Fig 1: CRBN is efficiently degraded by VHL-CRBN heterodimerizing PROTACs. (A) Schematic diagram of the PROTAC containing pomalidomide and the VHL ligand (VH032). (B) Structure of TD-158, TD-165, TD-343, and TD-487. (C) HEK293T cells were treated with TD-158, TD-165, TD-343 or TD-487 (0.1, 1, and 10 μM) for 24 h, and VHL protein levels were analyzed by immunoblotting. L.E. indicates long exposure of the Western blot. (D) DC50 graph of TD-158 and TD-165 compounds (TD- 165, DC50 = 20.4 nM; TD-158, DC50 = 44.5 nM). (E) HA-CRBN and Flag-VHL were expressed in HEK293T cells. After 24 h, the cells were treated with TD-158 (500 nM) for 24 h. Whole cell lysates were analyzed by immunoblotting for the indicated proteins. (F) HEK293T cells were treated with 500 nM TD-158 at different time points, and CRBN levels were analyzed by immunoblotting. L.E. indicates long exposure of the Western blot.
Fig 2: The N-terminal disordered region of CRBN is necessary for degradation, but not for ubiquitination, by VHL-CRBN heterodimerizing PROTACs. (A) Schematic diagram illustrating CRBN truncation mutants. LON, Lon protease domain; TB, thalidomide binding domain. (B) Xpress-tagged full-length CRBN or D1, D2, D3, or D4 deletion mutants were expressed in HEK293T cells. After 24 h, the cells were treated with TD-165 (3 µM) or DMSO for 24 h. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (C) Plasmids encoding Xpress-tagged D1 and His-SBP–tagged VHL were transfected into HEK293T cells. After 48 h, cells were treated with TD-165 (1 µM) or DMSO for 24 h. Whole-cell lysates and proteins pull-downed using streptavidin beads were analyzed by immunoblotting for the indicated proteins. (D) Plasmids encoding Xpress-tagged CRBN, K39R, K42/43 R, or K39/42/43 R mutants were transfected into HEK293T cells. After 24 h, the cells were treated with TD-158 (2 µM) or DMSO for 24 h. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins.
Fig 3: Crbn interacts with Orai1.a, b Phagocytes from the indicated mice a or Crbn deleted 293T cells by CRISPR/Cas9 b were lysed and proteins in the lysates were detected using immunoblotting. Data are representative of three independent experiments. c The transcript levels of Orai1 were analyzed by conventional PCR (bottom) or qRT-PCR (top) using cDNA synthesized from total mRNA extracted from the indicated BMDMs. n = 3 experiments, mean ± SEM. NS not significant (two-tailed unpaired Student t test). d LR73 cells were transfected with the indicated plasmid. At 1 day after transfection, the cells were lysed and proteins in the lysates were detected with the indicated antibodies. Data are representative of three independent experiments. e, f 293T cells transfected with the indicated plasmids e or BMDMs f were lysed and then the lysates were incubated with anti-FLAG antibody-conjugated agarose beads e or an anti-Orai1 antibody and protein A/G agarose beads f. Bead-bound proteins were detected with the indicated antibodies. Data are representative of five e or three f independent experiments. IP immunoprecipitation, TCL total cell lysates. g–i 293T cells were transfected with the indicated plasmids. At 2 days after transfection, the cells were lysed and the lysates were incubated with agarose beads conjugated with glutathione g, i or an anti-FLAG antibody h. Bead-bound proteins were detected with the indicated antibodies. Data are representative of four g or three h, i independent experiments. j Yeast cells transformed with the indicated plasmids were plated on selective or non-selective media. Cells on the non-selective media indicate the number of cells plated. BD binding domain, AD activation domain.
Fig 4: (A) The expression of CRBN at mRNA level was not restored after treatment of the resistant cells with 5‐Aza, EPZ‐6438, or their combination, suggesting that the downregulation of CRBN, which is observed in all the IMiD‐resistant cells with acquired resistance, might not have a direct causality to the development of resistance. (B) Western blot of IKZF1 in OPM2, OPM2‐PR, and OPM2‐PR cells after treatment with 5‐Aza and EPZ‐6438. All the cells were either untreated or treated with lenalidomide or pomalidomide (10 μm) for 24 h, at which point cell lysates were isolated. The CRBN‐dependent degradation of IKZF1 after treatment with IMiDs is reduced in OPM2‐PR, but is partly rescued in the resensitized OPM2‐PR cells. (C) Apoptosis response in primary IMiD‐resistant cell lines JJN3, LP1, and RPMI‐8226, showing that the combination of 5‐Aza and EPZ‐6438 – or EZH2 inhibition alone (in the case of JJN3), can also overcome intrinsic resistance to IMiDs. **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig 5: (A) qPCR results of the CRBN expression across the IMiD‐sensitive cell lines (OPM2, NCI‐H929: dark bars on the left and right plot, respectively) and their lenalidomide‐ and pomalidomide‐resistant counterparts (dark gray and light gray bars, respectively). There is a significant downregulation of CRBN mRNA in all four cell lines with acquired IMiD resistance, compared to the parental, sensitive cell lines. **P < 0.01, ***P < 0.001, and ****P < 0.0001.(B) Western blot for CRBN, confirming the reduction in CRBN expression at protein level in loss of IMiD sensitivity. (C) Cytospin and immunohistochemical staining for CRBN in OPM2, NCI‐H929, and their IMiD‐resistant counterparts, confirming the significant reduction in CRBN expression in the resistant cells.
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