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Cited by 106Multiple Myeloma Research and TreatmentsProtein Degradation and InhibitorsMonoclonal and Polyclonal Antibodies Research

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Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity

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Gandhi, Anita K., Mendy, Derek, Waldman, Michelle, Chen, Gengxin, Rychak, Emily, Miller, Karen, Gaidarova, Svetlana, Ren, Yan, Wang, Maria, Breider, Michael, Carmel, Gilles, Mahmoudi, Afshin, Jackson, Pilgrim, Abbasian, Mahan, Cathers, Brian E., Schafer, Peter H., Daniel, Tom O., Lopez‐Girona, Antonia, Thakurta, Anjan, Chopra, Rajesh·2014·British Journal of Haematology
DOI 10.1111/bjh.12622PMC PMC4253085
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Abstract

Summary: Cereblon, a member of the cullin 4 ring ligase complex ( CRL 4), is the molecular target of the immunomodulatory drugs ( IM i D s) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma ( MM ) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IM i D therapy. Our studies characterized a cereblon monoclonal antibody CRBN 65, with high sensitivity and specificity in W estern analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN 65 antibody and T aq M an quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

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Figures

Fig 1 The CRBN65 antibody detects cereblon (CRBN) protein with high specificity compared with commercial antibodies. DF15 and DF15R MM cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 (1:1000) or commercial antibodies (manufacturers' recommended dilution) against CRBN. Blots were analysed on LI-COR Oddysey and arrows denote the full-length CRBN protein band [51 kiloDalton (kDa)] that appears specifically in DF15 cells but not DF15R.
Fig 1 The CRBN65 antibody detects cereblon (CRBN) protein with high specificity compared with commercial antibodies. DF15 and DF15R MM cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 (1:1000) or commercial antibodies (manufacturers' recommended dilution) against CRBN. Blots were analysed on LI-COR Oddysey and arrows denote the full-length CRBN protein band [51 kiloDalton (kDa)] that appears specifically in DF15 cells but not DF15R.CC BY-NC-ND · PubMed Central
Fig 2 The CRBN65 antibody detects recombinant and endogenous CRBN protein with high sensitivity. (A) Recombinant human full-length CRBN (0·2–60 ng) along with DF15 and DF15R cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000). (B) Linear regression analysis using CRBN signal as measured on LI-COR Oddysey for rhCRBN is linear in the 0·2–60 ng range, and CRBN protein measured in DF15 cells falls within linear range of detection. CRBN, cereblon; kDa, kiloDalton; Mol W, molecular weight; R2, coefficient of determination.
Fig 2 The CRBN65 antibody detects recombinant and endogenous CRBN protein with high sensitivity. (A) Recombinant human full-length CRBN (0·2–60 ng) along with DF15 and DF15R cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000). (B) Linear regression analysis using CRBN signal as measured on LI-COR Oddysey for rhCRBN is linear in the 0·2–60 ng range, and CRBN protein measured in DF15 cells falls within linear range of detection. CRBN, cereblon; kDa, kiloDalton; Mol W, molecular weight; R2, coefficient of determination.CC BY-NC-ND · PubMed Central
Fig 3 The CRBN65 antibody detects CRBN protein across a wide range of MM cell lines with high specificity. A panel of MM cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000) or β-tubulin, without blocking peptide (upper panel) or with blocking peptide (lower panel). CRBN, cereblon; kDa, kiloDalton; MM, multiple myeloma; rCRBN, recombinant cereblon.
Fig 3 The CRBN65 antibody detects CRBN protein across a wide range of MM cell lines with high specificity. A panel of MM cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000) or β-tubulin, without blocking peptide (upper panel) or with blocking peptide (lower panel). CRBN, cereblon; kDa, kiloDalton; MM, multiple myeloma; rCRBN, recombinant cereblon.CC BY-NC-ND · PubMed Central
Fig 4 The CRBN65 antibody detects CRBN protein across a range of normal human cells and tumour cells. A panel of cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000) or actin antibody. Cell types of cell lines tested are as follows: KMS11 and KMS34, human multiple myeloma; Rec1, mantle cell lymphoma; OCI-LY10, activated B-cell–like diffuse large B-cell lymphoma; Jurkat, T-cell leukaemia; HS5, bone marrow stroma fibroblast; NHNP, normal human progenitor cells; G166, glioblastoma; Br231 and Br468, breast cancer cell lines; PC3, prostate carcinoma cell line; LNCAP, prostate adenocarcinoma cell line; CRBN, cereblon; kDa, kiloDalton.
Fig 4 The CRBN65 antibody detects CRBN protein across a range of normal human cells and tumour cells. A panel of cell extracts (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with CRBN65 antibody (1:1000) or actin antibody. Cell types of cell lines tested are as follows: KMS11 and KMS34, human multiple myeloma; Rec1, mantle cell lymphoma; OCI-LY10, activated B-cell–like diffuse large B-cell lymphoma; Jurkat, T-cell leukaemia; HS5, bone marrow stroma fibroblast; NHNP, normal human progenitor cells; G166, glioblastoma; Br231 and Br468, breast cancer cell lines; PC3, prostate carcinoma cell line; LNCAP, prostate adenocarcinoma cell line; CRBN, cereblon; kDa, kiloDalton.CC BY-NC-ND · PubMed Central
Fig 5 Cell model staining and peptide blocking experiments demonstrate the specificity of the CRBN65 antibody. (A) CRBN immunohistochemistry in DF15 and DF15R cell pellets. (B) DF15 cell pellet blocks and normal colon section was stained with CRBN65 with and without blocking peptide. (C) Representative photomicrographs from MM and adjacent normal bone marrow microarray. Objective = 40×. CRBN, cereblon.
Fig 5 Cell model staining and peptide blocking experiments demonstrate the specificity of the CRBN65 antibody. (A) CRBN immunohistochemistry in DF15 and DF15R cell pellets. (B) DF15 cell pellet blocks and normal colon section was stained with CRBN65 with and without blocking peptide. (C) Representative photomicrographs from MM and adjacent normal bone marrow microarray. Objective = 40×. CRBN, cereblon.CC BY-NC-ND · PubMed Central
Fig 6 A complex profile of CRBN splice variants exists in both MM cell lines and primary CD138+ MM tumour cells. (A) Nested polymerase chain reaction (PCR) reaction for CRBN splice variants in H929 MM cells were separated on an agarose gel and arrows indicate those bands that were gel purified and sequenced. (B) Nested PCR reaction for CRBN splice variants in 8 MM patient (pt) cells were separated on an agarose gel and multiple bands indicate complexity of CRBN mRNA profile. CRBN, cereblon; MM, multiple myeloma.
Fig 6 A complex profile of CRBN splice variants exists in both MM cell lines and primary CD138+ MM tumour cells. (A) Nested polymerase chain reaction (PCR) reaction for CRBN splice variants in H929 MM cells were separated on an agarose gel and arrows indicate those bands that were gel purified and sequenced. (B) Nested PCR reaction for CRBN splice variants in 8 MM patient (pt) cells were separated on an agarose gel and multiple bands indicate complexity of CRBN mRNA profile. CRBN, cereblon; MM, multiple myeloma.CC BY-NC-ND · PubMed Central
Fig 7 A schematic of CRBN splice variants crbncc-001 to crbncc-022 sequenced from MM cell lines with regions recognized by CRBN65 and TaqMan denoted. CRBN, cereblon; DDB1, DNA damage binding protein 1; IMiD, IMiD immunomodulatory drug; THAL, thalidomide.
Fig 7 A schematic of CRBN splice variants crbncc-001 to crbncc-022 sequenced from MM cell lines with regions recognized by CRBN65 and TaqMan denoted. CRBN, cereblon; DDB1, DNA damage binding protein 1; IMiD, IMiD immunomodulatory drug; THAL, thalidomide.CC BY-NC-ND · PubMed Central
Fig 8 CRBN mRNA and CRBN protein levels do not correlate in MM cell lines with intrinsic sensitivity or resistance to the IMiD agent lenalidomide. CRBN mRNA and CRBN protein levels were measured in 12 human MM cell lines by TaqMan polymerase chain reaction and Western blot, respectively. mRNA and protein were normalized to HRPT1 and β-tubulin, respectively. Graph represents the average of 3–4 experiments. CRBN, cereblon; LEN, lenalidomide; MM, multiple myeloma; mRNA, messenger RNA.
Fig 8 CRBN mRNA and CRBN protein levels do not correlate in MM cell lines with intrinsic sensitivity or resistance to the IMiD agent lenalidomide. CRBN mRNA and CRBN protein levels were measured in 12 human MM cell lines by TaqMan polymerase chain reaction and Western blot, respectively. mRNA and protein were normalized to HRPT1 and β-tubulin, respectively. Graph represents the average of 3–4 experiments. CRBN, cereblon; LEN, lenalidomide; MM, multiple myeloma; mRNA, messenger RNA.CC BY-NC-ND · PubMed Central
Fig 9 Acquired resistance to immunomodulatory drugs in MM cell lines leads to less CRBN mRNA; however, a disconnect exists between CRBN mRNA and CRBN protein levels. Quantification of mRNA and protein, and immunohistochemistry (IHC) were performed as described in the text. (A) Comparison of CRBN protein and CRBN mRNA in DF15 and DF15R resistant cells. (B) Comparison of CRBN protein and CRBN mRNA in parental H929 versus lenalidomide-resistant H929 lines 1051, 1052, 1053 and 1054. (C) CRBN IHC in H929-parental cell versus H929 lenaliomide-resistant line 1054. CRBN, cereblon; MM, multiple myeloma; mRNA, messenger RNA.
Fig 9 Acquired resistance to immunomodulatory drugs in MM cell lines leads to less CRBN mRNA; however, a disconnect exists between CRBN mRNA and CRBN protein levels. Quantification of mRNA and protein, and immunohistochemistry (IHC) were performed as described in the text. (A) Comparison of CRBN protein and CRBN mRNA in DF15 and DF15R resistant cells. (B) Comparison of CRBN protein and CRBN mRNA in parental H929 versus lenalidomide-resistant H929 lines 1051, 1052, 1053 and 1054. (C) CRBN IHC in H929-parental cell versus H929 lenaliomide-resistant line 1054. CRBN, cereblon; MM, multiple myeloma; mRNA, messenger RNA.CC BY-NC-ND · PubMed Central

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Research works printed but not linked: 2

  1. Gandhi A.K., 2013, Exon mutations in cereblon (CRBN) and DNA damage binding protein 1 (DDB1) genes are rare in myeloma cells and patients, Clinical Lymphoma Myeloma and Leukemia, 13, S182
  2. Schuster S.R., 2012, Cereblon expression predicts response, progression free and overall survival after pomalidomide and dexamethasone therapy in multiple myeloma (oral presentation), Blood (ASH Annual Meeting Abstracts), 120, 194

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Metadata

Authors
Gandhi, A. K., Mendy, D., Waldman, M., Chen, G., Rychak, E., Miller, K., Gaidarova, S., Ren, Y., Wang, M., Breider, M., Carmel, G., Mahmoudi, A., Jackson, P., Abbasian, M., Cathers, B. E., Schafer, P. H., Daniel, T. O., Lopez‐Girona, A., Thakurta, A., & Chopra, R.
Affiliations
  • Celgene (United States) (US)
  • Celgene Corporation San Diego USA
  • Celgene Corporation San Francisco CA USA
Corresponding author
Gandhi, Anita K.
ORCID
Venue
British Journal of Haematology
Publisher
Wiley
Published
2013-10-28
Volume
164
Issue
2
Pages
233-244
ISSN
  • 0007-1048 (ISSN-L)
  • 1365-2141
DOI
10.1111/bjh.12622
PMC
PMC4253085
OpenAlex
W2076744879
Cited by
106
References
23
Citations by year
2026: 3 · 2025: 6 · 2024: 2 · 2023: 4 · 2022: 6 · 2021: 14 · 2020: 7 · 2019: 5 · 2018: 13 · 2017: 9 · 2016: 10 · 2015: 15 · 2014: 11 · 2013: 1
FWCI
8.85
Type
journal article
Language
en
Open access
Licence
Locations
  • British Journal of Haematology (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • Celgene
  • Cancer Research UK (11566)
Topics
  • Health Sciences › Medicine › Hematology › Multiple Myeloma Research and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
Keywords
cereblon, lenalidomide, pomalidomide, immunomodulators, multiple myeloma, predictive biomarkers, lenalidomide resistance, mRNA-protein correlation, assay standardization
MeSH terms
Antibodies, Monoclonal, Antibody Specificity, Humans, Multiple Myeloma, Peptide Hydrolases, Thalidomide, Biomarkers, Tumor, Alternative Splicing, Drug Resistance, Neoplasm, Ubiquitin-Protein Ligases, Cell Line, Tumor, Adaptor Proteins, Signal Transducing, RNA Isoforms

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