Viral lymphomas: can antivirals be used to treat cancer?

Abstract

Current knowledge suggests that EBV, KSHV and HTLV-1 contribute to lymphomagenesis by subverting the host-cell molecular signaling machinery to deregulate cell growth and survival. Some signaling pathways that are affected by these viruses are well characterized, such as the NF-kB pathway, which is activated by these three viruses to promote cellular survival by inhibiting apoptosis, thereby playing a critical role in tumorigenesis. Other pathways, such as MTOR and JAK-STAT are also likely involved in viral lymphomagenesis. This provides the opportunity to inhibit these cellular pathways using drugs developed for the treatment of other malignancies. However, since these compounds target cellular proteins, they always have the potential for toxicity. In the context of viral malignancies, we have the unique opportunity of targeting viral proteins, and developing completely specific therapies. Here we will examine the question of whether the pathobiology of EBV, KSHV and HTLV-1 will allow the use of such an approach.

Keywords:

• Lymphoma
• AIDS
• HIV
• virus
• EBV
• KSHV
• HHV-8
• HTLV-1

Epstein–Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), also called human herpesvirus 8, and human T-cell lymphotropic virus (HTLV-1) are viruses that are a well-documented cause of lymphoid malignancies in humans, and their genomes are found in the tumor cells. Other viral and bacterial infections are recognized to participate in the process of lymphomagenesis, but their role appears to be indirect. These include hepatitis C virus and helicobacter pilori.

EBV causes non-Hodgkin and Hodgkin lymphomas, as well as lymphoproliferative disorders, which are more common but not exclusive in individuals with immunodeficiency. KSHV causes primary effusion lymphomas and lymphomas arising from multicentric Castleman’s disease. HTLV-1 is the causative agent of adult T-cell leukemia/lymphoma (ATLL).

Current knowledge suggests that EBV, KSHV, and HTLV-1 contribute to lymphomagenesis by subverting the host-cell molecular signaling machinery to deregulate cell growth and survival. Some signaling pathways that are affected by these viruses are well characterized, such as the NF-kB pathway, which is activated by these three viruses to promote cellular survival by inhibiting apoptosis, thereby playing a critical role in tumorigenesis. Other pathways, such as mTOR and JAK-STAT are also likely involved in viral lymphomagenesis. This provides the opportunity to inhibit these cellular pathways using drugs developed for the treatment of other malignancies. However, since these compounds target cellular proteins, they always have the potential for toxicity. In the context of viral malignancies, we have the unique opportunity of targeting viral proteins, and developing completely specific therapies. Here we will examine the question of whether the pathobiology of EBV, KSHV, and HTLV-1 will allow the use of such an approach.

EBV and KSHV are human herpesviruses, while HTLV-1 is a retrovirus. Antivirals exist that inhibit both types of viruses. However, they are not useful for the treatment of lymphomas. This can be explained by the following observations:

1. All herpesviruses, including EBV and KSHV, have two main stages: latent and lytic. During latency, only a small number of viral RNAs and proteins are made. In general, these allow the virus to persist in the infected cells. Some latent herpesviral proteins also induce cellular proliferation and protect the cell from interferon and apoptotic signals, which is a mechanism of viral persistence. The lytic phase occurs during acute infection or upon reactivation. During this phase, a large number of viral gene products are made which are involved in viral DNA replication and production of infectious viral particles. During the lytic phase, the infected cell usually dies, so in general, only the latent phase is compatible with a malignancy. In some instances, the lytically infected cells may have paracrine effects on adjacent latent and uninfected cells, such as inflammation and angiogenesis as seen in Kaposi’s sarcoma. However, in viral lymphomas, latent infection is thought to be the main oncogenic driving force.

2. Antiherpesviral drugs include those such as acyclovir and gancyclovir, which are guanosine analogues that have to be phosphorylated by viral thymidine kinases to be activated. However, the viral thymidine kinases are only expressed during lytic infection. Thus, these compounds do not have any effect on latently infected tumor cells. Attempts have been made to use these antiviral compounds in conjunction with drugs that can induce lytic replication through epigenetic modifications with valproic acid or SAHA (Vorinostat), both histone deacetylase inhibitors.1,2 Azydothymidine (AZT), bortezomib, gemcitabine, and doxorubicin can also induce lytic replication and expression of viral thymidine kinases, so their use in combination with other treatment modalities has also been proposed as a form of oncolytic therapy. So far, these approaches have shown results in experimental systems, but results from clinical trials have not been published.3^–6

3. In the case of HTLV-1, the virus is integrated into the lymphoma cells and viral replication is not part of the oncogenic process. Thus, antiretrovirals, such as those effective for HIV, are not useful for the treatment of ATLL.

So if current antivirals cannot effectively cure viral lymphomas, can we develop new ones that might? The answer in probably yes, but perhaps only some subtypes of viral lymphomas would be amenable to this approach, and most likely these treatments will be best used in combination with other targeted or cytotoxic agents. The following paragraphs outline our current understanding of which viral proteins are expressed in specific viral lymphomas, and whether they would be promising therapeutic targets.

EBV

EBV establishes a lifelong infection in the vast majority of immunocompetent individuals without causing any disease. Recent studies based on careful analysis of expression patterns in different tissues from EBV-infected immunocompetent individuals led to the description of different transcription programs that are used to establish and maintain EBV infection. Some of the same transcriptional programs are recapitulated in lymphomas and lymphoproliferative disease. When EBV first infects a naive B cell, a transient ‘growth program’ is established, where EBV expresses EBNA 1-6, as well as LMP1, LMP2A, and LMP2B. These proteins force the infected cells to become proliferating B cell blasts, probably allowing EBV infection to be propagated. In vitro generated lymphoblastoid cells maintain this growth program indefinitely. However, since many of these proteins are antigenic, this state is very transient in immunocompetent individuals. As soon as an immune response is established, most of the cells with this program are eliminated, or otherwise switch to a ‘default program’ of EBV expression, where only EBNA1, LMP1, and LMP2A are expressed. This stage is also temporary because LMP1 and LMP2A mimick CD40 and antigen receptor signaling, thereby inducing the B cells in peripheral lymphoid organs to behave like germinal center B cells and differentiate into resting memory B cells. These infected cells in turn switch to a ‘latency program’ where no viral genes are expressed, allowing lifetime persistence of EBV. Dividing peripheral blood-infected memory B cells expresses only EBNA1, which is not immunogenic but allows the EBV genome which is episomal to segregate and be propagated in dividing cells.

Three main patterns of EBV expression have been described in infected cells in lymphoproliferative disorders, as Latency I, II, or III.7 Latency III corresponds to the ‘growth program’ and involves the unrestricted expression of all nine latent genes including six EBV-encoded nuclear antigens (EBNA 1–6) and three latent membrane proteins (LMP1, LMP2A, and LMP2B). In Latency I, EBNA1 is the major viral protein produced. Latency II corresponds to the ‘default program’, and consists of expression of EBNA1 and varying amounts of the three LMP proteins. The prototypic malignancy with Latency I is Burkitt lymphoma, while Latency II is seen in Hodgkin lymphoma and Latency III in AIDS-related diffuse large B cell lymphoma (immunoblastic subtype) and post-transplantation lymphoproliferative disorders. A number of microRNAs are also expressed in EBV during latency, which play very diverse roles in modulating many cellular functions.

In terms of how we may use this knowledge to develop new antivirals, it is evident that by inhibiting EBNA1, we may be able to diminish the EBV effects in the broadest range of viral lymphomas. Since this protein is also involved in episome maintenance, we could conceivably cure the lymphoma cells of viral genomes.8 However, dependency of infected tumor cells on EBNA1 is not very clear. When RNA interference was tested in a Burkitt lymphoma cell line, decrease proliferation was found.9 Recently, some Burkitt lymphomas were found to express a viral protein called BHRF1, which is a viral homolog of BCL2, an important antiapoptotic protein.10 It is possible that in this subset, inhibition of BHRF1 would be useful for treatment. Other viral gene products like LMP1 and LMP2 seem to be necessary for tumor cell survival by inducing expression of important signaling pathways.11 Therefore, inhibition of these proteins could be useful to treat the subset of lymphomas with type II and III latency where these viral proteins are expressed. These include Hodgkin’s lymphoma, post-transplant lymphoproliferative disorders, and a subset of AIDS-related lymphomas.

KSHV

Six major KSHV proteins have been confirmed to be produced in latently infected lymphoma cells. They are called LANA, vCyclin (vCYC), vFLIP, vIL-6, vIRF-3, and KaposinB. Many of these genes bear potential to participate in lymphomagenesis and the maintenance of the malignant phenotype by affecting cellular survival, and/or proliferation.

One may consider targeting the LANA protein, which like EBV EBNA1, tethers the viral episomes to the cellular chromosomes during cell division. This protein also binds and may inactivate several tumor suppressor genes. However, depletion of LANA has not been found to result in impaired viability or proliferation of PEL cells. In contrast, the viral gene vFLIP can activate signaling, and inhibiting expression of this viral protein can drastically induce PEL cell apoptosis.12 Therefore, vFLIP may be a good therapeutic target.

HTLV-1

HTLV-1 seems to play a role in the early steps of ATLL malignant progression and not necessarily in the maintenance of the transformed phenotype, because ATLL tumor cells lack expression of HTLV-1 genes and carry viral genomes that tend to be heavily deleted (reviewed in Ref. 13). ATLL tumor cells do have oncogenic alterations and chromosomal aberrations, the complexity of which correlates with tumor aggressiveness.

Therefore, oncogenesis of ATLL seems to involve an HTLV-1-dependent step in which transformation of T cells results in a polyclonal population of proliferating immortalized T cells, with subsequent acquisition of new oncogenic genetic alterations, clonal expansion, and progression to full malignancy and HTLV-1 independence. In contrast to other animal retroviruses that transform cells by transduction of oncogenes or activation of oncogenes adjacent to insertion sites, HTLV-1 transforms T cells by a rather complex mechanism. Two viral proteins are important for tumorigenesis. The first one is the transactivator protein Tax, which has been considered to be the major viral oncogene for some time. However, the fact that Tax transcripts are only detected in approximately 40% of ATLL, and it is frequently actually deleted from the HTLV-1 genome in this disease. More recently, a novel antisense transcript termed the HTLV-1 bZIP factor (Hbz) was discovered.14 Hbz is ubiquitously expressed in ATLL, and has also been found to affect proliferation. Thus, Hbz is a more likely therapeutic target than Tax.

In summary, EBV and KSHV are both lymphotropic herpesviruses, and as such, they are capable of infecting lymphocytes, and causing lymphoproliferative disorders. These are more frequently of B cell origin reflecting the natural viral tropism. HTLV-1 is a T-cell lymphotropic virus that causes T-cell malignancies. While evolution has led to a balance where many of us are infected with these viruses without any consequence, problems arise when this balance is broken. The mechanisms that EBV and KSHV have acquired to ensure the survival of the cell they call home, can also lead to the uncontrolled proliferation of this cells, leading to the development of lymphoproliferations that can range from controllable disease to a highly aggressive malignancy. Understanding the mechanisms involved in this fine balance, and developing tools to manipulate it, have already resulted in improved treatment of patients with EBV-associated malignancies. For example, adoptive immunotherapy is already in use for the treatment of patients with PTLD, where EBV-specific cytotoxic T cells are infused.15 Understanding the mechanisms whereby viral gene products affect tumor cell survival and proliferation is shedding light on important cellular proteins and pathways that could be targeted, such as NF-kB, mTOR, and notch, potentially leading to improved treatment. In addition, this understanding provides a unique opportunity to develop new therapies that directly target the virus. High throughput screening methodologies have begun to lead to the discovery of inhibitors of potential viral oncoproteins, such as for EBNA1.16 The approach of specifically targeting EBV, KSHV, or HTLV-1 hopefully eliminating all cellular toxicities, is likely to be at least part of the therapy in the future.