PLZF is a new substrate of CRBN with thalidomide and 5- hydroxythalidomide Satoshi Yamanaka1, Hidetaka Murai2, Daisuke Saito2#, Gembu Abe2, Etsuko Tokunaga3, Takahiro Iwasaki4, Hirotaka Takahashi1, Hiroyuki Takeda4, Takayuki Suzuki5, Norio Shibata3, Koji Tamura2 & Tatsuya Sawasaki1* 1Division of Cell-Free Sciences, Proteo-Science Center, Ehime University, Matsuyama, 790-8577 Japan, 2Department of Ecological Developmental Adaptability Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8578, Japan, 3Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Nagoya, 466-8555, Japan, 4Division of Proteo-Drug-Discovery Sciences, Proteo-Science Center, Ehime University, Matsuyama, 790-8577 Japan. 5Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan. #Present address. Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, 819-0395 Japan. *Corresponding Author: Tatsuya Sawasaki Proteo-Science Center, Ehime University, Matsuyama 790-8577, Japan Tel: 81-89-927-8530 Fax: 81-89-927-9941 E-mail address: [email protected]. 1 Supplementary Figures Supplementary Figure 1. Flowchart of in vitro high-throughput screening and validation assay of candidate clones a, Flowchart of in vitro high-throughput screening. b, Validation of screening using CRBN mutant. Interaction between FLAG-GST-SALL4, -PLZF, or IKZF1 and bls-CRB-WT or - CRBN-YW/AA in the presence of DMSO or 50 µM thalidomide was analysed by in vitro binding assay using AlphaScreen technology. c, In vitro binding assay using pull-down and immunoblot analysis. bls-CRBN-WT or -CRBN-YW/AA was used as bait protein, and thalidomide-dependent interactions between bls-CRBN and FLAG-GST-SALL4, PLZF, and IKZF were confirmed by procedures described in Methods. d, In vitro binding assay for ZBTB family proteins using AlphaScreen technology. FLAG-GST-ZBTB proteins (ZBTB17, ZBTB20, ZBTB38, ZBTB39, ZBTB48, and PLZF) were evaluated for thalidomide- dependent interactions with bls-CRBN by same procedures indicated in Supplementary Fig. 1b. e, In vitro binding assay for thalidomide, pomalidomide, and lenalidomide. Interaction between bls-CRBN and FLAG-GST-SALL4 in the presence of DMSO, (3.125, 6.25, 12.5, 25, 50, 100, or 200 µM) thalidomide, pomalidomide, or lenalidomide was analysed using AlphaScreen technology. All relative AS (AlphaScreen) signals were expressed as relative luminescent signal with luminescent signal of DMSO as one, and error bars mean ± standard deviation (n = 3). 2 a b c Thalidomide Pomalidomide Lenalidomide Thalidomide Pomalidomide Lenalidomide Thalidomide Pomalidomide Lenalidomide 0 1 10 50 1 10 50 1 10 50 IMiD (µM) 0 1 10 50 1 10 50 1 10 50 IMiD (µM) 0 1 10 50 1 10 50 1 10 50 IMiD (µM) (kDa) (kDa) (kDa) 100 100 75 IB: AGIA (PLZF) IB: PLZF IB: PLZF 75 75 150 IB: FLAG (CRBN) 50 IB: CRBN IB: SALL4 50 100 IB: a-TuBulin IB: a-TuBulin 50 50 50 IB: CRBN 150 HEK293T cells IB: AGIA (SALL4) IB: a-TuBulin 50 100 IB: FLAG (CRBN) HuH7 cells 50 IB: a-TuBulin 50 d e CRBN-WT CRBN-YW/AA 0 1 3 6 12 24 Time (h) 0 0.1 1 10 50 0 0.1 1 10 50 Lenalidomide (µM) 0 0 50 0 50 0 50 0 50 0 50 Lenalidomide (µM) (kDa) (kDa) 100 100 IB: AGIA (PLZF) IB: AGIA (PLZF) 75 75 IB: FLAG (CRBN) IB: FLAG (CRBN) 50 50 50 IB: a-TuBulin 50 IB: a-TuBulin Supplementary Figure 2. Destabilization of PLZF in IMiD-treated cells a, Immunoblot analysis of AGIA-PLZF or AGIA-SALL4 protein levels in FLAG-CRBN expressing HEK293T cells treated with DMSO, thalidomide, pomalidomide, or lenalidomide for 16 h. b, Immunoblot analysis of endogenous PLZF or SALL4 protein levels in HuH7 cells treated with DMSO, thalidomide, pomalidomide or lenalidomide for 24 h. c, Immunoblot analysis of endogenous PLZF protein levels in HEK293T cells treated with DMSO, thalidomide, pomalidomide or lenalidomide for 24 h. d, Time course of DMSO or lenalidomide treatment in AGIA-PLZF and FLAG-CRBN expressing HEK293T cells. AGIA-PLZF protein levels were detected by immunoblot analysis. e, Immunoblot analysis of AGIA-PLZF protein levels in FLAG-CRBN-WT or FLAG-CRBN-YW expressing CRBN-/- HEK293T cells treated with DMSO or lenalidomide for 16 h. 3 a b HuH7 HEK293T HuH7 1.5 NS 1.5 NS 0 0.1 1 10 100 Lenalidomide (µM) NS NS (kDa) 100 1.0 IB: PLZF 1.0 75 0.5 IB: CRBN 0.5 50 50 IB: a-Tubulin Relative mRNA expression Relative mRNA 0.0 expression Relative mRNA 0.0 M M M M µ µ µ µ 10 DMSO 100 10 DMSO 100 Lenalidomide Lenalidomide c THP-1 THP-1 1.5 NS NS 0 0.1 1 10 100 Lenalidomide (µM) (kDa) 100 1.0 IB: PLZF 75 0.5 50 IB: CRBN 50 IB: a-Tubulin expression Relative mRNA 0.0 M M µ µ 10 DMSO 100 Lenalidomide Supplementary Figure 3. Analyses of expression of PLZF mRNA and degradation of endogenous PLZF in thalidomide-treated cells Extended Data Figure 3. Analyses of expression of PLZF mRNA and degradation of endogenous PLZF in lenalidomide-treated cells. a, HEK293T cells were treated with the indicated concentrations of lenalidomide for 24 h and PLZF mRNA expression levels were measured by quantitative RT-PCR. b, HuH7 cells were treated with the indicated concentrations of lenalidomide for 24 h. PLZF protein levels were analysed by immunoblot and PLZF mRNA expression levels were measured by quantitative RT-PCR. c, THP-1 cells were treated with the indicated concentrations of lenalidomide for 24 h. PLZF protein levels were analysed by immunoblot and PLZF mRNA expression levels were measured by quantitative RT-PCR. Relative mRNA expression used the expression level with DMSO treatment as one. Error bars mean ± standard deviation (n = 3) and P values were calculated by one-way ANOVA with Tukey’s post-hoc test (NS = Not Significant). 4 Supplementary Figure 4. PLZF is a substrate of CRL4CRBN with thalidomide and lenalidomide for E3 ubiquitin ligase. a, Immunoprecipitation of FLAG-CRBN in FLAG-CRBN and AGIA-PLZF expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide in the presence of DMSO or MG132 for 8 h. Components of CRLFLAG-CRBN and AGIA-PLZF were detected using each specific antibody, as indicated. b, Ubiquitination of AGIA-PLZF in AGIA-PLZF and FLAG- CRBN expressing HEK293T cells treated with DMSO or lenalidomide in the presence of DMSO or MG132 for 10 h. 5 a b HT TK BJAB SU-DHL-4 MT-4 Raji (kDa) DM Le Po DM Le Po DM Le Po DM Le Po DM Le Po IMiD (10 µM) DM 1 10 Pomalidomide (µM) (kDa) 75 75 IB: PLZF IB: PLZF (Short Exp.) 75 IB: CRBN IB: PLZF 50 (Long Exp.) 50 IB: Tubulin IB: CRBN 50 IB: Tubulin 50 Supplementary Figure 5. IMiD-induced protein degradation of PLZF in B cell lymphomas. a, HT, TK, BJAB, SY-DHL-4, and MT-4 cells were treated with DMSO, 10 µM lenalidomide, or pomalidomide for 24 h. PLZF protein levels were analysed by immunoblot. b, Raji cells were treated with DMSO, 10 µM lenalidomide, or pomalidomide for 24 h. PLZF protein levels were analysed by immunoblot. Extended Data Figure 5. IMiD-induced protein degradation of PLZF in B cell lymphomas. 6 Supplementary Figure 6. Sequence comparisons of thalidomide-related regions in vertebrate PLZF, SALL4 and CRBN. a, Alignment of amino acid sequence of ZNF1 and ZNF3 in PLZF among human (Hs), rabbit (Oc), mouse (Mm), chicken (Gg), and zebrafish (Dr). b, Alignment of amino acid sequence of ZNF2 in SALL4 among the species above. c, Alignment of amino acid sequence in CRBN among the species above. 7 a b HsPLZF HsSALL4 ✱✱✱✱ ✱✱✱✱ HsSALL4 HsPLZF MmSall4 MmPlzf GgSall4 GgPlzf ✱✱✱✱ ✱✱✱✱ 80 20 8 ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ 30 15 ✱✱✱✱ 60 15 6 ✱✱✱✱ ✱✱✱ 20 10 ✱✱✱✱ NS 40 10 4 ✱✱✱✱ ✱ NS 10 5 NS 20 5 2 NS Relative AS signal Relative Relative AS signal Relative Relative AS signal Relative Relative AS signal Relative Relative AS signal Relative 0 0 0 0 0 HsCRBN-WT HsCRBN-WT GgCrbn-WT HsCRBN-EV/VI HsCRBN-WT MmCrbn-WT HsCRBN-E377VHsCRBN-V388I HsCRBN-E377VHsCRBN-V388IHsCRBN-EV/VI HsCRBN-V388I MmCrbn-I391V GgCrbn-I390V c MmPlzf MmSALL4 d ✱✱✱✱ ✱✱✱✱ GgCrbn-WT GgCrbn-E379V ✱✱✱✱ ✱✱✱✱ 20 20 (kDa) 0 50 200 0 50 200 Thalidomide (µM) ✱✱✱✱ ✱✱✱✱ 100 15 ✱✱✱✱ 15 IB: AGIA (GgPlzf) 75 ✱✱✱✱ ✱ 10 10 ✱ IB: FLAG (GgCrbn) 50 5 NS 5 NS Relative AS signal Relative Relative AS signal Relative 50 IB: a-Tubulin 0 0 MmCRBN-WT MmCRBN-WT MmCRBN-I391VMmCRBN-VE/IV MmCRBN-V380EMmCRBN-I391VMmCRBN-VE/IV MmCRBN-V380E Supplementary Figure 7. Interaction and protein degradation analyses between PLZF or SALL4 and CRBN with thalidomide among human, mouse and chicken. a, In vitro binding assay using human, mouse, and chicken proteins. Thalidomide-dependent interaction between biotinylated-HsCRBN, -MmCrbn or -GgCrbn, and FLAG-GST-SALL4, or -PLZF (Hs, Mm or Gg) in the presence of DMSO or 200 µM thalidomide was analysed using AlphaScreen technology. b-c, In vitro binding assay using human or mouse proteins. Thalidomide-dependent interaction in the presence of DMSO or 50 µM thalidomide was analysed using same procedure in Supplementary Fig 7a. d, Immunoblot analysis of AGIA- GgPlzf in FLAG-GgCrbn-WT or -E379V expressing CRBN-/- HEK293T cells treated with DMSO, 50 µM or 200 µM thalidomide for 16 h. All relative AS (AlphaScreen) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars mean ± standard deviation (n = 3) and P values were calculated by one-way or two-way ANOVA with Tukey’s post-hoc test (NS = Not Significant, *P < 0.05, ***P < 0.001, and ****P < 0.0001).