Abstract Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer and develop novel therapeutic modalities. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. Upon ligation of sialoglycan to ITIM-containing Siglecs on immune cells, this pathway suppresses multiple facets of anti-cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation of anti-cancer T cells. However, a therapeutic intervention of this axis remains a great challenge due to the overlapping and promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle and block the glyco-immune checkpoint axis, we describe here a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is antibody-like, multi-functional, and comprised of a tumor-associated antigen-binding moiety and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from sialoglycans on tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses using multiple human system models mimicking immunosuppressive tumor microenvironment and immunocompetent syngeneic mouse tumor models. EAGLE treatment released cancer cell-mediated immunosuppression, restored dendritic cell functions, enhanced CD8+ T-cell proliferation/activation, and induced proinflammatory cytokines IFNγ, IL-17A, IL-2, IL-6, and TNFα in human coculture assays of cancer cells with dendritic cells or PBMC in the presence or absence of primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to significant anti-tumor activities with complete regressions as monotherapy in syngeneic mouse tumor models. Re-challenge experiments in cured mice from the EAGLE treatment resulted in a complete rejection of tumor cells, demonstrating that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8+ T-cells decreased or abolished its efficacy. Moreover, EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in syngeneic EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutic modality inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies. Citation Format: Lizhi Cao, Adam Petrone, Wayne Gatlin, Jenny Che, Abhishek Das, Robert LeBlanc, Zakir Siddiquee, Sujata Nerle, Michal Stanczak, Michele Mayo, Lihui Xu, Karl Normington, Jeff Brown, Wei Yao, Carolyn Bertozzi, James Broderick, Heinz Läubli, Li Peng. A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-109.
Immune checkpoint blockade (ICB) has substantially improved the prognosis of patients with cancer, but the majority experiences limited benefit, supporting the need for new therapeutic approaches. Up-regulation of sialic acid–containing glycans, termed hypersialylation, is a common feature of cancer-associated glycosylation, driving disease progression and immune escape through the engagement of Siglec receptors on tumor-infiltrating immune cells. Here, we show that tumor sialylation correlates with distinct immune states and reduced survival in human cancers. The targeted removal of Siglec ligands in the tumor microenvironment, using an antibody-sialidase conjugate, enhanced antitumor immunity and halted tumor progression in several murine models. Using single-cell RNA sequencing, we revealed that desialylation repolarized tumor-associated macrophages (TAMs). We also identified Siglec-E as the main receptor for hypersialylation on TAMs. Last, we found that genetic and therapeutic desialylation, as well as loss of Siglec-E, enhanced the efficacy of ICB. Thus, therapeutic desialylation represents an immunotherapeutic approach to reshape macrophage phenotypes and augment the adaptive antitumor immune response.
Significance Understanding the genetic basis of human disease can reveal mechanisms of disease pathology and guide the design of novel treatment strategies. Here, we leverage insights from genetic studies to create a blueprint for treatment of inflammatory bowel disease (IBD). We demonstrate the feasibility of using small-molecule inhibitors to recapitulate the antiinflammatory function of CARD9 mutations associated with protection from IBD.
Abstract PD1/PD-L1 and CTLA-4 checkpoint blockade have revolutionized cancer therapy and led to cures in metastatic melanoma, but most patients develop primary and acquired resistance to these therapies. Treating this refractory population requires the discovery of new immune escape mechanisms. Sialic acid–binding immunoglobulin-type lectins (Siglecs) are expressed on the majority of white blood cells of the immune system, play critical roles in immune cell signaling and serve as immune checkpoints to prevent unwanted immune responses. Sialic acid is a ligand for inhibitory Siglecs; hypersialyation is a hallmark of poor prognosis and is believed to help tumors escape from immune surveillance. However, the role of hypersialylation in resistance to immune checkpoint therapies remains unexplored. To study if hypersialylation drives immune escape in melanoma, we profiled the immunosuppressive sialoglycans using Siglec-based high-affinity sialoglycan-binding constructs called ‘HYDRAs'. The current study focuses on understanding Siglec-3, -7 and -9 sialoglycan ligand expression on tumors using the HYDRA-3, -7 and -9 platform, because these Siglecs are the major inhibitory Siglecs on both innate and adaptive immune cells among the fourteen Siglecs in humans. Serial sections from melanoma tumors and healthy tissues were stained with HYDRA-3, -7 or -9 and scored using the semi-qualitative H-score method by a blinded pathologist. HYDRA IHC on healthy and cancerous human tissues demonstrate unique binding patterns with melanomas having high signals for HYDRA-3, -7 and -9. A pre-treatment checkpoint inhibitor therapy cohort (n=53), which contained responders (n=30) and non-responders (n=23) to either aPD1 or aPD1 and aCTLA-4 combination therapy was further studied. Serial sections from each patient was stained with HYDRA-3, -7 or -9 and scored using the semi-qualitative H-score method by our blinded pathologist. Cutoffs were determined in an unbiased manner for each HYDRA individually and each possible HYDRA combination to obtain correlations with patient progression-free and overall survival. A significant tumor H-score cutoff of a combined HYDRA-3 and -7 correlated with poor outcomes. This HYDRA-3 and -7 cutoff did not correlate with other melanoma biomarkers such as BRAF-mutation, liver metastases, PD-L1, nor TILs, suggesting a unique biology independent of these markers. We discovered that melanoma patients with multi-Siglec ligands as profiled by HYDRAs tend to be resistant to PD-1 checkpoint blockade and can be candidates for novel treatments targeting the Siglec-Sialoglycan axis. A larger cohort and longitudinal study are currently underway to examine the Siglec-Sialoglycan axis of immunosuppression in melanoma and late-breaking results will be included in this poster. Citation Format: Adam Petrone, Dennie T. Frederick, Jillian M. Prendergast, James Broderick, Karl Normington, Genevieve Boland, Li Peng. Melanoma patients with multi-Siglec ligands as profiled by HYDRA technology are refractory to PD1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 491.
Abstract Immune checkpoint blockade (ICB) has significantly improved the prognosis of cancer patients, but the majority experience limited benefit, evidencing the need for new therapeutic approaches. Upregulation of sialic acid-containing glycans, termed hypersialylation, is a common feature of cancer-associated glycosylation, driving disease progression and immune escape via the engagement of Siglec-receptors on tumor-infiltrating immune cells. Here, we show that tumor sialylation correlates with distinct immune states and reduced survival in human cancers. The targeted removal of Siglec-ligands in the tumor microenvironment, using an antibody-sialidase conjugate, enhances anti-tumor immunity and halts tumor progression in several mouse tumor models. Using single-cell RNA sequencing, we reveal desialylation mechanistically to repolarize tumor-associated macrophages (TAMs) and identify Siglec-E on TAMs as the main receptor for hypersialylation. Finally, we show genetic and therapeutic desialylation, as well as loss of Siglec-E, to synergize with ICB. Thus, therapeutic desialylation represents a novel immunotherapeutic approach, shaping macrophage phenotypes and augmenting the adaptive anti-tumor immune response.
Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer and develop novel therapeutic modalities. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. Upon ligation of sialoglycan to ITIM-containing Siglecs on immune cells, this pathway suppresses multiple facets of anti-cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation of anti-cancer T cells. However, a therapeutic intervention of this axis remains a great challenge due to the overlapping and promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle and block the glyco-immune checkpoint axis, we describe here a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is antibody-like, multi-functional, and comprised of a tumor-associated antigen-binding moiety and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from sialoglycans on tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses using multiple human system models mimicking immunosuppressive tumor microenvironment and immunocompetent syngeneic mouse tumor models. EAGLE treatment released cancer cell-mediated immunosuppression, restored dendritic cell functions, enhanced CD8+ T-cell proliferation/activation, and induced proinflammatory cytokines IFNγ, IL-17A, IL-2, IL-6, and TNFα in human coculture assays of cancer cells with dendritic cells or PBMC in the presence or absence of primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to significant anti-tumor activities with complete regressions as monotherapy in syngeneic mouse tumor models. Re-challenge experiments in cured mice from the EAGLE treatment resulted in a complete rejection of tumor cells, demonstrating that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8+ T-cells decreased or abolished its efficacy. Moreover, EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in syngeneic EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutic modality inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies.Citation Format: Lizhi Cao, Adam Petrone, Wayne Gatlin, Jenny Che, Abhishek Das, Robert LeBlanc, Zakir Siddiquee, Sujata Nerle, Michal Stanczak, Michele Mayo, Lihui Xu, Karl Normington, Jeff Brown, Wei Yao, Carolyn Bertozzi, James Broderick, Heinz Läubli, Li Peng. A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-109.
Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R2 value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R2 value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. 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Because of the essential role of Cdk1 in the regulation of mitotic progression and the relatively high rate at which cancer cells cycle, small molecule inhibitors of Cdk1 activity have been developed as cancer chemotherapeutics (35Gray N. Detivaud L. Doerig C. Meijer L. ATP-site directed inhibitors of cyclin-dependent kinases.Curr. Med. Chem. 1999; 6: 859-875Crossref PubMed Google Scholar, 36Shapiro G.I. Preclinical and clinical development of the cyclin-dependent kinase inhibitor flavopiridol.Clin. Cancer Res. 2004; 10: 4270s-4275sCrossref PubMed Scopus (185) Google Scholar, 37Shapiro G.I. Cyclin-dependent kinase pathways as targets for cancer treatment.J. of Clin. Oncol. 2006; 24: 1770-1783Crossref PubMed Scopus (0) Google Scholar). Flavopiridol, an ATP-competitive inhibitor that targets Cdk1, Cdk2, Cdk4, Cdk6, and Cdk9, was the first Cdk inhibitor to be tested in clinical trials (36Shapiro G.I. Preclinical and clinical development of the cyclin-dependent kinase inhibitor flavopiridol.Clin. Cancer Res. 2004; 10: 4270s-4275sCrossref PubMed Scopus (185) Google Scholar, 38Raju U. Nakata E. Mason K.A. Ang K.K. Milas L. Flavopiridol, a cyclin-dependent kinase inhibitor, enhances radiosensitivity of ovarian carcinoma cells.Cancer Res. 2003; 63: 3263-3267PubMed Google Scholar). It induces G1 and G2 cell cycle arrests due to inhibition of Cdk2, Cdk4, Cdk6, and Cdk1 resulting in cytostatic growth arrest instead of cell death (36Shapiro G.I. Preclinical and clinical development of the cyclin-dependent kinase inhibitor flavopiridol.Clin. Cancer Res. 2004; 10: 4270s-4275sCrossref PubMed Scopus (185) Google Scholar). However, treatment with Flavopiridol after taxane-induced mitotic arrest is synergistic, leading to cytotoxicity by potentially promoting exit from abnormal mitosis and induction of cell death (36Shapiro G.I. 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Phase I study of the cyclin-dependent kinase inhibitor flavopiridol in combination with paclitaxel in patients with advanced solid tumors.J. Clin. Oncol. 2002; 20: 2157-2170Crossref PubMed Scopus (145) Google Scholar, 42George S. Kasimis B.S. Cogswell J. Schwarzenberger P. Shapiro G.I. Fidias P. Bukowski R.M. Phase I study of flavopiridol in combination with Paclitaxel and Carboplatin in patients with non-small-cell lung cancer.Clin. Lung Cancer. 2008; 9: 160-165Abstract Full Text PDF PubMed Scopus (40) Google Scholar) have been conducted, but failed to show the same preclinical efficacy observed in both in vitro and in vivo models. Recently, an inhibitor with greater reported selectivity for Cdk1, RO-3306, was developed, which arrests cells at the G2/M transition and induces a rapid mitotic exit in cells in mitosis (43Vassilev L.T. Tovar C. Chen S. Knezevic D. Zhao X. Sun H. Heimbrook D.C. Chen L. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.Proc. Natl. Acad. Sci. U S A. 2006; 103: 10660-10665Crossref PubMed Scopus (544) Google Scholar). To increase our understanding of how mitotic progression is regulated by Cdk1-dependent phosphorylation pathways, we set out to identify Cdk1 substrates by combining quantitative phosphoproteomics and small molecule inhibitors of Cdk activity (40Shapiro G.I. Supko J.G. Patterson A. Lynch C. Lucca J. Zacarola P.F. Muzikansky A. Wright J.J. Lynch Jr., T.J. Rollins B.J. A phase II trial of the cyclin-dependent kinase inhibitor flavopiridol in patients with previously untreated stage IV non-small cell lung cancer.Clin. Cancer Res. 2001; 7: 1590-1599PubMed Google Scholar, 43Vassilev L.T. Tovar C. Chen S. Knezevic D. Zhao X. Sun H. Heimbrook D.C. Chen L. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.Proc. Natl. Acad. Sci. U S A. 2006; 103: 10660-10665Crossref PubMed Scopus (544) Google Scholar, 44Shapiro G.I. Cyclin-dependent kinase pathways as targets for cancer treatment.J. Clin. Oncol. 2006; 24: 1770-1783Crossref PubMed Scopus (852) Google Scholar, 45De Azevedo Jr., W.F. Mueller-Dieckmann H.J. Schulze-Gahmen U. Worland P.J. Sausville E. Kim S.H. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase.Proc. Natl. Acad. Sci. U S A. 1996; 93: 2735-2740Crossref PubMed Scopus (511) Google Scholar) in mitotically-arrested HeLa cells. HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (FBS; Hyclone, Logan, Utah) and penicillin-streptomycin (100 U/ml and 100 μg/ml; Gibco). Cells were incubated at 37 °C in a humidified chamber with 5% CO2. HeLa cells were grown in heavy or light DMEM (GIBCO) supplemented with 10% dialyzed FBS (Hyclone) and penicillin-streptomycin. "Heavy" media contained 100 mg/L 13C615N2-lysine and 100 mg/L 13C615N4-arginine (Cambridge Isotope Laboratories, Tewksbury, MA), whereas "light" media contained 100 mg/L 12C614N2-lysine and 100 mg/L 12C614N4-arginine (Sigma, St. Louis, MO). Cells were grown for a minimum of six doublings in the respective medium. To synchronize cells in mitosis, thymidine (1 mm, Sigma) was added for 22 h to both conditions, followed by a 3 h washout with PBS (Corning, Tewksbury, MA) and subsequent addition of taxol (Sigma) to both conditions for 16 h. Both heavy and light conditions were then treated with MG132 at a concentration of 10 μm for 30 min. After 30 min, either Flavopiridol (2 μm for 30 min) or RO-3306 (5 μm for 30 min) were added to the heavy condition. Heavy and light HeLa cells were counted, mixed 1:1, snap-frozen in liquid nitrogen, and stored at −80 °C until lysis. Experiments were performed in biological triplicates. Frozen cell pellets were partially thawed on ice and immediately resuspended in lysis buffer containing 9 m urea (Sigma), 50 mm Tris pH 8.1 (Sigma), 2 mm sodium beta-glycerophosphate (Sigma), 2 mm sodium fluoride (Sigma), 2 mm sodium molybdate (Sigma), 1 mm sodium orthovanadate (Sigma), and protease inhibitors (1 tablet per 10 ml of lysis buffer) (Mini-Complete, Roche, Indianapolis, IN). Cells were sonicated on ice with a Branson microtip sonicator three times at 10 s bursts. Approximately 200 μl of lysate was removed to measure total protein using a BCA protein assay kit (Thermo Fisher Scientific, Grand Island, NY). Reduction of the lysate was performed by adding 5 mm dithiothreitol (DTT) (Sigma) to the remaining lysate with a 50 °C incubation period for 30 min with occasional swirling to distribute the heat. The sample was removed and cooled to room temperature, and 15 mm iodoacetamide (Sigma) was added (final concentration) and the lysate was incubated in the dark for 60 mins. The alkylation reaction was quenched with 5 mm DTT for 15 min. The sample was diluted sevenfold in 25 mm Tris pH 8.1 and 1 μg of trypsin was added for every 200 μg of protein in lysate. Samples were incubated at 37 °C overnight. Trifluoroacetic acid was added to the digested peptide lysate to a final concentration of 0.25%, followed by centrifugation of the lysate at 3000 × g for 5 min. Peptides were desalted using a C18 solid-phase extraction (SPE) cartridge (SepPak, Waters, Milford, MA) and placed in a vacuum centrifuge for 45 min to evaporate the organic solvent. Finally, the samples were snap frozen in liquid nitrogen and lyophilized overnight. Phosphopeptide purification was performed using titanium dioxide microspheres essentially as described (46Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar). Lyophilized peptides were dissolved in 2 m lactic acid (Sigma)/50% acetonitrile in water (Honeywell Burdick & Jackson, Morris Plains, NJ) and added to ∼350 μg TiO2 microspheres. The mixture was incubated for 1 h with agitation (46Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar). The TiO2 microspheres were removed by centrifugation and washed three times with 2 m lactic acid/50% acetonitrile and two times with 50% acetonitrile/0.1% TFA. Phosphopeptides were eluted twice with 50 mm potassium phosphate (Sigma) pH-adjusted to 11 with 1 m ammonium hydroxide (Sigma), dried, and desalted. Strong-cation exchange chromatography was carried out as previously described (47Villen J. Beausoleil S.A. Gerber S.A. Gygi S.P. Large-scale phosphorylation analysis of mouse liver.Proc. Natl. Acad. Sci. U S A. 2007; 104: 1488-1493Crossref PubMed Scopus (627) Google Scholar). Fractions were collected, lyophilized, and desalted on a 96-well OASIS C18 HLB desalting plate (Waters). Phosphopeptides were analyzed on a Q-Exactive Plus hybrid quadrupole Orbitrap mass spectrometer (ThermoFisher Scientific) equipped with an Easy-nLC 1000 (ThermoFisher Scientific) and nanospray source (ThermoFisher Scientific). Peptides were resuspended in 5% methanol/1% formic acid and loaded on to a trap column (1 cm length, 100 μm inner diameter, ReproSil, C18 AQ 5 μm 120 Å pore (Dr. Maisch, Ammerbuch, Germany)) vented to waste via a micro-tee and eluted across a fritless analytical resolving column (35 cm length, 100 μm inner diameter, ReproSil, C18 AQ 3 μm 120 Å pore) pulled in-house (Sutter P-2000, Sutter Instruments, San Francisco, CA) with a 60 min gradient of 5–30% LC-MS buffer B (LC-MS buffer A: 0.0625% formic acid, 3% ACN; LC-MS buffer B: 0.0625% formic acid, 95% ACN). The Q-Exactive Plus was set to perform an Orbitrap MS1 scan (r = 70K; AGC target = 3e6) from 350–1500 Thomson, followed by HCD MS2 spectra on the 10 most abundant precursor ions detected by Orbitrap scanning (r = 17.5K; AGC target = 1e5; max ion time = 75ms) before repeating the cycle. Precursor ions were isolated for HCD by quadrupole isolation at width = 0.8 Thomson and HCD fragmentation at 26 normalized collision energy (NCE). Charge state 2, 3, and 4 ions were selected for MS2. Precursor ions were added to a dynamic exclusion list ± 20ppm for 20 s. Raw data were searched using COMET (release version 2014.01) in high resolution mode (48Eng J.K. Jahan T.A. Hoopmann M.R. Comet: an open-source MS/MS sequence database search tool.Proteomics. 2013; 13: 22-24Crossref PubMed Scopus (781) Google Scholar) against a target-decoy (reversed) (49Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2827) Google Scholar) version of the human proteome sequence database (UniProt; downloaded 2/2013, 40,482 entries of forward and reverse protein sequences) with a precursor mass tolerance of ± 1 Da and a fragment ion mass tolerance of 0.02 Da, and requiring fully tryptic peptides (Lys, Arg; not preceding Pro K,R; not preceding P) with up to three mis-cleavages. Static modifications included carbamidomethylcysteine and variable modifications included: oxidized methionine, heavy lysine and arginine, phosphorylated serine, threonine, and tyrosine. Searches were filtered using orthogonal measures including mass measurement accuracy (± 3ppm), Xcorr for charges from +2 through +4, and dCn targeting a <1% FDR at the peptide level. The probability of phosphorylation site localization was assessed using PhosphoRS (50Taus T. Kocher T. Pichler P. Paschke C. Schmidt A. Henrich C. Mechtler K. Universal and confident phosphorylation site localization using phosphoRS.J. Proteome Res. 2011; 10: 5354-5362Crossref PubMed Scopus (568) Google Scholar). Quantification of LC-MS/MS spectra was performed using MassChroQ (51Valot B. Langella O. Nano E. Zivy M. MassChroQ: a versatile tool for mass spectrometry quantification.Proteomics. 2011; 11: 3572-3577Crossref PubMed Scopus (171) Google Scholar). Phosphopeptide ratios were adjusted for mixing errors based on the median of the log2 H/L distribution. Phosphopeptides were filtered by their H/L log2 ratio averages <-1.4 and the corresponding p values <0.05, which were calculated using a two tailed Student's t test assuming unequal variance. These peptides were then subjected to motif determination using an in-house modified version of the MMFPh algorithm (52Wang T. Kettenbach A.N. Gerber S.A. Bailey-Kellogg C. MMFPh: a maximal motif finder for phosphoproteomics datasets.Bioinformatics. 2012; 28: 1562-1570Crossref PubMed Scopus (26) Google Scholar). Saccharomyces cerevisiae homologs of human candidate Cdk1 substrates were identified using Ensemble BioMart. UniProt accession numbers were mapped to Ensemble protein IDs using the UniProt conversion tool. In BioMart, Ensemble Genes 83 and homo sapiens genome (GRCh38.p5) were selected under databases. Ensemble protein IDs were input under filters, gene, and input into BioMart. Under "attributes," homologs, yeast orthologs, yeast protein ID were selected. Yeast IDs were compared with previously identified Cdk1 phosphorylation sites in yeast (53Holt L.J. Tuch B.B. Villen J. Johnson A.D. Gygi S.P. Morgan D.O. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution.Science. 2009; 325: 1682-1686Crossref PubMed Scopus (664) Google Scholar), human and yeast homologs were aligned with Blast, and investigated for site conservation. Protein-protein interactions of proteins belonging to phosphopeptides with significant increase in phosphorylation occupancy were determined using the STRING database and analyzed in Cytoscape (54Shannon P. Markiel A. Ozier O. Baliga N.S. Wang J.T. Ramage D.
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