Rate of CRL4(CRBN) substrate Ikaros and Aiolos degradation underlies differential activity of lenalidomide and pomalidomide in multiple myeloma cells by regulation of c-Myc and IRF4

Abstract

Recent discoveries suggest that the critical events leading to the anti-proliferative activity of the IMiD immunomodulatory agents lenalidomide and pomalidomide in multiple myeloma (MM) cells are initiated by Cereblon-dependent ubiquitination and proteasomal degradation of substrate proteins Ikaros (IKZF1) and Aiolos (IKZF3). By performing kinetic analyses, we found that the downregulation or proteasomal degradation of Ikaros and Aiolos led to specific and sequential downregulation of c-Myc followed by IRF4 and subsequent growth inhibition and apoptosis. Notably, to ensure growth inhibition and cell death, sustained downregulation of Ikaros and Aiolos, c-Myc or IRF4 expression was required. In addition, we found that the half-maximal rate, rather than the final extent of Ikaros and Aiolos degradation, correlated to the relative efficacy of growth inhibition by lenalidomide or pomalidomide. Finally, we observed that all four transcription factors were elevated in primary MM samples compared with normal plasma cells. Taken together, our results suggest a functional link between Ikaros and Aiolos, and the pathological dysregulation of c-Myc and IRF4, and provide a new mechanistic understanding of the relative efficacy of lenalidomide and pomalidomide based on the kinetics of substrate degradation and downregulation of their downstream targets.

Figure 1

Ikaros, Aiolos, c-Myc and IRF4 are upregulated simultaneously in primary MM samples compared with normal bone marrow. (a) Microarray analysis of public data set GSE6477 showing the relative expression levels of IKZF1, IKKZF3, c-Myc and IRF4 in normal (n=15), monoclonal gammopathy of undetermined significance (MGUS, n=21), SMM (n=23), newly diagnosed MM (NDMM, n=75) and relapsed/refractory MM (RRMM, n=28). (b) Dual-stained (CD138⁺, red; target protein, brown) immunohistochemical analysis of normal or MM bone marrow tissue for Ikaros, Aiolos, c-Myc or IRF4. (c) Compiled H-score analysis (see Materials and Methods) comparing normal (n=10) versus MM (n=24) bone marrow tissue microarrays for Ikaros, Aiolos, c-Myc or IRF4 by immunohistochemical assays represented in b. The Student's unpaired t-test was used to determine statistical significance, where *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Figure 2

shRNA-mediated knockdown of IKZF1 or IKZF3 leads to the downregulation of c-Myc and IRF4. Decreased expression of Ikaros (a) or Aiolos (b) in stably transduced MM1.S and U266 MM cells after DOX induction (0.001–1 μg/ml) for 48 h of shRNAs targeting Ikaros (shIKZF1), Aiolos (shIKZF3) or luciferase as control (shLUC). (c) Western blot analysis of Ikaros, Aiolos, c-Myc and IRF4 in MM1.S shIKZF1 or shIKZF3 cells, which were cultured in the absence or the presence of DOX (Dox0.01 μg/ml), for 4 consecutive days (D1–D4). Asterik (*) by Ikaros indicates that only the bottom band was affected using this particular antibody (also see Materials and Methods).

Figure 3

shRNA-mediated knockdown of IKZF1 or IKZF3 inhibits proliferation and induces apoptosis in MM cells. (a) Cell population growth chart of MM1.S (left panel) or U266 (right panel) following DOX-induced shRNA knockdown of either IKZF1 or IKZF3 as compared with Vehicle (Veh) or shLUC control. (b) Apoptosis measured by flow cytometry using Annexin-V (AnnV⁺; x axis) and ToPro3⁺ (y axis) staining following either Veh or DOX (Dox, 0.01 μg/ml) treatment of shLUC (left panel), shIKZF1 (middle panel) or shIKZF3 (right panel). MM1.S cells on day 1 (top row), 3 (middle row) and 5 (bottom row). Figures shown are representatives of n=3 experiments. Prominent increases in apoptotic cell numbers compared with shLUC or uninduced Veh control samples are indicated in red as % of total cell numbers representing the sum of the three quadrants enriched for apoptotic cells.

Figure 4

DOX washout of shRNA knockdown of IKZF1 or IKZF3 but not c-MYC or IRF4 leads to a partial rescue of their respective anti-proliferative effects in MM cells. (a) Western blot analysis showing the relative expression of Ikaros, Aiolos, c-Myc and IRF4 in MM1.S shIKZF1 cells cultured with or without DOX (Dox, 0.01 μg/ml) for 4 consecutive (D4) days in five distinct cultures marked with superscripts 1 through 5 (see Results section for details). The five parallel cultures consisted of culture 1 (−DOX), culture 2 (+DOX), culture 3 (DOX was washed out after day 1 (+DOX/WashD1)), culture 4 (DOX was washed out after day 2 (+DOX/WashD2)), or culture 5 (DOX was washed out after day 3 (+DOX/WashD3)), followed by continuous culture up to 4 days while the samples were collected each day (D1^1–5 through D4¹⁻⁵). Maximum protein reductions are visualized by D4 (red arrow), or partial knockdown (green arrow) following washout after D1. (b) Measurement of proliferation by ³H-thymidine incorporation in MM1.S shIKZF1 cells in parallel samples to the experiment (see Results) described in part (a). Similar DOX-washout experiments as described in a followed by measurement of ³H-thymidine incorporation similar to part b were performed with shIKZF3 (c), shMYC (d) or shIRF4 (e) MM1.S cells.

Figure 5

Temporal kinetics of shRNA-induced knockdown of Ikaros or Aiolos protein followed by downregulation of c-Myc and then IRF4 protein levels. Quantified protein expression levels from western blot analysis of Ikaros, Aiolos, c-Myc and IRF4 in MM1.S shIKZF1 (a) or shIKZF3 (b) cells, which were cultured in the absence or the presence of DOX (Dox, 0.01 μg/ml) for the indicated time points (24–96 h) were normalized to Actin levels as a loading control and plotted as a function of time following DOX-induced knockdown.

Figure 6

Differential kinetics of lenalidomide- or pomalidomide-induced degradation of Ikaros or Aiolos, followed by downregulation of c-Myc and IRF4. (a) Western blot analysis showing the reduction of Ikaros and Aiolos protein levels in U266 cells following treatment with increasing concentrations of either lenalidomide or pomalidomide for 6 h, which is abrogated in the presence of MG-132. (b) The data in a were quantified by densitometry, followed by normalization to the Actin loading control and graphed as fraction affected versus drug concentration. (c) U266 cells were treated with either vehicle (Veh), lenalidomide (L1 or L10 μM) or pomalidomide (P1 or P10 μM) for 0.5, 1, 2, 4, 8, 24, 48 and 72 h, and protein lysates were analyzed by western blotting for Ikaros, Aiolos, c-Myc and IRF4. (d) The data in c were quantified by densitometry, followed by normalization to the Actin-loading control and transformed into fraction affected. These data were then fit to a rectangular hyperbolic model (see Materials and Methods) to determine the time point at which 50% (T_1/2) or the maximal amount (Max_red) of the relative input protein amount is either degraded, as in the case of Ikaros or Aiolos, or downregulated, as for c-Myc or IRF4 in lenalidomide (10 μM) (d) or pomalidomide treated (1 μM) (e) in U266 cells. Calculated T_1/2 (f) or Max_red (g) of Ikaros or Aiolos from either lenalidomide-treated (L1 or L10 μM) or pomalidomide-treated (P01 or P1 μM) cells plotted against the relative growth inhibition after 72 h for the indicated drug treatment.

Figure 7

Graphical model explaining the potential mechanism of lenalidomide- or pomalidomide-negative regulation of the c-Myc/IRF4 axis. Two possible explanations for the negative regulation of the c-Myc/IRF4 axis either directly (left panel) or indirectly (right panel). For direct regulation, Ikaros/Aiolos are bound as cis-acting elements in a generic activating complex (green shape; that is, switch/sucrose nonfermentable (SWI/SNF)), followed by the lenalidomide- or pomalidomide-induced degradation of Ikaros/Aiolos and subsequent blockade of c-Myc/IRF4 transcription. For indirect regulation, Ikaros/Aiolos are bound as trans-acting elements in a generic repressive complex (red shape; that is, nucleosomal remodeling deacetylase (NuRD)) on an intermediate repressor of c-Myc/IRF4, followed by lenalidomide- or pomalidomide-induced degradation of Ikaros/Aiolos, expression of the intermediate repressor and subsequent blockade of c-Myc/IRF4 transcription.