Newly Diagnosed, Untreated, Multiple Myeloma (MM) Patient Samples Already Harbor Cereblon (CRBN) Exon 10 Deletions Associated with Drug Resistance

Background: Acquisition of drug resistance, particularly immunomodulatory drugs (IMiDs) in MM is one of the most important challenges in patient treatment today. Early on in the disease, the plasma cells in the MM clone are often sensitive to chemotherapy. However, over time, after exposure to chemotherapy, the cells acquire increasing genomic complexity and mutations leading to resistance to therapy and ultimately patient death. Cereblon (CRBN) is a binding protein, which is required for the action of the widely used IMiDs such as lenalidomide and pomolidomide. CRBN is also essential to the newer CRBN E3 ligase modulator drugs and proteolysis targeting chimeras. We recently described an algorithm based on digital gene expression profiling of purified CD138(+) MM cells from patients at different clinical stages that we term the “MM-IP-7”, which correlated with clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs (XY Zhu et al, eHaem 2022). Given the recent description of genetic changes in the CRBN gene that are associated with IMiD resistance particularly the exon 10-spliced CRBN transcripts that increase in incidence in parallel with IMiD-refractory states (S. Gooding et al, Blood 2021), we sought to build an integrated, single-platform test to measure both gene expression levels and genetic deletions.

Methods: We designed a novel CRBN splicing isoform probe with homology to both exon 9 and exon 11. This probe will thus capture CRBN mRNA sequences in which exon 10 is deleted and portions of exons 9 and 11 and juxtaposed. We also included probes to CRBN overall (Exons 5 and 6). These probes were combined with the MM-IP-7 probes to create a dual gene expression and genetic assay that was used to analyze 30 cases of newly diagnosed MM, 30 cases of end stage MM, and 3 MM cell lines (MM1.S, OPM2, XG1). To define increased exon 10 deleted allele frequency, we calculated the ratio of exon 10 deletion/total exon 10. Cases with apparent exon 10 deletion by expression profiling, were further evaluated with RT-PCR.

Results: 40% MM samples in the end stage group versus 23% of samples in the newly diagnosed group were identified with more than 20% exon 10 deleted mRNA as compared to total CRBN mRNA levels. Three cases were shown to have more than 45% exon 10 deleted mRNA, including two newly diagnosed cases and one end stage case. RT-PCR confirmed exon 10 deletion in a newly diagnosed patient sample with sufficient remaining RNA that was tested (cloning and sequencing for confirmation are underway). Cell line results showed very low exon 10 splicing variant, which correlated with our mRNAseq data for these lines.

Conclusions: Our findings demonstrate that inclusion of probes bridging CRBN exon 10 deletion with homology to exons 9 and 11, is feasible approach to use on the gene expression platform, which allows for combined evaluation of expression and genetic changes. CRBN exon 10 deletion is a known treatment resistance feature in MM, which may already be present at diagnosis and selected by subsequent therapy with possible therapeutic planning implications. Additional studies to confirm the results and expand the study set are underway.