Abstract
BackgroundImmunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity.MethodsCereblon binding partners were identified from a MM cell line expressing histidine-tagged cereblon by pulling down cereblon and its binding partners and verified by co-immunoprecipitation. IMiD effects were determined by western blot analysis, cell viability assay, microRNA array and apoptosis analysis.ResultsWe identified argonaute 2 (AGO2) as a cereblon binding partner and found that the steady-state levels of AGO2 were regulated by cereblon. Upon treatment of IMiD-sensitive MM cells with lenalidomide, the steady-state levels of cereblon were significantly increased, whereas levels of AGO2 were significantly decreased. It has been reported that AGO2 plays a pivotal role in microRNA maturation and function. Interestingly, upon treatment of MM cells with lenalidomide, the steady-state levels of microRNAs were significantly altered. In addition, silencing of AGO2 in MM cells, regardless of sensitivity to IMiDs, significantly decreased the levels of AGO2 and microRNAs and massively induced cell death.ConclusionThese results support the notion that the cereblon binding partner AGO2 plays an important role in regulating MM cell growth and survival and AGO2 could be considered as a novel drug target for overcoming IMiD resistance in MM cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2331-0) contains supplementary material, which is available to authorized users.
Highlights
Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells
In order to better understand the response of MM cells to IMiD, lentiviral particle harboring human CRBN cDNA infected My5 cells (My5.CRBN.His) and lentivirus vector infected My5 cells (My5.LV) were treated with various concentrations of lenalidomide for several days and the survival of the cells was monitored by 3–(4,5-dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide dye (MTT) assay
CRBN, JJN3 and MM1.S (Fig. 1a and b), with 10 μM lenalidomide for one day did not have significant effect on them (Fig. 1c), suggesting that IMiD-induced degradation of IKZF1 and IKZF3 occurs within hours, the effects of the degradation of these transcription factors on the proteins associated with cell growth and death may take days
Summary
Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. It takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and we searched for other cereblon binding partners that participate in IMiD cytotoxicity. The expression of CRBNdownstream binding protein AGO2, by regulating miRNA levels, plays an important role for MM cell growth and survival
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