d), repre- senting stem cells, or by cytokine nonresponsiveness (Gy) were exposed to wild-type, Ba-L, or pseudotyped HIV within a VSV G envelope and evaluated for infection by PCR directed against late (SK19) or early (TC1) reverse tran- scription products. HI designates heat-inactivated control virus preparations. All products were confirmed by radiolabeled specific oligonucleotide hybridization as shown after phosphorimager analysis. 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Bahner I, Kearns K, Coutinho S, Leonard EH, Kohn DB. human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-inducted hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma. Blood 1997;90:1787-98. Koka PS, Fraser JK, Bryson Y, Bristol GC, Aldrovandi GM, Daar ES, et al. Human immunodeficiency virus inhibits multilineage hematopoiesis in vivo. J Virol 1998;72:5121-7. (8) Jenkins M, Hanley MB, Moreno MB, Wieder E, McCune JM. Human immunodeficiency virus-1 infection interrupts thymopoiesis and multilin- eage hematopoiesis in vivo. Blood 1998;91:2672-8. (9) Banda NK, Simon GR, Sipple JD, Terrell KL, Archer P, Shpall EJ, et al. Depletion of CD34+ CD4+ cells in bone marrow from HIV-1- infected individuals. Biol Blood Marrow Transplant 1999;5:162—72. (10) Adams GB, Pym AS, Poznansky MC, McClure MO, Weber JN. The in vivo (3 S (4 = (5. 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(29) Zack JA, Arigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 1990;61:213-22. (30) Matlin KS, Reggio H, Helenius A, Simons K. Pathway of vesicular sto- matitis virus entry leading to infection. J Mol Biol 1982;156:609-31. (31) Moriuchi H, Moriuchi M, Fauci AS. Differentiation of promonocytic U937 sublcones into macrophagelike phenotypes regulates a cellular factor(s) which modulates fusion/entry of macrophagetropic human immunodefi- ciency virus type |. J Virol 1998;72:3394—400. (32) Roederer M, Bigos M, Nozaki T, Stovel RT, Parks DR, Herzenberg LA. Heterogeneous calcium flux in peripheral T cell subsets revealed by five- color flow cytometry using log-ratio circuitry. Cytometry 1995;21:187-96. NOTES Presented at the International Symposium on HIV, Leukemia, and Opportu- nistic Cancers. Supported by Public Health Service grants HL44851 (National Heart, Lung, and Blood Institute) and DK50234 and DK02761 (National Institute of Diabetes and Digestive and Kidney Diseases), National Institutes of Health, Department of Health and Human Services; by the Richard Saltonstall Charitable Founda- tion; and by the Center for AIDS Research. 29 ''Regulation of Bcl2 Phosphorylation and Potential Significance for Leukemic Cell Chemoresistance Xingming Deng, Steven M. Kornblau, Peter P. Ruvolo, W. Stratford May, Jr. Although considered tightly linked, the linkage effectors for proliferation and antiapoptotic signaling pathways are not clear. Phosphorylation of Bcl2 at serine 70 is required for suppression of apoptosis in interleukin 3 (IL-3)-dependent myeloid cells deprived of IL-3 or treated with antileukemic drugs and can result from agonist activation of mitochon- drial protein kinase C « (PKCa). However, we have recently found that high concentrations of staurosporine up to 1 uM can only partially inhibit [L-3-stimulated Bcl2 phosphoryla- tion but completely block PKCa-mediated Bcl2 phosphory- lation in vitro, indicating the existence of a non-PKC, stau- rosporine-resistant Bcl2 kinase (SRK). Although the RAF-1- MEK-1-mitogen-activated protein kinase (MAPK) cascade is required for factor-dependent mitogenic signaling, a direct role in antiapoptosis signaling is not clear. In particular, the role of phosphorylation in the regulation of death substrates is not yet clear. Our findings indicate a potential role for the MEK/MAPK pathway in addition to PKC in antiapoptosis signaling, involving Bcl2 phosphorylation that features a role for extracellular signal-regulated kinase (ERK)1 and 2 as SRKs. These findings indicate a novel role for ERK1 and 2 as molecular links between proliferative and survival signal- ing and may, at least in part, explain the apparent paradox by which Bcl2 may suppress staurosporine-induced apopto- sis. Although the effect of phosphorylation on Bcl2 function is not clear, effector molecules that regulate Bcl2 phosphor- ylation may have clinical significance in patients with acute myelogenous leukemia (AML) who express detectable levels of Bcl2. Preliminary findings suggest that expression of PKCa, ERK2, and Bax in leukemic blast cells from patients with AML, although individually not prognostic, appears to have potential clinical value in predicting chemoresistance and survival outcomes. [J Natl Cancer Inst Monogr 2000; 28:30-7] Hematopoietic growth factors, such as interleukin 3 (IL-3), mediate cell growth by stimulating proliferation and by sup- pressing the process of programmed cell death (/—4). A great deal is known about the molecular components and mechanisms that regulate IL-3 and other cytokine superfamily receptor- mediated signal transduction pathways that result from receptor dimerization and activation of nonreceptor protein tyrosine ki- nases like JAK2, with coupling to the activation of cytoplasmic signal transducers and activators of transcription (3,5,6) or the activation of the Src-homology collagen, growth factor receptor- bound protein 2, son of sevenless—coupled RAS/RAF-MEK- I/ mitogen-activated protein kinase (MAPK) extracellular signal- regulated kinase (ERK) pathway (3,7—9). However, relatively little is understood about the postreceptor signaling mecha- nism(s) by which growth factors, such as IL-3, might also couple to and regulate apoptosis. Bcl2 and related family members are key regulators of pro- 30 grammed cell death or apoptosis, a natural process required for normal development, and they play a role in malignant transfor- mation and autoimmune diseases (/0—/4). Bcl2 was discovered in the oncogene hunt as the oncogene fusion product of the immunoglobulin H (IgH) promoter and full-length Bcl2 charac- terized by the t14;18 breakpoint translocation found in approxi- mately 80% of patients with indolent non-Hodgkin’s lymphoma (13-15). The survival function of Bcl2 as a potent suppressor of apoptosis was initially demonstrated when Bcl2 was shown to facilitate prolonged survival following exogenous expression in IL-3-dependent hematopoietic cells that were deprived of IL-3 (/6) and later through studies with transgenic and knockout mice (17-20). Thus, in the absence of IL-3, cells default to a suicidal apoptotic pathway involving intracellular proteolysis, which, in turn, can be inhibited by Bcl2 (/6). Subsequently, Bcl2’s ability to promote prolonged, but not indefinite, cell survival under various types of apoptotic stress (e.g., treatment with chemo- therapy drugs, irradiation, exposure to toxins, or viral infection) was also discovered (/6—/8,2/,22). Work in our laboratory has uncovered a novel regulatory role for IL-3 in post-translational regulation of induced Bel2 phos- phorylation (23,24). The mechanism(s) by which IL-3 and other survival agonists may induce Bcl2 phosphorylation and the po- tential regulatory role for this post-translational modification on Bcl2’s function will be the focus of this study. BcL2 FUNCTIONS AS A DOCKING PROTEIN WITH POTENTIAL PORE-FORMING PROPERTIES The Bcl2 family, which now numbers some 16 members, is made up of both suppressors (Bcl2, BCLy,, and MCL-1) and inducers (Bax, Bad, Bak, and Bid) of apoptosis [reviewed in (10-12)]. Briefly, Bcl2 has four conserved Bcl2 homology (BH) functional domains and seven a-helical regions providing struc- ture. The BHI, BH2, and BH3 domains are also contained in some pro-apoptotic death effector members, and mutational studies have shown these domains to be necessary for Bcl2-Bax heterodimerization and any potential Bcl2 or Bax pore-forming properties (25-29). The heterodimerization of Bcl2 and Bax re- cently has been formally demonstrated in vivo (30) and is cur- rently considered important, at least in part, for Bcl2’s ability to block Bax’s potent death effector properties (25,30). On the basis of the recent crystallographic and solution structure of Bcl-X, (3/) and a BH3-only Bax-derived peptide, the BH3 do- mains in Bel2, Bax, and other BH3-only members, such as Bid Affiliations of authors: X. Deng, P. P. Ruvolo, W. S. May, Jr., University of Florida Shands Cancer Center, Gainesville; S. M. Kornblau, The University of Texas M. D. Anderson Cancer Center, Houston. Correspondence to: W. Stratford May, Jr., M.D., Ph.D., University of Florida Shands Cancer Center, Box 100232, Gainesville, FL 32610-0232 (e-mail: smay @ufscc.ufl.edu). See “Note” following “References.” © Oxford University Press Journal of the National Cancer Institute Monographs No. 28, 2000 ''and Bim, are also modeled as death agonists (27,32,33). One popular model features their death-inducing effects occurring as a result of their potential membrane pore-forming properties that are potentially exerted as a result of binding to Bcl2 or Bcl-X, and/or integrating into mitochondrial membranes (25, 30,34). For example, Bax and the BH3-only Bid death effectors can bind to Bcl2 and become associated with the mitochondrial membranes. Apparently, when their death properties are revealed, they be- come integrally associated with mitochondrial membranes that potentially open megapore channel(s) (35). This presumably is accompanied by migration of Bax to the mitochondria in asso- ciation with leakage of caspase activators to the cytosol and collapse of the mitochondrial membrane potential (28-30). In support of this model, the crystal structure of Bcl-X,; and Bax predicts a similar structure with that of bacterial colicins and diphtheria pore-forming toxins that function by disrupting mem- brane function (3/,34—36). Furthermore, purified Bax can ap- parently directly induce mitochondrial membrane leakage in in- tact mitochondria in vitro by a process that can be blocked by Bcl2 (36). Thus, Bcl2 may function, at least in part, by docking with and/or somehow “neutralizing” Bax’s pore-forming prop- erties (25,3436). Alternatively, other functional properties of Bcl2 may result from its potential role as a multidocking site for other death regulators or components of mitogenic signal trans- duction pathways, including protein kinases (e.g., RAF-1) and phosphatases (e.g., protein phosphatase 2B [PP2B]) (Fig. 1) OT Ory iy leat OPT obs Caspase 3 D33 APOPTOSIS Fig. 1. Regulation of Bcl2 phosphorylation by interleukin 3 (IL-3) in factor- dependent myeloid cells. IL-3 activates two signal transduction pathways that can lead to Bcl2 phosphorylation at serine 70 by protein kinase C a (PKCa) and the RAF/MAPK/ERK-1, 2 (SRKs). PKCa can directly phosphorylate Bcl2 or indirectly through the activation of the SRKs ERK1 and ERK2 that also phos- phorylate Bcl2 at serine 70. Although postreceptor coupling of IL-3 can activate the well-described RAS/RAF/MAPK/ERK 1/2 pathway leading to direct phos- phorylation of Bcl2 by MAPKs, ERK1, and ERK2, this pathway can also be activated by PKCa via a direct effect on RAF-1 (90). Bcl2 phosphorylation at serine 70 occurs within the flexible loop (aa 29-80) that is between the BH4 domain and the BH3 domain. The protein phosphatase PP2A can dephosphory- late Bcl2, and C2-ceramide can stimulate dephosphorylation. Bcl2 can het- erodimerize with Bax or Bad, depending on the phosphorylation status of Bad affected by PI3-K or PKA. Bcl2 can regulate caspase 3 activation by regulating cytochrome C (CytoC) and apoptosis activation factor-1 (APaf-1) release from the mitochondria. Also shown is the Bcl2 aspartate34 (D34) site of caspase 3 cleavage and the putative Bax cleavage site, aspartate33 (D33). Journal of the National Cancer Institute Monographs No. 28, 2000 (37-39). Furthermore, in addition to Bcl2’s ability to act as a docking or scaffold assembly protein, it may alter the suscepti- bility of certain bound proteins to undergo posttranslational modifications, including proteolytic cleavage or phosphorylation that may potentially regulate their function. For example, Bcl2 can be inactivated following proteolytic cleavage (40,4/). How or whether Bcl2 can regulate these processes is not yet clear, but our preliminary findings suggest that the phosphorylation state of Bcl2 may play a role in proteolytic cleavage of both Bcl2 and Bax. These new findings will be discussed below. In addition, Bcl2 and Bcl-X, may also be regulated at the transcription level (42). Bci2 Is FUNCTIONALLY PHOSPHORYLATED ON SERINE 70 By IL-3 AND OTHER SURVIVAL AGONISTS Bcl2 was initially identified as a potential phosphoprotein when expressed in SF9 cells in which it was shown to prolong cell survival following baculovirus infection (43). Later studies in our laboratory (23,24) discovered that IL-3 could induce a rapid and robust serine phosphorylation of Bcl2. Importantly, this modification correlated closely with cell survival in factor- dependent cells and suggested a functional role for phosphory- lation. It is interesting that the potent protein kinase C (PKC) agonist and natural product bryostatin-1 (Bryo), which can also support survival of IL-3-dependent myeloid cells following IL-3 withdrawal, were found to induce Bcl2 phosphorylation, which initially suggested a functional role for PKC (23,24,44). Phos- phorylation of Bcl2 was found to occur at the same serine site, whether induced by Bryo, IL-3, or the related erythroid hema- topoietic hormone erythropoietin (23,24). Bcl2 mutational stud- ies confirmed a functional role for phosphorylation at the evo- lutionarily conserved ser70 site, which is located in a putative regulatory region known as the flexible loop domain (FLD) (30). Thus, only Bcl2 containing the serine 70 to alanine (S70A) mutation failed to undergo phosphorylation by either IL-3 or Bryo, and this mutant also displayed a severely reduced survival function when stably expressed in factor-dependent cells (23,24). However, cells expressing the S70A Bcl2 mutant did fare slightly better with respect to survival than vector-only transduced parental cells, indicating that the nonphosphorylat- able S70A Bcl2 mutant does retain some function under these conditions (24). This argues that phosphorylation may not be the only regulatory mechanism for Bcl2. In light of its multidocking and putative pore-channel properties, this is not surprising. By contrast, conversion of serine 70 to glutamate (S70E), a charged amino acid that could potentially mimic a phosphorylation site, resulted in an increased survival function. Thus, cells expressing S70E Bcl2 were more viable following the stress of either IL-3 withdrawal or etoposide chemotherapy treatment than cells ex- pressing similar amounts of exogenous wild-type (wt) Bel2 (24). These data strongly argue that ser70 is a regulatory site for Bcl2 (24) and allowed us to conclude that phosphorylation is neces- sary for Bcl2’s full and potent survival phenotype, at least in factor-dependent myeloid cells. Presumably, this extends to other growth factor-sensitive cells expressing Bcl2 because nerve growth factor (NGF) also induces Bcl2 phosphorylation in PC12 pheochromocytoma cells in association with survival (45). Of interest, dephosphorylation of Bcl2, even in the presence of NGF, is closely linked to apoptosis in these cells. The serine 70 site of Bcl2 is evolutionarily conserved and is located within the predicted unstructured FLD of Bcl2 (31,46). 31 ''The FLD is a stretch of approximately 50 amino acids (aa 30- 80) that resides between the putative a1 and «2 helical structures that separate the amino terminal BH4 and BH3 domains of Bcl2 (Fig. 1) (26,27,46). The potential loop domain is conserved be- tween Bcl2 and Bcl-X,, suggesting functional significance (46). It is interesting that deletion of this loop region from either Bel-X,, or Bcl2 results in a molecule with enhanced survival function under specific circumstances, such as when expressed in WEHI-231 cells that undergo apoptosis following exposure to IgM (46,47). It has, therefore, been proposed that the FLD may represent a negative regulatory region (46). However, one report (48) that uses the identical Bcl2 loop deletion mutant has found that this domain is required for its survival function, at least when cells are treated with certain chemotherapeutic agents, including paclitaxel (Taxol). One explanation for this apparent paradox is that deletion of the large loop domain may function- ally represent a “phosphorylation equivalent” mutation. Thus, if the FLD region were a negative regulatory region, phosphory- lation might somehow “inactivate” its negative effect on sur- vival. This possibility would be consistent with most reported findings in IL-3-dependent cells because, in the absence of IL-3 or a survival agonist, Bcl2 phosphorylation is not easily detected and the negative regulatory properties of the FLD may then dominate (24). Also consistent with this notion, forced overex- pression of a nonphosphorylatable Bcl2 mutant (S70A) was un- able to prolong cell survival following IL-3 deprivation or treat- ment with etoposide chemotherapy compared with wt or S70E Bcl2 (24). Bcu2 Is A SUBSTRATE FOR AT LEAST Two BcL2 KINASES: PKCa AND A STAUROSPORINE-RESISTANT Bcu2 KINASE We have previously reported that PKCa is a physiologic Bcl2 kinase (44). However, the existence of another, non-PKC Bcl2 kinase(s) was also indicated, as overexpression of exogenous Bcl2 is reported to protect cells from apoptosis that would nor- mally be induced by high concentrations of the potent PKC inhibitor staurosporine (49). Thus, a staurosporine-resistant Bcl2 kinase(s) (SRK) was sought. Involvement of an MAP kinase (i.e., ERK1 or ERK2) was considered likely according to reports that activation of the MAP kinase phosphatase-1 (MKP-1) was associated with Bcl2 dephosphorylation and apoptosis in NGF- dependent PC12 cells treated with angiotensin 2 (45). Because IL-3 can rapidly activate the RAF-MEK-1-MAPK pathway (50), we tested a role for an MAPK in Bcl2 phosphorylation. Prelimi- nary studies that used various protein kinase inhibitors indicated that PD98059, a specific MEK-1 inhibitor, could, like stauro- sporine, only partially block IL-3-induced Bcl2 phosphorylation. However, the combination of PD98059 and staurosporine could completely shut down IL-3-induced Bcl2 phosphorylation (5/). Thus, ERK1 (p44) and ERK2 (p42) were identified as potential candidate Bcl2 kinases. It is interesting that a distinct population of cytosolic ERKs was found to be located in the heavy mem- brane mitochondrial subcellular fraction, indicating potential as direct Bcl2 kinases. When individually tested, ERK1 and ERK2 were found to be potent, direct Bcl2 kinases (Fig. 1) (5/). Col- lectively then, although these findings identify ERK! and ERK2 as physiologic Bcl2 kinases, they cannot exclude the formal possibility that other SRKs may also exist (Fig. 1) (57). 32 Bci2 PHOSPHORYLATION IS A DYNAMIC PROCESS INVOLVING PHYSIOLOGIC BCL2 KINASE(S) AND A PHOSPHATASE Although Bcl2’s survival function can be regulated, at least in part, by phosphorylation at ser70, phosphorylation is not a static process (52). Rather, Bcl2 phosphorylation represents a balance between a Bcl2 kinase(s) and a phosphatase(s) (Fig. 1). Thus, the potential for perturbing Bcl2’s survival function through ago- nist-induced Bcl2 phosphorylation may also occur via phospha- tase activation (52). Concerning this possibility, it is interesting to note that ceramide, a potent PP2A activator (53,54), can be rapidly generated intracellularly after treatment of cells with various types of cell death stimuli, including cytotoxic cytokines like tumor necrosis factor a (55), chemotherapeutic drugs (56,57), ischemia/reperfusion injury (58), FAS antigen activa- tion (59), irradiation (60), and corticosteroids (6/). Indeed, the production of ceramide is so common during apoptosis that it has been considered a universal feature of this process (62,63). Whether ceramide is a trigger for cell death is not clear, but C2-ceramide can induce cell death when added directly to cells (55). We have discovered that C2-ceramide, but not the func- tionally inactive C2-dihydro-ceramide, can potently inhibit Bcl2 phosphorylation induced by either IL-3 or Bryo (64). Reversal of phosphorylation resulted from the rapid activation of a mito- chondrial-associated, okadaic acid-sensitive PP2A-like activity that was directly associated with Bcl2. Of interest, however, cells expressing the functionally potent S70E Bcl2 mutant fail to undergo apoptosis after treatment with high concentrations of C2-ceramide that can potently activate PP2A and would readily induce apoptosis in cells expressing wt or S70A Bcl2 (64). These findings indicate that inhibition of Bcl2 phosphorylation may be one mechanism by which C2-ceramide can induce ap- optosis in IL-3-dependent myeloid cells that express Bcl2. In support of this possibility, it was demonstrated that, although NGF can induce Bcl2 phosphorylation and survival in PC12W pheochromocytoma cells, NGF-induced Bcl2 phosphorylation could be inhibited and cells induced to undergo apoptosis after addition of angiotensin-2 (45). Angiotensin-2 was found to po- tently activate the MAPK-phosphatase, MKP-1, that resulted in apparent inhibition of MAPK/ERK activity and was associated with loss of phosphorylation of Bcl2 (45). These findings are consistent with the notion that inhibition of Bcl2 phosphoryla- tion is associated with apoptosis. However, because protein phosphatases and kinases seldom have a solitary substrate, it may be possible that the phosphorylation of other potential mol- ecule(s) may affect the survival status of the cell. Furthermore, other potential Bcl2 kinases and phosphatases may also exist. It has been reported that Bcl2 may bind and sequester the protein phosphatase PP2B in association with protection of Jurkat T cells from apoptosis induced by PP2B/calcinurin over- expression (39). Thus, although PP2B could be a potential Bcl2 phosphatase on this basis, we found that, at least in vitro, PP2B is a much weaker Bcl2 phosphatase than PP2A or PPI (52). This finding suggests that PP2B’s role in Bcl2’s binding may not have a direct effect on phosphorylation and function; alterna- tively, Bcl2 may regulate PP2B’s role in FAS-ligand-induced apoptosis of T cells by actively soaking up PP2B (65). Alterna- tively, because PP2B is, like BAD, located primarily in the cytosol, one other consequence of PP2B binding to Bcl2 may be to sequester this enzyme and to prevent dephosphorylation of Journal of the National Cancer Institute Monographs No. 28, 2000 ''cytosolic substrates such as BAD, which can help trigger apop- tosis under some circumstances (66-68). BcL2 PHOSPHORYLATION MAY POTENTIALLY AFFECT THE PROTEOLYTIC CLEAVAGE OF BoTtH BcL2 AND BAx It was reported that the N-terminal domain of Bcl2 could be proteolytically cleaved at a recognized caspase 3 proteolytic site at D34 (40). Furthermore, cleavage of Bcl2 renders a truncated form (A34N-Bcl2) that is nonfunctional in protecting cells from IL-3 deprivation. These data suggest that the cleaved N-terminal region of Bcl2, which contains the BH4 domain that is the dock- ing site for such signaling proteins as RAF-1, PP2B, and p53 BP2, is potentially required for its potent antiapoptotic activity. Thus, IL-3 postreceptor signaling may somehow protect Bcl2 from inactivation by caspase cleavage (40). Our preliminary findings (24) support this notion. We found that steady-state expression of Bcl2 is maintained and cell survival prolonged after IL-3 deprivation in cells that express wt but not S70A-Bcl2 (Fig. 2). This finding suggests that the ser70 site phosphoryla- tion of Bcl2, although not being required for Bax heterodimer- ization, may protect it from proteolytic cleavage. Precisely how phosphorylation may affect this process is not yet clear. It is interesting that phosphorylation can apparently protect pro- caspase 9 and presenilin-2 from proteolytic cleavage (69,70). Presenilin-2 is a transmembrane protein potentially involved in early onset of Alzheimer’s disease (70). Phosphorylation at a serine site residing c-terminal to an aspartate target site for caspase apparently retards the neuronal apoptotic process char- acteristic of this neurodegenerative disease (70). Although Bcl2 is an integral mitochondrial membrane protein that heterodimerizes with Bax, the majority of Bax is not an integral membrane protein, at least during normal cell growth (71,72). Rather, Bax is primarily cytosolic and/or only periph- erally associated (i.e., not membrane integrated) with the mito- chondria membranes (such that it can fractionate with mitochon- dria unless extracted by a pH 11.5 alkali treatment to remove peripherally associated proteins) (7/). Bax can be translocated during stress from the cytosol to the outer mitochondrial mem- brane, where it will apparently integrate into the membrane via its hydrophobic c-terminal transmembrane domain (7/—73). However, how Bax is cleaved and/or translocated from the cy- tosol to become an integral membrane protein that may trigger or be involved in apoptosis is not yet clear. Bax is a 21-kd protein. It was found that p21 Bax can be cleaved at the N- terminus to yield a p18 Bax form that apparently is more effi- cient at membrane insertion, at least in vitro (71). Our prelimi- nary data suggest that Bcl2 phosphorylation may enhance, at least in part, the stability of the interaction between Bcl2 and Bax (25) and potentially retard Bax cleavage. Our findings also indicate that an intact Bcl2 ser70 phosphorylation site is required to maintain the tight association between Bax and Bcl2 observed during co-immunoprecipitation from detergent lysates of cells (Fig. 2). Thus, the nonphosphorylatable, severe loss of function of S70A Bcl2 displays a significantly decreased association with Bax. Although Bcl2 phosphorylation is not required for Bax: Bcl2 association, it may stabilize such an association. Indeed, ceramide-induced Bcl2 dephosphorylation also appears to cor- relate with a similar decrease in Bcl2: Bax association (Fig. 3). Importantly, cells expressing the S70E Bcl2 mutant, which mimic phosphorylation but cannot be dephosphorylated, are vi- able even at elevated concentrations of ceramide (i.e., 50 uM). By contrast, cells expressing wt Bcl2 are killed at ceramide levels (10 4M) in which serine 70 is dephosphorylated (64). Furthermore, under conditions of IL-3 deprivation, Bax under- goes rapid proteolytic cleavage from a p21 to a p18 Bax form (Deng X, Ruvolo P, Carr BK, May WS: unpublished data). Thus, Bax cleavage is pronounced and occurs in cells that ex- press S70A Bcl2 after IL-3 withdrawal. This finding suggests A Fig. 2. Bcl2 phosphorylation at ser70 is necessary for maximal and stable association with Bax. A) NSFN1/H7 cells were stably transfected with wild-type (wt) or S70A mutant Bcl2 and grown in interleukin 3 (IL-3)-containing media. Cells were harvested, washed, and lysed in detergent buffer. The lysates were then immunoprecipitated with the use of a Bax antisera as described previously (23). The immunoprecipitates were resolved by so- dium dodecyl sulfate—polyacrylamide gel electrophoresis on a 10% gel, transferred to a PVDF membrane, and simultaneously analyzed by western blot analysis with a mixture of Bcl2 and B Bax antisera (lower panels). Total cell Bcl2 was determined by quantitative immunoprecipitation of Bcl2 from the cell lysate with the use of a Bcl2 antisera (upper panels). B) Bax- associated wt and S70A Bcl2 is calculated with the use of den- sitometry analysis and expressed as a percentage of the bound Bel2. IP: Bcl2 Blot: Bel2 IP: Bax Blot: Bcl2,Bax WT S70A a, <«- Total Bcl2 — |< Bound Bcl2 —— £2, <4 Bax Pre % Bound Bci2 S70A WT Journal of the National Cancer Institute Monographs No. 28, 2000 33 '' oO 1 10 50 C2-Ceramide (uM) 32p ee os —< Bcl2 Wester i, median, % P (Fisher’s exact test) Survival < median, wk Survival > median, wk P+ Bax 12 87 12 89 132.5 08 PKCa 82 Ta 61 124 92.5 48 Bcl2/Bax 88 69 04 141 80.5 007 PKCa/Bel2 82 Ta 60 132 88 38 PKCa - Bcl2/Bax 92 65 002 141.5 55 005 *From October 1991 through July 1995, 100 patients with newly diagnosed, untreated acute myelogenous leukemia (AML) with favorable or intermediate cytogenetics were evaluated at The University of Texas M. D. Anderson Cancer Center, Houston. Samples for this study were acquired during routine diagnostic assessment in accordance with regulations and protocols sanctioned by the Human Subjects Committee of The University of Texas M. D. Anderson Cancer Center. A Ficoll-generated mononuclear fraction of peripheral blood was obtained for analysis. Median protein levels were scored by densitometry from western blot analysis from patient samples and normalized against control signals from K562 or Y79 cells. PRKCa = cytogenetics. +Gehan—Wilcoxon test. variables, we found that the ratio of either Bcl2 to Bax (B2/Bx) or PKCa - B2/Bx (PK - B2/Bx; i.e., ratios of expression levels of the protein relative to the median level of expression of the individual protein) was highly prognostic for 100 patients with AML who exhibited FIPC (Table 1) (89). Results indicate that the AML samples that displayed a lower ratio of either B2/Bx or PKa:B2/Bx had a significantly higher initial remission- induction rate (88% versus 69%; P = .04) and a prolonged survival (median 141 weeks versus 80.5 weeks, P = .007) com- pared with patients whose blasts demonstrated higher ratios (89). Because a previous correlation was established for Bcl2 expres- sion, a poor outcome but no correlation was observed in these preliminary studies to indicate that expression of individual lev- els of PKCa or Bax affected outcomes. The expectation was that, when forming the interactive variable terms (i.e., ratios), any prognostic value of Bcl2 alone would be lost (because of the expression of essentially random levels of PKCa or Bax). Sur- prisingly, however, forming the interactive terms gave greater prognostic discrimination, suggesting that, although the relation- ships were not immediately apparent on the basis of raw expres- sion levels, a functional relationship among these variables ex- ists. More recent preliminary studies have also suggested that expression of higher levels of ERK2 may also affect Bcl2’s poor prognostic effect on AML (Kornblau SM, Ruvolo P, Deng X, May WS: unpublished data). A similar analysis of ERKI as another Bcl2 kinase is now pending. No definitive conclusions should be drawn at this point from this retrospective analysis because the actual Bcl2 phosphorylation state and the apoptosis rate of individual AML leukemic blast cells were not measured. However, these results were found to be statistically significant and thus suggest that a functional relationship may exist between these variables. Further studies are now in progress to test the role for these variables in a prospective study. If a correlation can be established between cell survival and increased Bcl2 phosphorylation, mitochondrial localization of PKCa and/or ERK1 and ERK2, and increased cell survival following expo- sure to induction-remission chemotherapy in vitro, these data would support the hypothesis that phosphorylation of Bcl2 may have clinical relevance. In this case, developing novel antineo- plastic strategies to block Bcl2 phosphorylation would be one novel strategy to improve both remission-induction success rates and survival for patients with AML. In summary, it now seems clear that, in addition to a requisite role in growth factor-induced proliferative signaling, the MEK- 1/MAPK (ERK1 and ERK2) pathway can functionally interface Journal of the National Cancer Institute Monographs No. 28, 2000 protein kinase C a; FIPC = favorable or intermediate prognosis with a survival signaling pathway induced by growth factors like IL-3 that feature Bcl2 phosphorylation. This finding now di- rectly links these two critical pathways (Fig. 1). Furthermore, by serving as an SRK, the ERKs can potentially cooperate with other survival signaling pathways, including PKC activation, to ensure Bcl2 survival function. 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The incidence varies from 1% in renal recipients to 5% in heart recipients, but can be markedly increased by the use of anti-T-cell therapies or by T-cell depletion in bone marrow transplan- tation. PTLD continues to arise, even many years after transplantation, and late T-cell lymphomas have recently been recognized. Pretransplant Epstein-Barr virus (EBV) se- ronegativity increases risk to as high as 30%-50%. PTLD has a highly variable clinical picture; certain patterns are, however, seen. Reversibility of PTLD with reduction in im- munosuppressives has long been recognized. Predicting re- versibility has been difficult. The presence or absence of bcl-6 mutations has recently been identified as being of pre- dictive value. Surgical resection can be curative. Cytotoxics, although problematic, can also be curative. Long-term re- mission has been achieved with anti CD21 and CD24 anti- bodies; efficacy has been reported for interferon alfa and for rituximab. In vitro expanded EBV-specific T cells have been effective as treatment and as prophylaxis in the setting of bone marrow transplantation. EBV viral load measured in blood appears to associate with the emergence of PTLD and may facilitate prophylactic studies. PTLD is a model of im- munodeficiency-related EBV lymphomagenesis. Pathoge- netic, therapeutic, and prophylactic insights gained from the study of PTLD are likely to be applicable to the acquired immunodeficiency syndrome setting. [J Natl Cancer Inst Monogr 2000;28:38-43] Malignancy following iatrogenic immunosuppression has been recognized since the beginning of organ transplantation (/). More than 20000 organ transplants are performed per year in the United States. Longer patient survival, the increasing use of organ transplantation in the pediatric age group, and the use of potent and highly T-cell-specific immunosuppressive agents all contribute to an increasing incidence of post-transplant malig- nancy (2-6). IMMUNOSUPPRESSIVE REGIMENS Immunosuppressive regimens vary from one institution to another, and a number of new agents has recently been intro- duced. Regimens are commonly based on an agent that inhibits T-cell function, such as cyclosporine or FK506, in combination with azathioprine and prednisone. Immunosuppression is most intense immediately after transplantation, with gradual reduction in dosage over the ensuing years. The incidence of post- transplant lymphoproliferative disorder (PTLD) is higher in non- 38 renal than in renal recipients. The most reliable estimates of incidence currently available are based on data obtained by the European and North American Collaborative Transplant Study. Those data are population based and multicenter and take length of follow-up into account. PTLD incidence among 45 141 renal recipients and 7634 cardiac recipients was determined. As had been noted in other series, incidence was highest in the first year after transplant. During that first year, 0.2% of renal and 1.2% of cardiac recipients developed PTLD, rates that were calculated to be 20 and 120 times higher than those seen in the general popu- lation. The incidence of PTLD in subsequent years was about 0.04% per year in renal and 0.3% per year in cardiac recipients (2). Ina subsequent report, analyzing 14284 heart recipients and 72 360 kidney recipients, a cumulative incidence of almost 5000 per 100000 by 7 years of follow-up was noted in heart recipients and slightly more than 1000 per 100000 by 10 years of follow- up in renal recipients, a cumulative incidence of about 5% for heart recipients and 1% for kidney recipients (3). A striking feature of this analysis is the fact that PTLD continues to arise even many years after transplantation, despite the fact that thera- peutic immunosuppression is reduced over time. Prolonged sur- vival in the face of immunodeficiency is, therefore, associated with a continued risk of lymphoid malignancy in the setting of organ transplantation. The effect of highly active antiretroviral therapy on the incidence of lymphoid malignancy in the setting of acquired immunodeficiency syndrome (AIDS) is as yet un- clear. Despite improvement in CD4 counts and relatively good immune function on such therapy, the transplant experience raises some concern that the problem of lymphoma may not be obviated. The risk of PTLD appears to be influenced by quantitative and qualitative differences in the degree of immunodeficiency. Statistically significantly higher doses of cyclosporin and aza- thioprine were found in heart than in kidney recipients on analy- sis of Collaborative Transplant Study data. A related observation was the significantly higher risk for PTLD among North Ameri- can recipients than for patients transplanted at European centers, amounting to a relative risk of 2.12, which was associated with, and attributed to, higher immunosuppressive dosage among North American recipients (2). Highly potent anti-T-cell therapy has furthermore been shown to markedly influence the incidence in both organ and allogeneic bone marrow recipients. A ninefold higher incidence of PTLD was noted among patients who had received induction therapy with the immunosuppressive anti- body OKT3 (11.4% versus 1.3%) in a series of cardiac transplant recipients (6). A strong dose response was observed, in that Correspondence to: Lode J. Swinnen, M.D., Division of Hematology/ Oncology, Loyola University Chicago, Cardinal Bernardin Cancer Center, Rm. 245, 2160 S. First Ave., Maywood, IL 60153 (e-mail: Iswinne @luc.edu). See “Note” following “References.” © Oxford University Press Journal of the National Cancer Institute Monographs No. 28, 2000 ''6.2% of the patients who had received one course of the drug and 35.7% of the patients who had received two courses devel- oped the disease. A murine monoclonal antibody directed against the human CD3 receptor-T-cell complex, OKT3 pro- foundly depletes circulating CD3* T lymphocytes. Similar ef- fects have been noted in other series (2,7,8) and with T-cell depletion of donor marrow to reduce graft-versus-host disease in allogeneic bone marrow transplantation. PTLD is relatively un- common (1 year after transplant) were identified as predic- tive for poorer response on multivariate analysis. Only 29% of patients with CNS involvement and 22% of patients presenting later than | year after transplant achieved complete remission. The implications of these observations for AIDS-related lym- phomas are unclear. Toxicity was mild, consisting of transient fever, hypotension, and neutropenia. The antibodies used are not currently clinically available. The commercially available anti- CD20 antibody rituximab has shown efficacy in PTLD, based on anecdotal reports (53), and on a retrospective study of 32 pa- tients with PTLD related to organ transplant (26 patients) or to bone marrow transplant (six patients) (54). Immunosuppressives were modified in 27 patients; it is not clear whether that occurred simultaneously with antibody treatment. Among the 26 evalu- able patients, 54% complete remission and 15% partial remis- sion were reported. Median duration of follow-up was 5 months. Two relapses were seen at approximately 9 months, which is about the time when the effect of this antibody is known to Journal of the National Cancer Institute Monographs No. 28, 2000 wane. In summary, monoclonal anti-B-cell antibodies appear to have significant activity in PTLD. Whether antibodies have use- ful activity against disease refractory to reduced immunosup- pression and particularly in tumors containing structural genetic alterations remains to be clearly defined. The latter cases would be of greatest interest in attempting to extrapolate to the setting of established AIDS-related lymphomas. Chemotherapy Cytotoxic chemotherapy has resulted in significantly greater toxic effects in organ transplant recipients than in the general population, mainly due to infectious complications. This is analogous to what has been seen with AIDS-related lymphomas. Cytotoxics have been considered as a treatment of last resort in PTLD, as other treatment options exist. A mortality of 70% has been reported for patients presenting at more than | year after transplant (4/,42). Septic and other complications of chemo- therapy have been the major problem in some centers, whereas others have found refractory disease to be common (/,4/,43). Those poor results have been obtained with a variety of full-dose or attenuated regimens, frequently combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and predni- sone. More encouraging results have been achieved in a small series of cardiac recipients treated with aggressive chemo- therapy, predominantly ProMACE-CytaBOM (40). Mortality during chemotherapy was 25% (sepsis, refractory disease), growth factor support was not used; the surviving patients all achieved complete remission. No patient has relapsed, at a me- dian follow-up of 64 months. This regimen allowed the discon- tinuation of all other immunosuppressives for the duration of chemotherapy and minimized exposure to doxorubicin in car- diac recipients. This approach is currently being tested in an intergroup study being conducted by the Southwest Oncology Group and the Eastern Cooperative Oncology Group. The ad- vent of better supportive care measures, granulocyte colony- stimulating factor, and preventive antibiotics may further reduce the toxicity of chemotherapy in this patient population. The con- cept that immunodeficiency-related lymphomas require aggres- sive rather than dose-attenuated chemotherapy is currently being studied in a number of clinical trials, including a phase II study of ProMACE-CytaBOM for AIDS-related lymphomas by the Southwest Oncology Group, and an EPOCH regimen at the Na- tional Cancer Institute (55). T-Cell Therapy EBV-specific immunocompetence has been rapidly restored in T-cell-depleted allogeneic bone marrow recipients by the in- fusion of a limited number of peripheral blood leukocytes from the donor (56). More recently, highly selective adoptive transfer of T-cell immunity has been achieved with the use of in vitro- expanded EBV-specific cytotoxic T cells as treatment and pro- phylaxis for PTLD in T-cell-depleted bone marrow transplant recipients (57,58). Polyclonal T-cell lines containing both CD4 and CD8 cells were generated, since it is not presently clear which antigens expressed by EBV-infected cells are important in generating an effector response. Adoptive transfer of EBV- specific T-cell immunity would clearly also lend itself to pro- phylaxis against PTLD. Whether exogenously expanded T cells would be effective in tumors refractory to reduced immunosup- pression is unknown. Using such approaches in the organ trans- plant or AIDS setting will require significant adaptations, in 41 ''view of the major histocompatibility complex-restricted nature of the T-cell response. The vast majority of organ transplant- related PTLDs is of recipient origin (59-6/), not of donor origin as is the case following bone marrow transplantation. T cells must, therefore, be generated from the patient. In summary, significant differences and similarities exist be- tween PTLD and AIDS-related lymphoma, in terms of viral association, histopathology and molecular pathology, clinical behavior, and response to treatment. PTLD can be a valuable model system for EBV-related lymphoid neoplasia in immuno- deficiency, allowing the development and testing of screening, prophylactic, and therapeutic measures that may be directly ap- plicable to the setting of AIDS-related lymphoma. REFERENCES (1) Nalesnik MA, Makowka L, Starzl TE. The diagnosis and treatment of posttransplant lymphoproliferative disorders. Curr Probl Surg 1988;25: 367-472. Opelz G, Henderson R. Incidence of non-Hodgkin lymphoma in kidney and heart transplant recipients. Lancet 1993;342:1514—6. Opelz G. 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VIlth International Symposium on Epstein-Barr Virus and Associated Dis- eases 1998; 85. NOTE Presented at the Third National AIDS Malignancy Conference. 43 ''Acquired Immunodeficiency Syndrome-Related Kaposi’s Sarcoma Regression After Highly Active Antiretroviral Therapy: Biologic Correlates of Clinical Outcome Anna Maria Cattelan, Maria Luisa Calabro, Paola Gasperini, Savina M. L. Aversa, Marisa Zanchetta, Francesco Meneghetti, Anita De Rossi, Luigi Chieco-Bianchi Background: Kaposi’s sarcoma (KS) is the most common cancer seen in subjects with acquired immunodeficiency syn- drome (AIDS). KS etiology and pathogenesis are still ill de- fined, and no definite improvement in survival has been ob- tained with current chemotherapeutic regimens. This open prospective study was aimed at evaluating the clinical re- sponse of AIDS-related KS to highly active antiretroviral therapy (HAART), a combination of protease and reverse transcriptase inhibitors, as well as the relationship between clinical response, human immunodeficiency virus type 1 (HIV-1) burden, and antibody titer against human herpes- virus 8 (HHV8) proteins. Patients and Methods: Fourteen KS patients were studied; 12 were in the poor-risk group. At given intervals, the patients underwent clinical examination, and their CD4* cell counts, plasma HIV-1 RNA levels, and antibody titers to lytic-phase ORF65 and latent-phase HHV8 proteins were determined. Results: When last seen, the over- all clinical response rate was 86% (median follow-up, 22 months); 10 complete and two partial responses were achieved, and two patients showed disease progression. All patients with complete or partial response showed a consis- tent decrease in HIV-1 RNA levels, with a corresponding increase in CD4* cell counts; HIV-1 RNA levels in the two progressors remained persistently high, despite a change in HAART. HHV8 ORF65 antibody titers were generally higher in patients with extensive skin or mucosal/visceral involvement versus patients with limited disease; no differ- ences in latent-phase HHV8 antibody titers were observed in relation to tumor burden. Conclusion: The findings indicate that antiretroviral therapy with protease inhibitors is effec- tive for AIDS-related KS; the clinical response was corre- lated with a decrease in plasma HIV-1 RNA levels and an increase in CD4* lymphocytes, whereas antibody levels to the lytic-phase HHV8 protein were influenced by the extent of tumor involvement. [J Natl Cancer Inst Monogr 2000;28: 44-9] The initial suggestion by Thomas (/) that an adaptive im- mune system evolved in vertebrate organisms with the principal aim of preserving cell type uniformity by eliminating spontane- ously arising tumor cells was further elaborated by Burnet (2) who advanced his theory of immune surveillance. Although this innovative idea triggered many studies on tumor immunology, it also met with much skepticism and eluded many expectations of a major therapeutic breakthrough. Nevertheless, circumstantial evidence indicates that primary or acquired immunodeficiencies greatly increase the risk of tumor development and, particularly, those tumors etiologically linked with a given virus infection (3). 44 Kaposi’s sarcoma (KS) is a good example of a tumor whose incidence is remarkably higher in immunocompromised hosts (4,5). Following its original description in 1872, KS was long regarded as a rare dermatologic condition appearing in patients of Mediterranean and eastern European origin (“classic” KS) as well as in endemic foci in sub-Saharan African areas (““endemic” KS). That KS was also associated with immunodeficiencies was noticed with the advent of therapeutically induced immunosup- pression in organ and marrow transplant recipients (“‘iatrogenic” KS). Its incidence was dramatically augmented, however, by the onset of the acquired immunodeficiency syndrome (AIDS) epi- demic in the early 1980s, and the epidemic KS variant became an AIDS-defining condition, since it was the most common ma- lignancy in people infected by the human immunodeficiency virus type | (HIV-1). Unlike classic KS, but similar to the iatrogenic variant, AIDS-related KS tends to progress rapidly and shows a wide spectrum of lesions, ranging from multiple skin patches and nodules to mucosal and visceral involvement (6). At the micro- scope, spindle cells separated by slits containing red blood cells are the hallmark of KS lesions; mitotic activity is moderate and a diploid profile is usually seen on flow cytometry analysis. The spindle cell phenotype recalls vascular endothelial cells, likely of lymph vessel origin, that may derive from vasoformative mesenchyme. KS tissue also contains admixed lymphocytes, he- mosiderin-laden macrophages, and other inflammatory cells (4). The etiology and pathogenesis of AIDS-related KS are still ill defined. On the basis of the natural history and histopathologic findings, it was advanced that, besides determining immunode- ficiency, HIV-1 might be involved through its transactivator Tat protein, which is released by infected cells and taken up by nearby cells (7), and whose angiogenetic properties are well established (8). Moreover, HIV-! infection might produce an increase in inflammatory cytokines, such as interleukin 1, inter- leukin 6, oncostatin M, and interferon gamma, which together with other angiogenic growth factors would, in turn, promote the growth of hyperplastic/neoplastic KS cells (9). A novel herpesvirus, termed “KS-associated herpesvirus” or Affiliations of authors: A. M. Cattelan, F. Meneghetti (Department of Infec- tious Diseases), S. M. L. Aversa (Department of Medical Oncology), General Hospital of Padova, Italy; M. L. Calabro, P. Gasperini, M. Zanchetta, A. De Rossi, L. Chieco-Bianchi, Department of Oncology and Surgical Sciences, On- cology Section, AIDS Reference Centre, University of Padova, Italy. Correspondence to; Luigi Chieco-Bianchi, M.D., Department of Oncology and Surgical Sciences, Oncology Section, AIDS Reference Centre, University of Padova, Via Gattamelata 64, 35128, Padova, Italy (e-mail chiecobl@ ux | .unipd.it). See “Notes” following “References.” © Oxford University Press Journal of the National Cancer Institute Monographs No. 28, 2000 ''“human herpesvirus 8” (HHV8), has also been linked to KS (10,11). HHV8 has been detected in the spindle cells, endothelial cells, and monocytes of almost all KS lesions and in about 50% of the peripheral blood mononuclear cells of KS patients (/2). Moreover, although almost 100% of the KS subjects studied had antibodies against HHV8, the HHV8 seroprevalence in the gen- eral population of the United States and Europe varies from 1% to 25%, depending on the geographic area and the methodology employed (/3). The finding that HIV-1 Tat protein exerts a positive effect on HHV8 replication suggests an interplay be- tween HIV-1 and HHV8 (/4). To define a chemotherapeutic strategy for AIDS-related KS, several studies have been conducted. Treatments with cytotoxic drugs, either as a single-agent or in combination, were found to have variable response rates and to increase the frequency of opportunistic infections with no substantial improvement in sur- vival (15,/6). The addition of antiretroviral zidovudine treat- ment to combination chemotherapy did not increase the response rates (17). However, the course of HIV-1 infection has been greatly modified by highly active antiretroviral therapy (HAART), a combination treatment that makes use of reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). HAART brings about a substantial and sustained decrease in peripheral blood HIV-1 RNA levels, as well as an increase in CD4* T cells, and significantly delays the development of AIDS-associated opportunistic infections and death (/8). Pre- liminary findings suggested that PIs might also determine a re- duction in KS lesions (/9-23), and complete remission was re- cently reported in patients during HAART (24). This study, which extends a previous report (25), was aimed at evaluating the clinical impact of HAART on AIDS-related KS lesions, as well as the relationship between the clinical response, HIV-1 viral load, and antibody titer against lytic and latent HHV8 proteins. PATIENTS AND METHODS Patients From October 1996 to October 1998, all HIV-1-seropositive patients with stable or progressive biopsy-proven KS and mea- surable disease and who were attending the Department of In- fectious Diseases of the General Hospital of Padova were en- rolled in an open prospective study. At study entry, the patients underwent a physical examination that included the measure- ment of all cutaneous and mucosal lesions as well as the com- pilation of a standard body diagram; patients with a clinical suspect of visceral KS underwent gastrointestinal/bronchial en- doscopy and chest tomography. The patients’ disease was clini- cally staged according to the AIDS Clinical Trial Group (ACTG) criteria based on tumor extent (T), severity of immu- nosuppression (I), and other systemic HIV-1-associated diseases (S) (26,27). Performance status was determined according to the criteria of the Eastern Cooperative Oncology Group. Study Treatment The HAART regimen consisted of a triple-drug combination, including two RTIs and one PI, according to current guidelines (28-30). During the study, a switch from one HAART regimen to another was allowed because of intolerance or failure to re- duce viral load. The drugs used in the different combinations were administered daily at the following doses: RTIs—600 mg Journal of the National Cancer Institute Monographs No. 28, 2000 zidovudine, 300 mg lamivudine, 2.15 mg zalcitabine, 400 mg didanosine, and 80 mg stavudine; PIs—2400 mg indinavir, 1200 mg ritonavir, 1800 mg saquinavir, and 2250 mg nelfinavir. Outcome End Points During the study, a complete physical examination was done every month; tumor measurements, blood cell counts, and CD4* lymphocyte counts were also recorded. If endoscopic and radio- graphic findings at study entry were abnormal, these examina- tions were repeated. Clinical responses were evaluated with the use of the ACTG criteria (26): A complete response (CR) was defined as the absence of new lesions and of any detectable residual disease, including tumor-associated lymphoedema, per- sisting for at least 4 weeks; for visceral KS, normal endoscopic and radiographic findings were considered a CR. A partial re- sponse (PR) was defined as the absence of new lesions and a 50% or greater decrease in the number of all pre-existing lesions, or complete flattening of 50% or more of the lesions, or a 50% or greater decrease in lesion size, determined by calculating the products of two perpendicular dimensions, for at least 4 weeks. Progressive disease (PD) was defined as the development of new lesions or an increase of 25% or more in the size of pre-existing lesions. Any response not meeting the criteria for CR, PR, or PD was considered stable disease. Quantitative HIV-1 RNA Assay EDTA peripheral blood samples were centrifuged at 800g for 30 minutes at 20°C over a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient. Plasma was recovered from the upper phase and centrifuged at 1000g for 10 minutes at 20 °C to ensure a cell-free specimen; 200 wL was employed for HIV-1 RNA determination, and the remainder was aliquoted and stored at -80°C. HIV-1 RNA was determined with the use of a quan- titative reverse transcription—polymerase chain reaction assay (Amplicor Monitor; Roche Diagnostic System, Branchburg, NJ), whose lower limit of detection is 200 HIV-1 RNA copies/mL. Analysis of HHV8 Antibodies Plasma samples were analyzed for antibodies to a latency- associated nuclear antigen (LANA) and a capsid-related protein encoded by ORF65, as previously described (3/,32). LANA antibodies were evaluated by an indirect immunofluorescence assay on paraformaldehyde-fixed BCP-1 cells; plasma samples were initially analyzed at a dilution of 1: 100 and subsequently at serial twofold dilutions. ORF65 antibodies were tested by the enzyme-linked immunosorbent assay (ELISA) at an initial plasma dilution of 1 : 100 and then at serial twofold dilutions; the cutoff value was the average of 10 HHV8-seronegative Italian blood donors plus 5 standard deviations. Purified recombinant dehydrofolate reductase (DHFR), the fusion partner of recom- binant ORF65 protein, was employed as the control antigen; plasma samples showing reactivity to this DHRF portion were considered nonspecific by ELISA. Antibody titers were calcu- lated as the reciprocal of the highest plasma dilution giving positive results. Statistical Analysis Immunologic and virologic data were analyzed by the non- parametric Mann—Whitney and Wilcoxon tests. Specimens in which the HIV-1 RNA load was below the lower detection limit of the assay were assigned a value of 100 copies/mL to include 45 ''the data in the statistical analyses. Statistical analyses were per- level was 75 500 copies/mL (range, 2500—1 870.000 copies/mL). formed with the use of SAS software (SAS Institute, Cary, NC). No statistically significant differences were observed between All P values are two-sided. patients with cutaneous or limited mucosal involvement (T9) and patients with more extensive disease (T,) regarding both CD4 cell number (Ty) median = 75 cells/wL and range = 11-443 cells/wL versus T, median = 21 cells/wL and range = 2-214 cells/wL; P = .38, Mann-Whitney test) and HIV-1 RNA copies/ Fourteen male patients (median age 41 years; range 28-57 mL (Ty median = 144000 copies/mL and range = 2500- years) were enrolled in this study; nine had a history of previous 1870000 copies/mL versus T, median = 72000 copies/mL and opportunistic infection, and KS was the AIDS-defining illness in range = 23000-315000; P = .90, Mann—Whitney test). the other five (PM, CL, AO, MA, and FM; Table 1). The median interval between KS diagnosis and study entry was 8.5 months (range, 1-47 months). At study entry, none of the patients had Three patients (MS, CL, and SL) concluded their previous been treated previously with PIs. Five patients (CA, AO, ZC, chemotherapies at 9, 5, and 10 months, respectively, following MA, and FM; Table 1) had never received any antiretroviral study entry. The initial HAART regimen was changed during the therapy; the other nine had been previously treated with RTIs, — study in four patients (PM, CL, MS, and AR) because of the lack and four of these patients (PM, SL, CL, and MS) had also of a satisfactory virologic response and in one patient (CG) received systemic KS chemotherapy (10 mg/m* bleomycin and _ because of intolerance. 6 mg/m? vinblastine on days 1 and 15 every 2 weeks). After six The median follow-up at last examination was 22 months bleomycin/vinblastine cycles, patients CL and MS were treated (range, 8-31 months). When last seen, the overall clinical re- with bleomycin (intravenous infusion of 10 mg/m? every 2 sponse rate was 86% (12 of 14), with 10 CRs and two PRs. The weeks) and liposomal daunorubicin (40 mg/m? every 2 weeks), median time to CR was 6 months (range, 2-23 months); four respectively. Patient PM concluded his chemotherapy 3 months _ patients with limited cutaneous lesions (MA, CW, FM, and AR) prior to study entry and, at the time of enrollment, had progres- achieved a CR in a median time of 3 months (range, 2-5 sive disease based on the appearance of new nodular and mu- months), whereas six patients with more extensive disease ob- cosal lesions; patients MS, CL, and SL were still under treatment tained a CR following a PR in a median time of 13 months RESULTS Patient Characteristics at Baseline Clinical and Biologic Responses at study entry. (range, 5—23 months). All patients who achieved a CR were still Twelve patients were in the poor-risk group, as defined by _ in this clinical condition when last seen (Table 2). any evidence of the following: visceral disease, tumor- Two patients (AO and SLu) who achieved a PR at 2 months associated edema, and CD4* cell count of fewer than 150 cells/ and at 1 month, respectively, were still in this clinical condition wL (27). Of the two patients with visceral disease, one patient at last examination (Table 2). Patient PM obtained a PR 6 (CL) had large lesions in the main left bronchial wall visualized months after HAART initiation but showed PD at 15 months. by bronchoscopy, and the other patient (SL) had multiple pul- Patient MS showed PD at 9 months, despite concomitant KS monary interstitial infiltrates confirmed by chest tomography. chemotherapy; he was then started on paclitaxel (30 mg/m? ev- One patient (AR; Table 1) fell into the ToIpS9 group; this patient ery week) and after 2 months achieved a PR, which was fol- had stable KS following a partial remission obtained during lowed 5 months later by PD (Table 2). treatment with a dual RTI therapy 3 months prior to study entry. At PR, CD4* cell counts were higher than baseline values At baseline, the median CD4* cell count was 58 cells/wL (median = 36 cells/wL and range = 2-214 cells/wL versus (range, 2-443 cells/wL), and the median plasma HIV-1 RNA median = 105 cells/wL and range = 27-350 cells/uwL; P = .11, Table 1. Characteristics of patients at baseline Kaposi’s sarcoma (KS) lesions Human immunodeficiency Patient code Age,y PS* KS staging Monthst Visceral Lymphoedema Mucosal Patch§ CD4cells/pL virus type | RNA copies/mL PM 30 | T,1,8, 19 + ++ 2 79 000 MS 45 2 T,1,8, 47 + + ++ 4 148 000 CL 4B I T,1,S, 9 + + it 98 67 000 SL 33 1 T,1,S; 16 + + ++ 21 72.000 CR 47 2 TLS, 8 + + 4 315 000 CA 49 | T,1oS, | + ++ 214 23 000 AO 40 2 T ToS, 12 + ++ 212 63 000 SLu 28 I Tol S, 8 + 40 37 000 ZC 33 I Tol, 8, 3 + 32 144.000 CG 4B \ Tol, 8, 10 ++ 98 162.000 MA 38 I Tol,S, 6 + 75 1 870.000 cw 57 I Tol S, 1 + i 220 000 FM 36 | ToloS, 5 + 443 2500 AR 42 0 ToloSo 36 + 206 29 000 *PS = performance status, determined according to Eastern Cooperative Oncology Group criteria. +Clinical staging according to the AIDS Clinical Trial Group criteria based on tumor extent (T), severity of immunosuppression (1), and other systemic HIV-1-associated diseases (S). £Time from the date of KS diagnosis. §+ = fewer than 10 patches; ++ = 10-30 patches; +++ = more than 30 patches. 46 Journal of the National Cancer Institute Monographs No. 28, 2000 ''Table 2. Clinical and biologic response to therapy* Clinical and biologic response Partial response (PR) Complete response (CR) HIV-1 RNA Progressive disease (PD) At last examination HIV-1 RNA Patient CD4 CD4 HIV-1 RNA CD4 Response CD4 HIV-1 RNA code Months — cells/wL copies/mL Months — cells/wL copies/mL Months — cells/wL copies/mL (months) — cells/pL copies/mL PM 6 106 228 000 15 23 634 000 PD (31) 6 660 000 MS 11 27 124 000 16 51 227 000 PD (20) 81 124 000 CL 5 292 31 000 13 423 <200 CR (31) 400 <200 SL 6 101 <200 23 448 <200 CR (31) 600 <200 CR 8 119 376 000 14 234 <200 CR (17) 290 <200 CA 3 142 <200 16 247 <200 CR (24) 300 <200 AO 2 350 1000 PR (8) 520 1400 SLu I 34 5300 PR (20) 110 7800 ZC I 87 <200 5 148 <200 CR (27) 620 <200 CG 2 104 2300 7 196 <200 CR (21) 230 <200 MA 3 287 240 CR (10) 350 14.500 CW 2 51 <200 CR (25) 220 <200 FM 5 430 <200 CR (8) 460 <200 AR 3 276 6500 CR (30) 290 29 500 *HIV-1 = human immunodeficiency virus type 1. Wilcoxon test), and HIV-1 RNA levels had decreased in most of | DISCUSSION the patients (median = 75000 copies/mL and range = 23 000- 315000 versus median = 3800 copies/mL and range 100- 376000 copies/mL; P = .15, Wilcoxon test), but the differences were not statistically significant. Furthermore, no statistically significant differences in the number of CD4* cells and HIV-1 RNA levels (P = .33 and P = .59, respectively, Mann—Whitley test) were observed between patients who subsequently achieved a CR and those with a PR or PD. At the time of CR, CD4* cell counts were statistically sig- nificantly higher than baseline values (median = 262 cells/wL and range = 51-448 cells/jL versus median = 87 cells/wL and range = 4-443 cells/wL; P = .006, Wilcoxon test), and the HIV-1 RNA load had dropped to undetectable levels in eight of 10 subjects (median 100 copies/mL and range = 100-6500 copies/mL versus baseline median 108 000 copies/mL and range = 2500-1 870000 copies/mL; P = .004, Wilcoxon test). At last examination, an additional upsurge in the number of CD4* cells was observed in almost all subjects who achieved a CR or a PR; plasma HIV-1 RNA levels were undetectable in eight subjects with a CR and relatively low in two others with a CR as well as in the two patients with a PR. However, the two patients with PD had persistently high plasma HIV-1 RNA lev- els and CD4 cell counts below 100/jL despite a change in HAART regimen (Table 2). Antibodies against HHV8 lytic-phase ORF65 protein and LANA were determined before HAART and at different time points during follow-up. At baseline, the patients with advanced KS showed high titers of ORF65 antibodies; during HAART, small variations with no apparent relationship to the course of disease were observed (Fig. 1, panel A). All patients with Ty disease except two (CG and AR) showed lower ORF65 antibody titers at baseline and throughout follow-up (Fig. 1, panel B). Of interest, patient CG had extensive cutaneous involvement at study entry (Table 1); he obtained a CR, and, when last seen, his titer was considerably reduced. LANA antibodies were detected in all patients at baseline (titer range, 200-64 000), with no difference between patients with KS in stages Ty or T,; small variations were observed at different time points, i.e., at the PR, CR, PD, and at last exami- nation (data not shown). Journal of the National Cancer Institute Monographs No. 28, 2000 The clinical response rate to systemic chemotherapy for AIDS-related KS is usually low and short-lived (/5,/6); despite the use of biologic response modifiers or differentiation agents, such as interferon alfa or retinoic acid derivatives, alone or in combination with standard chemotherapy, no substantial change has emerged (33). More recent trials (34-37) have employed antiangiogenic compounds or human chorionic gonadotropin preparations in the treatment schedule; although these ap- proaches are interesting, they need further evaluation. In reference to HIV-1 infection, the introduction of multidrug antiretroviral regimens, based on a combination of RTIs and PIs, has greatly improved the clinical outcome, as demonstrated by a substantial decline in AIDS incidence and mortality (/8). These potent antiretroviral agents induce a clearance of HIV-1 from the plasma and other biologic fluids, even if complete eradication is precluded by the persistence of latently infected cells. Moreover, immune responses to a variety of infectious pathogens are also restored in individuals who show an optimal virologic response to HAART, as evidenced by in vitro study findings (38) and the remarkable decrease in opportunistic infections (/8). HAART also appears to influence the clinical course of AIDS-related KS; partial or complete regressions were reported in KS patients (/9—24), and our present findings are in line with these observations. In our group of 14 KS patients treated with HAART, 10 CRs and two PRs were achieved and maintained up to the last examination. We observed a substantial decrease in the plasma HIV-1 RNA load associated with a rise in the CD4* cell count in all 12 patients with clinical remission as well as consistently high levels of viremia with low CD4* cell counts in the two patients with progressive KS. Thus, a good correlation between efficacy of HAART and KS clinical response was evi- dent. It is worth mentioning that eight of the 10 patients with a CR were never administered antitumor chemotherapy. It is also noteworthy that the addition of paclitaxel to the treatment sched- ule of patient MS caused a temporary shift from PD to a PR, even if the contemporaneous change in his HAART combination was not followed by a decrease in the plasma HIV-1 load. Our findings indicate that the evolution of AIDS-related KS greatly depends on the entity of the HIV-! burden and the en- 47 '' 10° g = 10 > 3 ° = c 3 = 10 wo oO ra oO. 102 Fig. 1. Antibody titers to ORF65 protein at baseline (Z), partial response (/%), complete response (24), progressive dis- ease (MM), and at last examination (§@ [gray]). Antibody titer was calculated as the reciprocal of the highest plasma dilution giving positive results. Panel A: patients with Kaposi’s sar- coma (KS) in stage T,. Panel B: patients with KS in stage Ty. 10° + & = 104 4 > 3 ° 2 < 3 J S 10 Te) oO ra oO 10? 5 S| x o% OOOO We xs OOK Wi Xs MO > LA suing degree of immunodeficiency. In addition, previous studies on the angiogenic properties of HIV-1 Tat regulatory protein (7) and the activation of the inflammatory cytokine cascade are consistent with the present findings, and they further emphasize the relevant, albeit indirect, role of HIV-1 infection in KS patho- genesis (9). However, the possibility that some antiretroviral PIs are also endowed with an intrinsic anti-KS activity cannot be ruled out. Following the identification of HHV8, an increasing body of evidence has pointed to an etiologic link between this virus and KS development. Like other gamma herpesviruses, 1.e., herpes- Virus saimiri and Epstein-Barr virus, HHV8 also seems to pos- sess an oncogenic potential; analysis of its genomic sequences revealed a set of genes that are structurally and functionally related to cellular genes known to interfere with cell cycle con- trol or are endowed with growth-promoting and antiapoptotic activity (39). Furthermore, two different viral genes, K1 and K12, produced morphologic changes and focus formation in- dicative of neoplastic transformation when expressed in rodent fibroblasts, and they were tumorigenic in vivo (40,41). We used available first-generation serologic assays to mea- sure plasma antibody titers to lytic (ORF65) and latency- associated nuclear (LANA) antigens in an attempt to discern an antibody trend that might be indicative of HHV8 behavior dur- ing KS evolution. The LANA antibody titer showed a variable pattern; ORF65 antibody levels in general seemed to be corre- lated to baseline tumor extension. However, on the basis of the 48 present data, we cannot draw any conclusions as to whether the ORF65 antibody titers reflect the clinical evolution of KS. We expect that direct evaluation of HHV8 viremia by molecular methods would be more informative in this regard; this approach might also be useful to determine whether HHV8 expression is directly influenced by antiretroviral agents. 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Cell-homologous genes in the Ka- posi’s sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? J Virol 1997;71:4187-92. (40) Lee H, Veazey R, Williams K, Li M, Guo J, Neipel F, et al. Deregulation of cell growth by the K1 gene of Kaposi’s sarcoma-associated herpesvirus. Nat Med 1998:4:435—40. (47) Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Kishishita M, Brady JN, et al. Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) transforming gene. J Virol 1998;72:4980-8. NOTES Presented at the International Symposium on HIV, Leukemia, and Opportu- nistic Cancers. Supported by Istituto Superiore di Sanita, Associazione Italiana per la Ricerca sul Cancro, Fondazione Italiana per la Ricerca sul Cancro, and the European Concerted Action on the Pathogenesis of Kaposi’s Sarcoma We thank Dr. Lolita Sasset for assistance with data collection, the nursing staff of the Department of Infectious Diseases of the General Hospital of Padova for their care of the patients, Pierantonio Gallo for graphic artwork, and Patricia Segato for help in preparing the manuscript. 49 ''Papillomavirus-Like Particle Vaccines John T. Schiller, Douglas R. Lowy Papillomavirus-like particle (VLP)-based subunit vaccines have undergone rapid development over the past 8 years. Three types are being investigated. The most basic type is composed of only the L1 major capsid protein and is de- signed to prevent genital human papillomavirus (HPV) in- fection by inducing virus-neutralizing antibodies. On the ba- sis of positive results in animal models, clinical trials of this type of vaccine for HPV16, and other types, are currently under way. Preliminary results have been encouraging in that systemic immunization with the L1 VLPs induced high serum titers of neutralizing antibodies without substantial adverse effects. The second type of vaccine incorporates other papillomavirus polypeptides into the VLPs as L1 or L2 fusion proteins. These chimeric VLPs are designed to in- crease the therapeutic potential of an HPV vaccine by in- ducing cell-mediated responses to nonstructural viral pro- teins, such as E7. Studies in mice indicate that these vaccines generate potent antitumor cytotoxic lymphocyte (CTL) re- sponses while retaining the ability to induce high-titer neu- tralizing antibodies. It is likely that prophylactic and thera- peutic clinical trials of chimeric VLPs will be initiated in the near future. The third type of VLP-based vaccine is designed to induce autoantibodies against central self-antigens by in- corporating self-peptides into the outer surface of VLPs, a process that could have therapeutic potential in various dis- ease settings unrelated to HPV infection. In a recent proof of concept study, a peptide from an external loop of mouse CCRS protein was inserted into a neutralizing epitope of L1. In mice, the particles generated by this chimeric L1 were able to induce high titers of CCR5 antibodies that specifi- cally recognized the surface of CCR5-transfected cells and blocked in vitro infection of an M-tropic human immunode- ficiency virus strain. [J Natl Cancer Inst Monogr 2000;28: 50-4] Very strong biologic, clinical, and epidemiologic evidence exists that sexually transmitted human papillomavirus (HPV) infections cause most cervical cancers (/). This infectious eti- ology provides an opportunity to prevent a major cause of cancer deaths in women through vaccination. The desire to prevent or treat genital HPV infection through immunization has led inves- tigators to employ a number of strategies to develop candidate HPV vaccines (2). This report will focus on the development of one of these strategies, papillomavirus-like particle (VLP)-based subunit vaccines. In general, VLP-based vaccines are attractive for combating viral infections because they retain the highly immunogenic array of repetitive epitopes found on the surface of authentic virions, yet VLPs are devoid of the potentially harmful viral genomes. Preclinical in vitro and animal studies of papil- lomavirus VLPs, composed of only the L1 major virion protein, have moved this candidate to the forefront of vaccines to prevent HPV infection [reviewed in (3)]. They have also prompted at- tempts to develop second-generation VLP-based vaccines that incorporate polypeptides of other viral and cellular proteins into 50 the VLPs. In these cases, the VLPs are used as vehicles to facilitate immune presentation of additional antigens to both the cellular and humoral arms of the immune system (Fig. 1). Some of these second-generation vaccines are being developed with the goal of improving the effectiveness against HPV infection, whereas others have the goal of combating other diseases. PROPHYLACTIC VACCINES Prophylactic vaccines against viruses are thought to function primarily through the induction of virion-neutralizing antibodies that prevent infection (4). It has been difficult to employ this strategy to develop an HPV vaccine. HPV virions cannot be propagated efficiently enough in cultured cells to serve as a source of antigen for a vaccine (5). Even if they could be easily propagated, they would be unattractive as a prophylactic vac- cine, because their genomes contain oncogenes. Subunit vac- cines that lack the viral genome are, therefore, much more at- tractive candidates. However, early attempts to develop virion protein-based subunit vaccines in animal papillomavirus models were only minimally successful. This minimal success is be- cause neutralizing antibodies predominantly recognize confor- mational epitopes of the L1 major capsid protein, and the early vaccines used denatured virion proteins or peptides [(6) and references therein]. The methodologic breakthrough in prophy- lactic vaccine development was the finding that L1 alone could self-assemble into VLPs that are structurally and antigenically very similar to authentic virions. This finding was first shown in a bovine papillomavirus type | (BPV1) model (7) and later confirmed for HPV VLPs as suitable serologic assays became available. VLPs have been generated in a variety of cultured cells, including those from mammals, insects, yeast, and even bacteria (3). Because HPVs do not infect animals, studies of protection from virus challenge after VLP vaccination were conducted with the use of animal-type viruses and VLPs in their animal host species. Three animal models have been used: cutaneous chal- lenge of domestic rabbits with cottontail rabbit papillomavirus (CRPV) (8—/0), oral mucosal challenge of dogs with canine oral papillomavirus (//), and oral mucosal challenge of cattle with bovine papillomavirus type 4 (BPV4) (/2). In these studies, purified VLPs were administered parenterally, and challenge virus was applied to an abraded epithelium to expose the pro- liferating basal keratinocytes to infection. In each model, vacci- nation with high nanogram to low microgram doses of L1 VLPs induced high titers of virion antibodies and protection from ex- perimental challenge with high-dose virus. In most experiments, approximately 90% of the control subjects developed papillomas Affiliation of authors: Laboratory of Cellular Oncology, National Cancer In- stitute, Bethesda, MD Correspondence to: John T. Schiller, Ph.D., National Institutes of Health, Bldg. 36, Rm. 1D-32, Besthesda, MD 20892 (e-mail: schillej@dc37a. nci.nih.gov). See “Notes” following “References.” Journal of the National Cancer Institute Monographs No. 28, 2000 '' Fig. 1. The type of papillomavirus-like particle is indicated above each particle. The applicable immune effector func- tions generated by each type of particle are indicated below. The non-virion poly- peptides are depicted in gray tone. VARIATIONS ON THE THEME OF PAPILLOMAVIRUS VLPS L1VLP a Early Viral Protein CTLs Virus Neutralizing ™, Antibodies L1/L2 Chimera L1 External Chimera + Virus Neutral. Abs —_—_—_—_—_—_——P L1 Internal Chimera Anti-Self Antibodies Early Viral Protein CTLs + Virus Neutral. Abs at the site of inoculation, whereas at least 90% of the vaccinated subjects showed no evidence of infection. High-titer antibodies and protection were seen even after vaccination in the absence of adjuvant (8,9,//). However, protection was obtained only after vaccination with the homologous VLP type (8,9,//). For in- stance, rabbits vaccinated with BPV1 VLPs were not protected from CRPV challenge. Although the L2 minor capsid protein is incorporated into VLPs when co-expressed with L1, there were no detectable differences in the titers of virion antibodies or in the degree of protection generated after vaccination with L1 or with LI and L2 VLPs (8,/2). Protection could be passively transferred to naive animals via immune sera or purified immu- noglobulin G, indicating that neutralizing antibodies were suf- ficient to confer protection from experimental challenge (8, //) (Table 1). CLINICAL TRIALS The positive results of the animal vaccine studies have prompted the National Institutes of Health (NIH), and at least two pharmaceutical companies, to begin clinical trials of HPV Table 1. Summary of papillomavirus-like particle (VLP) vaccine trials in animals* Vaccine Protection LI VLPs Yes LI/L2 VLPs Yes VLPs without adjuvant Yes Denatured VLPs No Heterologous VLPs No Immune serum Yes *Cottontail rabbit papillomavirus VLPs in rabbits, canine oral papillomavirus VLPs in dogs, and bovine papillomavirus type 4 VLPs in cattle. Journal of the National Cancer Institute Monographs No. 28, 2000 VLP vaccines. The early-phase NIH trials are a collaboration of the National Cancer Institute, the National Institute of Allergy and Infectious Diseases, and The Johns Hopkins Center for Im- munization Research. They use HPV16 LI VLPs generated in recombinant baculovirus-infected insect cells. A placebo- controlled, dose-escalation phase I trial compared intramuscular injection of the VLPs either alone, in alum, or in MF59 adjuvant (13). The vaccine was administered in three 10-g or 50-yg doses at 0, 1, and 4 months. Preliminary analyses of the results (unpublished) are encouraging in that the vaccine was consis- tently immunogenic and well tolerated. All of the 60 subjects who received the VLPs seroconverted by | month after the second dose, as measured in an HPV16 VLP-based enzyme- linked immunosorbent assay (ELISA), whereas none of the 12 control subjects seroconverted during the course of the study. Preliminary analysis suggests that both adjuvants increased the titers of VLP antibodies after low-dose (10 wg) VLP vaccina- tion. However, at the higher dose (50 pg), the highest geometric mean titer (GMT) was seen for the group injected with VLPs without adjuvant. The relative neutralizing titers obtained in an HPV16 pseudovirion neutralization assay appear to parallel ELISA titers for both group GMTs and individuals within groups (/4). In the individuals receiving VLPs alone or VLPs plus alum, reactogenicity to the vaccine was minimal, with tran- sient mild pain at the site of injection being the most frequent side effect. Reactogenicity did not increase with vaccine dose or boosting. The side effects in the individuals receiving VLPs plus MF59 were somewhat greater, with more frequent reports of mild or moderate transient pain at the site of injection. A phase II trial of the 50 wg VLPs without adjuvant formulation is cur- rently in progress. The appropriate valency of a prophylactic HPV vaccine is 51 ''currently under debate. On the basis of in vitro neutralization and hemagglutination assays of HPV VLP sera raised in ani- mals, it is assumed that protection in people will be predomi- nantly genotype specific (/4-/8). Because its goal is proof of concept, the NIH prophylactic vaccine program involves only VLPs of HPV 16, the type found in approximately 50% of cer- vical cancers. However, many other types are also detected in cervical cancer (/9), and an HPV vaccine for general distribu- tion will likely contain multiple VLP types. Types 18, 31, and 45, along with 16, account for approximately 80% of cancers worldwide (/9), so most or all of these types will likely be included in a commercial vaccine. The question of cross- interference in the elicitation of antibodies to specific VLP types in polyvalent formulations will need to be addressed during development of this type of vaccine. Good reasons exist to consider including VLPs of nononco- genic genital HPVs in a polyvalent prophylactic HPV vaccine as well. HPV6, and HPV11 to a lesser extent, induce most genital warts (20). Although genital warts very rarely undergo malig- nant progression, they cause substantial morbidity. A vaccine that targets genital warts would make the vaccine more attractive to men, because men, as well as women, suffer from these le- sions. In contrast, the overall incidence of HPV-induced cancers is much lower in men than in women, although a substantial proportion of penile and anal cancers in men are attributed to HPV infection (/). Vaccination of both men and women is likely to increase the effectiveness of a prophylactic vaccination pro- gram by increasing herd immunity and breaking the cycle of venereal transmission. THERAPEUTIC VLP VACCINES Studies in mice indicate that papillomavirus VLPs can induce Ll-specific cell-mediated immune (CMI) responses (2/), in ad- dition to inducing high titers of virion antibodies. However, the virion proteins are not expressed at a detectable level in the proliferating basal keratinocytes of virus producing lesions or in the dedifferentiated cells of HPV-induced dysplasias and can- cers (22). Therefore, it is unlikely that CMI responses to the virion proteins will induce regression of established lesions. In an attempt to generate effective CMI against papillomavirus- infected cells, papillomavirus VLPs have been generated in which polypeptides of nonstructure viral proteins are incorpo- rated into the VLPs as fusion proteins of L1 or L2 [reviewed in (23)]. Chimeric VLPs that contain the entire HPV 16 E7 oncoprotein fused to L2, or the N-terminus of E7 fused to LI, have been generated and shown to induce antigen-specific protection of mice from lethal challenge with E7-expressing tumor cells (24— 26). Protection was obtained after a single injection of 10 wg of VLPs in the absence of adjuvant. The chimeric VLPs could also act therapeutically to induce regression of established tumors (26). The antitumor immune response to the chimeric VLPs appears to be primarily mediated by CD8™ cytotoxic lympho- cytes. In vitro E7-specific cytotoxic lymphocyte (CTL) activity was detected in lymphocytes from chimeric VLP-vaccinated mice (25,26). Also, good protection was observed in major his- tocompatibility complex class II knockout or natural killer cell- depleted mice, but no protection was seen in B, microglobulin or perforin knockout mice (24). It is unclear how the VLPs are routed for class I presentation. It might involve an endocytic 52 pathway that the virus normally uses to enter the cell during the infectious process. LI and L2 chimeras for E7 produced similar results in mice, so it is unclear whether L1 or L2 chimeric VLPs would be preferable for testing in humans. LI chimeras have the theoret- ical advantage in delivering more copies of the target antigen per VLP than L2 chimeras (360 for L1 versus 12 for L2). L2 chi- meras have the theoretical advantage of being able to incorpo- rate larger polypeptides and thereby increasing the number of epitopes for immune recognition. It would seem reasonable to continue testing both types of chimeras. Several alternative strategies for generating CMI responses to E7 have been developed (2). From a safety standpoint, protein- based strategies for generating CTLs to oncoproteins, such as E7, are preferable to gene transfer-based strategies, because transfer of oncogenes might theoretically be tumorigenic. An attractive feature of VLPs is their ability to induce CTL re- sponses without the addition of strong nonspecific immune stimulators. It is likely that early-phase trials of chimeric VLPs that contain nonstructural papillomavirus polypeptides will be- gin shortly. Future efficacy trials could be done in several set- tings. The chimeric VLPs might be effective in treating clini- cally apparent HPV-induced neoplastic lesions. Although the initial safety studies may be done in cancer patients, there is also considerable interest in attempting to induce regression of HPV- induced premalignant cervical dysplasias by chimeric VLP vac- cination. Chimeric VLPs also have the potential to function as a combined prophylactic-therapeutic vaccine, because the inser- tion of the additional polypeptide did not appear to diminish the ability of the chimeric VLPs to induce high titers of virion- neutralizing antibodies (24). It is possible that chimeric VLPs could increase the effectiveness of a prophylactic vaccine by eliminating early subclinical infections that break through, de- spite the presence of neutralizing antibodies. The National Cancer Institute is contemplating a prophylactic vaccine trial of an HPV 16 chimera in which the entire E7 and E2 is fused to the C-terminus of L2. E2 was included because basal cells in benign lesions may express more E2 than E7, and be- cause it simply increases the number of viral epitopes for gen- erated CMI responses. To address concerns that a fusion protein that contains the two nonstructural viral proteins might have adverse effects on cells, mutations were introduced to inactivate the Rb binding activity of E7 and the sequence-specific tran- scription activating activity of E2. Fusion of E2 to E7 did not inhibit the ability of the chimeric VLPs to generate potent anti- tumor responses against E7 in a standard mouse tumor model (our unpublished results). A similar HPV6 chimera is being generated for eventual use in genital wart therapy trials. Chimeric papillomavirus VLPs containing polypeptides of nonpapillomavirus targets are also being investigated in preclini- cal studies. One approach is to incorporate polypeptides of other sexually transmitted diseases (STDs). With the provision that induction of neutralizing antibodies is sufficient for protection against genital HPV infection, this strategy could produce a vaccine that provides protection against both HPV and another STD at little or no increase in the cost of production or admin- istration. A second approach involves incorporating cellular tu- mor antigens into the VLPs. This strategy was recently shown to induce therapeutic antitumor immune responses in a mouse model (27). Immunization of mice with an immunodominant peptide derived from the P815 tumor-associated antigen PIA Journal of the National Cancer Institute Monographs No. 28, 2000 ''induces specific T-cell tolerance, resulting in progressive out- growth of a normally regressing P815 tumor line. In contrast, immunization with an LI chimera that contains this same P1A peptide did not induce tolerance. Rather, it protected mice from lethal challenge with a progressor P815 line. Vaccination with this chimeric VLP also functioned therapeutically to suppress the growth of established tumors and to increase survival of the tumor-bearing mice. AUTOANTIBODY-INDUCING VACCINES As exemplified above, the mammalian immune system has clearly evolved to produce a strong antibody response to viruses and VLPs that mimic them. In contrast, it has evolved to nor- mally be tolerant to self-antigens exposed to the circulating im- mune system. In part, the humoral immune system may distin- guish between self (safe) and nonself (dangerous) on the basis of epitope arrangement, with the highly ordered repetitive arrange- ment of virion surface determinants being especially immuno- genic (28). It was, therefore, of interest to determine whether a central self-antigen, to which the immune system was normally tolerant, could induce an antibody response if it was presented in the ordered context of a papillomavirus VLP. To test this pos- sibility, the first external loop of the mouse CCR5 chemokine receptor (which is primarily expressed on macrophages and memory T cells) was cloned into an immunodominant- neutralizing epitope of BPV1 LI (29). The chimeric VLPs as- sembled into particles, but they were smaller than those of wild- type LI VLPs, containing an estimated 12 capsomeres rather than 72, and they did not induce BPV-neutralizing antibodies. Nevertheless, vaccination of mice expressing an identical CCRS sequence resulted in high-titer antibodies that recognized the CCRS5 peptide in ELISA. The ability of the chimeric L1 to generate CCRS5 autoantibodies depended on the arrangement of the antigen, because no CCRS antibodies were generated if the chimeric particles were denatured prior to vaccination (Table 2). The antibodies generated against the chimeric particles recog- nized the native CCRS, because the sera specifically bound cells transfected with mouse CCRS5 and inhibited binding of RANTES, a CCR5 ligand. In contrast, antibodies generated against the same CCRS peptide coupled to keyhole-limpet he- mocyanin as a carrier bound the peptide in an ELISA but did not recognize cell surface CCRS and did not block ligand binding, indicating that autoantibodies to the native structure were not generated by the latter immunogen. Because human CCRS is the co-receptor for macrophage- tropic HIV strains, it was possible to determine if the antibodies generated to CCRS by the chimeric particles could inhibit HIV infection. Although mouse CCR5 cannot function as an HIV co-receptor, a hybrid CCRS in which the first external loop of the mouse protein replaces the corresponding loop in the human protein can function as a co-receptor. M-tropic HIV (BaL strain) infection of cells carrying this recombinant CCR5 was effec- tively neutralized by sera from the chimeric VLP-vaccinated mice but not by sera from wild-type VLP-vaccinated mice (Table 2). These results establish that, in principle, mammals can be induced to synthesize neutralizing autoantibodies to virus cell-surface receptors. Whether this strategy can be effective at preventing or controlling viral infection in vivo remains to be determined. The general safety of autoantibody induction as an approach to immunotherapy must obviously be considered and could vary Journal of the National Cancer Institute Monographs No. 28, 2000 Table 2. Induction of CCRS5 autoantibodies Antibody assay LI VLP) CCRS CCRS5 HIV BPV Sera to ELISA ELISA FACS neutralization neutralization LI VLP + - - - + CCRS-L1 VLP + + + ie = CCRS-L1 denatured + - NT NT = KLH-CCRS5 - + - - NT VLP = papillomavirus-like particle; ELISA = enzyme-linked immunosor- bent assay; FACS = fluorescence-activated cell sorter; HIV = human immu- nodeficiency virus; BPV = bovine papillomavirus; NT = not tested; KLH = keyhole-limpet hemocyanin. considerably, depending on the cellular target. A potential ad- vantage of targeting CCRS is that it appears to be a nonessential protein. Individuals who are homozygous for a defective CCR5 gene are phenotypically normal, except that they have a sub- stantially decreased risk of HIV infection (30,31). It is notewor- thy that the mice producing CCR5 autoantibodies were out- wardly healthy at 6 months after vaccination and did not exhibit signs of immunopathology at autopsy (29). There was also no decline in the numbers of macrophages or T-cell subsets that express CCR5 in comparison to control animals. Although we did not test for autoreactive T cells, we would not expect to break T-cell tolerance to CCRS. T cells that recognize central autoantigens are strongly selected against during development of the immune system. Of interest, the levels of CCRS5 antibodies had begun to slowly decline by 6 months postvaccination, and the relative decline paralleled the decline in L1 antibodies for individual animals. This result suggests that exposure of the vaccinated animals to self-CCR5 does not result in continuous stimulation of the CCR5-specific B cells, presumably because the cellular protein remains in a context that continues to be ignored. The parallel decline in antibodies to the viral antigen also suggests that the presence of the cellular CCR5 does not specifically attenuate the CCRS5-specific response generated against the chimeric VLPs. If autoantibody induction proves safe, this approach to immunotherapy could have diverse appli- cations. For instance, it could potentially be an effective alter- native to monoclonal antibody therapy in instances in which cell surface or soluble molecules, such as HER2/neu or tumor ne- crosis factor a, are known to be important mediators of disease. In summary, papillomavirus VLP-based vaccines are being developed according to the theory that the mammalian immune system has evolved to efficiently recognize the ordered surface of nonenveloped icosahedral virions as foreign or dangerous and to generate a variety of potent immune responses to them. There- fore, vaccines that mimic the outer structural features of virions should be highly antigenic. This concept is strongly supported by the studies described in this report. Low-dose vaccination with VLPs induced both high-titer antibodies and CTLs to viral antigens without the addition of an adjuvant (Fig. 1). 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The NIH clinical trials discussed here are supported by the NIH Office of Research on Women’s Health and the NIH Office of Research on Minority Health. Journal of the National Cancer Institute Monographs No. 28, 2000 4 ''Role of the National Cancer Institute in Acquired Immunodeficiency Syndrome-Related Drug Discovery Edward A. Sausville, Robert H. Shoemaker The primary role of the Developmental Therapeutics Program of the National Cancer Institute (NCI) is to facilitate drug dis- covery by the extramural cancer and acquired immunodeficien- cy syndrome (AIDS) research communities. This role is accom- plished in a variety of ways through grants programs, web-based informatics, provision of chemicals and natural product extracts, and screening services that will be described briefly in this ar- ticle. Recently, the NCI has begun efforts to bring together mo- lecular-targeted, high-throughput screening and extramural sites with chemical libraries of interest. This new initiative is de- signed to match emerging molecular targets and high-throughput assay technology with novel sources of chemical diversity in the extramural community. Shortly after recognition of the AIDS epidemic, the Devel- opmental Therapeutics Program (DTP) of the NCI was charged with developing a drug-screening program that might give rise to the discovery of novel therapeutics for the treatment of human immunodeficiency virus (HIV) disease. Beginning in 1987 and continuing through 1997, a functional screen for primary anti- viral treatments with the use of a cell-based assay system was in place. Details of the colorimetric-assay methodology and screen- ing strategy have been published (/,2). This screening program supported discovery of numerous lead compounds, from both natural and synthetic sources, many of which were subsequently demonstrated to be nucleoside and non-nucleoside reverse tran- scriptase inhibitors. A substantial number of novel natural prod- uct agents with anti-HIV activity have been isolated, including cyanovirin, a novel gp120-binding protein derived from a cul- tured blue-green alga (3). Detailed information on these mol- ecules may be found on the DTP web site (http://dtp.nci-nih. gov), which is described below in detail. Several novel molecules identified by the screen, or deriva- tives of screening leads, have been developed to the point of clinical trials. The nucleoside analog 3TC was submitted to the screen as a racemic mixture by IAF Biochem International, Inc. (Ville de Laval, Quebec), a Canadian pharmaceutical company, and subsequently licensed to Glaxo-Wellcome (Research Tri- angle Park, NC) for clinical development. This drug and Ziagen, a prodrug form of carbovir (4) also licensed by Glaxo- Wellcome, have been approved by the U.S. Food and Drug Administration for use in the treatment of HIV disease. PROGRAM REVIEWS In 1995, the National Institutes of Health (NIH) Office of AIDS Research (OAR) initiated a wide-ranging review of re- search activities that pertain to HIV at the National Institutes of Health. This review resulted in a number of recommendations pertinent to NCI. Prominent recommendations were that there should no longer be a focus on the mechanistically unselective cell-based screen for discovery of new AIDS-directed therapeu- tics and that there should be a review of the management struc- ture and the directions that the program would be taking in the Journal of the National Cancer Institute Monographs No. 28, 2000 future. The full text of this review is available at the OAR web site (http://www.nih.gov/od/oat/). After receiving that report, NCI discontinued large-scale screening of synthetic compounds and natural product extracts with the cell-based assay. Information derived from this screen- ing effort has been cataloged and made available on the DTP web site. Data for approximately 32000 compounds are avail- able and may be searched by biologic activity, as well as by chemical class. The DTP AIDS program has recently undergone an addi- tional review that was chaired by Dr. Jack Edwards of the Uni- versity of California at Los Angeles and included experts from academia, industry, and the community with expertise in virol- ogy, chemistry, biology, pharmaceutical development, and clini- cal trials. The recommendations from this group will help shape the form of future NCI efforts in the area of HIV and AIDS- associated malignancies. This additional review follows a com- prehensive review of DTP’s cancer program that was initiated in 1997. The recommendations of that group, which was chaired by Dr. Susan Horwitz of Albert Einstein College of Medicine, have led to wide-ranging changes, including an emphasis on molecu- lar targets for drug discovery and on increased interaction with the extramural drug discovery and development community. The full text of the committee’s report is available on request. CURRENT RESOURCES FOR DRUG DISCOVERY Resources currently available from DTP to support drug dis- covery are summarized in Table |. Grant support is available in the form of traditional investigator-initiated grants as well as special programs, such as the National Cooperative Drug Dis- covery Grants. New initiatives designed to exploit molecular targets will be discussed below. DTP maintains a web site (http://dtp.nci.nih.gov) that provides a wealth of information about NCI programs, chemical compounds that have been ac- quired and screened in anticancer and anti-HIV assays, as well as access to screening results. Tools are available on the web site to facilitate use of the information. Chemical analogue searching is possible, as is use of the COMPARE algorithm (5) for pattern recognition analysis of the anticancer drug screening database. The COMPARE program performs a correlation analysis with the use of patterns of relative in vitro sensitivity of 60 tumor cell lines and has been shown to be useful in helping to define the mechanism of cytotoxicity of compounds tested in the screen. The web site supports the interactive use of this program and also provides information on the chemical and natural product Affiliation of authors: E. A. Sausville, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD; R. H. Shoemaker, Screening Technologies Branch, Developmen- tal Therapeutics Program, National Cancer Institute-Frederick, Frederick, MD. Correspondence to; Robert H. Shoemaker, Ph.D., Screening Technologies Branch, Bldg. 440, National Cancer Institute-Frederick, Frederick, MD 21702- 1201 (e-mail: shoemaker @ dtpax2.nciferf.gov). See “Note” following “References.” 35 ''Table 1. National Cancer Institute’s resources for drug discovery @ Grants program © Web-based informatics (http://dtp.nci.nih.gov) @ Chemical compounds and natural product extracts © Cell-based screening services extract libraries maintained by NCI. More than 100000 crude natural product extracts derived from a wide variety of natural sources, including terrestrial plants, cultured fungi, marine or- ganisms, and other microorganisms, are described. Two- and three-dimensional structures for more than 200000 synthetic compounds are accessible as well as information on how to obtain samples from the repositories. A limited capacity for anti-HIV testing in the cell-based assay has been maintained and is available primarily to support research by NIH grantees. FUTURE DIRECTIONS During the next few years, the focus of screening and dis- covery activities will shift away from primarily intramurally based programs to a focus on extramurally driven activities. NCI will work toward partnering extramural principal investigators with molecular targets and sources of screening expertise with investigators developing chemical libraries. It is hoped that this partnering will occur in concert with new grant programs that DTP expects to be announced and phased in within the next year. These new grant programs will establish centers of excel- lence in chemical diversity as well as centers of excellence in molecular targets. They can begin to define the effects of these molecules on in vitro systems and then utilize NCI resources for the later stage preclinical studies that lead ultimately to clinical trial. NCI anticipates the need to operate selected high-throughput, molecular-targeted screens at the Frederick Cancer Research and Development Center or through other contract mechanisms, ei- ther as service functions to the extramural community or in collaboration with extramural investigators with unique molecu- lar targets. The Collaborative Research and Development Agree- ment (CRADA) provides a formal mechanism for defining a research plan agreeable to both NCI and a university- or indus- try-based investigator. The type of resources that DTP would bring to these areas would include screening expertise, a diverse repository of chemical compounds, natural products repository, expertise in bioassay-directed isolation, and characterization of leads obtained from natural products, as well as medicinal chem- istry, molecular modeling, and bioinformatics resources. The resources the CRADA partner would bring to NCI would be access to state-of-the-art molecular targets and an in-depth knowledge of the biology pertinent to these systems. As described above, the prior focus had been on HIV as a primary screening target in a cell-based assay. In the future, NCI anticipates focusing on selected molecular targets presented by HIV and extending efforts in the area of AIDS-related malig- nancies. These targets, associated with particular diseases, for example, angiogenesis and Kaposi’s sarcoma, could also extend to aspects of immunity implicated in the expression or in the occurrence of malignancies as well as targets intrinsic to viruses associated with particular malignancies. Presentations at this conference have alluded to potential mo- lecular targets related to human herpesvirus 8 (HHV8) and Ka- posi’s sarcoma, Epstein-Barr virus and associated lymphomas, and human papillomavirus as related to the occurrence of cer- vical and anogenital tumors. Because of the prevalence of Kaposi’s sarcoma in AIDS pa- tients, its clear linkage with HHV8, and the availability of com- Potential Interactions Between NCI and Extramural Investigators Combinatorial - Libraries DEY Academia Academia/NCI NCI Industry Industry N a Seine ESS a Conduct of Lead Clinical arge ») Screening HTS Campaigns Optimization Identification Assay Development _ Development Natural Product NCI Extracts Fig. 1. The example of interactions between the National Cancer Institute (NCI) and extramural investigators depicted here is based on the University of Penn- sylvania-NCI Collaborative Research and Development Agreement described in the text. Many alternative scenarios are possible. For example, academic labo- ratories may choose to partner with industry for conduct of high-throughput 56 screening campaigns. In that situation, NCI could play a role in identifying and suggesting sources of chemical diversity for input to the screen. Through the Rapid Access to Intervention Development program, an academic investigator with an advanced project might be able to obtain critical data to support clinical investigation of a new drug. Journal of the National Cancer Institute Monographs No. 28, 2000 ''plete genomic sequence information, DTP has focused on HHV8 for the first initiatives in the area of AIDS-associated malignan- cies. As reported elsewhere at this conference, DTP has devel- oped and begun to characterize a cell-based assay for testing potential anti-HHV8 agents. This assay is intended for use in conjunction with high-throughput, molecular-targeted screens. In collaboration with Dr. Robert Ricciardi of the University of Pennsylvania (Philadelphia, PA), DTP has begun develop- ment of methods for screening for selective inhibitors of the HHV8 DNA polymerase and processivity factor. Dr. Ricciardi’s group has recently cloned and characterized these genes (6), which have no counterpart in uninfected human cells and thus represent a target with potential for development of highly se- lective therapeutic agents. This collaboration has been structured as a CRADA in which NCI will scale-up recombinant protein expression and purification and then screen the natural product and synthetic compound repositories to identify lead structures that affect the polymerase and processivity factor. Then, in col- laboration with Dr. Ricciardi’s group, DTP will characterize the leads biologically in appropriate biochemical and in vivo mod- els. Qualified leads may then be licensed to industry for opti- mization and for development to clinical trials. This collabora- tion illustrates one of several ways in which NCI may interact with extramural investigators who have unique molecular tar- gets, screening technologies, or sources of chemical diversity for screening. This and other potential types of interactions are shown schematically in Fig. 1. In addition to interaction with the extramural community, DTP anticipates continuing the association with the National Institute of Allergy and Infectious Diseases for drug discovery and development initiatives related to topical microbicides, an- timicrobial leads, as well as interactions with intramural NCI laboratories engaged in research directed against relevant mo- lecular targets, such as integrase. NEw INITIATIVES IN DRUG DEVELOPMENT In the area of preclinical drug development, NCI has recently launched a new program to facilitate entry of novel therapies into the clinic. The Rapid Access to Intervention Development Journal of the National Cancer Institute Monographs No. 28, 2000 (RAID) program provides extramural academic investigators ac- cess to the same DTP contract resources used for development of compounds through the traditional NCI Decision Network pro- cess. Among the activities that can be supported by RAID are bulk production of drugs that would be suitable for clinical use, development of clinical formulations, or conduct of pharmacol- ogy and toxicology studies to support investigational new drug filing. For this program, NCI does not hold the investigational new drug. The intent is to remove preclinical barriers to clinical research. Additional details on the RAID program may be found on the DTP web site (http://dtp.nci.nih.gov). REFERENCES (1) Weislow OS, Kiser R, Fine DL, Bader J, Shoemaker RH, Boyd MR. New soluble-formazan assay for HIV-1 cytopathic effects: application to high- flux screening of synthetic and natural products for AIDS-antiviral activity. J Natl Cancer Inst 1989;81:577-86. Boyd MR. Strategies for the identification of new agents for the treat- ment of AIDS: a national program to facilitate the discovery and pre- clinical development of new drug candidates for clinical evaluation. In: DeVita VT, Hellman S, Rosenberg SA, editors. AIDS, etiology, diag- nosis, treatment and prevention. Philadelphia (PA), Lippincott; 1988, p. 305-19. (3) Boyd MR, Gustafson KR, McMahon JB, Shoemaker RH, O’ Keefe BR, Mori T, et al. Discovery of cyanovirin-N, a novel human immunodeficien- cy virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother 1997;41:1521-30. Vince R, Hua M, Brownell J, Daluge S, Lee F, Shannon WM, et al. Potent and selective activity of a new carbocyclic nucleoside (carbovir: NSC 614846) against human immunodeficiency virus in vitro. Biochem Biophys Res Commun 1988;156:1046-53. Paull KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, et al. Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J Natl Cancer Inst 1989;81:1088—92. (6) Lin K, Dai CY, Ricciardi RP. Cloning and functional analysis of Kaposi’s sarcoma-associated herpesvirus DNA polymerase and its processivity fac- tor. J Virol 1998;72:6228-32. (2 SS (4 < (5 SS NOTE Presented at the Third National AIDS Malignancy Conference. 57 ''''U. C. BERKELEY LIBRARIES ARKIN) ~COb9471302 ''