Abstract
The immunomodulatory drug (IMiD) thalidomide and its derivatives lenalidomide and pomalidomide are therapeutic agents used in the treatment of multiple myeloma. Although pomalidomide offers considerable clinical benefits to patients with lenalidomide-resistant multiple myeloma, the molecular mechanisms underlying its superior efficacy remain unclear. Here we show that ARID2, a component of the polybromo-associated BAF (PBAF) chromatin-remodeling complex, is a pomalidomide-induced neosubstrate of CRL4CRBN. BRD7, another subunit of PBAF, is critical for pomalidomide-induced ARID2 degradation. ARID2 is involved in transcriptional regulation of pomalidomide target genes including MYC. Pomalidomide is more effective than lenalidomide in degrading ARID2 and is capable of inhibiting MYC expression and proliferation in lenalidomide-resistant cell lines. Notably, ARID2 expression is associated with a poor prognosis and is higher in chemoresistant minimal residual disease (MRD) populations, and in patients with relapsed/refractory multiple myeloma. These findings suggest that ARID2 is a promising target for overcoming lenalidomide resistance in patients with multiple myeloma.
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Data availability
RNA-seq data are available at Gene Expression Omnibus (GEO) (accession no. GSE126463). Source data are provided with this paper.
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Acknowledgements
We thank J. Kato for technical assistance. We thank K. Kataoka for useful discussion. This work was supported by JSPS KAKENHI (grant nos. 17H06112 to H.H. and Y.Y., 17H04213 and 18H05502 to T.I. and 15K18418 and 17K14996 to J.Y.). This work was also supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (no. S1411011 to H.H.) and by PRESTO (no. JST JPMJPR1531 to T.I.).
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Contributions
J.Y. performed most of the experiments. T.S. prepared recombinant proteins and performed biochemical experiments. T.I. prepared a number of DNA constructs, cell lines and antibodies. Y.M. and H.M. performed proliferation analysis and prepared DNA constructs. S.T. contributed knockdown experiments. T.K. performed yeast two-hybrid screening. S.M. performed immunohistochemistry. J.Y., T.I., T.A.-O., N.S., M.K., Y.Y. and H.H. interpreted data. J.Y., T.I., Y.Y. and H.H. planned the study. J.Y., Y.Y. and H.H. wrote the manuscript. Y.Y. and H.H. supervised the research.
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The authors declare the following competing interests: H.H. received research support from Celgene/Bristol Myers Squibb.
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Extended data
Extended Data Fig. 1 Evidence that ARID2 is ubiquitinated and degraded by CRL4CRBN in a pomalidomide-dependent manner.
a, Schematic representation of yeast two-hybrid screening. b–e, Immunoblot analysis of MM.1S cells treated with DMSO or the indicated concentration of thalidomide (Thal, b) or pomalidomide (Pom, c-e) for 36 h. f, qRT-PCR analysis of MM.1S cells treated with DMSO or 0.2 µM pomalidomide for the indicated time. Expression data were normalized to the level of DMSO-treated MM.1S cells and represent mean ± s.d. (n = 3, biologically independent replicates). g, Immunoblot analysis of MM.1S cells expressing shRNA #2 against CRBN. Cells were treated with DMSO or 1 µM pomalidomide for 36 h prior to harvest. h, Immunoblot analysis of OPM2 cells treated with DMSO or the indicated concentrations of pomalidomide for 72 h. Where indicated, 1 µM MLN4924 was added 24 h prior to harvest. i, Immunoblot analysis of OPM2 cells expressing shRNA #1 against CRBN. Cells were treated with DMSO or 1 µM pomalidomide for 72 h. j, In vivo ubiquitination assay of Flag-tagged ARID2. WT or CRBN−/− 293T cells transiently expressing the indicated proteins were treated with DMSO or 10 µM pomalidomide for 18 h. Where indicated, 10 µM MG132 was added 4 h prior to harvest. k, In vivo ubiquitination assay. MM.1S cells expressing exogenous CRBN in a doxycycline-dependent manner were treated with 10 µM doxycycline for 72 h or left untreated. Then, 1 µM pomalidomide was added indicated prior to harvest. Ubiquitinated proteins were captured using agarose-TUBE1 and subjected to immunoblotting. l, In vitro ubiquitination assay of V5-tagged ARID2 expressed in 293T cells. Co-expressed E3-substrate protein complexes were purified using anti-Flag beads in the presence or absence of 30 µM pomalidomide and incubated with E1, E2, and ubiquitin with or without ATP. V5-ARID2 was then isolated using anti-V5 beads and subjected to immunoblotting to visualize ubiquitinated ARID2 using anti-ubiquitin antibody. Data in b–e and g–l were independently repeated twice with similar results.
Extended Data Fig. 2 Roles of ARID2 and Ikaros/Aiolos in the growth and gene expression of multiple myeloma cell lines.
a, Proliferation (left) and immunoblot (right) analyses of MM.1S cells expressing shRNA against Ikaros. Cells were subjected to immunoblotting 3 days post infection of shRNA expression vectors. Data represent mean ± s.d. (n=3, biologically independent replicates). b,c, Proliferation (left) and immunoblot (right) analyses of OPM2 (b) and H929 (c) cells expressing shRNA against ARID2. Cells were subjected to immunoblotting 3 days (b) or 2 days (c) post infection of shRNA expression vectors. Data represent mean ± s.d. (n=3, biologically independent replicates). d, Analysis of the effect of Aiolos knockdown on ARID2 mRNA (left, 72 h) and protein (right) levels in MM.1S. The Aiolos and beta-actin blots shown here are identical to those shown in Fig. 2b. e, Heat map showing log10-scaled p-values. Fisher’s exact test (two-sided) was used to compare genes up- or down-regulated by pomalidomide treatment, ARID2 knockdown, or Aiolos knockdown (related to Fig. 2c). f, 1,300 pomalidomide-affected genes were grouped into seven clusters based on their expression values are shown as a heat map (related to Fig. 2d). Data in a–d were independently repeated twice with similar results.
Extended Data Fig. 3 Detailed analysis of GSEA.
a, Overview of GSEA results of interferon gamma signaling. Proportions highlighted in magenta indicate the fractions of core enrichment genes (genes that contribute most to the gene set’s enrichment) of pomalidomide treatment. Cyan rectangles indicate gene sets that contain core enrichment genes of ARID2 knockdown. b, GSEA using the Reactome pathway database. Normalized enrichment scores of pathways negatively correlated with pomalidomide treatment are shown as a heat map. c, Visualization of the results of Gene Ontology (GO) analysis using Cytoscape. Nodes indicate GO terms that were negatively correlated with pomalidomide treatment (FDR q<0.01). Cyan-edged nodes indicate GO terms that were also enriched in ARID2 knockdown but not in Aiolos knockdown. Green-edged nodes indicate GO terms that were also enriched in Aiolos knockdown but not in ARID2 knockdown. Yellow-edged nodes indicate GO terms that were enriched in all three perturbations. d, Heat map showing expression changes of cyclins (left panel) and mitosis-related genes (right panel).
Extended Data Fig. 4 Evidence supporting the trimeric interactions among BRD7, ARID2, and CRBN.
a, Co-immunoprecipitation analysis of endogenous proteins in OPM2 cells. Cells were treated with DMSO or 10 µM pomalidomide together with 5 µM MG132 for 3 h prior to harvest. b, 293T cells transiently overexpressing the indicated proteins were treated with DMSO or 10 µM pomalidomide (Pom) in the presence or absence of 1 µM MLN4924 for 18 h prior to harvest for anti-Flag immunoprecipitation. c, Co-immunoprecipitation analysis using full-length CRBN and its deletion mutants. 293T cells transiently overexpressing Flag-CRBN or one of its deletion mutants and V5-BRD7 were subjected to anti-Flag immunoprecipitation 48 h post transfection. d, Co-immunoprecipitation analysis using full-length (FL) ARID2 and its internal deletion mutant lacking zinc-finger motif (ΔZNF). 293T cells transiently overexpressing Flag-CRBN, V5-BRD7, and the indicated Halo-ARID2 proteins were treated with DMSO or 10 µM pomalidomide (Pom) together with 1 µM MLN4924 for 18 h prior to harvest for anti-Flag immunoprecipitation. e, Co-immunoprecipitation analysis using full-length (FL) ARID2 and its C-terminal deletion mutants. f, The summary of the results in d and e. ARID, AT-rich interaction domain; ZNF, zinc-finger motif. Data in a–e were independently repeated twice with similar results.
Extended Data Fig. 5 Evidence that BRD7 directly interacts with CRBN while ARID2 interacts with CRBN in BRD7- and pomalidomide-dependent manners.
a, Pull-down assays using recombinant (rec.) proteins expressed in insect cells. Recombinant PBAF subunits, purified using Ni-NTA agarose, were incubated with recombinant Flag-tagged CRBN (fCRBN) immobilized to Flag M2 agarose. After several washing steps, input and bound proteins were analyzed by immunoblotting. b, Pull-down assays were carried out using recombinant proteins as in a in the presence or absence of 100 µM pomalidomide (Pom). c, Recombinant Flag-CRBN expressed alone or co-expressed with BRD7 in insect cells was immobilized to anti-Flag beads and then incubated with recombinant ARID2 in the presence or absence of 100 µM pomalidomide (Pom). Data in a–c were independently repeated twice with similar results.
Extended Data Fig. 6 Evidence that BRD7 mediates pomalidomide-induced ARID2 degradation and growth inhibition of OPM2 cells.
a, The effect of the MDM2 inhibitor nutlin-3a on myeloma cell growth. MM.1S and OPM2 cells were treated with DMSO or 10 µM nutlin-3a for 48 h and then subjected to proliferation assay. Data were normalized to the values of DMSO-treated cells and represent mean ± s.d. (n=3, biologically independent replicates). b, The effect of the topoisomerase II inhibitor etoposide on myeloma cell growth. OPM2 cells expressing shRNA #3 against BRD7 or control OPM2 cells were treated with 50 µM etoposide for 12 h. Then, BrdU incorporation was measured. Data were normalized to the values of undamaged cells and represent mean ± s.d. (n=3, biologically independent replicates). c,d, Proliferation analysis of MM.1S (c) and OPM2 (d) cells. Lentiviruses expressing shRNAs against BRD7 were infected on day 0, and proliferation assays were carried out on days 3, 5, and 7. Data were normalized to the value of control cells at each time point and represent mean ± s.d. (n=3, biologically independent replicates). e, Co-immunoprecipitation analysis using full-length BRD7 and its deletion mutants. 293T cells transiently overexpressing CRBN, Myc-ARID2, and the indicated Flag-BRD7 proteins were subjected to anti-Flag immunoprecipitation 48 h post transfection. The results are summarized in the right panel. NLS, nuclear localization signal; BD, bromodomain. f,g, BRD7 knockdown and rescue experiments. OPM2 cells expressing RNAi-resistant full-length BRD7 or its deletion mutant deficient in CRBN binding (Δ400–480) were infected with lentivirus expressing shRNA #3 against BRD7. The resulting cells were treated with DMSO or 1 µM pomalidomide (Pom) and subjected to proliferation assay on days 3, 4, and 6 (f) and to immunoblotting on day 3 (g). Data were normalized to the values on day 0 and represent mean ± s.d. (n=3, biologically independent replicates). Data in e and g were independently repeated twice with similar results.
Extended Data Fig. 7 Evidence for non-overlapping functions of the BRD7/ARID2 and Ikaros/Aiolos axes.
a, qRT-PCR analysis of MYC and IRF4 in MM.1S and OPM2 cells. Cells were harvested 62 h post infection of lentivirus expressing shRNA against ARID2 or Aiolos. Data were normalized to the values of control cells and represent mean ± s.d. (n=3, biologically independent replicates). b, qRT-PCR analysis of MYC and IRF4 in OPM2 cells treated with or without 1 µM pomalidomide for 48 h. Data were normalized to the values of non-treated cells and represent mean ± s.d. (n=3, biologically independent replicates). c,d, Time-course analysis of MYC and IRF4 downregulation at the protein level in MM.1S cells expressing shRNA against ARID2 (c), Aiolos (d, left), or Ikaros (d, right). MM.1S cells infected with lentivirus expressing the indicated shRNA were incubated for the indicated time and then harvested for immunoblotting. The ARID2 and b-actin blots shown in panel c are identical to that shown in Fig. 2b. e,f, MM.1S cells were harvested for immunoblot analysis at the indicated time post infection of indicated shRNA expression vectors. (g) A schematic showing how OPM2 cells expressing wild-type (WT) or IMiD-resistant (GA) Ikaros and Aiolos were established. Ikaros and Aiolos were coexpressed as a proteolytically cleavable fusion protein. h,i, The effect of blocking the Ikaros/Aiolos axis on pomalidomide-induced growth inhibition. OPM2 cells coexpressing wild-type (WT) or IMiD-resistant (GA) Ikaros and Aiolos were treated with DMSO or 10 µM pomalidomide (Pom) for 7 days and then subjected to proliferation assay (n=2, biologically independent replicates) (h). Alternatively, the number of viable cells was counted on days 5, 9, and 14 (n=3, biologically independent replicates) (i). Data in c–f were independently repeated twice with similar results.
Extended Data Fig. 8 Evidence that ARID2 is important for MYC-dependent transcriptional regulation in MM.1S cells.
a, Expression changes of the MYC-activated genes by ARID2 knockdown in MM.1S cells were analyzed by GSEA. b, Heat map showing expression changes of 10 representative MYC-activated genes caused by pomalidomide treatment, ARID2 knockdown, or Aiolos knockdown in MM.1S cells. c, Pomalidomide-induced expression changes of 10 MYC-activated genes with individual data points. Data represent mean ± s.d. (n=3, biologically independent replicates).
Extended Data Fig. 9 Evidence supporting the role of PBAF subunits in the prognosis of multiple myeloma patients.
a, Survival analysis of multiple myeloma patients (GSE57317) using probe sets for PBAF subunits other than those used in Fig. 5a (top) and using representative probe sets for Ikaros, Aiolos, and IRF4 (bottom). P-values were calculated by log rank test (two-sided). b, Survival analysis of different multiple myeloma patients (GSE2658, n=351) using representative probe sets for PBAF subunits. P-values were calculated by log rank test (two-sided). c, Hematoxylin-eosin staining of five paired samples (the first diagnosis and relapse) from multiple myeloma patients (related to Fig. 5e). The scale bars indicate 100 µm. d, Validation of antibody specificity of anti-ARID2 used for immunohistochemical analysis in Fig. 5e. Control OPM2 cells and OPM2 cells expressing shRNA #1 against ARID2 were subjected to immunofluorescence staining. The scale bars indicate 30 µm. This experiment was performed only once. e, Semi-quantitative analysis of ARID2 signals using IHC profiler. For boxplot, each center line, box limits, whiskers, and points represent median, upper and lower quartiles, the maximum or minimum value within 1.5x interquartile range from hinges, and outliers, respectively.
Extended Data Fig. 10 Mixed effects of IMiD drugs and ARID2, Aiolos, or Ikaros knockdown on five multiple myeloma cell lines.
a, Proliferation assay of five multiple myeloma cell lines (MM.1S, OPM2, RPMI8226, U266B1, and KMS12PE) treated with DMSO or the indicated concentrations of lenalidomide (Len) or pomalidomide (Pom) for 5 days. Data were normalized to the values of DMSO-treated cells and represent mean ± s.d. (n=3, biologically independent replicates). b, Proliferation assay (left) and immunoblotting (right) of RPMI8226 expressing shRNAs against CRBN. For proliferation assay, DMSO or 1 µM pomalidomide was added 2 days post infection of shRNA expression vectors, and proliferation assay was performed 7 days post infection. Data were normalized to the values of DMSO-treated cells and represent mean ± s.d. (n=3, biologically independent replicates). For immunoblotting, cells were treated with DMSO or 1 µM pomalidomide for 72 h prior to harvest. c, Dose-response analysis of lenalidomide-induced ARID2 degradation. RPMI8226 cells treated with DMSO or the indicated concentrations of lenalidomide or pomalidomide for 3 days were subjected to immunoblotting. d, Co-immunoprecipitation analysis in 293T cells. Cells transiently overexpressing the indicated proteins were treated with DMSO or the indicated concentrations of lenalidomide or pomalidomide together with 1 µM MLN4924 for 18 h. (e and f) Immunoblot analysis of the five cell lines expressing shRNAs against Aiolos (e) or Ikaros (f). Cells were harvested 4 days post infection of shRNA expression vectors. g, Proliferation assay of the five multiple myeloma cell lines expressing shRNAs against ARID2, Aiolos, or Ikaros. Proliferation assay was performed on days 2, 5, and 7 post infection of shRNA expression vectors. Data were normalized to the values of control knockdown cells at each time point and represent mean ± s.d. (n=3, biologically independent replicates). h, Heat map showing GR values of the five cell lines expressing shRNAs against Ikaros. GR values were calculated from the results of proliferation assay at days 2 and 7. Data in b–f were independently repeated twice with similar results.
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Yamamoto, J., Suwa, T., Murase, Y. et al. ARID2 is a pomalidomide-dependent CRL4CRBN substrate in multiple myeloma cells. Nat Chem Biol 16, 1208–1217 (2020). https://doi.org/10.1038/s41589-020-0645-3
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DOI: https://doi.org/10.1038/s41589-020-0645-3
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