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[Preprint]. 2023 Jan 19:rs.3.rs-2447544.

doi: 10.21203/rs.3.rs-2447544/v1.

Reciprocal inhibition of PIN1 and APC/C_CDH1 controls timely G1/S transition and creates therapeutic vulnerability

Shizhong Ke^{1

2}, Fabin Dang^{3

2}, Lin Wang^{1

2}, Jia-Yun Chen^{4

5

2}, Mandar T Naik⁶, Abhishek Thavamani¹, Yansheng Liu^{7

8}, Wenxue Li⁷, Nami Kim¹, Nandita M Naik⁶, Huaxiu Sui⁹, Wei Tang¹⁰, Chenxi Qiu^{1

11}, Kazuhiro Koikawa¹, Felipe Batalini^{1

12}, Xiaodong Wang¹³, John G Clohessy¹⁴, Yujing Jan Heng³, Galit Lahav⁴, Nathanael S Gray¹⁵, Xiao Zhen Zho^{1

16}, Wenyi Wei³, Gerburg M Wulf¹, Kun Ping Lu^{1

16

17}

Affiliations

¹ Division of Hematology/Oncology, Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
² These authors contributed equally to this work.
³ Department of Pathology, Beth Israel Deaconess Medical Center and Cancer Research Institute, Harvard Medical School, Boston, MA 02215, USA.
⁴ Department of Systems Biology, Harvard Medical School, Boston, MA 02215, USA.
⁵ Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA 02215, USA.
⁶ Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, RI 02912, USA.
⁷ Yale Cancer Biology Institute, West Haven, CT 06516, USA.
⁸ Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.
⁹ Key Laboratory of Functional and Clinical Translational Medicine, Fujian Province University, Xiamen Medical College, Xiamen 361023, China.
¹⁰ Data Science & Artificial Intelligence, R&D, AstraZeneca, Gaithersburg, USA.
¹¹ Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
¹² Department of Medicine, Division of Medical Oncology, Mayo Clinic, Arizona, USA.
¹³ Molecular and Integrative Physiological Sciences, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA 02215, USA.
¹⁴ Preclinical Murine Pharmacogenetics Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
¹⁵ Department of Chemical and Systems Biology, Chem-H and Stanford Cancer Institute, Stanford University, Stanford, CA 94305, USA.
¹⁶ Departments of Biochemistry & Oncology, Schulich School of Medicine and Dentistry, and Robarts Research Institute, Western University, London, ON N6A 3K7, Canada.
¹⁷ Lead Contact.

PMID: 36711754
PMCID: PMC9882653
DOI: 10.21203/rs.3.rs-2447544/v1

Reciprocal inhibition of PIN1 and APC/C_CDH1 controls timely G1/S transition and creates therapeutic vulnerability

Shizhong Ke et al. Res Sq. 2023.

[Preprint]. 2023 Jan 19:rs.3.rs-2447544.

doi: 10.21203/rs.3.rs-2447544/v1.

Authors

Affiliations

¹ Division of Hematology/Oncology, Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
² These authors contributed equally to this work.
³ Department of Pathology, Beth Israel Deaconess Medical Center and Cancer Research Institute, Harvard Medical School, Boston, MA 02215, USA.
⁴ Department of Systems Biology, Harvard Medical School, Boston, MA 02215, USA.
⁵ Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA 02215, USA.
⁶ Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, RI 02912, USA.
⁷ Yale Cancer Biology Institute, West Haven, CT 06516, USA.
⁸ Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.
⁹ Key Laboratory of Functional and Clinical Translational Medicine, Fujian Province University, Xiamen Medical College, Xiamen 361023, China.
¹⁰ Data Science & Artificial Intelligence, R&D, AstraZeneca, Gaithersburg, USA.
¹¹ Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
¹² Department of Medicine, Division of Medical Oncology, Mayo Clinic, Arizona, USA.
¹³ Molecular and Integrative Physiological Sciences, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA 02215, USA.
¹⁴ Preclinical Murine Pharmacogenetics Facility, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
¹⁵ Department of Chemical and Systems Biology, Chem-H and Stanford Cancer Institute, Stanford University, Stanford, CA 94305, USA.
¹⁶ Departments of Biochemistry & Oncology, Schulich School of Medicine and Dentistry, and Robarts Research Institute, Western University, London, ON N6A 3K7, Canada.
¹⁷ Lead Contact.

PMID: 36711754
PMCID: PMC9882653
DOI: 10.21203/rs.3.rs-2447544/v1

Abstract

Cyclin-dependent kinases (CDKs) mediated phosphorylation inactivates the anaphase-promoting complex (APC/C^CDH1), an E3 ubiquitin ligase that contains the co-activator CDH1, to promote G1/S transition. PIN1 is a phosphorylation-directed proline isomerase and a master cancer signaling regulator. However, little are known about APC/C^CDH1 regulation after phosphorylation and about PIN1 ubiquitin ligases. Here we uncover a domain-oriented reciprocal inhibition that controls the timely G1/S transition: The non-phosphorylated APC/C^CDH1 E3 ligase targets PIN1 for degradation in G1 phase, restraining G1/S transition; APC/C^CDH1 itself, after phosphorylation by CDKs, is inactivated by PIN1-catalyzed isomerization, promoting G1/S transition. In cancer, PIN1 overexpression and APC/C^CDH1 inactivation reinforce each other to promote uncontrolled proliferation and tumorigenesis. Importantly, combined PIN1- and CDK4/6-inhibition reactivates APC/C^CDH1 resulting in PIN1 degradation and an insurmountable G1 arrest that translates into synergistic anti-tumor activity against triple-negative breast cancer in vivo. Reciprocal inhibition of PIN1 and APC/C^CDH1 is a novel mechanism to control timely G1/S transition that can be harnessed for synergistic anti-cancer therapy.

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Figures

**Extended Data Fig. 1 |. APC/C^CDH1 is a physiological E3 ubiquitin ligase for PIN1.**
a, Overall survival for BC with low and high PIN1 protein abundance, dichotomized by median expression. Median survival was 13 months for the PIN1-high, but 48 months for the PIN1-low group. b, Overall survival for BC with low and high PIN1 mRNA levels, dichotomized by top quartile. **c, d,** IB analysis for indicated proteins derived from MDA-MB-231 cells treated with increasing concentrations of AApin (ATO (0.5, 1, 1.5, 2 μM) plus ATRA (5, 10, 15, 20 μM)) in 1:10 ratio) (c) or Sulfopin (5, 10, 15 μM) (d) for 3 days and 10 μM MG132 or 1 μM Bortezomib for last 12 hrs before harvesting. e, Data from PIN1 affinity purification-mass spectrometry experiment was used to generate the interaction networks by Cystoscape. Heatmap represented log₂-transformed DIA-MS signal changes of GST-PIN1 versus GST. f, Coomassie blue stain of Pin1-interacting proteins after immunoprecipitation (IP) resolved by SDS-PAGE. Proteins were identified by mass spectrometric peptide sequencing. MDA-MB-231 cells were transfected with 3XFlag-Pin1 and treated with 10 μM MG132 for 12 hrs. Lysates were immunoprecipitated with M2 (anti-Flag) or control (IgG) beads. g, Glutathione S-transferase (GST) pull-down of GST-PIN1 and haemagglutinin (HA)-tagged CDH1. 293T cells were transfected with indicated constructs for 36 hrs and GST-PIN1 were pulled down with glutathione beads. **h, i,** WT or *CDH1* KO MEFs were treated with increasing concentrations of PIN1 inhibitors for 3 days. IB analysis for PIN1. j, MDA-MB-231 cells were co-treated with increasing concentration of MLN4924 and AApin (1.5 μM ATO plus 15 μM ATRA) for 3 days. IB analysis of PIN1. **k-t,** Validation of potential PIN1-interacting E3 ligases identified by mass spectrometry. IB of PIN1 from MDA-MB-231 cells stably expressing indicated shRNAs and treated with AApin (1.5 μM ATO plus 15 μM ATRA) for 3 days.

**Extended Data Fig. 2 |. *CDH1* KO stabilizes APC/C^CDH1 substrates and PIN1 across cell cycle.**
a, RT-PCR analysis of indicated mRNA from WT and *Cdh1* KO MEFs synchronized in G1 phase by serum starvation, followed by releasing back into the cell cycle before harvesting cells at indicated time points. b, IB analysis of WT and *CDH1* KO MCF-10A cells synchronized in G1 phase followed by releasing back into the cell cycle before harvesting cells at indicated time points. c, RT-PCR analysis of indicated mRNA of WT and *CDH1* KO MCF-10A cells treated as in **b. d,** Schematic diagram of the APC-degron reporter (Geminin: aa1–110). e, Overall survival for BRCA tumors in TCGA with low and high *CDH1* mRNA levels.

**Extended Data Fig. 3 |. *PIN1* KO restores APC/C^CDH1 E3 ligase activity**
a, IB analysis for indicated proteins derived from multiple BC cell lines. b, Normalized counts of *PIN1* KO versus WT cells were used for GSEA analysis against the biological process related gene sets. Normalized enrichment scores (NES) were used to generate bar graphs for visualization of the functional transcriptional outputs of the three cell lines. *P < 0.001. c, Enrichment plots of indicated up-regulated and down-regulated gene sets analyzed by GSEA in *PIN1* KO versus WT cells. d, Cell division frequency in WT and *PIN1* KO MCF-7 cells stably expressing the APC-degron reporter from Fig. 1k. e, Frequency of G1 arrest (ratio of G1 arrested cells to total cells). The error bar indicates 95% confidence interval determined by bootstrapping. Data in graphs are mean ± s.d., analyzed by unpaired two-sided t-test. ***P < 0.001.

**Extended Data Fig. 4 |. Reciprocal inhibition of PIN1 and APC/C^CDH1 E3 ligase**
a, Co-IP of endogenous CDH1 with endogenous CDK4. MDA-MB-231 and MCF-7 cells were treated with 1 μM Palbociclib for 24 hrs and precipitated with IgG or anti-CDK4 antibodies. Input is 5% of the total lysates used in IP. b, IB analysis of indicated proteins derived from MCF-10A transfected with CDK4 or CDK6 and cultured in serum-free condition for 36 hrs. c, IB analysis for indicated proteins derived from WT or RB KO MDA-MB-231 cells treated with indicated concentration of Palbociclib for 3 days. d, IB analysis for indicated proteins derived from shCON or shRB/RBL1/RBL2 MDA-MB-231 cells treated with 5 μM Palbociclib for 3 days. Knockdown of *RB/RBL1/RBL2* was validated by RT-PCR (right). e, IB analysis of GST pull-down precipitates derived from 293T cells transfected with HA-CDH1 and GST-PIN1 mutants as indicated for 36 hrs. f, IB analysis of GST pull-down precipitates derived from 293T cells transfected with GST-PIN1 and HA-CDH1 mutants for 36 hrs. **g, h,** IB analysis of indicated proteins derived from 293T cells transfected with wild-type CDH1, S163A (g) or S163E (h) mutants CDH1 and treated with 50 μg/ml CHX for the indicated time. i, Domain architecture of PIN1 containing an N-terminal WW domain binding specific pSer/Thr-Pro motifs, and a C-terminal peptidyl-prolyl cis/trans isomerase (PPIase) domain that catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs. Note the sequence homology of D-box motifs (RXXL) in human and mouse. **j, k,** IB analysis of immunoprecipitates from MDA-MB-231 cells stably co-expressing Flag-PIN1-WT or disabling mutations in the WW domain (Flag-PIN1-W34A) or the PPIase/D-box domain (Flag-PIN1-RLAA) or the dual mutant (Flag-PIN1-W34A; RLAA), and HA-CDH1-WT (left) or the phosphosite-deficient mutant CDH1-7A (right) treated with 10 μM MG132 for 12 hrs and pulled down using Flag-M2 beads. Non-specific bands for IgG_H marked with asterisks (*). l, IB analysis of for indicated proteins derived from 293T cells transfected with indicated constructs. The graphs were one representative experiment out of three independent experiments. m, A structural modeling illustration of docking the PIN1 PPIase domain (cyan; PDB: 1PIN) to the CDH1-WD40 domain (chartreuse; PDB: 4UI9_R). D-box motif is marked in red. The structure model was generated using HADDOCK. n, Superposition of free PIN1 (Green; PDB: 1PIN) and PIN1 in complex with Sulfopin (Cyan; PDB: 6VAJ). The PIN1 PPIase domain in complex with Sulfopin closely resembles free PIN1 with a Root Mean Square Deviation (RMSD) of 0.231 Å. K117 is one of the very few residues that have significant different conformation between free PIN1 and PIN1-Sulfopin complex.

**Extended Data Fig. 5 |. Pharmacologic inhibition of PIN1 and CDK4 restores APC/C^CDH1 E3 ligase activity inducing an insurmountable G1 arrest.**
a, IB analysis of immunoprecipitates derived from MDA-MB-468 cells treated with 5 μM Palbociclib and pulled down by anti-CDH1 antibody. Input is 5% of the total lysates used in IP. b, IB analysis of immunoprecipitates derived from MDA-MB-231 cells stably expressing HA-CDH1 treated with Palbociclib or PIN1 inhibitors and pulled down by anti-HA antibody. HA-CDH1-7A expressed MDA-MB-231 cells were used along with a control. **bc,** IB analysis of indicated GST pull-down precipitates from MDA-MB-231 cells stably expressing HA-CDH1 and treated with vehicle or 10 μM Sulfopin for 3 days and 10 μM MG132 for the last 12 hrs before harvesting. d, CHX chase assay for indicated proteins derived from 293T cells transfected with EV or Flag-PIN1 constructs for 36 hrs and treated with 50 μg/ml CHX for the indicated time. e, APC-degron reporter levels in MCF-7 cells stably expressing EV or wild-type PIN1 as determined by FACS. f, IB analysis for indicated proteins from MCF-7 cells stably co-expressing the APC-degron reporter (mCherry-Geminin (1-110)) and empty vector (EV) or Flag-PIN1 and treated with increasing concentrations of Palbociclib (0.5, 1, 2 μM) for 48 hours. g, Percentage of cells in G1 from Fig. 5d. Data in graphs are mean ± s.d., analyzed by unpaired two-sided t-test. ***P < 0.001. h, Time course cell cycle regulator expression in MCF-7 cells treated with AApin (10 μM ATRA plus 1 μM ATO). **i, i,** DNA histogram in time course in MCF-7 cells from h and quantification of cells in G1 and S/G2/M. The graphs were one representative experiment out of three independent experiments. k, IB analysis of indicated proteins from MDA-MB-231 cells treated with vehicle or 1 μM Palbociclib for 3 days and 10 μM MG132 for the last 12 hrs before harvesting. l, Distributions of S/G2 duration in DMSO-, Palbociclib- and PIN1 inhibitors-treated MCF-7 cells stably expressing the APC-degron reporter from Fig. 5e. m, Frequency of G1 arrest in MCF-7 cells from Fig. 5e (ratio of G1 arrested cells, the last 24 hours, to total cells). The error bar indicates 95% confidence interval determined by bootstrapping. Data in graphs are mean ± s.d., analyzed by unpaired two-sided t-test. **P < 0.01, ***P < 0.001. n, Cell division frequency in MCF-7 cells from Fig. 5e. o, Immunofluorescence for MDA-MB-231 cells stably expressing the APC-degron reporter (mCherry-Geminin fusion protein) and PIN1 (Venus-PIN1 fusion protein) and treated with different inhibitors for indicated periods.

**Extended Data Fig. 6 |. Pharmacologic inhibition of PIN1 and CDK4 restores APC/C^CDH1 E3 ligase activity inducing an insurmountable G1 arrest.**
a, Schematic diagrams showing the mechanism of APC/C^CDH1 inactivation by CDKs and EMI1 for cell cycle commitment. b, WT and *Cyclin D1/D2/D3* TKO MEFs were treated with Palbociclib or Sulfopin for 4 days and cell viability were assessed by CellTiter-Glo. Data in graphs are mean ± s.d. c, IB analysis for indicated proteins derived from shCON and shCDK2 MDA-MB-231 cells treated with 10 μM Sulfopin, 0.5 μM Palbociclib or a combination of Palbociclib and Sulfopin for 3 days. d, IB analysis for indicated proteins derived from shCON and shEMI1 MDA-MB-231 cells treated with increasing concentrations of Sulfopin (5, 10 μM) for 3 days. **e-g,** IB analysis for indicated proteins derived from WT and *CDH1* KO MCF-7 cells treated with increasing concentrations of Sulfopin (2, 4, 8, 10 μM) (e), AApin (ATO (0.5, 1, 1.5, 2 μM) plus ATRA (5, 10, 15, 20 μM)) in 1:10 ratio (f) or Palbociclib (0.5, 1, 2, 4 μM) (g) for 3 days. i, RT-PCR analysis of indicated mRNA of WT and *CDH1* KO MDA-MB-231 cells treated with 10 μM Sulfopin, 1 μM Palbociclib or their combination for 3 days. j, RT-PCR analysis of indicated mRNA of WT and *CDH1* KO BT-549 cells treated with 5 μM Sulfopin, 2.5 μM Palbociclib or their combination for 3 days. Heatmap represented relative mRNA expression. k, CHX chase assay for indicated proteins derived from WT and *CDH1* KO MDA-MB-468 cells treated with 50 μg/ml CHX for the indicated time. The graphs were one representative experiment out of three independent experiments. l, IB analysis of ubiquitinated proteins derived from WT and *CDH1* KO MDA-MB-231 cells transfected with the indicated constructs and treated with 1 μM Palbociclib and 10 μM Sulfopin for 3 days and 2 μM MG132 for last 12 hrs and pulled down under denaturing conditions by nickel-nitrilotriacetic acid (Ni-NTA) agarose.

Extended Data Fig. 7 |. PIN1 inhibitors synergize with CDK4 inhibitors against TNBC *in vitro.*
a, The heatmap of growth inhibition matrices and synergy scores for Sulfopin in combination with Palbociclib in MDA-MB-231 cells, higher scores (darker red) denoting stronger synergy. b, Long-term colony formation assays (left) and growth inhibition matrices (middle) of RB KO MDA-MB-231 cells treated with indicated concentrations of Sulfopin and Palbociclib for two weeks. Synergy scores for Sulfopin in combination with Palbociclib in RB KO MDA-MB-231 cells (right). c, The heatmap of growth inhibition matrices and synergy scores for Sulfopin in combination with Palbociclib in SUM-159 cells. d, Long-term colony formation assays (left) and growth inhibition matrices (middle) of MDA-MB-468 cells treated with indicated concentrations of Sulfopin and Palbociclib for two weeks and stained with crystal violet. Synergy scores for Sulfopin in combination with Palbociclib in MDA-MB-468 cells (right). e, Mammosphere formation assay of MDA-MB-231 cells treated with 1 μM Palbociclib, 10 MM Sulfopin, AApin (10 μM ATRA plus 1 μM ATO) or a combination of both drugs for two weeks. f, Senescence was assessed by staining for SA-β-gal activity for SUM-159 cells treated with AApin (10 μM ATRA plus 1 μM ATO), 10 μM Sulfopin, 1 μM Palbociclib or combinations for 3 days. g, WT and *Cdh1* KO MEFs were treated with indicated drugs for 3 days and cell viability were assessed by CellTiter-Glo. Data in graphs are mean ± s.d., analyzed by unpaired two-sided t-test. ***P < 0.001, ns, not significant. h, Long-term colony formation assays of WT and *CDH1* KO MDA-MB-231 and BT-549 cells treated with indicated concentrations of Sulfopin for two weeks and stained with crystal violet. The graphs were one representative experiment out of three independent experiments.

**Extended Data Fig. 8 |. PIN1 inhibitors synergize with CDK4 inhibitors against TNBC in mouse models.**
**a, b,** Tumor growth in mice with established TNBC PDOX treated with Sulfopin, CDK4 inhibitors or their combination. Tumor sizes (a) and tumor weights (b) were measured when mice were euthanized after 45 days. Data in graphs are mean ± s.e.m., analyzed by unpaired two-sided t-test. **P < 0.01, *** P < 0.001, **** P < 0.0001. c, Representative immunofluorescence images for PDOX tumors stained with Cyclin B1 (cyan), Geminin (purple) and PLK1 (yellow). Scale bars, 50 μm. **d-l,** Hematological parameters were analyzed in TNBC PDOX nude mice treated with indicated inhibitors for 45 days. m, Body weights were monitored in TNBC PDOX nude mice during the treatments. Data in graphs are mean ± s.e.m. n, Representative images of H&E staining in K14cre; p53wt/f; Brca1wt/f. Scale bars, 50 μm. o, IB analysis for indicated proteins derived from K14cre; p53wt/f; Brca1wt/f. tumors or MEFs. p, IB analysis of immunoprecipitates derived from K14cre; p53wt/f; Brca1wt/f mouse tumors and pulled down by anti-Pin1 or anti-Cdk4 antibody. Input is 5% of the total lysates used in IP.

**Fig. 1 |. Cell cycle regulator APC/C^CDH1 is a physiological E3 ubiquitin ligase for PIN1.**
a, Overall survival for BC with low and high PIN1 protein abundance, dichotomized by top quartile expression. Median survival was 13 months for the PIN1-high, but 100 months for the PIN1-low group. b, Data from PIN1 affinity purification-mass spectrometry experiment was used to generate the interaction networks by Cystoscape. Heatmap represented log₂-transformed DIA-MS signal changes of GST-PIN1 versus GST. c, Co-IP of endogenous CDH1 with endogenous PIN1. MDA-MB-231 cells were treated with 10 μM MG132 for 12 hrs and precipitated with IgG or anti-PIN1 antibodies. Input is 5% of the total lysates used in IP. d, Confocal images of the colocalization of endogenous PIN1 (red) and CDH1 (green). Colocalization rates were calculated by LAS X software. Scale bars, 5 μm. e, MDA-MB-231 and MCF-7 cells stably expressing shCDH1 or shCDC20 were treated with increasing concentrations of ATRA (5 μM, 20 μM) for 3 days, followed by IB of cell lysates. f, IB of WT and *CDH1* KO MEFs synchronized in G1 phase by serum starvation, followed by releasing back into the cell cycle before harvesting cells at indicated time points. g, Cell-cycle profiles corresponding to (f) monitored by fluorescence-activated cell sorting (FACS). h, Intensity plots of PIN1 and mCherry-Geminin (aa1-110) reporter in MCF-10A cells synchronized in G1 phase, followed by releasing back into the cell cycle before fixing cells at indicated time points. PIN1 protein was stained with Cy5 (left panel). Fluorescent intensity of PIN1 and Geminin (aa1-110) were quantified and log2 transformed across the time courses (right panel). i, Correlation between PIN1 protein levels and CDH1 protein levels across 105 BRCA samples. Protein level z-scores measured with mass spectrometry by the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The graphs were one representative experiment out of three independent experiments. The quantifications of related critical WB were shown in Supplementary Data 2.

**Fig. 2 |. Active APC/C^CDH1 targets PIN1 for degradation.**
a, IB analysis for indicated proteins derived from MCF-7 cells stably expressing shCDH1 or HA-CDH1 and cultured in 10% serum or serum-free conditions. b, Domain architecture of CDH1 contains previously identified serine/threonine sites in the N-terminus that are potential phosphorylation sites for CDKs. The C-box and KLLR motifs are critical for CDH1 association with the APC core complex. The C-terminal IR motif of CDH1 mediates interaction with the TPR subunits APC7 and APC3. c, Morphology of MCF-7 stably expressing the CDH1-7A lentiviral construct. d, SA-β-gal stain in MDA-MB-231 and SUM-159 cells infected with the CDH1-7A lentiviral constructs. Quantification of SA-β-gal positive cells (percent of total). Data in graphs are mean ± s.d. analyzed by unpaired two-sided t-test. **** P < 0.0001. e, IB analysis of indicated proteins derived from 293T cells transfected with indicated constructs. f, IB analysis of indicated proteins derived from MDA-MB-231 or MDA-MB-468 cells stably expressing the CDH1-7A lentiviral construct and treated with DMSO or 10 μM MG132 for 12 hrs. **g, h,** RT-PCR analysis of indicated mRNA of WT and CDH1-7A MDA-MB-231 and MDA-MB-468 cells. i, CHX chase assay for indicated proteins derived from WT and CDH1-7A MDA-MB-231 cells treated with 50 μ/ml CHX for the indicated time. j, IB analysis of immunoprecipitates from 293T cells stably co-expressing Flag-PIN1-WT or disabling mutations and the phosphosite-deficient mutant CDH1-7A treated with 10 μM MG132 for 12 hrs and pulled down using Flag-M2 beads. k, IB analysis for indicated proteins derived from 293T cells stably co-expressing CDH1-7A or empty vector (EV) in the presence of Flag-PIN1 and its mutants. I, IB analysis of the ubiquitinated proteins derived from 293T cells transfected with the indicated constructs for 48 hours and treated with 2 μM MG132 for 12 hrs and pulled down under denaturing conditions by nickel-nitrilotriacetic acid (Ni-NTA) agarose. The graphs were one representative experiment out of three independent experiments.

**Fig. 3 |. PIN1 regulates APC/C^CDH1 E3 ligase activity at post-translational levels to ensure the timely G1/S transition.**
a, Long-term colony-formation assay of four BC wild-type and *P1N1* KO cell lines. Cells were grown for about 2 weeks, fixed and stained with crystal violet. b, Gene Ontology (GO) enrichment analysis was applied to proteomics of *P1N1* KO versus WT MDA-MB-231 cells. Color codes for p-value (brighter purple is more significant) and symbol size codes for the ratio of proteins related to specific GO term/total number of proteins significantly altered. c, IB analysis for indicated proteins derived from WT and *P1N1* KO MDA-MB-231 cells synchronized in M phase by nocodazole and then released back into the cell cycle for the indicated periods of time. d, Cell-cycle profiles of WT (blue) and *P1N1* KO (pink) in c as determined by FACS. e, Release into mitosis after double-thymidine block in the absence or presence of PIN1. DNA contents were measured by FACS in WT and *P1N1* KO MDA-MB-231 cells synchronized at the G1/S boundary by double thymidine block and then released back into the cell cycle for 4 hours. f, Cell cycle phase distribution of WT and *P1N1* KO MDA-MB-231 cells from **e. g,** Tracking cell division and cell death on the single cell level. Asynchronous cultures of MCF-7 WT and *P1N1* KO cells expressing the APC-degron reporter were followed for 72 hours for single cell expression of mCherry-Geminin (shades of blue). h, Cycloheximide (CHX) chase assay for indicated proteins derived from WT and *P1N1* KO MDA-MB-231 cells (left) or WT and *P1N1* KO MDA-MB-468 cells (right) treated with 50 μg/ml CHX for the indicated time. The graphs were one representative experiment out of three independent experiments. The quantifications of related critical WB were shown in Supplementary Data 2.

**Fig. 4 |. Reciprocal inhibition of PIN1 and APC/C^CDH1 E3 ligase.**
a, 293T cells were transfected with indicated constructs for 36 hrs. Input is 5% of the total lysates used in IP. b, *In vitro* kinase assay showing that CDK4 phosphorylates CDH1 at Ser163. c, IB analysis of GST pull-down precipitates derived from 293T cells transfected with HA-CDH1 mutants and GST-PIN1 as indicated for 36 hrs. d, GST-PIN1 pull-down precipitates derived from MDA-MB-231 cells stably expressing HA-CDH1 or HA-CDH1-7A, treated with 10 μM MG132 for 12 hrs. The graphs were one representative experiment out of three independent experiments. e, NMR analysis of phosphorylated peptide bound to PIN1. Average chemical shift perturbation in PIN1 backbone amide resonances on the binding of the CDH1 phosphopeptide. This data is acquired at pH 6.6 and 25��C with 1:13 molar excess of the CDH1 phosphorylated peptide shown. f, Two dimensional (2D) 1H-¹⁵N Heteronuclear single quantum coherence (HSQC) spectrum of ¹⁵N-labeled PIN1 protein. The phosphopeptide binding appears fast on the NMR timescale as seen by the movement of representative 15N-HSQC peaks from the backbone of R17, S18, W34, and E35, and the sidechain of W34. g, HADDOCK model demonstrating putative interaction between the CDH1 phosphopeptide shown as red sticks and PIN1 WW (magenta) and PPIase domain (cyan; PDB: 1PIN). h, Overlay of ¹³C-HSQC spectra acquired on 58 μM free peptide (red) and its 1:4 complex with PIN1 (green). The peak volumes were used to derive isomer population estimates. The graphs were one representative experiment out of two independent experiments. i, Schematic diagram illustrating PIN1-catalyzed *trans* to *cis* prolyl-isomerization of the CDH1-pS163-P motif. j, IB analysis for indicated proteins derived from MDA-MB-231 cells stably co-expressing phosphosite-deficient CDH1 (CDH1-7A) or empty vector (EV) in the presence of Flag-PIN1 and its mutants. k, R119 of PPIase domain forms the electrostatic interaction with CDH1 residues D180 and E465. K117 and G123 of PPIase domain mediate hydrogen bonds with the backbone of V219 and the side chain of W212, respectively. L122 of PPIase domain occupies the hydrophobic pocket formed by CDH1 residues L179, A181 and L467. The quantifications of related critical WB were shown in Supplementary Data 2.

**Fig. 5 |. Pharmacologic inhibition of PIN1 and CDK4 restores APC/C^CDH1 E3 ligase activity inducing an insurmountable G1 arrest.**
a, IB analysis of immunoprecipitates derived from WT and *P1N1* KO MDA-MB-231 cells treated with Palbociclib and pulled down by anti-CDH1 antibody. Input is 5% of the total lysates used in IP. **b, c,** IB analysis of indicated GST pull-down precipitates derived from MCF-7 cells stably expressing HA-CDH1 and treated with vehicle or Sulfopin for 3 days and 10 μM MG132 for last 12 hrs before harvesting. d, Dot plots of FACS for WT or *PIN1*-KO MCF-7 cells stably expressing the APC-degron reporter (mCherry-Geminin fusion protein) and treated with DMSO or AApin (1.5 μM ATO plus 15 μM ATRA) for 3 days. DNA were labeled with Hoechst live cell dye. Y-axis, mCherry-Geminin; X-axis, DNA; color code of dots, reporter levels. e, Tracking cell division and cell death in response to Palbociclib (4 μM), Sulfopin (10 μM) or AApin (1.5 μM ATO plus 15 μM ATRA) on the single cell level. Asynchronous cultures of MCF-7 cells expressing the APC-degron reporter were followed for 4 days (96 hours) for single cell expression of mCherry-Geminin (shades of blue), mitosis (green), cell death (dark rhomboid). f, IB analysis for indicated proteins derived from WT and *CDH1* KO MCF-7 cells treated with a combination of 1 μM Palbociclib and AApin (ATO (0.5, 1, 1.5, 2 μM) plus ATRA (5, 10, 15, 20 μM)) for 3 days. g, IB analysis for indicated proteins derived from WT and *CDH1* KO BT-549 cells treated with 5 μM Sulfopin, 2.5 μM Palbociclib or their combination for 3 days. The quantifications of related critical WB were shown in Supplementary Data 2. h, CHX chase assay for indicated proteins derived from WT and *CDH1* KO MDA-MB-231 cells pre-treated with combination of 10 μM Sulfopin and 1 μM Palbociclib for 36 hours followed by 50 μ/ml CHX treatment for the indicated time. The graphs were one representative experiment out of three independent experiments. **i, j,** Schematic diagrams showing the reciprocal inhibition of PIN1 and APC/C^CDH1 controls cell cycle progression.

**Fig. 6 |. PIN1 inhibitors synergize with CDK4 inhibitors against TNBC *in vitro* and *in vivo*.**
**a, b,** Colony formation matrices of MDA-MB-231 and SUM-159 cells treated with indicated concentrations of Sulfopin and Palbociclib for two weeks. c, IB analysis of PIN1 in SUM-159 cells treated as in **b. d,** Correlation of PIN1 abundance and cell growth inhibition in SUM-159 cells from b and **c. e,** Cell counts of MDA-MB-231 cells treated with 1 μM Palbociclib, 10 μM Sulfopin or a combination of both drugs for 4 days. f, MDA-MB-231 cells were treated with increasing concentrations of indicated drugs for 3 days, followed by analyzing apoptotic and necrotic cells by Annexin V and PI. Data in graphs are mean ± s.d., analyzed by unpaired two-sided t-test. *P < 0.05, **P < 0.01, ***P < 0.001. g, Tumor growth in mice with established TNBC PDOX treated with Sulfopin, CDK4 inhibitors or their combination. h, Tumor sizes were measured when mice were euthanized after 45 days. NT, no tumor detectable. Data in graphs are mean ± s.e.m., analyzed by unpaired two-sided t-test. **P < 0.01, **** P < 0.0001. i, Representative immunofluorescence images for PDOX tumors stained with PIN1 (green) and Ki67 (red). Scale bars, 50 μm. j, Tumor growth in NCG mice with established MDA-MB-468 xenografts treated with Sulfopin, Palbociclib or their combination. k, Tumor weights were measured when mice were euthanized after 7 weeks, n=5 mice per group. Data in graphs are mean ± s.e.m., analyzed by unpaired two-sided t-test. *P < 0.05, **P < 0.01. l, Growth curve generated from nude mice bearing K14cre; *p53wt/f; Brcalwt/f*_BT3 tumors treated with vehicle, Sulfopin, Palbociclib or their combination, n=6 mice per group. m, Growth curve generated from nude mice bearing K14cre; p53wt/f; Brcalwt/f_BT1 tumors treated with vehicle, Sulfopin, Palbociclib or their combination, n=5 mice per group. **n, o,** Growth curve (n) and survival curve (o) generated from FVB mice bearing K14cre; *p53wt/f; Brcalwt/f*_BT3 tumors treated with vehicle (median survival of 18 days), Sulfopin (median survival of 21 days), Palbociclib (median survival of 21 days) or their combination (median survival of 34.5 days), n=10 mice per group. **p, q,** Growth curve (p) and survival curve (q) generated from FVB mice bearing K14cre; *p53wt/f; Brcalwt/f*_BT3 tumors treated with vehicle (median survival of 19 days), Sulfopin (median survival of 25 days), Abemaciclib median survival of 25 days) or their combination (median survival of 55 days), n=10 mice per group. Data are mean ± s.e.m. P values were determined by two-sided unpaired student’s t-test or log-rank test. **P < 0.01, ***P < 0.001, **** P < 0.0001.

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Reciprocal inhibition of PIN1 and APC/C_CDH1 controls timely G1/S transition and creates therapeutic vulnerability

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Reciprocal inhibition of PIN1 and APC/C_CDH1 controls timely G1/S transition and creates therapeutic vulnerability

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