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Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

Gandhi AK, Kang J, Havens CG, Conklin T, Ning Y, Wu L, Ito T, Ando H, Waldman MF, Thakurta A, Klippel A, Handa H, Daniel TO, Schafer PH, Chopra R - Br. J. Haematol. (2013)

Bottom Line: We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression.The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation.In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Celgene Corporation, Summit, NJ, USA.

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Lenalidomide and pomalidomide stimulate Aiolos degradation via ubiquitination and protein turnover. (A) Jurkat cells were transfected with control empty vector, wildtype IKZF3 or mutant IKZF3 for 6 h then treated with either DMSO, 1 or 10 μmol/l lenalidomide or pomalidomide for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos and Actin. (B) U266 cells were pulsed with 10 μg/ml cycloheximide and treated with either DMSO, 10 μmol/l lenalidomide or 1 μmol/l pomalidomide for 1·5, 3 or 6 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin. Densitometry analysis of Aiolos and Ikaros expression normalized to Actin; data shown is mean ± SEM of three experiments. Len, lenalidomide; Pom, pomalidomide; CHX, cycloheximide; Mt, mutant; WT, wildtype; DMSO, dimethyl sulfoxide; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
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fig04: Lenalidomide and pomalidomide stimulate Aiolos degradation via ubiquitination and protein turnover. (A) Jurkat cells were transfected with control empty vector, wildtype IKZF3 or mutant IKZF3 for 6 h then treated with either DMSO, 1 or 10 μmol/l lenalidomide or pomalidomide for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos and Actin. (B) U266 cells were pulsed with 10 μg/ml cycloheximide and treated with either DMSO, 10 μmol/l lenalidomide or 1 μmol/l pomalidomide for 1·5, 3 or 6 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin. Densitometry analysis of Aiolos and Ikaros expression normalized to Actin; data shown is mean ± SEM of three experiments. Len, lenalidomide; Pom, pomalidomide; CHX, cycloheximide; Mt, mutant; WT, wildtype; DMSO, dimethyl sulfoxide; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Mentions: CRL4CRBN is an E3 ubiquitin ligase complex known to modify target substrates with ubiquitin. Therefore, because lenalidomide- or pomalidomide-mediated degradation of Aiolos and Ikaros requires the proteosome (Fig 2B), we investigated whether lysine ubiquitination was required for drug-mediated Aiolos degradation. To prevent ubiquitination of Aiolos, a Flag-tagged mutant IKZF3 (Flag-MT-IKZF3), in which all 30 lysine residues were conservatively substituted with arginines, was constructed. Jurkat cells were transfected with either a Flag-tagged WT IKZF3, Flag-MT-IKZF3, or an empty vector control. Cells were treated with lenalidomide or pomalidomide for 24 h and extracts were analysed by Western blot for Aiolos. Drug treatment caused degradation of endogenous Aiolos and recombinant Flag-WT-Aiolos, but not of Flag-MT-Aiolos, consistent with a mechanism of degradation that involves drug-induced lysine ubiquitination (Fig 4A).


Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

Gandhi AK, Kang J, Havens CG, Conklin T, Ning Y, Wu L, Ito T, Ando H, Waldman MF, Thakurta A, Klippel A, Handa H, Daniel TO, Schafer PH, Chopra R - Br. J. Haematol. (2013)

Lenalidomide and pomalidomide stimulate Aiolos degradation via ubiquitination and protein turnover. (A) Jurkat cells were transfected with control empty vector, wildtype IKZF3 or mutant IKZF3 for 6 h then treated with either DMSO, 1 or 10 μmol/l lenalidomide or pomalidomide for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos and Actin. (B) U266 cells were pulsed with 10 μg/ml cycloheximide and treated with either DMSO, 10 μmol/l lenalidomide or 1 μmol/l pomalidomide for 1·5, 3 or 6 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin. Densitometry analysis of Aiolos and Ikaros expression normalized to Actin; data shown is mean ± SEM of three experiments. Len, lenalidomide; Pom, pomalidomide; CHX, cycloheximide; Mt, mutant; WT, wildtype; DMSO, dimethyl sulfoxide; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
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Related In: Results  -  Collection

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fig04: Lenalidomide and pomalidomide stimulate Aiolos degradation via ubiquitination and protein turnover. (A) Jurkat cells were transfected with control empty vector, wildtype IKZF3 or mutant IKZF3 for 6 h then treated with either DMSO, 1 or 10 μmol/l lenalidomide or pomalidomide for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos and Actin. (B) U266 cells were pulsed with 10 μg/ml cycloheximide and treated with either DMSO, 10 μmol/l lenalidomide or 1 μmol/l pomalidomide for 1·5, 3 or 6 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin. Densitometry analysis of Aiolos and Ikaros expression normalized to Actin; data shown is mean ± SEM of three experiments. Len, lenalidomide; Pom, pomalidomide; CHX, cycloheximide; Mt, mutant; WT, wildtype; DMSO, dimethyl sulfoxide; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Mentions: CRL4CRBN is an E3 ubiquitin ligase complex known to modify target substrates with ubiquitin. Therefore, because lenalidomide- or pomalidomide-mediated degradation of Aiolos and Ikaros requires the proteosome (Fig 2B), we investigated whether lysine ubiquitination was required for drug-mediated Aiolos degradation. To prevent ubiquitination of Aiolos, a Flag-tagged mutant IKZF3 (Flag-MT-IKZF3), in which all 30 lysine residues were conservatively substituted with arginines, was constructed. Jurkat cells were transfected with either a Flag-tagged WT IKZF3, Flag-MT-IKZF3, or an empty vector control. Cells were treated with lenalidomide or pomalidomide for 24 h and extracts were analysed by Western blot for Aiolos. Drug treatment caused degradation of endogenous Aiolos and recombinant Flag-WT-Aiolos, but not of Flag-MT-Aiolos, consistent with a mechanism of degradation that involves drug-induced lysine ubiquitination (Fig 4A).

Bottom Line: We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression.The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation.In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Celgene Corporation, Summit, NJ, USA.

Show MeSH