Viruses as potential targets for therapy in HIV-associated malignancies

Hematol Oncol Clin N Am 17 (2003) 697 – 702

Viruses as potential targets for therapy in HIV-associated malignancies Richard F. Ambinder, MD, PhD Hematologic Malignancies Division, Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, 1650 Orleans Street, Bunting Blaustein Building, Room 389, Baltimore, MD 21231, USA

Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) are gamma herpesviruses. Both were discovered first in association with tumors. EBV is associated with a variety of lymphoid malignancies (posttransplant lymphoma, some Hodgkin’s disease, natural killer/T-cell nasal lymphoma, and AIDS lymphomas), smooth muscle malignancies in the immunocompromised, and epithelial malignancies including nasopharyngeal and gastric carcinoma [1,2]. KSHV discovered in Kaposi’s sarcoma also is associated with primary effusion lymphoma and Castleman’s disease [3]. In patients infected with HIV, approximately 40% of non-Hodgkin’s lymphomas (including all primary central nervous system lymphomas) and 90% of Hodgkin’s lymphomas are associated with EBV [4]; all Kaposi’s sarcoma lesions are associated with KSHV; and primary effusion lymphomas are associated with EBV and KSHV [5,6]. The presence of the viruses in tumor cells is evidenced by the presence of viral DNA, RNA, or protein in tumor cells. Although both viruses are associated with a variety of tumors and probably play important roles in pathogenesis, only a tiny percentage of those individuals who are infected develop tumors, even in high-risk populations. The pathogenesis of all of the virus-associated tumors remains poorly understood. Although several genes in both viruses have transforming properties in tissue culture systems, the pathogenesis of clinical disease is complex, as evidenced by the observation that transforming genes (eg, latency membrane protein-1 [LMP1] identified in EBV) are not expressed in most tumor cells in EBV-associated Burkitt’s lymphoma, and transforming genes in KSHV (eg, K1) are not expressed in most tumor cells in Kaposi’s sarcoma. Uncertainties related to pathogenesis not withstanding, the presence of virus in tumor cells offers potentially attractive targets for therapy.

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EBV and KSHV as tumor targets EBV and KSHV may be considered as tumor markers. Although EBV viral DNA is present in approximately 1 in 100,000 lymphocytes, it generally is not detected in other nonmalignant tissues [7]. The one well-documented exception is oral hairy leukoplakia, a disease that is predominantly found in HIV patients in which lytic EBV is found in lingual epithelium [8]. If indicated, oral hairy leukoplakia can be treated with acyclovir. Similarly, KSHV is harbored in rare lymphocytes, but has not been demonstrated convincingly in other nonmalignant tissues. Thus, the specific destruction of cells that harbor EBV and KSHV in patients should be well tolerated and have much less impact on nonmalignant cells than does conventional cytotoxic chemotherapy or an anti-B –cell antibody therapy such as rituximab. The first and still most compelling demonstration that targeting virus can be used to treat or prevent tumors came from an experience with posttransplant lymphoproliferative disease in allogeneic bone marrow transplant recipients. In transplant recipients after T-cell depletion, there was a high rate of EBV lymphoproliferative disease [9]. Infusion of EBV-specific donor T cells led to regression of tumors in some instances and prevented tumor development in a high-risk population. Similar approaches have not been applied yet to KSHV-associated malignancies, but the observation that in organ transplant recipients with Kaposi’s sarcoma lesions often will regress with reduction or withdrawal of immunosuppression suggests that there is a similar sensitivity to immune manipulations [10]. Therapeutic targeting of viruses might be achieved by adoptive or pharmacologic therapies. In the following sections, each of these approaches is discussed briefly, with emphasis placed on how these therapies might relate to HIV-associated malignancies.

Immunotherapy Several problems arise when considering the application of immunotherapy to HIV patients, one of which is the source of the T cells. Outside of the bone marrow transplant setting—in which successes have been achieved with cells expanded from the bone marrow donor—there are two sources of T cells to be considered: the patient’s own or partially matched T cells. Autologous T cells can be expanded, but present techniques typically require weeks [11]. That timeframe might be adequate for slow-growing Kaposi’s sarcoma, but is unlikely to be acceptable for a rapidly growing lymphoma. In addition, expansion of autologous T cells from patients with HIV may be technically more challenging than is expansion of T cells from other donors. Partially mismatched cells from healthy donors have been expanded and well tolerated in organ transplant recipients [12]. Banks of cells from healthy donors have been expanded and frozen, and were available almost immediately when partial matching at human leukocyte antigen loci was acceptable. T cells from healthy donor banks lyse target cells in vitro [13].

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Their longevity and efficacy in patients, however, remains to be determined. Other approaches that are in the early stages of development include genetic modification of T cells to express T-cell receptors for appropriate target antigens, in order to obviate the need for prolonged growth of lines or clones of T cells in culture [13]. A second concern with regard to the use of immunotherapy for tumors is the sensitivity of particular tumor types to immune manipulations. An example of the problem of sensitivity to immune manipulations is found in Burkitt’s lymphoma, which is inherently resistant to killing by CD8(+) cytotoxic T cells. Indeed class I molecules often are lacking from Burkitt’s lymphoma as antigen processing machinery and adhesion molecules that make cells susceptible to T cell killing. The sensitivity of Kaposi’s sarcoma to CD8(+) T cells has not been investigated, in part because of a lack of suitable cell lines to study. Primary effusion lymphomas, however, show impaired antigen presentation that reflects downregulation of class I MHC molecules and TAP1 [14,15]. In contrast, EBV-associated Hodgkin’s disease and immunoblastic lymphoma would appear to be ideal targets for CD8(+)-based immune strategies. A third concern is the spectrum of viral antigen expression in tumors. Often immundominant epitopes are not expressed in tumors. Burkitt’s lymphoma is again an extreme example of immune resistance in this regard, expressing only a single viral antigen (EB nuclear antigen 1). That antigen inhibits its own antigen presentation in the MHC class I pathway by virtue of a repeated GLY-GLY-ALA sequence in the protein [16]. In many other EBV-associated lymphoid malignancies, there is restricted expression of latency antigens. The now-established techniques for growing EBV-specific T cells for adoptive therapy of EBV-associated posttransplant lymphoproliferative disease use EBV-immortalized B cells as stimulators [17,18]. This technique mainly yields cells that target the immunodominant nuclear antigens. Most of the T cells in these T-cell preparations will not target tumor. Subpopulations of cells in these T-cell expansions may target tumor antigens such as LMP2 (which are expressed in most of these types of tumors), and it is possible that adoptive immunotherapy using such preparations may be useful [19]. Technical improvements in T-cell expansion are on the horizon. Massive expansion of T-cell populations in vitro now can be accomplished [20]. Even if only a small fraction of T cells in the expanded cell line targets antigens expressed in tumors, their absolute numbers may be adequate for effective therapy. A second possibility is to specifically expand the T cells that target particular antigens. LMP1 and LMP2 are attractive target EBV antigens and Kaposin is an attractive KSHV target antigen. These proteins are expressed in latency in most of the relevant virusassociated malignancies and have CD8(+) T-cell epitopes defined [14,21,22]. Among the many sources of antigen that might be employed are synthetic peptide, recombinant adenovirus, vaccinia, or recombinant lentivirus. Alternatively, T cells might be modified genetically with a cloned specific T-cell receptor [23]. Even if T cells with appropriate specificity can be expanded from patients or methods to achieve at least short-term viability of infused T cells from allogeneic donors are devised, the process of ex vivo T-cell expansion will remain expensive

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and its applicability will be limited. The same targets for adoptive immunotherapy discussed above also might be targets for therapeutic or prophylactic vaccines. Recently, interesting results have been reported with a peptide vaccine in nasopharyngeal carcinoma—an EBV-associated tumor [24]. Although HIV patients often have diminished responses to vaccination, they do respond to vaccines [25]. Hodgkin’s disease may be an especially attractive place to use a vaccine because HIV patients with Hodgkin’s disease have relatively wellpreserved CD4(+) T-cell counts.

Pharmacologic therapy There are many potential pharmacologic targets associated with the presence of EBV or KSHV in tumor cells. Induction of viral gene expression—particularly lytic gene expression—is one approach. ‘‘Curing’’ tumor cells of their viral DNA episomes is a second approach. With regard to induction of viral gene expression, the great majority of viral proteins are not expressed in tumor cells. Most are ‘‘lytic cycle’’ proteins; that is, they are expressed when new virus is being packaged into virions. Some of these proteins are transcriptional regulators, others are important in nucleic acid metabolism, and still others make up the structure of the viral capsid itself. Induction of such proteins may have several potential therapeutic consequences.  

Lytic induction may directly kill cells. Lytic induction with expression of immunodominant viral antigens may make cells susceptible to powerful immune responses that generally are not active against tumors.  Lytic induction with expression of viral kinases including viral thymidine kinase and phosphotransferase (also known as protein kinase)—both of which phosphorylate ganciclovir and other nucleoside analogs—might make cells susceptible to killing with ganciclovir or similar analogs [26,27]. Is it possible that lytic induction is bad for a patient? The answer to this question is not known, but I would argue probably not. All of the most serious diseases that are associated with EBV or KSHV are associated with latent infection. The only disease that is associated with pure lytic infection is oral hairy leukoplakia, which is more an annoyance than a health threat. The most important barrier to exploring these therapeutic approaches involving lytic gene expression is the difficulty in inducing lytic infection. Agents such as butyrate, valproic acid, phorbol esters, and DNA methyltransferase inhibitors all are inducers in some settings in vitro. Several of these agents are being evaluated further in ongoing clinical trials. The persistence of EBV and KSHV as episomes offers the rationale for a different strategy altogether. Whereas many tumor viruses, such as papillomavirus or HTLV1, are found integrated into the DNA of tumor cells, EBV and KSHV

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generally persist as closed circular DNA molecules or episomes tethered to chromosomes by viral proteins. In the absence of those tethers, the viral genomes cannot persist. Experiments with Burkitt’s cell lines suggest that a loss of episomes is associated with a loss of the malignant phenotype [28]. The viral tethers potentially are targets for a small molecule therapeutic approach. Small molecules might disrupt DNA binding, protein oligomerization, or chromatin binding with loss of viral DNA from tumor cells and loss of the malignant phenotype. The existence of well-defined molecular targets should facilitate screening or rational drug design.

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