CRBN Structural Changes, Copy Number Changes and COP9 Signalosome Subunits Gene Expression Mediate Sensitivity to New Celmod Compound CC-92480 in Multiple Myeloma Patients

Background: Immunomodulatory agents (IMiDs) hijack the Cullin-4A (CUL4A) / DNA damage-binding protein 1 (DDB1) / small RING protein (RBX) ubiquitin E3 ligase complex through their interaction with the substrate recognition protein Cereblon (CRBN), thereby triggering the proteasomal degradation of Ikaros (IKZF1) and Aiolos (IKZF3). We and others have previously shown that CRBN is essential for IMiDs activity and that resistance may be driven by CRBN genetic alteration, such as mutations or copy loss. Another resistance mechanism is alternative splicing, where the spliced isoform CRBN (CRBN-005, ENST00000424814) lacking exon 10 results in the loss of the IMIDs binding domain. Recent reports have also identified other components critical to IMiDs sensitivity in MM cells such as the COP9 signalosome, an eight-subunit protein complex that regulates the ubiquitin proteasome system.

The new generation of cereblon E3 ligase modulators (CELMoDs) have greater affinity to CRBN, induce more potent degradation of Ikaros/Aiolos and are effective in relapsed/refractory myeloma patients (RRMM) who are refractory to lenalidomide and pomalidomide. However, there is still little information on the mechanism of resistance of MM cells to these new CELMoDs. Here we investigated at the single cell level the transcriptome (scRNA) and genome copy number alterations (CNV) of primary MM cells from patients treated with the novel CELMoD compound CC-92480

Methods and results: Serial bone marrow aspirates were collected from 5 RRMM patients included in the CC-92480-MM-001 trial (NCT03374085) at our center, at inclusion (pre) and relapse (post). Three patients (P1, P2 and P3), were classified as responders, and 2 patients (P4 and P5) were identified as primary refractory. All 5 patients were exposed in previous lines to lenalidomide and pomalidomide. All patients were treated with CC-92480 in combination with dexamethasone. Bone marrow mononuclear fractions were isolated by Ficoll density gradients coupled with magnetic sorting of CD138^pos cells. Single cell gene expression analyses of CD138^pos cells were then performed using 3'Chromium single cell RNA-Seq (10x Genomics) in each sample and paired-end sequencing was done on Illumina platform. Cell Ranger was used for sample de-multiplexing, barcode processing, peaks counting and genome alignment (hg38). Seurat, scVelo and SCEVAN were used for gene expression, spliced junctions, RNA velocity and pseudo-bulk copy number analyses respectively.

In the responder group, we identified the presence of CRBN mRNA splice variants lacking exon 10 and comparative RNA velocities analyses showed an increase of the CRBN spliced/unspliced transcript ratio at progression in all 3 patients. Furthermore, P1 and P3 showed a monoallelic 3p26 loss (encompassing CRBN) in "pre” and "post” samples, whereas P3 also presented a 2q37 loss, resulting in low COPS7B and COPS8 gene expression in both samples. P2 manifested a monoallelic 3p26 loss only in the "post” sample and lower expression of COPS2, 3, 5 and 7A genes after treatment.

In the primary refractory group, P5 showed biallelic loss of 3p26, 17p11, 17p13 and 17q25 resulting in low CRBN, TP53, COPS3 andGPS1(COPS1) gene expression in both samples. P4 had a monoallelic 3p26 loss and a clonal C-T substitution mutation at position 3,153,948 in both samples, resulting in arginine-histidine substitution at amino acid 309 [p.(R309H)] in CRBN DDB1 binding domain within exon 8. To assess the role of this mutation in resistance to IMiDs, we cloned R309H-mutant CRBN or wild-type (WT) CRBN into pLX304 plasmid and transfected them into OCI-MY5 cells. Streptavidin pulldown and immunoprecipitation assays showed decreased binding of thalidomide, DDB1 and CUL4A to R309H-CRBN mutant compared to WT-CRBN. Whereas treatment of WT-CRBN expressing cells with CC-92480 resulted in complete degradation of Aiolos and downregulation of MYC, expression of R309H-CRBN significantly attenuated theses effects and nearly abrogated CC-92480 cytotoxicity.

Conclusion: We here defined in primary patient plasma cells the mechanisms of resistance to the novel CELMoD CC-92480 to be driven by biallelic CRBN copy number loss or alteration of its structure, resulting from CRBN mutations or splicing (exon 10) coupled with monoallelic 3p loss. COP9 signalosome subunits gene expression decreased in resistant patients and may also play a critical role in CELMoD sensitivity.

Neri:Pfizer: Consultancy, Honoraria; Sanofi-Aventis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Bahlis:Pfizer: Research Funding; AbbVie, Amgen, Bristol Myers Squibb, Celgene, Forus, Janssen, Genentech, GSK, Karyopharm, Novartis, Pfizer, Takeda, Sanofi: Consultancy.