Thalidomide and the IMiD immunomodulatory drugs, lenalidomide and pomalidomide, are widely used in the treatment of multiple myeloma (MM), del (5q) myelodysplastic syndromes and other hematologic malignancies, including mantle cell lymphoma. Ito et al. 1 recently identified cereblon as a key target of thalidomide. Subsequent studies confirmed cereblon to be a common target for lenalidomide and pomalidomide, and established its essential role in mediating anticancer and immunomodulatory effects of these drugs. 2, 3 Cereblon is encoded by the CRBN gene on chromosome 3 containing 11 exons, and the fully spliced transcript produces a 51-kDa protein. Cereblon is a component of the cullin ring E3 ubiquitin ligase complex (CRL4CRBN) that also contains DNA damage-binding protein 1 (DDB1), cullin (Cul) 4a and regulator of cullins (Roc) 1. 1 E3 ligases attach ubiquitin moieties to specific substrate proteins in the cell that can mark them for proteasomal degradation. The putative role of cereblon within the E3 ligase complex is that of a substrate receptor. With the discovery of cereblon, as a target of IMiD therapy, there has been considerable interest in defining whether expression of cereblon protein or the presence of CRBN mutations will impact clinical responses to these drugs. 2, 4–7 There are limited available data on CRBN gene mutation in the literature. Originally, a nonsense mutation (R419X) of CRBN was described to be associated with autosomal recessive non-syndromic mental retardation. 8 However, the functional link between the mutation in CRBN and the onset of mental retardation has not been demonstrated. Sequencing analyses of CRBN in MM cells from patients identified a truncating mutation (Q99X) and a point mutation (R283K) in 1 of 30 MM patients. 9 In addition, an A/G polymorphism has been identified at À 29 nucleotide from the transcriptional start site of the CRBN transcript. 10 So far, mutations in other components of the CRL4 E3 ligase complex (DDB1, Cul4a or Roc1) in MM cell lines or patients have not been described in the limited genome-wide sequencing of MM patients. 11 Here, we focused on sequencing the exons of CRBN with a goal of identifying missense or nonsense mutations with likely functional and clinical consequence. We analyzed IMiD-sensitive, intrinsically IMiD-resistant, as well as isogenic-sensitive or acquired lenalidomide-and/or pomalidomide-resistant MM cell lines. In addition, 90 MM patient samples, including those from 36 lenalidomide-resistant patients, were evaluated for the presence of CRBN mutations. As shown in Table 1, we found that the vast majority of IMiD-sensitive cell lines harbored the wild-type CRBN gene sequence. Also, none of the three intrinsically resistant cell lines (LP1, RPMI 8226 or JJN3) carried any mutation within the CRBN exons. As described previously, all three cell lines express high levels of cereblon transcript and protein. 12 Thus, the lack of mutation in the CRBN gene strongly suggests the existence of a cereblon-independent mechanism (s) of intrinsic resistance to IMiD drugs in the LP1, RPMI 8226 and JJN3 cell lines. In contrast, a heterozygous CRBN mutation (D249Y) was detected in the lenalidomide-resistant ANBL-6 cell line, while its sensitive parental line did not harbor the mutation. The location of the mutation suggests that it may impact the binding of cereblon to the drug or interacting partner DDB1. However, it is not clear whether the resistant phenotype in the lenalidomide-resistant ANBL-6 cell line is a direct result of the CRBN mutation alone and therefore it requires further evaluation. One copy of CRBN gene was shown to be deleted in the …