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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

KPT-185 does not inhibit viral production in CRISPR-Cas9 genome-edited cells expressing the resistant XPO1C258S mutant protein.(A) Sequencing chromatogram of genomic DNA of the XPO1 region around the targeted cysteine codon from homozygous mutant XPO1C528S cells. Mutated codon (528 TGT ➔ TCA) is indicated in bold. The sgRNA sequence of the CRISPR is underlined and the PAM motif is given in magenta.(B) Wild-type and mutant XPO1C258S HEK293T cells were transfected with HIV-1 molecular clone NL4-3 and treated with different concentrations of KPT-185. Virus production was analyzed by virus-associated p24 Gag protein in the supernatant. Error bars represent standard deviations, n = 3.
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f0015: KPT-185 does not inhibit viral production in CRISPR-Cas9 genome-edited cells expressing the resistant XPO1C258S mutant protein.(A) Sequencing chromatogram of genomic DNA of the XPO1 region around the targeted cysteine codon from homozygous mutant XPO1C528S cells. Mutated codon (528 TGT ➔ TCA) is indicated in bold. The sgRNA sequence of the CRISPR is underlined and the PAM motif is given in magenta.(B) Wild-type and mutant XPO1C258S HEK293T cells were transfected with HIV-1 molecular clone NL4-3 and treated with different concentrations of KPT-185. Virus production was analyzed by virus-associated p24 Gag protein in the supernatant. Error bars represent standard deviations, n = 3.

Mentions: To further prove that the observed inhibition of the viral replication by KPT-185 is exclusively caused by inhibition of XPO1 and not by off-target or unspecific effects, we generated a HEK293T cell-line expressing the resistant XPO1C528S protein. Mutating the Cys528 in the hydrophobic cargo-binding groove of XPO1 to a serine residue confers resistance to KPT-185 (Neggers et al., 2015). We used CRISPR-Cas9 genome editing in combination with homology directed repair to introduce a single Cys528Ser point mutation in the XPO1 gene of HEK293T cells. HEK293T cells were transfected with Cas9 endonuclease, a 23-bp guide RNA and a 135 base single stranded oligodeoxynucleotide repair donor template containing the TCA mutant codon to introduce the serine at position 528 in the XPO1 gene. Single cell derived colonies were analyzed for the mutation in their genomic DNA by Sanger sequencing (Fig. 3A). A homozygous mutant colony was selected for further experiments. The mutant HEK293T-XPO1C528S cell line or wild-type HEK293T cells were transfected with the HIV-1 molecular clone NL4-3 and treated with different concentrations of KPT-185. Twenty-four hours after treatment, culture supernatants were analyzed for the presence of virus (Fig. 3B). Absolute virus production was about 3 times lower in mutant cells as compared with wild-type cells. KPT-185 suppressed virus production from transfected wild-type HEK239T cells, while it had no effect on virus production from the mutant HEK293T-XPO1C528S cells. These results unambiguously demonstrate that the anti-HIV activity of KPT-185 is exclusively caused by inhibition of XPO1 and confirm that cysteine528 is involved in the mechanism of action of KPT-185.


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)

KPT-185 does not inhibit viral production in CRISPR-Cas9 genome-edited cells expressing the resistant XPO1C258S mutant protein.(A) Sequencing chromatogram of genomic DNA of the XPO1 region around the targeted cysteine codon from homozygous mutant XPO1C528S cells. Mutated codon (528 TGT ➔ TCA) is indicated in bold. The sgRNA sequence of the CRISPR is underlined and the PAM motif is given in magenta.(B) Wild-type and mutant XPO1C258S HEK293T cells were transfected with HIV-1 molecular clone NL4-3 and treated with different concentrations of KPT-185. Virus production was analyzed by virus-associated p24 Gag protein in the supernatant. Error bars represent standard deviations, n = 3.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588406&req=5

f0015: KPT-185 does not inhibit viral production in CRISPR-Cas9 genome-edited cells expressing the resistant XPO1C258S mutant protein.(A) Sequencing chromatogram of genomic DNA of the XPO1 region around the targeted cysteine codon from homozygous mutant XPO1C528S cells. Mutated codon (528 TGT ➔ TCA) is indicated in bold. The sgRNA sequence of the CRISPR is underlined and the PAM motif is given in magenta.(B) Wild-type and mutant XPO1C258S HEK293T cells were transfected with HIV-1 molecular clone NL4-3 and treated with different concentrations of KPT-185. Virus production was analyzed by virus-associated p24 Gag protein in the supernatant. Error bars represent standard deviations, n = 3.
Mentions: To further prove that the observed inhibition of the viral replication by KPT-185 is exclusively caused by inhibition of XPO1 and not by off-target or unspecific effects, we generated a HEK293T cell-line expressing the resistant XPO1C528S protein. Mutating the Cys528 in the hydrophobic cargo-binding groove of XPO1 to a serine residue confers resistance to KPT-185 (Neggers et al., 2015). We used CRISPR-Cas9 genome editing in combination with homology directed repair to introduce a single Cys528Ser point mutation in the XPO1 gene of HEK293T cells. HEK293T cells were transfected with Cas9 endonuclease, a 23-bp guide RNA and a 135 base single stranded oligodeoxynucleotide repair donor template containing the TCA mutant codon to introduce the serine at position 528 in the XPO1 gene. Single cell derived colonies were analyzed for the mutation in their genomic DNA by Sanger sequencing (Fig. 3A). A homozygous mutant colony was selected for further experiments. The mutant HEK293T-XPO1C528S cell line or wild-type HEK293T cells were transfected with the HIV-1 molecular clone NL4-3 and treated with different concentrations of KPT-185. Twenty-four hours after treatment, culture supernatants were analyzed for the presence of virus (Fig. 3B). Absolute virus production was about 3 times lower in mutant cells as compared with wild-type cells. KPT-185 suppressed virus production from transfected wild-type HEK239T cells, while it had no effect on virus production from the mutant HEK293T-XPO1C528S cells. These results unambiguously demonstrate that the anti-HIV activity of KPT-185 is exclusively caused by inhibition of XPO1 and confirm that cysteine528 is involved in the mechanism of action of KPT-185.

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