Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance

Abstract

Treatments that target immune checkpoints, such as the one mediated by programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been approved for treating human cancers with durable clinical benefit. However, many patients with cancer fail to respond to compounds that target the PD-1 and PD-L1 interaction, and the underlying mechanism(s) is not well understood. Recent studies revealed that response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumour cells. Hence, it is important to understand the mechanistic pathways that control PD-L1 protein expression and stability, which can offer a molecular basis to improve the clinical response rate and efficacy of PD-1-PD-L1 blockade in patients with cancer. Here we show that PD-L1 protein abundance is regulated by cyclin D-CDK4 and the cullin 3-SPOP E3 ligase via proteasome-mediated degradation. Inhibition of CDK4 and CDK6 (hereafter CDK4/6) in vivo increases PD-L1 protein levels by impeding cyclin D-CDK4-mediated phosphorylation of speckle-type POZ protein (SPOP) and thereby promoting SPOP degradation by the anaphase-promoting complex activator FZR1. Loss-of-function mutations in SPOP compromise ubiquitination-mediated PD-L1 degradation, leading to increased PD-L1 levels and reduced numbers of tumour-infiltrating lymphocytes in mouse tumours and in primary human prostate cancer specimens. Notably, combining CDK4/6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumour regression and markedly improves overall survival rates in mouse tumour models. Our study uncovers a novel molecular mechanism for regulating PD-L1 protein stability by a cell cycle kinase and reveals the potential for using combination treatment with CDK4/6 inhibitors and PD-1-PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human cancers.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

GF has patents/pending royalties on the PD-1 pathway from Roche, Merck, Bristol-Myers-Squibb, EMD-Serono, Boehringer-Ingelheim, AstraZeneca, Dako and Novartis. GF has served on advisory boards for CoStim, Novartis, Roche, Eli Lilly, Bristol-Myers-Squibb, Seattle Genetics, Bethyl Laboratories, Xios, and Quiet. PS in a consultant and a recipient of a research grant from Novartis. No potential conflicts of interests were disclosed by other authors.

The authors declare no competing financial interests.

Figures

Extended Data Figure 1. PD-L1 fluctuates during cell cycle progression

a, b, Immunoblot (IB) of whole cell lysates (WCL) derived from MDA-MB-231 or HCC1954 cells synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times. c, d, Quantitative real-time PCR (qRT-PCR) analyses of relative mRNA levels of PD-L1 and GAPDH from samples derived from HeLa cells synchronized in M phase by nocodazole treatment prior to releasing back to the cell cycle for the indicated time points. e, IB of WCL derived from HeLa cells pre-treated with/without IFNγ (10 ng/ml) for 12 hours and then synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times. f, IB of WCL derived from HeLa cells stably expressing HA-c-Myc WT, or HA-T58A/S62A-c-Myc as well as empty vector (EV) as a negative control. g, IB of WCL derived from HeLa cells with/without stably expressing HA-c-Myc WT synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times. h–j, IB of WCL derived from MC38, CT26, 4T1, or B16-F10 mouse tumor cells treated with the indicated concentration of nocodazole for 20 hours before harvesting. (k–m) IB of WCL derived from B16-F10, 4T1, or CT26 mouse tumor cells treated with the indicated concentration of taxol for 20 hours before harvesting.

Extended Data Figure 2. Cyclin D/CDK4 negatively regulates PD-L1 protein stability

a, b, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from wild type (WT), cyclin A1−/−A2−/− or WT, cyclin E1−/−E2−/− MEFs. c, Quantitative real-time PCR (qRT-PCR) analysis of relative mRNA levels of PD-L1 from wild type MEFs and cyclin D1−/−D2−/−D3−/− MEFs. Data were represented as mean ± s.d, n = 5. d, Cell cycle profiles for WT and cyclin D1−/−D2−/−D3−/− MEFs, which were labeled with BrdU and analyzed by FACS. e, IB analysis of WCL derived from cyclin D1fl/flD2−/−D3fl/fl MEFs with or without depleting cyclin D1 and cyclin D3 by pLenti-Cre via viral infection (pLenti-EGFP as a negative control), selected with puromycin (1 μg/ml) for 72 hours before harvesting. f, IB analysis of WCL derived from cyclin D1−/−D2−/−D3−/− MEFs stably reintroducing cyclin D1, cyclin D2, or cyclin D3, respectively, with empty vector (EV) as a negative control. g, IB analysis of WCL derived from mouse mammary tumors induced by MMTV-c-Myc with/without genetic depletion of cyclin D1. n = 5 mice per experimental group. h, IB analysis of WCL derived from WCL derived from wild type and cdk6−/− MEFs. i, j, IB analysis of WCL derived from MDA-MB-231 cells stably expressing shCDK6 or shCDK2 as well as shScr as a negative control, respectively. k, l, IB analysis of WCL derived from MDA-MB-231 cells transfected with indicated constructs (k) and the intensity of PD-L1 band was quantified by the ImageJ software (l). m, IB analysis of WCL derived from MDA-MB-231 cells depleted of Rb (with shScr as a negative control) treated with the CDK4/6 inhibitor, palbociclib, where indicated. n, o, IB analysis of WCL derived from mouse CT26 or 4T1 tumor cell lines treated with or without the CDK4/6 inhibitor, palbociclib or ribociclib, respectively. p, q, IB analysis of WCL derived from MDA-MB-231 cells pre-treated with palbociclib (1 μM) for 36 hours before treatment with cycloheximide (CHX) for the indicated time points (p) and PD-L1 protein abundance was quantified by the ImageJ and plotted as indicated (q). r, IB analysis of WCL derived from 19 different cancer cell lines with indicated antibodies. s–u, IB analysis of WCL derived from MCF7, T47D or HLF stably expressing p16 as well as EV as a negative control. v–x, IB analysis of WCL derived from MDA-MB-436, BT549 or HCC1937 stably expressing three independent shRNAs against p16 as well as shScr as a negative control.

Extended Data Figure 3. CDK4/6 inhibitor, palbociclib, treatment elevated PD-L1 levels in vivo

a, b, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from MC38 or B16-F10 mouse tumor cell line implanted tumors treated with palbociclib (150 mg/kg 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 MC38 implanted tumors treated with vehicle or palbociclib for 7 days. n = 5 mice per experimental group. d, IB analysis of WCL derived from multiple organs in mice treated with palbociclib (150 mg/kg body weight, by gastric gavage) or vehicle for 7 days. n = 5 mice per experimental group. e, Quantification of PD-L1 protein bands intensity in Extended Data Fig. 3d by using the ImageJ software. n = 5 mice per experimental group. f, IB analysis of WCL derived from 15 different tissues with/without palbociclib treatment and MMTV-c-Myc induced breast tumors. g, Quantification of PD-L1 protein bands intensity in Extended Data Fig. 3f by using the ImageJ software. n = 3 biological replicates h,In vitro kinase assay for Rb through using immunoprecipitated CDK4/cyclin D kinase complex from liver or brain by anti-CDK4 antibody IP. Note that cyclin D-CDK4 complex in non-dividing organs (livers and brains) displayed kinase activity, which might explain why CDK4/6 inhibitor elevated PD-L1 in these organs. Error bars, ± s.d., two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Figure 4. Cullin 3SPOP promotes PD-L1 ubiquitination and subsequent degradation largely through interaction with the cytoplasmic tail of PD-L1

a, A schematic illustration of PD-L1 with N-terminal signal peptide, extracellular domain, trans-membrane domain, cytoplasmic tail and the potential SPOP-binding motif in PD-L1. b, d, Immunoblot (IB) analysis of whole cell lysates (WCL) and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. c, IB analysis of WCL derived from PC3 stably expressing shCullin 3. e, g, IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. f, IB 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 hours before harvesting and lysed in the denature buffer. h, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. i, IB of WCL derived from MDA-MB-231 PD-L1 KO cells stably expressing PD-L1 WT, delta 283-290, T290M as well as EV as a negative control. j, IB analysis of WCL derived from 293T cells transfected with HA-PD-L1 WT and the T290M mutant, which were treated with cycloheximide (CHX) for indicated time points before harvesting. k, IB 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 hours before harvesting and lysed in the denaturing buffer. l, IB of WCL derived from 293T cells transfected with indicated constructs.

Extended Data Figure 5. SPOP negatively regulates PD-L1 protein stability in a poly-ubiquitination dependent manner

a–c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with indicated constructs. d, e, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with 20 μg/ml cycloheximide (CHX) at indicated time points (d). The PD-L1 protein abundance were quantified by the ImageJ software and plotted (e). f, IB 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 hours before harvesting and lysed in the denaturing buffer. g, A schematic illustration of SPOP with MATH and BTB domain to interact with substrate and Cullin 3, respectively. h, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. i IB analysis of WCL derived from 293T cells transfected with indicated constructs. j, qRT-PCR analysis of relative mRNA levels of PD-L1 from Spop+/+ and Spop−/− MEFs. Data were represented as mean ± s.d, n=5. k, IB analysis of WCL derived from PC3 cells infected with indicated lentiviral shRNAs against SPOP and selected with puromycin (1 μg/ml) for 72 hours before harvesting. l–m, IB analysis of WCL derived from C42 cells with depletion of SPOP using sgRNA and treated with cycloheximide (CHX) for indicated time points before harvesting (l). The PD-L1 protein abundance were quantified by the ImageJ software and plotted (m). n, o, IB analysis of WCL derived from LNCaP cells stably expressing shAR or shERG as well as shScr as a negative control. p, q, IB analysis of WCL derived from DU145 cells stably expressing shTrim24 or shDEK as well as shScr as a negative control. r–u, IB analysis of WCL derived from C42 SPOP WT and SPOP−/− cells that stably expressed shAR, shERG, shTrim24, or shDEK as well as shScr, respectively.

Extended Data Figure 6. Cancer-derived SPOP mutations fail to promote PD-L1 degradation

a, The mutation frequency (mutated cases/total cases) of SPOP across 24 cancer types from the TCGA database. Mutations are categorized as happening in the MATH domain, in the BTB domain or at any other position of the gene, including UTRs. Because some patient cases contain mutations of two or three categories, the proportion of three colors 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 with low translational consequences have been discarded. c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with indicated constructs. d, The mutation frequency (mutated cases/total cases) of PD-L1 (CD274) across 19 cancer types from the TCGA database. e, Oncoplot of PD-L1 (CD274) and SPOP across all 39 cancer types in the TCGA database. Only mutations or truncations in the C terminal tail of PD-L1 or in the MATH domain of SPOP are counted. f, IB of WCL derived from B16-F10 mouse tumor cell line stably expressing the indicated SPOP constructs. g, h, Growth curve and cell cycle profile of B16-F10 cells stably expressing SPOP WT and the F102C mutant as well as EV as a negative control. i, Cell cycle profile of 22Rv1 cells stably expressing SPOP WT and the F102C mutant as well as EV as a negative control. j, Relative cell surface PD-L1 expression of 4T1 implanted tumors ectopically expressing SPOP-WT or the SPOP-F102C mutant were subjected to FACS analysis. n = 5 mice per experimental group. k, B16-F10 cells stably expressing SPOP-WT or the SPOP-F102C mutant implanted tumors from C57BL/6 mice were dissected and taken a picture after euthanizing the mice. l, The number of CD3+ T-cell populations from the isolated tumor-infiltrating lymphocytes in 4T1 cells stably expressing SPOP-WT or the SPOP-F102C mutants implanted tumors were subjected to FACS analysis. n = 5 mice per experimental group. m, B16-F10 cells stably expressing SPOP-WT or the SPOP-F102C mutant implanted tumors from C57BL/6 mice treated with anti-PD-L1 antibody were dissected and taken a picture after euthanizing the mice. n = 7 mice per experimental group. n, The weight of B16-F10 cells implanted tumors from C57BL/6 mice treated with anti-PD-L1 antibody. 12 mice per experimental group. o, Relative cell surface PD-L1 expression of B16-F10 cells implanted tumors ectopically expressing SPOP-WT or the SPOP-F102C mutant treated with anti-PD-L1 antibody were subjected to FACS analysis. n = 5 mice per experimental group. p, The number of CD3+ T-cell populations from the isolated tumor-infiltrating lymphocytes in B16-F10 cells implanted tumors ectopically expressing SPOP-WT or the SPOP-F102C mutant treated with control IgG or anti-PD-L1 antibody were subjected to FACS analysis. n = 7 mice per experimental group. q, B16-F10 cells stably expressing SPOP-WT or the SPOP-F102C mutant implanted tumors from Tcrα−/− mice were dissected and taken a picture after euthanizing the mice. n = 7 mice per experimental group. r, Relative cell surface PD-L1 expression of B16-F10 cells stably ectopically expressing SPOP-WT or the SPOP-F102C mutant implanted tumors from Tcrα−/− mice were subjected to FACS analysis. n = 7 mice per experimental group. s, The number of CD3+ T-cell populations from the isolated tumor-infiltrating lymphocytes in B16-F10 cells stably ectopically expressing SPOP-WT or the SPOP-F102C mutant implanted tumors from Tcrα−/− mice were subjected to FACS analysis. n = 7 mice per experimental group. Error bars, ± s.d., two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance.

Extended Data Figure 7. Validation of anti-PD-L1 and anti-CD8 antibodies through using PD-L1 KO or shCD8 cells

a, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from MDA-MB-231 cells depleted PD-L1 through the CRISPR-Cas9 system. b, Immunofluorescence (IF) for MDA-MB-231 PD-L1 WT and KO cells using the anti-PD-L1 antibody. The scale bar represents 50 μm. c, d, Immunochemistry (IHC) for MDA-MB-231 PD-L1 WT and KO cells from cultured on glass slides (c) or implanted tumors (d) using the anti-PD-L1 antibody. The scale bar represents 50 μm. e, f, IB analysis of WCL derived from HBP-ALL (e) or KE37 (f) cells stably expressing shCD8 as well as shScr as a negative control using the anti-CD8 antibody. g, h, IHC for HBP-ALL (g) or KE37 (h) cell pellets stably expressing shCD8 as well as shScr as a negative control using the anti-CD8 antibody. The scale bar represents 50 μm.

Extended Data Figure 8. Depletion of Cdh1, but not Cdc20, prolongs SPOP proteins stability, which is simultaneously coupled with a decrease in PD-L1 protein level

a–c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa depleted SPOP through the CRISPR-Cas9 system (a) or depleted Cdc20 or Cdh1 through multiple independent shRNAs (b, c). d, IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. e, IB analysis of WCL and IP derived from HeLa cells treated with MG132 (10 μM) for 12 hours before harvesting. f, A sequence comparison of D-box motif (RxxLxxxxN) in SPOP derived from different species. g, IB analysis of WCL derived from HeLa cells transfected with indicated constructs. h, i, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with cycloheximide (CHX) as indicated time points before harvesting (h). The protein abundance of SPOP-WT and deletion of RxxL mutant were quantified by the ImageJ software (i).

Extended Data Figure 9. Cyclin D/CDK4-mediated phosphorylation of SPOP at the Ser6 residue promotes its binding with 14-3-3γ to reduce its poly-ubiquitination and subsequent degradation by APC/Cdh1

a, A sequence comparison of conserved SP sites and putative 14-3-3γ binding motif in SPOP. b, Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. 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 complex were performed. BSA was used as a negative control where indicated. e, IB analysis of WCL and immunoprecipitation (IP) derived from MDA-MB-231 cells transfected with indicated constructs, which were treated with/without palbociclib (1 μM) for 12 hours. f, Streptavidin beads pull-down assay for biotin-labeled SPOP peptide with/without phosphorylation at the Ser6 residue to examine its in vitro association with 14-3-3γ. g, IB analysis of WCL and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. h, i, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting. j, k, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with 20 μg/ml cycloheximide (CHX) as indicated time points (j). The protein abundance of SPOP-WT and S6A mutant were quantified by the ImageJ software and plotted accordingly (k). l, p, IB 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 hours before harvesting and lysed in the denaturing buffer for following assay. m–o, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) and with/without palbociclib (1 μM) for 12 hours before harvesting. q–s, IB of WCLs derived from PC3, BT549 and HeLa cells stably expressing sh14-3-3γ as well as shScr as a negative control. t, IB of WCL derived from HeLa cells stably expressing shScr or sh14-3-3γ synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times.

Extended Data Figure 10. Combination therapy of anti-PD-1 mAb and CDK4/6 inhibitor in MC38 colon cancer mouse model

a, A schematic model that illustrates the treatment plan for mice bearing subcutaneous MC38 tumors. Female C57BL/6 mice were implanted with 0.1 × 106 MC38 cells subcutaneously and treated with four arms: control antibody treatment, anti-PD-1 mAb treatment, CDK4/6 inhibitor treatment, anti-PD-1 mAb plus CDK4/6 inhibitor combination treatment. b, MC38 implanted tumor-bearing mice were enrolled in different treatment groups as indicated. Tumor volumes of mice treated with control antibody (n = 15), anti-PD-1 mAb (n = 15), the CDK4/6 inhibitor, palbociclib (n = 14) or combined therapy (n = 12) were measured every three days and plotted individually. We repeated this experiment twice. c, Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-1 mAb with the CDK4/6 inhibitor, palbociclib. *P < 0.05. (Gehan-Breslow-Wilcoxo test). We repeated this experiment twice. d, e, g, i, The absolute number of CD3+, CD4+, CD8+, Granzyme B+, or IFNγ+ TILs cells of implanted MC38 tumors treated with indicated agents was analyzed by FACS. Control: n = 8, palbociclib: n = 10, PD-1 Ab: n = 9, Palbociclib & PD-1 Ab: n = 8. f, h, j, The percentage of CD4+, CD8+ in CD3+ TILs cells of implanted MC38 tumors treated with indicated agents was analyzed by FACS. Control: n = 8, palbociclib: n = 10, PD-1 Ab: n = 9, Palbociclib & PD-1 Ab: n = 8. k, A proposed working model to illustrate how PD-L1 protein stability is regulated by the cyclin D/CDK4-SPOP-Cdh1 signaling pathway. The cyclin D/CDK4 negatively regulates PD-L1 protein stability largely through phosphorylating its upstream physiological E3 ligase SPOP to promote SPOP binding with 14-3-3γ, which subsequently disrupts Cdh1-mediated destruction of SPOP. As such, CDK4/6 inhibitor treatment could unexpectedly elevate PD-L1 protein levels largely through inhibiting cyclin D/CDK4-mediated phosphorylation of SPOP to promote its degradation by APC/CCdh1. The unexpected rise of 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 evading immune surveillance checkpoint. Hence, our work provides a novel molecular mechanism as well as the rationale for the combinational treatment of PD-L1 blockage treatment and the CDK4/6 inhibitors as a more efficient anti-cancer clinical option. Error bars, ± s.d., two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance.

Figure 1. The protein abundance of PD-L1 fluctuates during cell cycle progression

a, c, Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells synchronized in M phase by nocodazole (a) or in late G1/S phase by double thymidine (b) following by releasing back into the cell cycle. b, d, The cell-cycle profiles in (a) or (c) were monitored by fluorescence-activated cell sorting (FACS).

Figure 2. Cyclin D-CDK4 negatively regulates PD-L1 protein stability

a–d, IB analysis of WCL derived from wild type versus combinational (cyclin D1−/−D2−/−D3−/−) (a) or single isoform cyclin D knockout MEFs (b), MDA-MB-231 cells depleted cyclin D1 or cyclin D3 using shRNAs (c), or MMTV-Wnt1 induced mouse mammary tumors with/without genetic depletion of cyclin D1 (d). e–h, IB analysis of WCL derived from wild type versus cdk4−/− MEFs (e), MDA-MB-231 cells depleted CDK4 using shRNAs (f), or multiple breast cancer cell lines treated with palbociclib (0.5, 1 μM) for 48 hours (g, h). i, j, Immunofluorescence staining of PD-L1 and CD3 in mouse mammary tumors induced by MMTV-ErbB2 treated with vehicle or palbociclib as described in Method (i) and the quantification of CD3+ T cell population (j). The scale bar: 50 μm. k, FACS analysis for PD-L1 or CD3+ T-cell populations from MC38 implanted tumors treated with vehicle or palbociclib for 7 days. Vehicle, n = 4 for (i, j) or 7 mice for (k); palbociclib, n = 4 for (i, j) or 7 mice for (k). Error bars, ± s.d., two-tailed t-test, **P < 0.01, ***P < 0.001 (two-tailed t-test).

Figure 3. Cullin 3SPOP is the physiological E3 ubiquitin ligase for PD-L1

a–d, IB analysis of WCL derived from C4-2 cells treated with MG132 (10 μM) or MLN4924 (1 μM) for 12 hours (a), immunoprecipitates (IP) and WCL derived from 293T cells transfected with indicated constructs (b, d), or anti-PD-L1 IP and WCL derived from PC3 cells (e). Cells were treated with MG132 (10 μM) for 12 hours in b, c. e–g, IB analysis of WCL derived from Spop+/+ versus Spop−/− MEFs (e), C4-2 cells depleted SPOP with sgRNAs (f), or C4-2 cells stably expressing indicated SPOP WT and mutants (g). h, i, IB analysis of IP and WCL derived from 293T cells (h), or Ni-NTA pull-down products derived from PC3 cells transfected with indicated constructs and treated with 30 μM MG132 for 6 hours. j–l, FACS analysis for PD-L1 (j) or CD3+ T-cell population (l) of the B16-F10 implanted tumors ectopically expressing SPOP-WT or F102C mutant (n = 6 mice each group). Tumor weight were recorded at the time of sacrifice (k) (n = 5 mice each group). m, Representative images of PD-L1 and CD8 immunohistochemistry (IHC) staining in SPOP wild-type or mutant primary human prostate cancer samples. The scale bar: 400 μm or 100 μm. n, o Quantification of IHC analysis for PD-L1 (n) and CD8+ T cells (o) in SPOP wild-type versus mutant human prostate tumor specimens. (n =15 for SPOP mutant, n = 82 for SPOP WT). Error bars, ± s.d., two-tailed t-test, except (n) Mann-Whitney test, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Cyclin D-CDK4-mediated phosphorylation of SPOP stabilizes SPOP largely through recruiting 14-3-3γ to disrupt its binding with Cdh1

a–e, IB of WCL derived from HeLa cells with/without depletion of SPOP (a) or Cdh1 (e) synchronized in M phase by nocodazole treatment prior to releasing for the indicated times, IP and WCL derived from MDA-MB-231 (b) or 293T (c) cells, or Ni-NTA pull-down products derived from HeLa cells transfected with the indicated constructs (d). Cells were treated with MG132 (30 μM) for 6 hours in b–d. f,In vitro kinase assays showing that cyclin D1/CDK4 phosphorylates recombinant SPOP at Ser6, not Ser222. g–j, IB analysis of IP and WCL derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) or with/without palbociclib (1 μM) for 12 hours (g–i), or HeLa cells with/without depletion of SPOP treated with palbociclib (0.5, 1 μM) for 48 hours (j). k, CT26 implanted tumor-bearing mice were enrolled in different treatment groups as indicated. Tumor volumes of mice treated with control antibody (n = 13), anti-PD-1 mAb (n = 14), the CDK4/6 inhibitor, palbociclib (n = 12) or combined therapy (n = 12) were measured every three days and plotted individually. We repeated this experiment twice. l, Kaplan-Meier survival curves for each treatment group demonstrate the improved efficacy of combining PD-1 mAb with the CDK4/6 inhibitor, palbociclib. ***P < 0.001. (Gehan-Breslow-Wilcoxo test). We repeated this experiment twice.