The Mechanistic Study Of Thalidomide And Its Structure Analogs In Regulating CRBN And Substrates Of The Cullin 4 RING-CRBN E3 Ligase

Background and purpose:The immunomodulatory drugs (IMiDs) thalidomide (Thal) and its structural analogs have significant outcomes in the treatment for multiple myeloma. Since the primary target of Thal and its structure analogs was identified as cereblon (CRBN) and CRBN is a substrate receptor of a cullin-4 RING E3 ligase (CRL4) complex, studies have focused on the effect of these drugs on the ubiquitination of the substrates of the CRBN associated E3 ligase. However, it is still unknown that how Thal and its analogs affect the activity of the CRL4-CRBN E3 ligase. Here, we studied the effect of IMiDs on the CRBN ubiquitination and the function of the E3 ligase in multiple myeloma cell lines. Our results reveal a novel mechanism of Thal and its analogs and provide an alternative model for the modulation of E3 ligase activity.Methods:We overexpressed FLAG-Strep-CRBN (FS-CRBN) in HEK293T cells. FS-CRBN was affinity purified under denaturing conditions after the treatment with Thal and its analogs. CRBN ubiquitination was detected with an anti-ubiquitin immunoblotting and protein complex was detected by immunoblotting after purification under native conditions. Multiple myeloma cells were treated with Thal and its analogs and the endogenous CRBN and its interacting partner BKCa protein levels were detected by immunoblotting. We further overexpressed GFP or CRBN in MM1.S cells and examined the effect of the increased CRBN on the sensitivity of MM1.S cells to Pom.Results:(1) Thal and its analogs inhibit the ubiquitination of both human and mouse CRBN.(2) Thal and Len do not disrupt the formation of the CRL4-CRBN E3 ligase complex.(3)Thal and Len increase the endogenous CRBN protein levels in multiple myeloma cell lines.(4) Increased CRBN protein level results in the enhanced activity of the CRL4-CRBN E3 ligase and thus decreases its substrate BKCa level in multiple myeloma cells.(5) The increase of the CRBN level improves the sensitivity of MM1.S cells to Pom.(6)The protein levels of IKZF1 and IKZF3 are also associated with the sensitivity of MM1.S cells to Pom.Conclusions:Thal and its analogs increase endogenous CRBN protein level in multiple myeloma cell lines. The increase of CRBN enhances the activity of CRL4-CRBN E3 ligase, promotes the ubiquitination and degradation of its substrates, and potentiates the inhibition of the multiple myeloma cell growth by IMiDs. These results suggested a new strategy for developing chemical compounds that regulate E3 ligase activity.