Elucidation of Molecular Mechanisms of Iberdomide and Mezigdomide Resistance in Cell Line Models of Multiple Myeloma

Introduction

Iberdomide/CC-220 (IBER) and mezigdomide/CC-92480 (MEZI) are two novel oral Cereblon (CRBN) E3 ubiquitin ligase modulating drugs (CELMoDs) currently undergoing clinical trials for the treatment of multiple myeloma (MM) patients in various settings. As acquired drug resistance mechanisms contribute to myeloma pathobiology and patient outcomes, we sought to develop cell line models of resistance to these agents and characterize the underlying mechanisms.

Methods

XG-1, MM.1S, and H929 myeloma cell lines were exposed to increasing IBER concentrations up to 20 nM to develop IBER-resistant models (XR220, MR220 and HR220). Similarly, MM.1S, and H929 cells were exposed to MEZI at up to 5 nM, with target concentrations determined by the known pharmacokinetics of these drugs in early phase human trials.

Results

XR220, MR220, and HR220 were deemed resistant based on the failure of IBER at up to 20 nM to reduce Ikaros, Aiolos, IRF4, and c-Myc protein levels, attenuate cell proliferation, and/or induce apoptosis compared with the parental controls. Moreover, they were found to be cross-resistant to pomalidomide (POM), lenalidomide (LEN), and MEZI treatment. Bulk RNA-Seq and hallmark pathway analysis comparing IBER-resistant to wild-type cells identified up-regulation in the former of c-MYC targets, and down-regulation of mitotic spindle, protein secretion, and interferon gamma response gene sets. Across the three model systems, 37 genes were commonly up-regulated, and 52 genes were down-regulated, with both CRBN and Ubiquitin specific peptidase 15 (USP15) among the latter, though no other CRBN pathway genes were dysregulated. Genomic studies in XR220 cells found decreased CRBN expression was associated with gene copy number loss, while a nonsense variant (SNV)-NM_016302.4: c.1006C>A (i. e, p. Glu336X) was found in MR220 cells. This site is located upstream of the CRBN 3’ exon-exon junction at 143 nucleotides and was confirmed to elicit nonsense-mediated mRNA decay to decrease CRBN mRNA stability. Notably, in the HR220 model, no CRBN mutation variants were seen by genomic DNA sequencing, but decreased CRBN levels were detected by TaqMan assay based on genomic DNA. Interestingly, in HR480, cells we found decreased CRBN levels associated with gene copy loss and two intronic variants: one at Chr3:3152588 C>A (GRCh38), and one at Chr3:3172931 A>T (GRCh38). RNA splicing analysis in HR480 cells showed that the (Chr3:3152588) variant caused CRBN exon 10 skipping. Single clones with this variant showed that it was by itself sufficient to significantly decrease CRBN levels and no truncated CRBN variants were detected. Finally, in MR480 cells, similar SNV patterns were seen as in MR220 cells, with a higher variant allele fraction in MR480 leading to a lower coverage level of CRBN mRNA.

To understand potential therapeutic and treatment sequencing implications, we tested the efficacy of several drug classes. The proteasome inhibitors bortezomib and carfilzomib showed efficacy against both the IBER- and MEZI-resistant cells, though this was slightly decreased compared with their parental cells. In contrast, chemotherapy drugs, such as melphalan and 4-hydroperoxy cyclophosphamide, and inhibitors of c-MYC, such as 10058-F4 and 10074-G5, showed enhanced efficacy towards IBER-resistant cells.

Conclusions

Dysregulation of CRBN through copy number loss or splicing alterations underly the mechanisms of IBER and MEZI resistance in cell line-derived model systems. Additional studies are needed to determine if these mechanisms play roles in clinical resistance to CELMoDs, and to validate hypotheses about optimal sequencing of therapies and approaches to overcome resistance.