Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide

Abstract

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN-DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN(YW/AA). Overexpression of CRBN wild-type protein, but not CRBN(YW/AA) mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21(WAF-1) expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Figures

Figure 1

Binding of lenalidomide and pomalidomide to CRBN within the CRBN–DDB1 complex. (a) Fluorescence-based thermal shift assay of binding of lenalidomide (▪), pomalidomide (▴) and thalidomide (○) but not pthalimide (•) to a recombinant human CRBN–DDB1 complex. Representative curves from one of two independent experiments with similar results. (b) Structures of compounds utilized in these studies and thalidomide analog magnetic affinity beads (FG beads). The glutarimide moiety in each compound is highlighted in red. (c) Immunoblot analyses of rabbit anti-hCRBN and mouse anti-hDDB1 from U266 myeloma cell extract bound to thalidomide analog beads; In=U266 total protein input before affinity bead binding; DMSO=U266 extract control (1% DMSO preincubation); Len=U266 extract preincubated with lenalidomide (100 μℳ), Pom=U266 extract preincubated with pomalidomide (100 μℳ). Fluorescent labeled donkey anti-mouse antisera (green, DDB1) and goat anti-rabbit (red, CRBN) were used to identify proteins. Representative immunoblot from at least five independent experiments with similar results. (d) Immunoblot analysis of HEK293T extracts incubated with thalidomide analog affinity beads and eluted with 1 mℳ of thalidomide, phthalimide or glutarimide. Input extract before bead purification =In. (e) Immunoblot analysis of HEK293T extracts pre-incubated with thalidomide (Thal), lenalidomide (Len) or pomalidomide (Pom) at indicated concentrations and bound to thalidomide analog affinity beads, washed and eluted with SDS buffer as described in Materials and Methods. Input extract before bead purification (In) also shown in the immunoblot. (f) CRBN binding dose–response to lenalidomide in U266 extracts; Insert shows immunoblot of CRBN of dose–response competition of U266 extract CRBN by preincubation of varying concentrations of lenalidomide. (g) U266 extract CRBN binding dose–response of pomalidomide. Insert shows immunoblot of CRBN of dose–response competition of U266 extract CRBN by preincubation with varying concentrations of pomalidomide. Data for Figure 1f and g are representative of two similar dose–response experiments with IC50 values of ∼1 and ∼2 μℳ, respectively.

Figure 2

Reduction of CRBN gene expression in U266 myeloma cells modulates antiproliferative response to lenalidomide and pomalidomide. (a) Lenalidomide dose–response inhibition of proliferation parental U266 versus lentiviral 60 and 75% CRBN-reduced cell lines. All points are average of quadruplicates. CRBN immunoblot of parental U266 cells and two clones of lentiviral transduced CRBN short hairpin RNA cells (insert Figure 2a). (b) Pomalidomide dose–response inhibition of proliferation of parental versus lentiviral 60 and 75% CRBN-reduced cell line. All points are average of quadruplicates. (c) Immunoblot analysis of CRBN, p21WAF-1 IRF4 and β-actin protein expression in U266-CRBN60 and U266-CRBN75 treated with DMSO (0.1%), 1 μℳ pomalidomide (Pom) or 10 μℳ lenalidomide (Len) for 48 h. (d) Percent change in expression of p21 (green bars) and IRF4 (red/orange bars) proteins from compound-treated cells to control-treated baselines were determined by densitometry of bands relative to β-actin in parental U266 and CRBN75 cells (as indicated in graph).

Figure 3

Expression of FH-CRBNYW/AA prevents pomalidomide, lenalidomide or thalidomide inhibition of CRBN autoubiquitination in HEK293 cells and pomalidomide induction of protein changes in KMS12 cells. (a) Pomalidomide, lenalidomide and thalidomide inhibit autoubiquitination of CRBN. HEK293T cells stably expressing FLAG-HA CRBN (FH)-CRBN were established by lentiviral transduction. Cells were treated with the indicated concentrations of compounds for 3 h before harvest. FH-CRBN was immunoprecipitated (IP) with anti-FLAG antibody under stringent wash conditions and immunoblotted (IB) with anti-HA antibody. (b) HEK293T cells stably expressing FLAG-HA CRBN or FH-CRBNYW/AA were treated and processed as in Figure 3a. (c) Cartoon of CRBN E3 ligase complex. CRBN is bound to the adapter protein DDB1 that binds to Cul4A a scaffold protein for Roc1 and the E2 ubiquitin-conjugating enzyme. E2 adds ubiquitin protein either to CRBN itself (autoubiquitination) or to a putative substrate protein (S) that binds CRBN. X marks the inhibition of autoubiquitination of CRBN by thalidomide, lenalidomide or pomalidomide. (d) KMS12 cells constitutively expressing FH-CRBN or FH-CRBNYW/AA were established by lentiviral transduction and expression of CRBN determined in whole-cell extracts by immunoblot analysis with mouse anti-CRBN. (e) Control KMS12 cells (−) or cells stably expressing wild-type FH-CRBN or FH-CRBNYW/AA were treated with the indicated concentrations of pomalidomide and incubated for 48 h before extract preparation, SDS–PAGE and immunoblotting for c-myc or IRF4. (f) Control KMS12 cells or cells stably expressing FH-CRBNYW/AA were treated with the indicated concentrations of pomalidomide and incubated for 48 h before extract preparation, SDS–PAGE and immunoblotting for p21WAF-1 (p21).

Figure 4

Effects of S and R enantiomers of methyl-pomalidomide on T-cell cytokine production and U266 cell CRBN binding to thalidomide analog affinity beads. (a) Structures of S methyl-pomalidomide and R methyl-pomalidomide. (b) T cell activation of interleukin-2 production by S or R methyl-pomalidomide enantiomers; (?) S methyl-pomalidomide (Me-PomS) (•) R methyl-pomalidomide (Me-PomR). (c) CRBN immunoblot quantitation of S or R methyl-pomalidomide competition for thalidomide analog bead binding to CRBN from U266 extracts; (?) S methyl-pomalidomide (•) R methyl-pomalidomide; (d) Immunoblot of T cell CRBN protein reduction by siCRBN treatment for 72 h. (e) Activated T-cell interleukin-2-induction and (f) tumor necrosis factor-α-induction by lenalidomide and pomalidomide at the indicated concentrations in the presence of control siRNA or CRBN siRNA for 72 h.

Figure 5

Prolonged exposure of myeloma cells in culture to high-dose lenalidomide induces resistance to antiproliferative effect of lenalidomide correlating with decreases in CRBN. (a) Time course of acquisition of lenalidomide resistance in H929 cells and concurrent reduction in CRBN mRNA determined by reverse transcription--PCR (data for one clone R10-4 is shown, representative of generation of four independent lenalidomide resistant cells lines, H929 R10-1, R10-2, R10-3 and R10-4 ). H929 cells were treated with control (final 0.1% DMSO) (D) or lenalidomide (1 μℳ) (L1) for 2 months until the proliferation of cells was no longer inhibited by 1 μℳ lenalidomide. The lenalidomide treated cultures were subsequently treated with 10 μℳ lenalidomide for a further 4 months to fully establish resistance to high-dose lenalidomide (30 μℳ). Arbitary units for CRBN mRNA are shown. (b) Immunoblot of CRBN in H929 cells treated with DMSO for 6 months (H929/D1) and H929 lenalidomide-resistant cells lines (R10-1, R 10-2, R 10-3 and R 10-4). (c) Dose-dependent inhibition of proliferation of parental H929 cells (•) and H929/D1 cells grown continuously in 0.1% DMSO (▪) cells by lenalidomide. Lack of inhibition of proliferation by lenalidomide, up to 10 μℳ concentration, of four H929 cell lines (R10-1, R10-2, R10-3, R10-4; ○, □,∇, ◊) made resistant to high-dose lenalidomide over 6 months in culture. (d) Dose-dependent inhibition of proliferation of parental H929 cells (•) and H929/D1 cells grown continuously in 0.1% DMSO (▪) cells by pomalidomide. Dose-dependent inhibition of proliferation by pomalidomide of four H929 cell lines made resistant to high-dose lenalidomide over 6 months in culture.

Figure 6

Comparison of lenalidomide and pomalidomide effects in DF15 and DF15R myeloma cells. (A) Dose-dependent inhibition of proliferation of parental DF15 cells by lenalidomide (•) and pomalidomide (▪). No proliferation inhibition of DF15R cells by lenalidomide (○) or pomalidomide (□). Insert is immunoblot analysis of protein lysates from DF15 and DF15R cells demonstrating minimal CRBN protein in DF15R compared with DF15 cells. (B) CRBN analysis in DF15 and DF15R cells by immunofluorescence confocal microscopy in the absence (panels a) or presence (panels b) of anti-CRBN blocking peptide (Supplemental Figure S2). Images were obtained using a Nikon E800 confocal microscope (Nikon Instruments, Melville, NY, USA) at a 60 × magnification. CRBN immunofluorescence is shown as green color and DAPI blue staining identifies the nucleus of cells. (C) Immunoblot of thalidomide analog affinity bead binding to CRBN in DF15 and DF15R cell extracts. Lane description in order left to right: In=DF15 input before bead purification; DMSO=DF15 extract control (1% DMSO preincubation); Len=DF15 extract preincubated with lenalidomide (100 μℳ); In=DF15R input before bead purification; DMSO=DF15R control (1% DMSO preincubation); Len=DF15R extract preincubated with lenalidomide (100 μℳ). Representative immunoblot from two independent experiments with similar results.