Fig 2: 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.

Fig 3: Pharmacologic inhibition of PIN1 and CDK4 restores APC/CCDH1 E3 ligase activity inducing an insurmountable G1 arrest.a, Schematic diagrams showing the mechanism of APC/CCDH1 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.

Fig 4: Reciprocal inhibition of PIN1 and APC/CCDH1 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: Regulation of pyrimidine synthesis and CAD by cyclin D1. Hepatocytes were cultured as in Figure 3. A, amount of 13C-UXP (UMP, UDP, and UTP combined) at 5 and 6 positions by NMR. B, diagram of the pyrimidine synthesis pathway. C, abundance of 13C isotopologues of UTP by MS. D, concentration of N- carbamoyl-L-aspartate isotopologues by MS. E, abundance of 13C-aspartate by NMR (left) and MS (right). F, expression of total CAD, phospho-Ser1859 CAD, and ph-S6K1 by Western blot in AML12 hepatocytes. G, nonradioactive in vitro kinase assay using recombinant cyclin D1/Cdk4 along with CAD-HA-FLAG isolated from fasting mouse liver and ATPγS. Western blot was performed using an antibody to thiophosphate ester. H, Western blot analysis of resting (0 h) and regenerating liver 42 h after PH in control (GFP) or hepatocyte-specific KO (Cre) mice. The relative expression of total and phospho-Ser1859 CAD are shown. I, Phos-Tag Western blot of total and phospho-Ser1859 CAD from the PH model. Cyclin D1 KO (Cre) was associated with a less phosphorylated form of total and phospho-Ser1859 CAD compared to control mice (GFP). CAD, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; Cdk4, cyclin-dependent kinase 4; HA, hemagglutinin; MS, mass spectrometry; PH, partial hepatectomy.