a,b, Immunoblot of WCL derived from MDA-MB-231 (a) or HCC1954 (b) cells synchronized in M phase by nocodazole treatment before release back into the cell cycle for the indicated times.c,d, Quantitative reverse transcription PCR (qRT–PCR) analyses of relative mRNA levels ofPD-L1 (also known asCD274) andGAPDH from samples derived from HeLa cells synchronized in M phase by nocodazole treatment before release back to the cell cycle for the indicated times.e, Immunoblot of WCL derived from untreated HeLa cells or HeLa cells pre-treated with IFNγ (10 ng ml⁻¹) for 12 h and then synchronized in M phase by nocodazole treatment before release back into the cell cycle for the indicated times.f, Immunoblot of WCL derived from HeLa cells stably expressing wild-type HA–MYC, or HA–MYC(T58A/S62A). Empty vector was used as a negative control.g, Immunoblot of WCL derived from wild-type HeLa cells or HeLa cells stably expressing HA–MYC; cells were synchronized in M phase by nocodazole treatment before release back into the cell cycle for the indicated times.h–k, Immunoblot of WCL derived from MC38, CT26, 4T1 or B16-F10 mouse tumour cells treated with the indicated concentration of nocodazole for 20 h before collecting.l–m, Immunoblot of WCL derived from 4T1 or CT26 mouse tumour cells treated with the indicated concentration of taxol for 20 h before collecting.

a,b, Immunoblot analysis of WCL derived from wild-type andCcna1^−/−Ccna2^−/− (a) orCcne1^−/−Ccne2^−/− (b) MEFs.c, qRT–PCR analysis of relative PD-L1 mRNA levels from wild-type andCcnd1^−/−Ccnd2^−/−Ccnd3^−/− MEFs. Data are mean ± s.d.,n = 5. NS, not significant, two-tailedt-test.d, Cell cycle profiles for wild-type andCcnd1^−/−Ccnd2^−/−Ccnd3^−/− MEFs, which were labelled with BrdU and propidium iodide and analysed by FACS.e, Immunoblot analysis of WCL derived fromCcnd1^fl/flCcnd2^−/−Ccnd3^fl/fl MEFs upon expression of Cre resulting in ablation ofCcnd1 andCcnd3. Cre was delivered via viral infection with pLenti-Cre (pLenti-EGFP used as a negative control). Cells were selected with puromycin (1 μg ml⁻¹) for 72 h before collecting.f, Immunoblot analysis of WCL derived fromCcnd1^−/−Ccnd2^−/−Ccnd3^−/− MEFs with stable reexpression ofCcnd1,Ccnd2 orCcnd3. Empty vector was used as a negative control.g, Immunoblot analysis of WCL derived from mouse mammary tumours induced by MMTV-Myc inCcnd1^+/+ orCcnd1^−/− females.n = 5 mice per experimental group.h, Immunoblot analysis of WCL derived from wild-type andCdk6^−/− MEFs.i,j, Immunoblot analysis of WCL derived from MDA-MB-231 cells stably expressing shCDK6 or shCDK2 as well as shScramble as a negative control.k,l, Immunoblot analysis of WCL derived from MDA-MB-231 cells transfected with indicated constructs (k), the intensity of PD-L1 band was quantified by the ImageJ software (l).m, Immunoblot analysis of WCL derived fromRB1 depleted MDA-MB-231 cells (with shScramble as a negative control) treated with palbociclib where indicated.n,o, Immunoblot analysis of WCL derived from mouse CT26 or 4T1 tumour cell lines treated with or without palbociclib or ribociclib, respectively.p,q, Immunoblot analysis of WCL derived from MDA-MB-231 cells pre-treated with palbociclib (1 μM) for 36 h before treatment with cycloheximide (CHX) for the indicated time (p) and PD-L1 protein abundance was quantified by ImageJ as indicated (q).r, Immunoblot analysis of WCL derived from 19 different cancer cell lines with indicated antibodies.s–u, Immunoblot analysis of WCL derived from MCF7 (s), T47D (t) or HLF (u) stably expressing p16 as well as empty vector as a negative control.v–x, Immunoblot analysis of WCL derived from MDA-MB-436 (v), BT549 (w) or HCC1937 (x) stably expressing three independent shRNAs targetingp16 (also known asCdkn2a) as well as shScramble as a negative control.

a,b, Immunoblot analysis of WCL derived from MC38 or B16-F10 implanted tumours, in mice treated with palbociclib (150 mg kg⁻¹ body weight, by gastric gavage) or vehicle for 7 days.n = 5 mice per experimental group.c, FACS analysis for PD-L1 or CD3⁺ T cell populations from B16-F10-implanted tumours in mice treated with vehicle or palbociclib for 7 days.n = 5 mice per experimental group.d, Immunoblot analysis of WCL derived from the indicated organs in mice treated with palbociclib (150 mg kg⁻¹ body weight, by gastric gavage) or vehicle for 7 days.n = 5 mice per experimental group.e, Quantification of PD-L1 protein bands intensity ind using ImageJ.n = 5 mice per experimental group.f, Immunoblot analysis of WCL derived from 15 different organs dissected from mice with or without palbociclib treatment and MMTV-c-Myc induced breast tumours.g, Quantification of PD-L1 protein bands intensity inf using ImageJ.n = 3 biological replicates.h,In vitro kinase assay using immunoprecipitated cyclin D1–CDK4 kinase complex from livers or brains by anti-CDK4 antibody and recombinant Rb as a substrate. Note that cyclin D1–CDK4 complex in non-dividing organs (livers and brains) displayed kinase activity, which might explain why the CDK4/6 inhibitor increased PD-L1 in these organs. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001,. two-tailedt-test.

Source data

a, A schematic illustration of PD-L1 with N-terminal signal peptide, extracellular domain, transmembrane domain, cytoplasmic tail and the potential SPOP-binding motif in PD-L1.b,d, Immunoblot analysis of WCL and glutathioneS-transferase (GST) pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.c, Immunoblot analysis of WCL derived from PC3 stably expressing shCullin 3.e,g, Immunoblot analysis of WCL and immunoprecipitate derived from HEK293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.f, Immunoblot of WCL and Ni–NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 h before collecting and lysis in denaturing buffer.h, Immunoblot analysis of WCL and immunoprecipitate derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.i, Immunoblot of WCL derived from MDA-MB-231PD-L1-knockout cells stably expressing wild-type PD-L1 or Δ283–290 and T290M PD-L1 mutants, with empty vector used as a negative control.j, Immunoblot analysis of WCL derived from HEK293T cells transfected with wild-type HA–PD-L1 or the T290M mutant. Cells which were treated with CHX for indicated times before collecting.k, Immunoblot of WCL and Ni–NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 h before collecting and lysis in denaturing buffer.l, Immunoblot of WCL derived from 293T cells transfected with indicated constructs.

a–c, Immunoblot analysis of WCL derived from 293T cells transfected with indicated constructs.d,e, Immunoblot analysis of WCL derived from 293T cells transfected with indicated constructs. Thirty-six hours after transfection, cells were treated with 20 μg ml⁻¹ CHX for indicated length of time (d). The PD-L1 protein abundances were quantified using ImageJ (e).f, Immunoblot of WCL and Ni–NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 h before collecting and lysis in the denaturing buffer.g, A schematic illustration of SPOP with MATH and BTB domains interacting with substrate and cullin 3, respectively.h, Immunoblot analysis of WCL and immunoprecipitate derived from HEK293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.i, Immunoblot analysis of WCL derived from HEK293T cells transfected with indicated constructs.j, qRT–PCR analysis of relative mRNA levels ofPD-L1 (also known asCd274) fromSpop^+/+ andSpop^−/− MEFs. Data are mean ± s.d.,n = 5. NS, not significant, two-tailedt-test.k, Immunoblot analysis of WCL derived from PC3 cells infected with indicated lentiviral shRNAs againstSPOP and selected with puromycin (1 μg ml⁻¹) for 72 h before collecting.l,m, Immunoblot analysis of WCL derived from C4-2 cells with depletion ofSPOP using sgRNA, treated with CHX for indicated time points before collecting (l). The PD-L1 protein abundance was quantified using ImageJ (m).n,o, Immunoblot analysis of WCL derived from LNCaP cells stably expressing shAR or shERG as well as shScr as a negative control.p,q, Immunoblot analysis of WCL derived from DU145 cells stably expressing shTRIM24 or shDEK as well as shScr as a negative control.r–u, Immunoblot analysis of WCL derived from C4-2 wild-typeSPOP andSPOP^−/− cells that stably expressed shAR, shERG, shTRIM24, or shDEK as well as shScramble.

a, The mutation frequency (mutated cases/total cases) of SPOP across 24 cancer types from The Cancer Genome Atlas (TCGA) database. Mutations are categorized as occuring in the MATH domain, in the BTB domain or at any other position of the gene, including untranslated regions (UTRs). Because some patient cases contain mutations of two or three categories, the proportions of the colours are allocated mutation-wise, instead of case-wise.b, The distribution of mutation positions of SPOP in 24 cancer types from the TCGA database. Mutations without altering amino acid sequence have been discarded.c, Immunoblot analysis of WCL derived from HEK293T cells transfected with indicated constructs.d, The mutation frequency (mutated cases/total cases) of PD-L1 across 19 cancer types from the TCGA database.e, Oncoplot of PD-L1 and SPOP across all 39 cancer types in the TCGA database. Only PD-L1 or SPOP mutant tumours are shown; only mutations or truncations in the C-terminal tail of PD-L1 or in the MATH domain of SPOP are counted.f, Immunoblot of WCL derived from B16-F10 mouse tumour cell line stably expressing the indicated SPOP constructs.g,h, Growth curve and cell cycle profile of B16-F10 cells stably expressing wild-type SPOP and the F102C mutant, empty vector was used as a negative control.i, Cell cycle profile of 22Rv1 cells stably expressing wild-type SPOP and the F102C mutant, empty vector was used as a negative control.j, Relative cell-surface PD-L1 expression in 4T1-implanted tumours expressing wild-type SPOP or the F102C mutant measured using FACS analysis.n = 5 mice per experimental group.k, B16-F10 tumours derived from cells stably expressing wild-type SPOP or the F102C mutant and implanted in C57BL/6 mice were dissected and photographed after euthanization of mice.l, FACS analysis of the absolute number of CD3⁺ TILs from 4T1 tumours stably expressing wild-type SPOP or the F102C mutant.n = 5 mice per experimental group.m, B16-F10 tumour cells stably expressing wild-type SPOP or the F102C mutant were implanted into C57BL/6 mice. Tumour-bearing mice were treated with anti-PD-L1 antibody; tumours were then dissected and photographed.n = 7 mice per experimental group.n, The weight of B16-F10 cell derived tumours implanted in C57BL/6 mice that were subsequently treated with anti-PD-L1 antibody. 12 mice per experimental group.o, FACS analysis of relative cell surface expression of PD-L1 in implanted tumours derived from B16-F10 cells expressing wild-type SPOP or the F102C mutant. Animals were treated with anti-PD-L1 antibody.n = 5 mice per experimental group.p, FACS analysis of the absolute number of CD3⁺ TILs from B16-F10 tumours stably expressing wild-type SPOP or the F102C mutant in mice treated with anti-PD-L1 antibody.n = 7 mice per experimental group.q, B16-F10 cells stably expressing wild-type SPOP or the F102C mutant were inoculated intoTcra^−/− mice. After two weeks, tumours were dissected and photographed.n = 7 mice per experimental group.r, Tumour weights of B16-F10 cells stably expressing wild-type SPOP or the F102C mutant and implanted intoTcra^−/− mice were dissected and recorded.n = 7 mice per experimental group.s, FACS analysis of relative cell surface expression of PD-L1 from B16-F10 tumours stably expressing wild-type SPOP or the F102C mutant arising inTcra^−/− mice.n = 7 mice per experimental group. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant, two-tailedt-test.

Source data

a, Immunoblot analysis of WCL derived fromPD-L1 knockout MDA-MB-231 cells.b, Immunofluorescence of wild-type andPD-L1 knockout MDA-MB-231 cells using the anti-PD-L1 antibody.c,d, Immunochemistry using the anti-PD-L1 antibody of wild-type andPD-L1 knockout MDA-MB-231 cells from cultures on glass slides (c) or from implanted tumours (d).e,f, Immunoblot analysis using the anti-CD8 antibody of WCL derived from HBP-ALL (e) or KE37 (f) cells stably expressing shCD8 as well as shScr as a negative control.g,h, Immunohistochemistry using the anti-CD8 antibody for HBP-ALL (g) or KE37 (h) cell pellets stably expressing shCD8 or shScr as a negative control. Scale bars, 50 μm.

a–c, Immunoblot analysis of WCL derived fromSPOP-knockout (a),CDC20-knockdown (b) orFZR1-knockdown (c) HeLa cells.d, Immunoblot analysis of WCL and immunoprecipitate derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.e, Immunoblot analysis of WCL and immunoprecipitate derived from HeLa cells treated with MG132 (10 μM) for 12 h before collecting.f, A sequence comparison of D-box motifs (RXXLXXXXN) in SPOP derived from different species.g, Immunoblot analysis of WCL derived from HeLa cells transfected with indicated constructs.h,i, Immunoblot analysis of WCL derived from 293T cells transfected with indicated constructs. Thirty-six hours after transfection, cells were treated with CHX at indicated time points before collecting (h). The protein abundances of wild-type SPOP and ΔRXXL mutant were quantified using ImageJ (i).

a, A sequence comparison of conserved SP sites and putative 14-3-3γ binding motif in SPOP.b, Immunoblot analysis of WCL and immunoprecipitate derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.c,d,In vitro kinase assays with recombinant Rb and SPOP as substrates and cyclin D1–CDK4, cyclin D2–CDK4 and cyclin D3–CDK4 as kinase complexes were performed. BSA was used as a negative control where indicated.e, Immunoblot analysis of WCL and immunoprecipitate derived from MDA-MB-231 cells transfected with indicated constructs, with and without palbociclib treatment (1 μM) for 12 h.f, Streptavidin bead pull-down assay for biotin-labelled SPOP peptide with and without phosphorylation at the Ser6 residue to examine itsin vitro association with 14-3-3γ.g, Immunoblot analysis of WCL and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.h,i, Immunoblot analysis of WCL and immunoprecipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 h before collecting.j,k, Immunoblot analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h after transfection, cells were treated with 20 μg ml⁻¹ CHX at indicated time points (j). The protein abundance of wild-type SPOP and the S6A mutant were quantified using ImageJ (k).l,p, Immunoblot of WCL and Ni–NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 h before collecting and lysis in denaturing buffer for subsequent assays.m–o, Immunoblot analysis of WCL and immunoprecipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) with or without palbociclib (1 μM) for 12 h before collecting.q–s, Immunoblot of WCLs derived from PC3 (q), BT549 (r) and HeLa (s) cells stably expressing sh14-3-3γ as well as shScr as a negative control.t, Immunoblot of WCL derived from HeLa cells stably expressing shScr or sh14-3-3γ synchronized in M phase by nocodazole treatment before release back into the cell cycle for the indicated times.

a, A schematic of the treatment plan for mice bearing subcutaneous MC38 and CT26 tumours. Female C57BL/6 mice (for MC38) or BALB/c mice (for CT26) were implanted with 1 × 10⁵ MC38 or CT26 cells subcutaneously and received one of four treatments: control antibody treatment, anti-PD-1 antibody treatment, CDK4/6 inhibitor (palbociclib) treatment, or anti-PD-1 antibody plus CDK4/6 inhibitor combination treatment.b, MC38 implanted tumour-bearing mice were enrolled in different treatment groups as indicated. Tumour volumes of mice treated with control antibody (n = 15), anti-PD-1 antibody (n = 15), palbociclib (n = 14) or anti-PD-1 antibody plus palbociclib combined therapy (n = 12) were measured every three days and plotted individually. Experiment repeated twice.c, Kaplan–Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-1 antibody with palbociclib. *P < 0.05, Gehan–Breslow–Wilcoxon test. Experiment repeated twice.d,e,g,i, The absolute numbers of CD3⁺, CD4⁺, CD8⁺, granzyme B⁺ or IFNγ⁺ TILs in implanted MC38 tumours from mice treated with indicated agents was analysed by FACS. Control:n = 8, palbociclib:n = 10, anti-PD-1 antibody:n = 9, palbociclib and anti-PD-1 antibody:n = 8.f,h,j, The percentage of CD4⁺, CD8⁺ in CD3⁺ TILs cells in implanted MC38 tumours from mice treated with indicated agents was analysed by FACS. Control:n = 8, palbociclib:n = 10, PD-1 antibody:n = 9, palbociclib and anti-PD-1 antibody:n = 8.k, A working model of how PD-L1 protein stability is regulated by the cyclin D–CDK4–SPOP–FZR1 signalling pathway. Cyclin D–CDK4 negatively regulates PD-L1 protein stability largely through phosphorylating its upstream physiological E3 ligase SPOP. This phosphorylation promotes SPOP binding to 14-3-3γ, which subsequently disrupts FZR1-mediated destruction of SPOP. Thus, CDK4/6 inhibitor treatment increases PD-L1 protein levels by inhibiting cyclin D–CDK4-mediated phosphorylation of SPOP to promote its degradation by APC/C. The rise in PD-L1 could present a severe clinical problem for patients receiving CDK4 inhibitor treatment and could be one of the underlying mechanisms accounting for CDK4 inhibitor resistance via evasion of immune surveillance checkpoints. Hence, our work provides a molecular mechanism as well as the rationale for the combination of PD-L1 blockade treatment and CDK4/6 inhibitors as a more efficient anti-cancer clinical option. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailedt-test.

Source data

This file contains the source data for gels in Figures 1-4 and Extended Data Figures 1-9. (PDF 4329 kb)