Abstract
Acquisition of drug resistance, particularly to immunomodulatory drugs (IMiDs) in multiple myeloma (MM), is an important challenge in patient treatment. IMiDs are the canonical molecular glue that bind to cereblon (CRBN) and redirect its E3 ubiquitin ligase activity to neo-substrates, such as IKZF1/IFZF3. Genetic changes in the CRBN gene have been associated with IMiD resistance, including the exon 10-spliced CRBN transcripts that increase in incidence in parallel with IMiD-refractory states. Herein, we designed a new probe against CRBN splice isoform to detect exon 10 deletion and validated the variant expression using NanoString and messenger RNA (mRNA) sequencing analysis. We analyzed 74 MM samples, including 28 cases of newly diagnosed MM and 46 cases of relapsed and end-stage MM. Four cases were identified with 40% exon 10-deleted mRNA as compared with total CRBN mRNA (1 from newly diagnosed, 3 from relapsed and end-stage). Sequencing of complementary DNAs from 2 samples unexpectedly demonstrated additional deletion of exon 8. Extensive molecular-modeling and coimmunoprecipitation studies identified the mechanisms of CRBN dysfunction in protein structures, including impact on the interaction between CRBN with the E3 ligase machinery on deletion of exon 8 or dual deletion of exons 8 and 10. In summary, we developed a feasible approach to detect exon 10-deleted CRBN isoform on a gene expression platform and unexpectedly identified a variant with alterations of exon 10 and 8 transcripts in both newly diagnosed and refractory cases. This variant is present at diagnosis and could have separate, yet likely additive, effects on drug resistance.