Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells

Abstract

Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.

Fig. 1

Proteomic analysis of lenalidomide-induced changes in ubiquitination, protein abundance and CRBN interaction in MM1S cells. (A) Experimental design for SILAC-based assessment of global changes in ubiquitination and protein levels. Cells were treated for 12 hours with DMSO, lenalidomide, or thalidomide. For ubiquitination analysis 5μM MG132 were added for the last 3 hours. (B) Log₂ ratios for individual K-ε-GG sites of lenalidomide versus DMSO treated cells for replicate 1 and 2. Each dot represents a unique K-ε-GG site. (C) Log₂ ratios of changes of protein abundance of lenalidomide versus DMSO treated cells. Each dot represents a distinct protein group. (D) CRBN interaction analysis in cells treated for 6 hours with 1 μM lenalidomide. Scatter plot shows log₂ changes of proteins pulled down by HA-CBRN in lenalidomide versus DMSO treated control cells.

Fig. 2

Effect of lenalidomide on IKZF1 and IKZF3 protein levels. (A) 293T cells transfected with vectors expressing the indicated cDNA fused to firefly luciferase and control renilla luciferase were treated with DMSO or 1 μM lenalidomide for 24 hours. Bars represent the firefly to renilla luciferase ratio, normalized to DMSO-treated cells. (B) Effects of lenalidomide on endogenous IKZF1 and IKZF3 in MM1S cells treated for 24 hours. (C) Time course of lenalidomide treatment in MM1S cells for IKZF1 and IKZF3 protein levels and (D) mRNA levels. (E) Primary multiple myeloma samples were treated for 6 hours and analyzed by immunoblot. (H) In vivo ubiquitination analysis of HA-tagged IKZF1 and IKZF3 expressed in MM1S cells treated for 1.5 hours with 100 nM Epoxomicin and the indicated concentrations of lenalidomide. The FK2 antibody detects covalently linked ubiquitin.

Fig. 3

CRBN is a substrate receptor for IKZF1 and IKZF3. (A) Immunoprecipitiation of endogenous CRBN in MM1S cells treated for 1 hour with the indicated drugs. (B) In vitro ubiquitination reaction of HA-IKZF3 co-immunoprecipitated by FLAG-CRBN from 293T cells and incubated in the presence or absence of E1 and E2 ubiquitin conjugating enzymes. (C) Mapping of the degron that confers lenalidomide sensitivity. Blue boxes in the IKZF3 protein represent zinc finger domains. (D) Sequence alignment of the core lenalidomide degron between the 5 Ikaros proteins. Western blots of 293T cells lysates 48 hours after co-transfection of FLAG-tagged IKZF3 or IKZF4 with HA-tagged CRBN and 24 hours drug treatment.

Fig. 4

Biological role of IKZF1 and IKZF3 in multiple myeloma cell lines and T cells. (A) Lenalidomide-sensitive and insensitive cell lines were infected with lentivirus expressing IKZF1 or IKZF3 shRNAs and GFP. Relative depletion was assessed by flow cytometry and normalized to day 2 post-infection. (B) MM1S cells were transduced with retrovirus expressing GFP and wild-type IKZF3 or a dominant negative IKZF3 isoform with deletion of the complete DNA binding region. (C) MM1S cells were infected with retrovirus expressing IKZF3^Q147H/GFP or IKZF3^wt/dTomato and competed against each other in media containing DMSO or lenalidomide. (D) Human CD3+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 and treated with different concentrations of lenalidomide for 24 hours. (E+F) T cells were infected with lentiviral vectors expressing shRNAs targeting the indicated genes. After selection with puromycin, T cells were stimulated with anti-CD3/CD28 Dynabeads and treated with DMSO or 1 μM lenalidomide for 12 hours before lysis. IL-2 RNA expression levels were analyzed by quantitative RT-PCR using GAPDH expression as an internal control.