The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins

Abstract

Thalidomide-like drugs such as lenalidomide are clinically important treatments for multiple myeloma and show promise for other B cell malignancies. The biochemical mechanisms underlying their antitumor activity are unknown. Thalidomide was recently shown to bind to, and inhibit, the cereblon ubiquitin ligase. Cereblon loss in zebrafish causes fin defects reminiscent of the limb defects seen in children exposed to thalidomide in utero. Here we show that lenalidomide-bound cereblon acquires the ability to target for proteasomal degradation two specific B cell transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3). Analysis of myeloma cell lines revealed that loss of IKZF1 and IKZF3 is both necessary and sufficient for lenalidomide's therapeutic effect, suggesting that the antitumor and teratogenic activities of thalidomide-like drugs are dissociable.

Fig. 1. Down-regulation of IKZF1 and IKZF3 by lenalidomide

(A) Vector schematic. (B) Distribution of fold change in Fluc/Rluc ratios after lenalidomide (LEN) (2 μM) treatment. (C and D) Fluc/Rluc ratios (top panels) and immunoblots (bottom panels) of 293FT cells transfected to produce the indicated IKZF proteins fused to Fluc (top panels) or HA tag (bottom panels). Where indicated, cells were treated with lenalidomide (2 μM), MLN4924 (1 μM), or MG132 (10 μM) for 12 hours. Fluc/Rluc ratios were normalized to corresponding dimethyl sulfoxide (DMSO)–treated cells. Data are presented as mean ± SD (n = 4). (E) Immunoblot analysis of MM1S and L363 cells treated with LEN (2 μM) and MLN4924 (1 μM), as indicated, for 12 hours.

Fig. 2. Down-regulation of IKZF1 and IKZF3 by lenalidomide requires cereblon

(A) Immunoblot analysis of 293FT cells stably infected with lentiviral vectors expressing the indicated IKZF-HA proteins and a doxycycline-inducible CRBN shRNA. Where indicated, LEN (2 μM) and doxcycyline (Dox) (1 μg/ml) were added for 12 and 60 hours, respectively. (B) Fluc/Rluc ratios (top panels) and immunoblots (bottom panels) of CRBN+/+ and CRBN−/− 293FT cells transfected to produce IKZF1 fused to Fluc (top panel) or HA tag (bottom panels). Where indicated cells were treated with LEN (2 μM) for 12 hours. Fluc/Rluc ratios were normalized to corresponding DMSO-treated cells. Data are presented as mean ± SD (n = 4). (C and D) Immunoblot analysis of CRBN+/+ and CRBN–/– MM1S myeloma cells. Where indicated, cells were treated with LEN (2 μM) for 24 hours (C) or 1 hour before the addition of cyclohexamide (CHX) (100 μg/ml) for the indicated periods (D).

Fig. 3. Lenalidomide promotes ubiquitylation of IKZF1 and IKZF3 by cereblon

(A and B) FLAG-IKZF was immunoprecipitated from CRBN−/− 293FT cells stably infected to produce the indicated IKZF proteins and used to capture cereblon from CRBN+/+ 293FT cells (A) or CRBN−/− 293FT cells transfected to produce the indicated CRBN variants (B). Cells were treated with LEN (2 μM) for 12 hours before lysis, as indicated. Bound proteins were detected by immunoblot analysis. (C) Immunoblot analysis of proteins captured with nickel Sepharose from 293FT cells transfected to produce the indicated FLAG-, His-, and V5-tagged proteins. The cells were treated with MG132 (10 μM) and, where indicated, with LEN (2 μM) for 12 hours. (D) CRBN−/− 293FT cells were transfected to produce IKZF1-HA and the indicated Myc-cereblon variants and lysed. The extracts were mixed, treated with LEN (2 μM) or DMSO, and immunoprecipitated with antibodies against HA (anti-HA) or anti-Myc. The immunoprecipitates were incubated with recombinant E1, E2, and ubiquitin (Ub) and subjected to immunoblot analysis.

Fig. 4. Antimyeloma activity of lenalidomide linked to loss of IKZF1 and IKZF3

(A and B) Immunoblot analysis (A) and proliferation (B) of myeloma cell lines treated with LEN (2 μM) for the indicated periods. In (B), data are presented as mean ± SD (n = 4). (C) Change in % red fluorescent protein (RFP) positivity over time in MM1S cells infected with viruses encoding RFP and the indicated shRNAs. The day 2% RFP for each virus was normalized to 1, and subsequent values were expressed relative to cells infected with a virus encoding RFP and a control (CNTL) shRNA. (D) Immunoblot analysis of MM1S cells transiently infected with lentiviruses expressing the indicated shRNAs for 72 hours. (E) MM1S cells were infected with lentiviral vectors encoding GFP and the indicated FLAG-tagged proteins. Shown for each protein is the percentage of GFP positivity for cells treated with LEN (2 μM) for the indicated duration compared to DMSO. (F) Immunoblot analysis of MM1S cells infected as in (E) and treated with DMSO or LEN (2 μM) for 24 hours.