Small molecule ONC201 inhibits HIV-1 replication in macrophages via FOXO3a and TRAIL

Abstract

Despite the success of antiretroviral therapy (ART), eradication of HIV-1 from brain reservoirs remains elusive. HIV-1 brain reservoirs include perivascular macrophages that are behind the blood-brain barrier and difficult to access by ART. Macrophages express transcription factor FOXO3a and the TNF superfamily cytokine TRAIL, which are known to target HIV-1-infected macrophages for viral inhibition. ONC201 is a novel and potent FOXO3a activator capable of inducing TRAIL. It can cross the blood-brain barrier, and has shown antitumor effects in clinical trials. We hypothesized that activation of FOXO3a/TRAIL by ONC201 will inhibit HIV-1 replication in macrophages. Using primary human monocyte-derived macrophages, we demonstrated that ONC201 dose-dependently decreased replication levels of both HIV-1 laboratory strain and primary strains as determined by HIV-1 reverse transcriptase activity assay. Consistent with data on HIV-1 replication, ONC201 also reduced intracellular and extracellular p24, viral RNA, and integrated HIV-1 DNA in infected macrophages. Blocking TRAIL or knockdown of FOXO3a with siRNA reversed ONC201-mediated HIV-1 suppression, suggesting that ONC201 inhibits HIV-1 through FOXO3a and TRAIL. The anti-HIV-1 effect of ONC201 was further validated in vivo in NOD/scid-IL-2Rgcnull mice. After intracranial injection of HIV-1-infected macrophages into the basal ganglia, we treated the mice daily with ONC201 through intraperitoneal injection for six days. ONC201 significantly decreased p24 levels in both the macrophages and the brain tissues, suggesting that ONC201 suppresses HIV-1 in vivo. Therefore, ONC201 can be a promising drug candidate to combat persistent HIV-1 infection in the brain.

Keywords: FOXO3a; HIV-1; Macrophages; ONC201; Reservoir; TRAIL.

Figure 1.. ONC201 has an antiviral effect on macrophages.

A) Human MDM were plated in 96-well plates and infected with HIV-1 for 24 hours before incubation with doses of ONC201 or its isomer ranging from 0.03 to 30 μM. Uninfected MDM were treated with the same ONC201 and isomer dosages in parallel for comparison. At 5-day post ONC201 and isomer treatments, cell viability was determined by the MTS assays. Data were analyzed by two-way ANOVA, and results shown are the means ± SD of 3 experiments. B) At the same experimental end point as the MTS assays, supernatants were removed and the replication levels of HIV-1 were monitored by RTase activity assay. Results shown are from representative experiments performed with three different donors. Data were analyzed by two-way ANOVA: ** denotes p < 0.01, *** denotes p < 0.001, compared to the HIV-1+ONC201 isomer group of the same treatment dosage.

Figure 2.. ONC201 has an antiviral effect against different primary HIV strains.

A-C) Human MDM were plated in 96-well plates and infected with different primary HIV strains, including 2873MVC (HIV-1 Clade C strain, A), G0048CPX (HIV Clade B strain, B) and 2562BG (HIV Clade B strain, C) for 24 hours before incubation with doses of ONC201 or its isomer ranging from 3 to 30 μM. At 2-, 6-, 10-, and 14-day post ONC201 and isomer treatments, supernatants were collected and the replication levels of HIV-1 were monitored by RTase activity assay. Values represent SEM of biological replicates. Data were analyzed by two-way ANOVA: ** denotes p < 0.01, *** denotes p < 0.001, compared to the HIV-1+ONC201 isomer group of the same treatment dosage.

Figure 3.. ONC201 inhibits HIV-1 p24, gag RNA, DNA, and integrated LTR DNA in macrophages.

Human MDM were plated in 24-well plates in triplicate and then infected with HIV-1_ADA for 24 hours before incubation with ONC201 or ONC201 isomer at 30 μM for 5 days. A, B) Cell lysates were collected and subjected to SDS-PAGE and immunoblotting for HIV-1 p24. Actin was used as the loading control. Densitometric quantifications of p24 in MDM were presented as a ratio to actin and normalized as fold changes to the vehicle control DMSO group. C) Supernatants were collected and subjected to ELISA for HIV-1 p24. Values represent means ± SEM of three biological replicates. D-F) RNA (D) and DNA (E, F) were isolated from the samples. HIV-1 gag was detected through quantitative real time RT-PCR and real time PCR, respectively. Relative HIV-1 gag and LTR DNA levels were determined and standardized with 18S rRNA or GAPDH internal control. Values represent means ± SEM of three biological replicates. ANOVA analysis: * denotes p < 0.05 compared to the vehicle control DMSO group.

Figure 4.. ONC201 reduces HIV-1 infection levels in NSG mouse brains engrafted with infected human macrophages.

HIV-1-infected human macrophages were intracranially injected into the basal ganglia of NOD/scid-IL-2Rgcnull (NSG) mice. Mice were administrated daily with ONC201 (50 mg/kg) or solvent control DMSO through intraperitoneal injections. A) Timeline of experimental procedures and sample collection in the HIV-1-infected NSG mouse model. B-J) Immunostaining of p24 and CD68 in the injection sites of mouse brains: co-immunostaining of p24 and DAPI (B-G); co-immunostaining of CD68 and DAPI (H-I); quantification of the p24/CD68 ratio per area (J). K-M) Brain tissues that contained the injection sites were homogenized for detection of HIV-1 p24 and human CD68 in Western blots. Data were evaluated statistically by an unpaired Student’s t-test, n = 6 per treatment group.

Figure 5.. ONC201 activates FOXO3a and induces TRAIL expression in HIV-1 infected macrophages.

Human MDM were plated in 24-well plates and then infected with HIV-1_ADA for 24 hours before incubation with ONC201 or ONC201 isomer at 3, 10, or 30 μM for five days. A) Cell lysates were collected and subsequently subjected to SDS-PAGE and immunoblotting for the detection of phospho-FOXO3a, total FOXO3a, and p24. Actin was used as the loading control. Densitometric quantifications of phospho-FOXO3a in MDM were presented as a ratio to total FOXO3a and normalized as fold changes to the vehicle control DMSO group. B) Soluble TRAIL concentrations after ONC201 or ONC201 isomer treatment were determined from cell culture supernatants using ELISA. Data were normalized with total cellular protein concentrations. Results shown are from representative experiments performed with three different donors. ANOVA analysis: * denotes p <0.05, ** denotes p < 0.01, compared to the vehicle control DMSO group.

Figure 6.. Antiviral effect of ONC201 is dependent upon FOXO3a in HIV-1-infected macrophages.

Human MDM were plated in 24-well plates and then infected with HIV-1_ADA for 24 hours before incubation with ONC201 or ONC201 isomer at 30 μM for five days. At two days after HIV-1 infection, MDM were transfected with either non-targeting control siRNA or FOXO3a siRNA. A-B) At the experimental end point, FOXO3a and TRAIL mRNA were detected in total cellular RNA through real time RT-PCR. Data were normalized to 18S rRNA and presented as fold change compared to control siRNA group with isomer treatment. C) HIV-1 infection levels were determined by the RTase activity assay. Values represent means ± SEM of three biological replicates. ANOVA analysis: *** denotes p < 0.001, compared to the control siRNA with isomer treatment group; # denotes p < 0.05, ### denotes p < 0.001, compared to the control siRNA with ONC201 treatment group.

Figure 7.. Antiviral effect of ONC201 in HIV-1 infected macrophages is dependent on TRAIL expression.

HIV-1-infected MDM were incubated with ONC201 or ONC201 isomer at 30 μM, along with 100ng/ml, 50ng/ml soluble TRAIL receptor, or 100ng/ml soluble TNF receptor as a control for 5 days. A) HIV-1 infection levels were determined by RTase activity assay. B) Cell lysates were subjected to SDS-PAGE and immunoblotting for HIV-1 p24. Actin was used as the loading control. Densitometric quantifications of p24 in MDM were presented as a ratio to actin and normalized as fold changes to the control group. ANOVA analysis: *** denotes p < 0.001, compared to the control group with isomer treatment; # denotes p < 0.05, ## denotes p < 0.01, compared to the control group with ONC201 treatment.

Figure 8.. ONC201 pretreatment increases its efficacies against HIV-1 infection in macrophages.

Human MDM were plated in 96-well plates and incubated with doses of ONC201 or its isomer ranging from 3 to 30 μM for 5 days before infection with HIV-1. ONC201 and isomer treatments continued during and after the infection. A) At three-day post infection, cell viability was determined by the MTS assays. B) Cell lysates were collected and subsequently subjected to SDS-PAGE and immunoblotting for the detection of cleaved caspase-3 and pro-caspase-3. Actin was used as the loading control. C) Densitometric quantifications of cleaved caspase-3 in MDM were presented as a ratio to pro-caspase-3 and normalized as fold changes to the vehicle control DMSO group. D) Supernatants were collected and the replication levels of HIV-1 were monitored by RTase activity assay. Results shown are from representative experiments performed with three different donors. Data were analyzed by two-way ANOVA: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, compared to the vehicle control DMSO group.