A novel cereblon variant with both exon 8 and 10 deletions in newly diagnosed and relapsed multiple myeloma

1. Identified a novel CRBN variant with both exon 8 and 10 transcript deletions in myeloma using a novel digital gene expression assay.

2. Functional and molecular modeling demonstrated significance of the combined deletion including impact on the binding pocket of the drug and compactness of CRBN protein.

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 redirects 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 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 to total CRBN mRNA (1 from newly diagnosed, 3 from relapsed and end-stage). Sequencing of cDNAs from two samples unexpectedly demonstrated additional deletion of exon 8. Extensive molecular-modeling and co-immunoprecipitation 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 exon 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, impacts on drug resistance.