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.