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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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Aiolos and Ikaros degradation by lenalidomide and pomalidomide is Cereblon-dependent. (A) Primary human CD3+ T cells were transfected with control siRNA or CRBN siRNA for 48 h and CRBN knockdown efficiency was measured 24 h post-transfection by qRT-PCR. (B) CRBN siRNA knockdown T cells were treated with lenalidomide or pomalidomide at the indicated concentrations for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin expression. Len, lenalidomide; Pom, pomalidomide; CRBN, Cereblon gene; siCRBN, CRBN siRNA; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
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fig05: Aiolos and Ikaros degradation by lenalidomide and pomalidomide is Cereblon-dependent. (A) Primary human CD3+ T cells were transfected with control siRNA or CRBN siRNA for 48 h and CRBN knockdown efficiency was measured 24 h post-transfection by qRT-PCR. (B) CRBN siRNA knockdown T cells were treated with lenalidomide or pomalidomide at the indicated concentrations for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin expression. Len, lenalidomide; Pom, pomalidomide; CRBN, Cereblon gene; siCRBN, CRBN siRNA; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Mentions: The results described above suggest that lenalidomide or pomalidomide binding enables CRL4CRBN to associate with Ikaros and Aiolos, thus enabling their ubiquitination. To investigate whether drug-induced degradation of Aiolos and Ikaros in T cells occurs in a Cereblon-dependent manner, primary T cells were transfected with an siRNA against CRBN or control siRNA. CRBN siRNA resulted in a 75% decrease in CRBN mRNA levels compared with a non-targeting siRNA control (Fig 5A). Previous studies have established that a similar degree of knockdown leads to abrogation of lenalidomide- or pomalidomide-induced IL2 upregulation in T cells (Lopez-Girona et al, 2012). Figure5B demonstrates that the ability of lenalidomide or pomalidomide to induce Aiolos and Ikaros degradation in T cells was abrogated in the presence of CRBN siRNA, but not a non-targeting control siRNA. These data show that lenalidomide and pomalidomide induce degradation of Aiolos and Ikaros in a Cereblon-dependent manner.


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)

Aiolos and Ikaros degradation by lenalidomide and pomalidomide is Cereblon-dependent. (A) Primary human CD3+ T cells were transfected with control siRNA or CRBN siRNA for 48 h and CRBN knockdown efficiency was measured 24 h post-transfection by qRT-PCR. (B) CRBN siRNA knockdown T cells were treated with lenalidomide or pomalidomide at the indicated concentrations for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin expression. Len, lenalidomide; Pom, pomalidomide; CRBN, Cereblon gene; siCRBN, CRBN siRNA; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232904&req=5

fig05: Aiolos and Ikaros degradation by lenalidomide and pomalidomide is Cereblon-dependent. (A) Primary human CD3+ T cells were transfected with control siRNA or CRBN siRNA for 48 h and CRBN knockdown efficiency was measured 24 h post-transfection by qRT-PCR. (B) CRBN siRNA knockdown T cells were treated with lenalidomide or pomalidomide at the indicated concentrations for 24 h. Cell lysates were separated by SDS-PAGE and immunoblotted for Aiolos, Ikaros and Actin expression. Len, lenalidomide; Pom, pomalidomide; CRBN, Cereblon gene; siCRBN, CRBN siRNA; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Mentions: The results described above suggest that lenalidomide or pomalidomide binding enables CRL4CRBN to associate with Ikaros and Aiolos, thus enabling their ubiquitination. To investigate whether drug-induced degradation of Aiolos and Ikaros in T cells occurs in a Cereblon-dependent manner, primary T cells were transfected with an siRNA against CRBN or control siRNA. CRBN siRNA resulted in a 75% decrease in CRBN mRNA levels compared with a non-targeting siRNA control (Fig 5A). Previous studies have established that a similar degree of knockdown leads to abrogation of lenalidomide- or pomalidomide-induced IL2 upregulation in T cells (Lopez-Girona et al, 2012). Figure5B demonstrates that the ability of lenalidomide or pomalidomide to induce Aiolos and Ikaros degradation in T cells was abrogated in the presence of CRBN siRNA, but not a non-targeting control siRNA. These data show that lenalidomide and pomalidomide induce degradation of Aiolos and Ikaros in a Cereblon-dependent manner.

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