Criteria for selecting treatment according to cereblon protein expression in multiple myeloma

Abstract

The treatment of Multiple Myeloma (MM) prioritizes combination therapy due to its effectiveness. Among of them, Immune-modulating drugs (IMiDs) commonly used as the anticancer agents for MM. The mechanism of action for IMiDs involves binding to the CRBN protein, a subunit of the E3 ubiquitin ligase complex by altering its substrate such as Ikaros (IKZF1) and Aiolos (IKZF3). These proteins leading to MM cell death or the regulation of the cell cycle and then inhibiting tumor growth. To investigate the expression of CRBN protein, a monoclonal antibody that specifically binds to CRBN was produced using hybridoma cells, and the expression level of CRBN protein in various tissues was examined. As the results, the expression of CRBN protein was found in various tissues including heart, brain, lung, liver, skeletal muscle, kidney, and testis. Moreover, it was checked that the brain and immune like cell highly expressed CRBN protein. To investigate the function of the CRBN protein in animal model, CRBN knock-out (KO) mice were generated and then the 2D gel electrophoresis was investigated to separate and analyze proteins with differential expression by using brain tissues from both normal and knockout mice. As the result, five proteins exhibiting differential expression were identified. Additionally, using these samples, the protein kinase assay was assayed to investigated the mechanism related to CRBN protein. As the previous reports, we confirmed that the CRBN protein is associated with the E3 ubiquitin ligase pathway. We investigated that CRBN protein expression and cytotoxicity of IMDs in multiple myeloma cell lines. As the results, the CRBN expression rate was higher in the order of RPMI-8226, U226 H929, and also increased the cytotoxic efficacy of IMiDs. Additionally, we found that CRBN expression was higher HCC827 cells compared to HCC827/CLR cells, and the cytotoxic efficacy of IMiDs was also increased HCC827 cells compared to HCC827/CLR cells. Taken together, these results suggested that MMs and Lung cancers with highly CRBN expression were increased the cytotoxicity of IMiDs. Expanding on previous findings that indicated an absence of cytotoxic efficacy of IMDs in rodents compared to humans, we created humanized CRBN knock-in (KI) mice with a two-amino acid modification. We also established an animal model of multiple myeloma using the mouse multiple myeloma cell line MOPC315-Luc (humanized CRBN). We transplanted the MOPC315-Luc cell line into CRBN KO and KI mouse models for multiple myeloma and treated them with IMD for 47 days. As a result, we observed a significant reduction in the size of multiple myeloma in humanized CRBN KI mice compared to CRBN KO mice. These results suggest that the degree of CRBN protein expression during treatment in MM influences the efficacy of IMiDs and that MM patients with high CRBN expression may benefit from treatment with a combination therapy containing IMiDs. These data might be providing a basis for CRBN to be used as a novel target protein in MM to screen for therapeutics. Keywords: Multiple myeloma, Cereblon, immunomodulatory drugs