The challenge to eradicate the latent HIV-1 reservoir, a prerequisite for a curative therapy, is probably best compared to leukemia treatment. A subset of cells needs to be systemically and completely, but selectively, eradicated. A single cell that escapes therapy will likely result in the reoccurrence of the tumor. Combination therapies where several drugs that target different mechanisms are combined to maximize the therapeutic effect of therapy have proven to be the most successful way forward to treat various forms of leukemia.
Previous unsuccessful HIV-1 eradication attempts used single drugs. On the basis of the success of combination therapy in the treatment of several forms of leukemia, drug combinations that target latent HIV-1 infection at several levels of molecular control will likely also be the most promising way forward to HIV-1 eradication. One of the major hurdles to overcome is that other than in the case of leukemia cells, latently HIV-1-infected T cells are phenotypically not distinguishable from uninfected CD4+ memory T cells that serve as the cellular main reservoir of latent HIV-1 infection. Latently HIV-1-infected cells can thus not be specifically targeted. Thus, a therapy form based on a systemic stimulus that ideally is selective for T cells and most importantly will trigger HIV-1 reactivation without causing detrimental side effects such as a cytokine storm or immune hyperactivation will have to be developed. Dissociation of HIV-1 reactivation from high-level cell activation will likely be essential. We hypothesized that this may be achievable if we can identify combinations of drugs that first lower the activation threshold for latent HIV-1 infection and then trigger HIV-1 reactivation with a low-level activating stimulus.
In a drug screen designed to directly identify drug combinations that would reactivate latent HIV-1 infection, we initially identified two FDA-approved drugs, aclacinomycin and dactinomycin, as compounds that lower the activation threshold required to achieve full reactivation at the population level and that directly trigger HIV-1 reactivation in primary T cells. Our studies revealed that the drugs do not act by their primary mechanism as topoisomerase inhibitors or as DNA intercalators but rather target latent HIV-1 infection by their ability to trigger cell differentiation at subtherapeutic concentrations. To this end, we also demonstrate that cytarabine, a third FDA-approved anticancer drug with cell-differentiating capacity, and aphidicolin prime latent infection for reactivation. Our findings suggest that repositioning of a subgroup of FDA-approved anticancer drugs that exert cell-differentiating effects could be a promising way forward to a novel therapeutic approach to eradicate latent HIV-1 infection.
A major consideration for the development of a curative therapy is the current success of antiretroviral therapy (ART). For most patients who have access to care, ART provides the ability to treat HIV-1 infection like a chronic disease. Based on the data available at this time, ART may provide patients with the possibility to live a relatively normal life with a close-to-normal life expectancy. As beneficial as a curative treatment for HIV-1 would be for many reasons, other than for the treatment of leukemia, given the success of ART, the acceptable side effects need to be minor and should be limited during the treatment.
In the light of these requirements, it needs to be emphasized that aclacinomycin, dactinomycin, and cytarabine in vitro exert their priming or reactivating effects on latent HIV-1 infection at concentrations that are not cytotoxic to T cell lines or primary T cells. This raises the possibility of using these drugs at subtherapeutic doses relative to cancer therapy.
Other than being used at lower concentrations, the three drugs are clinically extremely well defined. Aclarubicin was first described in 1975 as a product of Streptomyces galilaeus
). Relative to other anthracycline antibiotics, such as daunorubicin or doxorubicin, aclacinomycin has been described to have reduced cardiotoxicity (49
). While its primary anticancer activity is linked to its ability to act as a DNA intercalator, the mechanisms by which it induces cell differentiation are ill defined. It has been suggested that the differentiating ability of aclacinomycin would be linked to its ability to block the synthesis of asparagine-linked glycoproteins, which is not affected by anthracyclines that have monosaccharide side chains, but overall, it remains unclear how aclacinomycin treatment results in cell differentiation.
The primary dactinomycin effect during cancer treatment is also based on its ability to intercalate into DNA and to inhibit RNA polymerase (45
). Dactinomycin is being used in the treatment of childhood leukemia. How it actually triggers cell differentiation remains largely unclear.
Cytarabine is classified as an antimetabolite and seems to primarily act by inhibiting DNA polymerase. Incorporation of the cytosine analog into the nascent DNA and nascent RNA has been reported. Cytarabine used as part of an HIV-1 eradication treatment may be particularly interesting. In a study comparing low-dose cytarabine with other treatment regimens for acute myeloid leukemia and high-risk myelodysplastic syndrome in patients not considered fit for intensive treatment, cytarabine proved to be most efficient (15
). This indicates that cytarabine can exert effects at low doses and is tolerated well at low doses. Cytarabine was actually used in the induction therapy for the “Berlin patient,” an HIV-1-infected individual who was cured of HIV-1 infection in the course of a bone marrow transplant required as the result of reoccurring leukemia (3
A fourth cell-differentiating agent, which is the first to be reported to trigger HIV-1 expression, is N
). More recently, Contreras et al. demonstrated that this effect is linked to the ability of HMBA to release P-TEFb from its inactive complex with HEXIM-1 (19
). However, transfer of HMBA as an HIV-1-reactivating agent into the clinical situation is unlikely to be successful. HMBA caused severe thrombocytopenia, which limits the amount of drug that can be administered. Furthermore, continuous drug exposure is required to cause the cell-differentiating effect. As the biological half-life of HMBA in the patient is very short (about 1.5 h), HMBA must be administered by continuous infusion to maintain a clinical effect (4
We demonstrated that just like HMBA, dactinomycin and aclacinomycin release P-TEFb from its inactive complex with HEXIM-1. Given previous findings that P-TEFb restriction is important for the transition into latency (73
) and given the overall importance of P-TEFb for HIV-1 transcriptional elongation (47
), our results suggest that induced P-TEFb release is an essential part in the effect of these drugs to lower the reactivation threshold. However, it is unlikely that P-TEFb release is the only target of these drugs in the context of HIV-1 reactivation. Rather, it is likely that the drugs alter the cellular transcription factor profile and provide a more permissive cellular environment for viral transcription, also at other levels. Unfortunately, to date the molecular mechanisms underlying the cell-differentiating effects of the various drugs/compounds are ill defined. There are, however, some candidate genes that are reported to be regulated by these cell-differentiating drugs, which also have been implied in the regulation of HIV-1 expression. Aclacinomycin has been reported to induce GATA expression (26
), and GATA has been described to induce LTR activity (71
). Aclacinomycin also was noted to trigger a rapid but transient decrease in the levels of c-myc
downregulation has also been reported for dactinomycin (12
) and for cytarabine. For cytarabine, the first peak of c-myc
downregulation in K562 human erythroleukemia cells was correlated with the onset of cell differentiation (8
). In the context of latent HIV-1 infection, this is interesting, as valproic acid, an HDAC inhibitor reported to trigger HIV-1 reactivation (85
), has also been reported to downregulate c-Myc. Inhibition of c-Myc was shown to reduce HDAC1 occupancy of the HIV-1 LTR, to relieve c-Myc-imposed repression of Tat activation, and to increase LTR expression (31
). Interestingly, valproic acid is not only an HDAC inhibitor but also a cell-differentiating agent (27
). We are currently investigating whether there is a correlation between the ability of cell-differentiating drugs to trigger HIV-1 reactivation and their ability to downregulate c-myc
In the context that HMBA, dactinomycin, and aclacinomycin have all been reported to act as cell-differentiating agents, it is further interesting that the HDAC inhibitor SAHA (vorinostat), reported to reactivate latent HIV-1 infection (7
), was initially also developed as a very potent cell-differentiating polar agent, a second-generation HMBA (60
). Its ability to act as a potent HDAC inhibitor was described only later (59
). To generate SAHA, the structures of HMBA and the HDAC inhibitor trichostatin A (TSA) were used as the templates (60
). However, while SAHA acts to directly trigger reactivation of latent HIV-1 infection and primes latent HIV-1 infection for reactivation, the HDAC inhibitor TSA exhibits no HIV-1-reactivating capacity in our experimental systems. This raises the question whether the HIV-1-reactivating capacity of SAHA is actually a function of its ability to drive cell differentiation or of its ability to inhibit HDACs.
Of note, while many dispute the value of T cell lines for HIV-1 latency research, a previous study by the Karn laboratory could identify only a single difference. Latent infection in T cell lines, other than in primary T cells, could be reactivated by TNF-α. No other differences at the molecular level were revealed (54
). Planelles, Bosque, and their collaborators have recently demonstrated that latently HIV-1-infected primary T cells can even be induced to proliferate by IL-7 without triggering comprehensive HIV-1 reactivation, voiding the argument that proliferating T cell lines must control latent HIV-1 infection by a fundamentally different mechanism than that of resting memory T cells (13
). We recently reported that AS601245 would prevent stimulation-induced HIV-1 reactivation despite the induction of high levels of NF-κB activity in either cell type, T cell lines and primary T cells, another example that latently infected T cell lines in many ways are a good experimental substitute for primary T cells (80
). Our finding that the priming capacity of aclacinomycin or dactinomycin acting on latent HIV-1 infection that we identified using latently HIV-1-infected T cell lines can also be observed in latently infected primary T cells adds further support to the notion that the more recently developed models of latent HIV-1 infection in T cell lines (23
) are representative of the molecular mechanisms that control latent infection in primary T cells. Combining research in the two systems is likely the most promising way forward to understanding latent HIV-1 infection.
From a drug-screening point of view, the identification of these drugs during a screening effort is somewhat remarkable. Drugs such as cytarabine that act only to reactivate latent HIV-1 infection in conjunction with a low-level activating stimulus validate the approach of directly screening for drug combinations rather than screening for single magic-bullet compounds. Obviously, it could be argued that drug screening should exclusively be performed using in vitro latently HIV-1-infected primary T cells. This is likely possible for drug-repositioning efforts with very limited amounts of compounds to be tested. However, screening larger compound libraries that are comprised of >1 × 105 compounds is unlikely to become a reality due to the scarcity of the cell material that can be reliably generated.
Screening other anticancer drug/compound collections, in particular nucleoside analog libraries, now seems a promising approach to identify more priming agents for the treatment of latent HIV-1 infection. It is conceivable that some of these compounds lack potent anticancer effects but remain potent cell-differentiating agents. Screens for low-level activating agents that act in conjunction with identified priming agents would be the logical next step forward. Our data provide concept validation that this approach could be a promising move toward the development of a curative therapy for HIV-1 infection.