Identifying a novel CRBN transcript with both exon 10 and 8 deletions. (A) CRBN contains several domains, including LON and CULT domains. The exon 10 encoding region is a part of CULT domain and the exon 8 encodes parts of LON-C domain. The cDNAs from 2 patients with MM (R13 and N17) were generated, cloned, and sequenced. The sequence results were compared with the WT CRBN mRNA sequence (National Center for Biotechnology Information [NCBI] reference sequence: NM_00117382.1) in GenBank, indicating exon 10 (between 1043 and 1175) and exon 8 (between 862 and 979) were both deleted from 2 clones isolated. (B) The cloned cDNA from 1 patient (R13) was introduced into OCIMY5 cell line along with WT CRBN and vector alone. The expression of CRBN in those cells was detected by western blot. MTT assay demonstrated this novel isoform cannot mediate pomalidomide-induced antimyeloma activity. (C) OCIMY5 with vector, WT CRBN, R13 were treated with pomalidomide and analyzed using MTT assay. (D) R13 (GFP⁺) and WT (GFP⁻) expressing cells were mixed, treated, and analyzed by flow cytometry. The percentage of R13 expressing cells increased after exposure to Pom. Vec = Vector, POM = pomalidomide, WT = wild type, R13= OCIMY5 cell line with cloned cDNA.