CRBN enhancer DNA methylation correlates with gene expression, affects IMiD sensitivity in vitro, and impacts clinical outcome of MM patients exposed to IMiDs. (A) Methylation significantly increased from NDMM to IMiD rrMM (P < .002 ). (B) CRBN enhancer methylation and gene expression showed a significant negative correlation (Spearman’s rho = −0.407; P = .008). For patients with high methylation, gene expression was suppressed. (C) Significantly decreased CRBN enhancer methylation, CRBN expression and lenalidomide (Len) sensitization of KMS-11 (upper panel) and OPM2 cells (lower panel) after exposure to DNMTi’s (500 nM 5-azacytidine [Aza] for 48 hours or 100 nM 5-aza-2′-deoxycytidine [Dec] for 72 hours). Left: CRBN enhancer methylation, measured by pyrosequencing. Center: CRBN expression 5 days after incubation with DNA methyltranferase inhibitors (DNMTi) analyzed by using RT-PCR TaqMan assays, ΔΔCt method, and GUSB as housekeeping control. Right: after pretreatment with DNMTi for 48 hours with 500 nM 5-azacytidine or 72 hours with 100 nM 5-aza-2′-deoxycytidine, cells were treated for 5 days with 10 µM lenalidomide. Cell survival was measured by annexin V-propidium iodide (PI) fluorescence-activated cell sorting. Significantly restored IMiD sensitivity was observed in both cell lines. (D) Functional luciferase activity of CpG-free pCpGL vector containing in vitro methylated or unmethylated CRBN enhancer (Firefly), normalized to internal Renilla control activity in the L363 cell line. Empty MP -pCpGL vector served as a negative control. (E) Kaplan-Meier analysis of NDMM patients who underwent IMiD containing therapies (thalidomide or lenalidomide). Patients with CRBN enhancer (enh.) methylation (meth.) >26% (red) showed significantly inferior PFS compared with patients with methylation levels <26% (blue). Significance level: *P < .05; **P < .01; ***P < .001. DMSO, dimethylsulfoxide; n.s., not significant.