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Human Exportin-1 is a Target for Combined Therapy of HIV and AIDS Related Lymphoma.

Boons E, Vanstreels E, Jacquemyn M, Nogueira TC, Neggers JE, Vercruysse T, van den Oord J, Tamir S, Shacham S, Landesman Y, Snoeck R, Pannecouque C, Andrei G, Daelemans D - EBioMedicine (2015)

Bottom Line: Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases.At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction.Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.

View Article: PubMed Central - PubMed

Affiliation: KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium.

ABSTRACT
Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL) is an aggressive non-Hodgkin lymphoma with very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1) mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE) prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.

No MeSH data available.


Related in: MedlinePlus

Anti-HIV mechanism of action of KPT-185.(A) Time-of-addition experiment. C8166 cells were infected with HIV-1 at time 0 and inhibitors were added at different time points post infection. Virus production was determined by virus associated p24 production in the supernatant at 31 h after infection. Control: mock treated, nevirapine (7.5 μM): reverse transcriptase (RT) inhibitor; L870, 810 (1.6 μM): integrase (IN) inhibitor; WP7-5 (0.32 μM): transcription inhibitor; ritonavir (2.8 μM): protease (PR) inhibitor; KPT-185 (0.125 μM). Representative of data for 2 independent experiments.(B) KPT-185 suppresses expression of intron-containing late viral RNA species. Northern blot analysis of the viral mRNA species (fully spliced: 2 kb; partially spliced: 4 kb; unspliced: 9 kb) in PBMCs infected with HIV-1 IIIB and treated with different concentrations of KPT-185.(C) KPT-185 blocks the Rev-XPO1-mediated nuclear export of intron-containing viral RNA. Top: Schematic view of the MS2-tagged Rev-dependent viral-like RNA, pLTR-p57-24xMS2-RRE. Bottom: HeLa cells were co-transfected with plasmids encoding MS2-GFP and Rev-BFP and an RRE containing viral-like RNA construct carrying 24 MS2 recognition sites, as indicated. After overnight incubation, the sub-cellular localization of fluorescent proteins was visualized by confocal fluorescence microscopy in both GFP and BFP channels. The right column shows overlays of both channels together with DIC (differential interference contrast) images. The insets show a magnification of the 10 μm × 10 μm boxed area in ‘glow’ color lookup table. Scale bar, 25 μm.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.
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f0010: Anti-HIV mechanism of action of KPT-185.(A) Time-of-addition experiment. C8166 cells were infected with HIV-1 at time 0 and inhibitors were added at different time points post infection. Virus production was determined by virus associated p24 production in the supernatant at 31 h after infection. Control: mock treated, nevirapine (7.5 μM): reverse transcriptase (RT) inhibitor; L870, 810 (1.6 μM): integrase (IN) inhibitor; WP7-5 (0.32 μM): transcription inhibitor; ritonavir (2.8 μM): protease (PR) inhibitor; KPT-185 (0.125 μM). Representative of data for 2 independent experiments.(B) KPT-185 suppresses expression of intron-containing late viral RNA species. Northern blot analysis of the viral mRNA species (fully spliced: 2 kb; partially spliced: 4 kb; unspliced: 9 kb) in PBMCs infected with HIV-1 IIIB and treated with different concentrations of KPT-185.(C) KPT-185 blocks the Rev-XPO1-mediated nuclear export of intron-containing viral RNA. Top: Schematic view of the MS2-tagged Rev-dependent viral-like RNA, pLTR-p57-24xMS2-RRE. Bottom: HeLa cells were co-transfected with plasmids encoding MS2-GFP and Rev-BFP and an RRE containing viral-like RNA construct carrying 24 MS2 recognition sites, as indicated. After overnight incubation, the sub-cellular localization of fluorescent proteins was visualized by confocal fluorescence microscopy in both GFP and BFP channels. The right column shows overlays of both channels together with DIC (differential interference contrast) images. The insets show a magnification of the 10 μm × 10 μm boxed area in ‘glow’ color lookup table. Scale bar, 25 μm.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.

Mentions: To ascertain that the mechanism of the observed inhibition of virus replication by KPT-185 is caused by the inhibition of XPO1 function we engaged in detailed mechanism of action studies. First we performed a time-of-addition experiment (Daelemans et al., 2011). This experiment is based on the fact that HIV undergoes several well-established successive chronological processes and for almost each of these processes well-characterized inhibitors exist. In this experiment, in which a single replication cycle of the virus is followed, it is determined how long the addition of a drug can be postponed before it loses its anti-HIV activity. Comparing the time-of-addition profile of an investigational drug to that of classical anti-HIV inhibitors with known target of action, narrows down the time frame of action and thus the possible target of action of the investigational drug. For instance, an inhibitor interfering with the reverse transcription process will suppress virus replication when added at a time point before the fulfillment of the reverse transcription process (i.e. approximately 4–5 h post infection), but not if added at a time point after the reverse transcription process has already occurred (Fig. 2A). In this experiment, KPT-185 shows the same profile as the viral transcription inhibitor WP7-5, suggesting that KPT-185 interferes with a process coinciding with viral transcription. Of note, it has been suggested that the nuclear export of late HIV RNAs occurs co-transcriptionally (Nawroth et al., 2014).


Human Exportin-1 is a Target for Combined Therapy of HIV and AIDS Related Lymphoma.

Boons E, Vanstreels E, Jacquemyn M, Nogueira TC, Neggers JE, Vercruysse T, van den Oord J, Tamir S, Shacham S, Landesman Y, Snoeck R, Pannecouque C, Andrei G, Daelemans D - EBioMedicine (2015)

Anti-HIV mechanism of action of KPT-185.(A) Time-of-addition experiment. C8166 cells were infected with HIV-1 at time 0 and inhibitors were added at different time points post infection. Virus production was determined by virus associated p24 production in the supernatant at 31 h after infection. Control: mock treated, nevirapine (7.5 μM): reverse transcriptase (RT) inhibitor; L870, 810 (1.6 μM): integrase (IN) inhibitor; WP7-5 (0.32 μM): transcription inhibitor; ritonavir (2.8 μM): protease (PR) inhibitor; KPT-185 (0.125 μM). Representative of data for 2 independent experiments.(B) KPT-185 suppresses expression of intron-containing late viral RNA species. Northern blot analysis of the viral mRNA species (fully spliced: 2 kb; partially spliced: 4 kb; unspliced: 9 kb) in PBMCs infected with HIV-1 IIIB and treated with different concentrations of KPT-185.(C) KPT-185 blocks the Rev-XPO1-mediated nuclear export of intron-containing viral RNA. Top: Schematic view of the MS2-tagged Rev-dependent viral-like RNA, pLTR-p57-24xMS2-RRE. Bottom: HeLa cells were co-transfected with plasmids encoding MS2-GFP and Rev-BFP and an RRE containing viral-like RNA construct carrying 24 MS2 recognition sites, as indicated. After overnight incubation, the sub-cellular localization of fluorescent proteins was visualized by confocal fluorescence microscopy in both GFP and BFP channels. The right column shows overlays of both channels together with DIC (differential interference contrast) images. The insets show a magnification of the 10 μm × 10 μm boxed area in ‘glow’ color lookup table. Scale bar, 25 μm.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.
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f0010: Anti-HIV mechanism of action of KPT-185.(A) Time-of-addition experiment. C8166 cells were infected with HIV-1 at time 0 and inhibitors were added at different time points post infection. Virus production was determined by virus associated p24 production in the supernatant at 31 h after infection. Control: mock treated, nevirapine (7.5 μM): reverse transcriptase (RT) inhibitor; L870, 810 (1.6 μM): integrase (IN) inhibitor; WP7-5 (0.32 μM): transcription inhibitor; ritonavir (2.8 μM): protease (PR) inhibitor; KPT-185 (0.125 μM). Representative of data for 2 independent experiments.(B) KPT-185 suppresses expression of intron-containing late viral RNA species. Northern blot analysis of the viral mRNA species (fully spliced: 2 kb; partially spliced: 4 kb; unspliced: 9 kb) in PBMCs infected with HIV-1 IIIB and treated with different concentrations of KPT-185.(C) KPT-185 blocks the Rev-XPO1-mediated nuclear export of intron-containing viral RNA. Top: Schematic view of the MS2-tagged Rev-dependent viral-like RNA, pLTR-p57-24xMS2-RRE. Bottom: HeLa cells were co-transfected with plasmids encoding MS2-GFP and Rev-BFP and an RRE containing viral-like RNA construct carrying 24 MS2 recognition sites, as indicated. After overnight incubation, the sub-cellular localization of fluorescent proteins was visualized by confocal fluorescence microscopy in both GFP and BFP channels. The right column shows overlays of both channels together with DIC (differential interference contrast) images. The insets show a magnification of the 10 μm × 10 μm boxed area in ‘glow’ color lookup table. Scale bar, 25 μm.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(D) KPT-185 inhibits the transport of HIV-1 Rev protein. HeLa cells transfected with RevM5-GFP, a mutant of Rev, were analyzed by confocal microscopy. RevM5-GFP is found in the cytoplasm of the cells. Inhibition of nuclear export by siRNA knock down of XPO1 causes the RevM5-GFP protein to accumulate in the nucleus. Similarly, treatment with KPT-185 results in a redistribution of RevM5-GFP to the nucleus. See also Figure S1 and Movie S1.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.(E) KPT-185 disrupts the XPO1-Rev interaction in living cells. HeLa cells expressing Rev-BFP and/or XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP is found in the nucleoli of the cells, while XPO1-YFP concentrates at the nuclear membrane. In cells co-expressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and co-localizes with Rev-BFP. Two hours after addition of compound the co-localization of wild-type XPO1-YFP with Rev-BFP in the nucleoli was disrupted. See also Movie S2.
Mentions: To ascertain that the mechanism of the observed inhibition of virus replication by KPT-185 is caused by the inhibition of XPO1 function we engaged in detailed mechanism of action studies. First we performed a time-of-addition experiment (Daelemans et al., 2011). This experiment is based on the fact that HIV undergoes several well-established successive chronological processes and for almost each of these processes well-characterized inhibitors exist. In this experiment, in which a single replication cycle of the virus is followed, it is determined how long the addition of a drug can be postponed before it loses its anti-HIV activity. Comparing the time-of-addition profile of an investigational drug to that of classical anti-HIV inhibitors with known target of action, narrows down the time frame of action and thus the possible target of action of the investigational drug. For instance, an inhibitor interfering with the reverse transcription process will suppress virus replication when added at a time point before the fulfillment of the reverse transcription process (i.e. approximately 4–5 h post infection), but not if added at a time point after the reverse transcription process has already occurred (Fig. 2A). In this experiment, KPT-185 shows the same profile as the viral transcription inhibitor WP7-5, suggesting that KPT-185 interferes with a process coinciding with viral transcription. Of note, it has been suggested that the nuclear export of late HIV RNAs occurs co-transcriptionally (Nawroth et al., 2014).

Bottom Line: Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases.At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction.Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.

View Article: PubMed Central - PubMed

Affiliation: KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium.

ABSTRACT
Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL) is an aggressive non-Hodgkin lymphoma with very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1) mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE) prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.

No MeSH data available.


Related in: MedlinePlus