Cereblon Modulator CC-885 Induces CRBN-dependent Ubiquitination And Degradation Of CDK4 In Multiple Myeloma

Multiple myeloma is a hematologic malignancy that accounts for about 1percent of all cancers and 13 percent of hematologic malignancies and is currently incurable.With the application of immunomodulatory drugs,the prognosis of MM patients has been significantly improved.Molecular glue degraders that hijack cellular E3 ubiquitin ligases to target disease-driven proteins for proteosome-dependent degradation are emerging as a promising treatment.Immunomodulatory drugs are classical molecular glue that bind to cereblon(CRBN)to repurpose the function of the CRL4(CRBN)E3 ubiquitin ligase and developed to treat various hematological malignancies.Recently,a novel cereblon modulator CC-885 was developed to elicit broad antitumor activity.Although the degradation of GSPT1 is essential for the broad in vitro antitumor activity of CC-885,it is unclear whether other neosubstrates also contribute to the pharmacological effects of CC-885,especially in multiple myeloma(MM).Here,we show that CC-885 treatment caused growth retardant of MM cells via impairment of cell cycle progression and cell death both in vitro and in vivo.Mechanically,CC-885 selectively induced the ubiquitination and degradation of CDK4 in MM cells in a CRBN-dependent manner.CC-885-mediated CDK4 destruction decreased the phosphorylation of the tumor suppressor retinoblastoma(RB)and prevented the expression of E2F downstream genes.Importantly,genetic ablation or pharmacological inhibition of CDK4 enhances CC-885-induced cytotoxicity in MM cells,suggesting CDK4destruction contributed to the cytotoxicity of CC-885 in MM cells.Methods1.Cytotoxicity assay:RPMI8226 cells cultured in 96-well plates were treated with CC-885 at different doses(6.25-50 n M)for 1-3 days.Add CCK-8solution to each well and incubate for another 4h.The absorbance at 450 nm was then measured with a microplate reader.2.Cell cycle assay:Flow cytometry was used to detect the cell cycle distribution of dimethyl sulfoxide(DMSO)and 100 n M CC-885 after 24 h treatment of RPMI8226 and MM1S cells.3.Apoptosis test:RPMI8226 and MM1S cells were treated with 50 or100 n M CC-885 for 72h.The effect of CC-885 on apoptosis and necrosis of myeloma cells was detected by Annexin-V/PI double staining.4.Tumor formation in nude mice:1x10~7RPMI8226 cells were suspended in serum-free medium and subcutanically injected into BALB/Ca mice.After obvious growth of the transplanted tumor,the nude mice were divided into two groups and given DMSO or CC-885(30 mg/kg)daily,respectively.Use a caliper to measure the size of the tumor every few days.5.Half-life experiment:MM1S cells were pretreated with 100n MCC-885for 2h,then treated with actinomycetes(CHX)and CC-885 for western blot analysis.6.Western blot assay:MM1S cells were treated with different concentrations of CC-885 for 12 hours,and CDK4 changes were detected by Western blot assay.7.Real-time fluorescence quantitative PCR:The relative expression levels of CDK4 and 5 downstream E2F genes in MM1S cells were detected after treatment with 1μM CC-885 for 24h.8.Luciferase reporter assay:Transcriptional regulation components of E2F gene were constructed into reporter gene vectors,and the luciferase activity,namely the response of downstream signaling pathway,was detected after 24h treatment with different concentrations of CC-885.9.CDK4 knockdown test:CDK4 knockdown myeloma cells were obtained by sh RNA method,CDK4 protein levels were detected by Western blot,and CDK4m RNA levels were detected by real-time quantitative PCR.MM1S cells after CDK4 knockdown were treated with dimethyl sulfoxide or 1μM CC-885 for24 h.Then CCK-8 was added and the absorbance of A450 was measured by microplate reader.10.Immunoprecipitation:293T cells were transfected with Flag-CDK4 for24 h and then treated with dimethyl sulfoxide,1μm or 10μm CC-885 in the presence of 1μm MLN4924 for another 24 h.The cells were then collected and immunoprecipitated with marker M2 beads,and the protein levels of CDK4 and downstream p-Rb were detected by western blotting assay.Results1.Cytotoxicity assay:CCK8 analysis showed that CC-885 treatment inhibited the growth of MM cells in a dose-dependent and time-dependent manner.2.Cell cycle assay:Compared with dimethylsulfoxide(DMSO)treatment,CC-885 induced cell cycle arrest in the G1 phase.3.Apoptosis test:CC-885 induced dose-dependent myeloma cell death.4.Tumor formation experiment in nude mice:Compared with the control group,CC-885 treatment inhibited tumor growth and reduced tumor weight.5.Half-life experiment:The half-life of CDK4 protein was shortened in the presence of CC-885.6.Western blot assay:CC-885 can down-regulate endogenous CDK4protein expression in RPMI8226 and U266 cells without affecting their m RNA levels.7.Real-time fluorescence quantitative PCR:CC-885 significantly reduced the m RNA expression of several downstream target genes of E2F in MM1S cells.8.Luciferase reporter experiment:Treatment with CC-885 reduced E2F activity in a dose-dependent manner.9.CDK4 knockdown test:Silencing of CDK4 in MM1S cells with two different sh RNAs increased the sensitivity of myeloma cells to CC-885 treatment.10.Immunoprecipitation:The interaction between CDK4 and CRBN was not detectable in untreated cells.However,CC-885 promoted the interaction between CDK4 and CRBN in a dose-dependent manner after treatment with CC-885.Conclusion1.CC-885 shows anti-multiple myeloma activity in both in vitro and in vivo xenograft models2.CC-885 selectively induces CDK4 degradation in MM cells3.CDK4 is a CRBN-dependent neosubstrate of CC-8854.Genetic ablation or pharmacological inhibition of CDK4 enhances CC-885-induced cytotoxicity in MM cells.